CN118726215A - A recombinant microorganism producing pantoic acid and its application - Google Patents

Abstract

The present invention provides a genetically engineered pantoic acid producing strain with or having an enhanced NADH dependent acetylhydroxy acid reductase, a method of producing the same, a method of producing D-pantoic acid using the same, and use thereof in producing D-pantoic acid.

Description

A recombinant microorganism producing pantoic acid and its application

Technical Field

The present invention relates to the field of biotechnology, and in particular to a recombinant microorganism producing pantoic acid and a construction method thereof, as well as application of the recombinant microorganism in fermentation preparation of D-pantoic acid.

Background Art

Calcium pantothenate, also known as vitamin B5, is one of the 13 essential vitamins for the human body. It can only be synthesized in microorganisms and plants. It is an important precursor for the synthesis of coenzyme A and an essential vitamin for maintaining normal physiological functions of organisms. At the same time, calcium pantothenate is also widely used as a food additive and pharmaceutical raw material. At present, calcium pantothenate is mainly obtained by biological or chemical methods to split DL-pantolactone to obtain high-purity D-pantolactone and then react with β-alanine. Due to the use of highly toxic or strong acid/alkali reagents in the process of preparing pantolactone by petrochemical technology, the large amount of wastewater/gas generated in the production process, etc., which causes great damage to the environment, and the chiral removal of DL-pantolactone increases costs, etc., calcium pantothenate synthesis companies are often subject to environmental protection inspections, and the price of calcium pantothenate ranges from tens of thousands to hundreds of thousands of yuan per ton.

The rapid development of synthetic biology and metabolic engineering has made it possible to replace non-renewable petrochemical resources with renewable raw materials, replace petrochemical processes with green and environmentally friendly fermentation processes, and replace high-cost production of chemicals with low costs by designing and constructing microbial cell factories, and has achieved great success in many products.

Pantoic acid obtained through microbial fermentation is itself a high-purity D-pantoic acid. It does not need to be subsequently split and can be directly used to react with β-alanine to produce calcium pantothenate, which will greatly reduce the production cost and environmental pressure of calcium pantothenate. The synthesis of D-pantoic acid is also the limiting factor in the current industrial biosynthesis of calcium pantothenate. Escherichia coli has its own pantoic acid synthesis pathway, but due to the complex metabolic network regulation of the cell itself, wild-type cells have difficulty accumulating detectable concentrations of pantothenic acid.

Therefore, providing microbial cells that are genetically stable, can produce high-purity pantothenic acid through biofermentation using renewable resources as raw materials without the need for the addition of inducers and antibiotics will be of great significance for promoting the production of calcium pantothenate, reducing production costs, and alleviating environmental pressures.

Summary of the invention

In one aspect, the present invention provides a genetically engineered pantoate-producing strain having or having enhanced NADH-dependent acetohydroxyacid reductoisomerase.

In one embodiment, the present invention provides a genetically modified pantoate producing strain, which:

having or having increased activity of: acetolactate synthase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase, phosphoserine phosphatase, and NADH-dependent acetohydroxyacid reductoisomerase; and

Optionally, having reduced activity or inactivation of: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase and/or branched-chain amino acid aminotransferase; preferably, wherein the branched-chain amino acid aminotransferase is attenuated.

In one aspect, the present invention provides a method for producing a pantoate-producing strain, comprising imparting or enhancing the activity of NADH-dependent acetohydroxyacid reductoisomerase in the pantoate-producing strain.

In one embodiment, the present invention provides a method for producing a genetically modified pantothenate-producing strain, comprising conferring or enhancing in the strain: the activities of acetolactate synthase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase, phosphoserine phosphatase, and NADH-dependent acetohydroxyacid reductoisomerase; and

Optionally, the activity of L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase and/or branched-chain amino acid aminotransferase is reduced or inactivated; preferably, the activity of branched-chain amino acid aminotransferase is weakened.

In one aspect, the present invention provides a method for producing a genetically modified pantothenate-producing strain, comprising: replacing a gene encoding an NADPH-dependent acetohydroxyacid reductoisomerase with a gene encoding an NADH-dependent acetohydroxyacid reductoisomerase in Escherichia coli with a deposit number of CGMCC No. 21699 at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China.

In one aspect, the present invention provides a method for producing D-pantoic acid, comprising culturing the genetically modified pantoic acid-producing strain of the present invention or the genetically modified pantoic acid-producing strain prepared according to the method for producing a genetically modified pantoic acid-producing strain of the present invention under conditions suitable for the fermentation production of D-pantoic acid, optionally comprising isolating and purifying the produced D-pantoic acid.

In one aspect, the present invention provides use of the genetically modified pantoic acid-producing strain of the present invention or the genetically modified pantoic acid-producing strain prepared according to the method for producing a genetically modified pantoic acid-producing strain of the present invention in producing D-pantoic acid.

In one aspect, the present invention provides a method for improving the production of D-pantoic acid in a pantoic acid-producing strain, comprising: imparting or enhancing the activity of NADH-dependent acetohydroxyacid reductoisomerase in the pantoic acid-producing strain.

DETAILED DESCRIPTION

Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

As used herein, "genetically modified" refers to a strain artificially altered by biological means, which has one or more changes compared to the initial strain before modification, such as gene deletion, amplification or mutation, thereby having a changed biological property such as improved production performance. As used herein, the initial strain can be a natural strain to which the genetic modification is to be performed or a strain with other genetic modifications.

As used herein, the term "pantoate-producing strain" or "pantoate-producing strain" refers to a strain capable of producing D-pantoate that accumulates to a detectable level.

As used herein, the terms "polypeptide", "amino acid sequence", "peptide" and "protein" are used interchangeably herein to refer to amino acid chains of any length, which may contain modified amino acids and/or may be interrupted by non-amino acids. The term also encompasses amino acid chains modified by natural or human intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component.

As used herein, the expressions "gene", "nucleic acid sequence", "polynucleotide" and "nucleotide sequence" are used interchangeably and refer to nucleotide chains, including DNA and RNA. "Gene expression" refers to the transcription of a DNA region operably linked to an appropriate regulatory region, especially a promoter, into a biologically active RNA and the ability of the RNA to be translated into a biologically active protein or peptide.

As used herein, a degenerate sequence refers to a nucleotide sequence that encodes the same amino acid sequence as a specified sequence but has a different nucleotide sequence due to the degeneracy of the genetic code.

As used herein, the terms "homology", "sequence identity", etc. are used interchangeably herein. Sequence identity can be detected by comparing the number of identical nucleotide bases between a polynucleotide and a reference polynucleotide, for example, by a standard alignment algorithm program using a default gap penalty determined by each supplier. Whether two nucleic acid molecules have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" nucleotide sequences can be determined using known computer algorithms, such as BLASTN, FASTA, DNAStar and Gap (University of Wisconsin Genetics Computer Group (UWG), Madison WI, USA). For example, the percent identity of nucleic acid molecules can be determined, for example, by comparing sequence information using the GAP computer program (e.g., Needleman et al. J. Mol. Biol. 48:443 (1970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 (1981)). Briefly, the GAP program defines similarity based on the number of similar aligned symbols (i.e., nucleotides) divided by the total number of symbols in the shorter of the two sequences.

As used herein, acetolactate synthase (EC 2.2.1.6) is responsible for catalyzing two molecules of pyruvate to generate one acetolactate. There are acetolactate synthases from a variety of different sources in nature, such as three acetolactate synthases in Escherichia coli, namely acetolactate synthase I (encoded by the ilvBN gene), acetolactate synthase II (encoded by the ilvGM gene) and acetolactate synthase III (encoded by the ilvIH gene). In microorganisms such as Corynebacterium glutamicum, only one acetolactate synthase is contained, which is encoded by the ilvBN gene and encoded by the alsS gene in Bacillus subtilis. As used herein, having or having enhanced acetolactate synthase activity refers to that the strain has or has enhanced acetolactate synthase activity that converts pyruvate into acetolactate. Herein, L-valine feedback-resistant acetolactate synthase III refers to that the activity of the enzyme is not inhibited by L-valine or the degree of inhibition is reduced compared with wild-type acetolactate synthase III.

As used herein, acetohydroxy acid reductoisomerase (EC 1.1.1.382, EC 1.1.1.383, EC1.1.1.86) is the key enzyme responsible for catalyzing acetolactate to produce 2,3-dihydroxyisovalerate. The commonly used acetohydroxy acid reductoisomerases are mostly derived from microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. As used herein, having or having enhanced acetohydroxy acid reductoisomerase activity means that the strain has or has enhanced acetohydroxy acid reductoisomerase activity that converts acetolactate into 2,3-dihydroxyisovalerate.

As used herein, dihydroxy-acid dehydratase (EC 4.2.1.9) is the key enzyme responsible for catalyzing the conversion of 2,3-dihydroxyisovalerate into 2-ketoisovalerate. The commonly used dihydroxy-acid dehydratase is mostly derived from microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. As used herein, having or having enhanced dihydroxy-acid dehydratase activity refers to that the strain has or has enhanced dihydroxy-acid dehydratase activity that converts 2,3-dihydroxyisovalerate into 2-ketoisovalerate.

As used herein, 3-methyl-2-oxobutyrate hydroxymethyltransferase (EC 2.1.2.11) is responsible for converting 3-methyl-2-ketobutyrate into 2-dehydropantothenate in the presence of 5,10-methylenetetrahydrofolate. 3-methyl-2-oxobutyrate hydroxymethyltransferase is included in most microorganisms and plants, and the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene generally used is mostly derived from microorganisms such as Escherichia coli and Corynebacterium glutamicum. As used herein, having or having enhanced 3-methyl-2-oxobutyrate hydroxymethyltransferase activity refers to that the strain has or has enhanced 3-methyl-2-oxobutyrate hydroxymethyltransferase activity that converts 3-methyl-2-ketobutyrate into 2-dehydropantothenate.

As used herein, 2-dehydropantothenate-2-reductase (EC 1.1.1.169) is responsible for catalyzing the conversion of 2-dehydropantothenate to pantoic acid. 2-dehydropantothenate-2-reductase is contained in most microorganisms and plants. As used herein, having or having enhanced 2-dehydropantothenate-2-reductase activity means that the strain has or has enhanced activity of converting 2-dehydropantothenate to pantoic acid.

As used herein, serine hydroxymethyltransferase (EC 2.1.2.1) is encoded by glyA, which is responsible for catalyzing the production of glycine from L-serine and converting tetrahydrofolate into 5,10-methylenetetrahydrofolate. The enzyme is widely present in plants, animals and microorganisms. The serine hydroxymethyltransferase genes generally used are mostly derived from Escherichia coli, Corynebacterium glutamicum, yeast, etc. As used herein, having or having enhanced serine hydroxymethyltransferase activity refers to a strain having or having enhanced serine hydroxymethyltransferase activity that converts L-serine into glycine and simultaneously generates 5,10-methylenetetrahydrofolate.

As used herein, aminomethyltransferase (EC 2.1.2.10) is encoded by gcvT, glycine cleavage system H protein (EC1.4.1.27) is encoded by gcvH, and glycine decarboxylase (EC 1.4.1.27) is encoded by gcvP. In Escherichia coli, these three enzymes together with the lipoamide dehydrogenase subunit encoded by the lpdA gene constitute the glycine cleavage multienzyme system, which is responsible for catalyzing the cleavage of glycine to generate carbon dioxide while generating NADH and 5,10-methylenetetrahydrofolate. As used herein, having or having enhanced glycine cleavage enzyme system activity means that the strain has or has enhanced enzyme activity that cleaves glycine to generate carbon dioxide and generates NADH and 5,10-methylenetetrahydrofolate.

As used herein, phosphoglycerate dehydrogenase (EC 1.1.1.95) is encoded by the serA gene and catalyzes 3-phosphoglycerate to produce 3-phosphohydroxypyruvate. Herein, having or having enhanced phosphoglycerate dehydrogenase activity means that the strain has or has enhanced enzyme activity that catalyzes 3-phosphoglycerate to produce 3-phosphohydroxypyruvate.

As used herein, phosphoserine phosphorylase (EC 3.1.3.3) is encoded by the SerB gene and catalyzes phospho-L-serine to produce L-serine. Herein, having or having enhanced phosphoserine phosphorylase activity means that the strain has or has enhanced enzyme activity that catalyzes phospho-L-serine to produce L-serine.

As used herein, phosphoserine/phosphohydroxythreonine aminotransferase (EC 2.6.1.52) is encoded by the serC gene and catalyzes 3-phosphohydroxypyruvate to generate phospho-L-serine. Herein, having or having enhanced phosphoserine/phosphohydroxythreonine aminotransferase activity means that the strain has or has enhanced enzyme activity that catalyzes 3-phosphohydroxypyruvate to generate phospho-L-serine.

As used herein, L-serine deaminase I (EC 4.3.1.17) is encoded by the sdaA gene, and the enzyme catalyzes the deamination of L-serine to produce pyruvate. Herein, the reduced activity or inactivated L-serine deaminase I refers to the enzyme's reduced or lost activity in catalyzing the deamination of L-serine to produce pyruvate.

As used herein, acetate kinase (EC 2.7.2.1) is encoded by the ackA gene, and the enzyme catalyzes the conversion of acetyl phosphate to acetate while generating ATP. Herein, reduced activity or inactivated acetate kinase means that the activity of the enzyme in catalyzing the conversion of acetyl phosphate to acetate is reduced or lost.

As used herein, propionate kinase (EC 2.7.2.15) is encoded by the tdcD gene, and the enzyme catalyzes the reversible conversion between propionate and propionyl phosphate. Herein, reduced activity or inactivated propionate kinase means that the activity of the enzyme catalyzing the reversible conversion between propionate and propionyl phosphate is reduced or lost.

As used herein, formate acetyltransferase (EC 2.3.1.54) is encoded by the tdcE gene, and the enzyme catalyzes pyruvate to produce formate. Herein, the reduced activity or inactivated formate acetyltransferase means that the activity of the enzyme in catalyzing pyruvate to produce formate is reduced or lost.

As used herein, phosphate acetyltransferase (EC 2.3.1.8) is encoded by the pta gene and catalyzes the conversion of acetyl-CoA into acetyl phosphate. Herein, the reduced activity or inactivated phosphate acetyltransferase refers to a reduction or loss of the activity of the enzyme in catalyzing the conversion of acetyl-CoA into acetyl phosphate.

As used herein, alcohol dehydrogenase (EC 1.1.1.1) is encoded by adhE gene and catalyzes acetaldehyde to ethanol. Herein, alcohol dehydrogenase with reduced activity or inactivated refers to the enzyme having reduced or lost activity in catalyzing acetaldehyde to ethanol.

As used herein, pyruvate formate lyase (EC 2.3.1.54) is encoded by the pflB gene and catalyzes the cleavage of pyruvate to form formate and acetyl-CoA. Herein, reduced activity or inactivated pyruvate formate lyase means that the activity of the enzyme in catalyzing the cleavage of pyruvate to form formate and acetyl-CoA is reduced or lost.

As used herein, fumarate reductase (EC 1.3.5.1) is encoded by frdABCD and catalyzes fumarate to produce succinate. Herein, reduced activity or inactivated fumarate reductase means that the activity of the enzyme in catalyzing fumarate to produce succinate is reduced or lost.

As used herein, lactate dehydrogenase (EC 1.1.1.28) is encoded by ldhA and catalyzes pyruvate to produce D-lactate. Herein, the activity-reduced or inactivated lactate dehydrogenase means that the activity of the enzyme in catalyzing pyruvate to produce D-lactate is reduced or lost.

As used herein, methylglyoxal synthase (EC 4.2.3.3) is encoded by the mgsA gene and catalyzes dihydroxyacetone phosphate to produce methylglyoxal. Herein, reduced or inactivated methylglyoxal synthase means that the activity of the enzyme in catalyzing dihydroxyacetone phosphate to produce methylglyoxal is reduced or lost.

As used herein, ribokinase (EC 2.7.1.16) is encoded by the ara gene and catalyzes L-ribulose to generate 5-phosphate ribulose. Herein, reduced activity or inactivated ribokinase means that the activity of the enzyme in catalyzing L-ribulose to generate 5-phosphate ribulose is reduced or lost.

As used herein, valine-pyruvate aminotransferase (EC 2.6.1.66) is encoded by avtA gene, catalyzing L-alanine and 3-methyl-2-ketobutyrate to produce pyruvate and L-valine. Herein, reduced or inactivated valine-pyruvate aminotransferase refers to the enzyme catalyzing L-alanine and 3-methyl-2-ketobutyrate to produce pyruvate and L-valine activity reduction or loss.

As used herein, branched-chain amino acid aminotransferase (EC 2.6.1.42) is encoded by the ilvE gene, catalyzing the corresponding keto acid to generate three branched-chain amino acids, including L-valine, L-leucine and L-isoleucine. Herein, the reduced activity or inactivated branched-chain amino acid aminotransferase refers to the enzyme catalyzing the corresponding keto acid to generate three branched-chain amino acids. Activity is reduced or lost.

As used herein, "having an activity" means having a detectable activity compared to a reference (eg, an initial strain or a wild-type strain) that does not have the activity.

As used herein, "having an enhanced ... activity" means that the activity is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more compared to a reference (e.g., an initial strain or a wild-type strain) having the activity.

As used herein, "reduced activity or inactivation" means that the activity is reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more compared to a reference activity (e.g., the corresponding activity in an initial strain or a wild-type strain).

As used herein, "inactivated" means having no detectable activity compared to a reference activity (eg, a corresponding activity in an initial strain or a wild-type strain).

In this article, the reference can be a wild-type microorganism or a microorganism before the desired genetic manipulation (e.g., for performing genetic manipulation to increase the initial microorganism of gene activity). In this article, parental microorganism and initial microorganism are used interchangeably to refer to a microorganism to which a desired genetic manipulation (e.g., enhancing or weakening gene or protein activity) is performed.

The activity of a protein (e.g., an enzyme) can be produced or enhanced by any appropriate means known in the art, including, but not limited to, expressing or overexpressing (e.g., via a vector such as a plasmid) a corresponding gene encoding the protein in a strain, introducing a mutation that results in increased activity of the protein, etc.

The activity of a protein (e.g., an enzyme) can be reduced or inactivated by any appropriate means known in the art, including, but not limited to, using a weakened or inactivated corresponding gene encoding the protein, introducing a mutation that results in reduced or inactivated activity of the protein, and using an antagonist or inhibitor of the protein (e.g., an antibody, a ligand, etc.).

As used herein, "weakened or inactivated gene" refers to a gene activity, such as expression level (as a protein coding gene) or regulation performance (as a regulatory element) reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable, compared to a reference (e.g., a corresponding gene in an initial strain or wild-type strain). In the case of a gene encoding a protein, such as an enzyme, "weakened or inactivated gene" also encompasses a protein activity level expressed by the gene that is reduced, such as reduced by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even 100%, compared to the activity level of the corresponding protein in the initial strain or wild-type strain.

As used herein, "imparting an activity" means producing in a genetically engineered 2-hydroxyisovaleric acid-producing strain an activity that was not present in the original strain prior to genetic engineering.

As used herein, "enhancing the activity of" refers to increasing the activity, for example, by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more.

A variety of methods are known in the art for conferring or enhancing desired protein activity, including, for example, but not limited to, expression or overexpression of protein encoding genes and mutations or other modifications that increase protein activity.

As used herein, "overexpression" refers to an increase in the expression level of a gene relative to the level before genetic manipulation, such as an increase of at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300% or more. Methods for overexpressing genes are well known in the art, such as, but not limited to, using strong promoters, increasing gene copy number, enhancers, etc. Increasing gene copy number can be, for example, but not limited to, achieved by introducing one or more copies of an exogenous gene or endogenous gene, such as, for example, by an expression vector or by integration into a genome.

As used herein, "foreign gene" refers to a gene from another cell or organism, such as a gene from the same species or a different species.

As used herein, "endogenous gene" refers to a cell's or organism's own gene.

As used herein, reducing or inactivating the activity of a protein, such as an enzyme, refers to reducing the activity of the protein by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable. A variety of reduction or inactivation methods are known in the art, including, for example, inhibition of gene expression such as knockdown (knockdown) (e.g., using small interfering RNA), use of a weak promoter (when the gene is a polypeptide encoding gene), etc.; gene knockout, deletion of part or all of a gene or polypeptide sequence; mutation of certain sites in a gene or polypeptide, such as a coding sequence or an active domain, to reduce gene expression or regulate activity or the activity of an expression product; and use of antagonists or inhibitors (e.g., including but not limited to antibodies, interfering RNA, etc.).

As used herein, weakening or inactivating a gene refers to reducing the expression level of the gene (when it is a protein coding gene) or the regulatory performance (when it is a regulatory element) by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or more, or even undetectable. A variety of means of weakening or inactivating genes are known in the art, including, for example, inhibiting gene expression such as knocking down (e.g., using small interfering RNA), using a weak promoter (when the gene is a polypeptide coding gene), etc.; knocking out a gene, deleting part or all of a gene sequence; mutating certain sites in a gene, such as a coding sequence, to reduce gene expression or regulatory activity or the activity of an expression product, etc.

In one aspect, the present invention provides a genetically engineered pantoate-producing strain having or having enhanced NADH-dependent acetohydroxyacid reductoisomerase activity.

In one embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is derived from a microorganism, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. In a preferred embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity.

In one embodiment, the genetically modified pantoic acid producing strain expresses or overexpresses a gene encoding the NADH-dependent acetohydroxy acid reductoisomerase. In one embodiment, the gene encoding the NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO: 9, for example, comprising the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably under the control of a strong promoter such as the RBS5 artificial regulatory element (SEQ ID NO: 170).

Herein, the genetically modified pantoate-producing strain may contain, in addition to the NADH-dependent acetohydroxyacid reductoisomerase, other genetic modifications required for producing D-pantoate, such as modified enzymes or regulatory molecules involved in the pantoate production pathway.

In one embodiment, the genetically engineered pantothenate-producing strain further has or has enhanced activity of: acetolactate synthase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase, and phosphoserine phosphatase.

In one embodiment, the acetolactate synthase comprises an acetolactate synthase from Bacillus subtilis. In particular, the acetolactate synthase from Bacillus subtilis comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase activity.

In one embodiment, the acetolactate synthase comprises acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli. In one embodiment, the acetolactate synthase I comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase I activity. In one embodiment, the acetolactate synthase II comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase II activity. In one embodiment, the L-valine feedback-resistant acetolactate synthase III comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having L-valine feedback-resistant acetolactate synthase III activity.

In one embodiment, the acetolactate synthase includes acetolactate synthase from Bacillus subtilis, and acetolactate synthase I, acetolactate synthase II, and L-valine feedback-resistant acetolactate synthase III from Escherichia coli.

In one embodiment, the dihydroxyacid dehydratase, 2-dehydropantothenate-2-reductase, glycine lyase system (eg, aminomethyltransferase and/or glycine decarboxylase), phosphoserine/phosphohydroxythreonine aminotransferase, and phosphoserine phosphatase are from Escherichia coli.

In one embodiment, the dihydroxy-acid dehydratase comprises the amino acid sequence of SEQ ID NO:11, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has dihydroxy-acid dehydratase activity.

In one embodiment, the 2-dehydropantothenate-2-reductase comprises the amino acid sequence of SEQ ID NO:17, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has 2-dehydropantothenate-2-reductase activity.

In one embodiment, the aminomethyltransferase comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has aminomethyltransferase activity.

In one embodiment, the glycine decarboxylase comprises the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has glycine decarboxylase activity.

In one embodiment, the phosphoserine/phosphohydroxythreonine aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine/phosphohydroxythreonine aminotransferase activity.

In one embodiment, the phosphoserine phosphatase comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine phosphatase activity.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase includes 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum and/or Escherichia coli.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 13, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.

In one embodiment, the serine hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has serine hydroxymethyltransferase activity.

In one embodiment, the phosphoglycerate dehydrogenase is from Corynebacterium glutamicum. Particularly, the phosphoglycerate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homogeneity therewith and having phosphoglycerate dehydrogenase activity.

As used herein, the "enzyme activity having or having enhanced" means that in the genetically modified pantoic acid producing strain, each of the enzymes is independently active or has enhanced activity, i.e., one or more enzymes may be active, while others have enhanced activity, including all active or all have enhanced activity. In a preferred embodiment, the enzymes are all enhanced in activity.

In some embodiments, in the genetically modified pantoate producing strains described in the present invention, one or more copies of the target gene or its homologous gene can be integrated into the genome (e.g., by homologous recombination), optionally at any site in the genome, (as long as such integration does not significantly negatively affect the growth and production of the strain), for example, one copy of any gene in the genome is replaced by one or more copies of the target gene or its homologous gene. Those skilled in the art know how to integrate transgenes and select strains that have integrated transgenes.

In some embodiments, the activity of the enzyme is produced or enhanced by expressing or overexpressing genes encoding the enzyme in the strain, which genes can be inserted into the strain genome to encode genes that are not required for strain survival and production of pantoic acid, thereby obtaining a genetically modified strain that can produce pantoic acid. In some embodiments, these loci include but are not limited to loci encoding the following enzymes: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase, branched-chain amino acid aminotransferase.

In one embodiment, the gene encoding acetolactate synthase (for example, including a gene encoding acetolactate synthase from Bacillus subtilis, and/or a gene encoding acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli), a gene encoding an NADH-dependent acetohydroxyacid reductoisomerase from a Thermacetogenium phaeum strain, a gene encoding a dihydroxyacid dehydratase, a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum, a gene encoding a 2-dehydropantothenate-2-reductase, a gene encoding a serine hydroxymethyltransferase, a gene encoding a glycine lyase system (for example, a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase), a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Escherichia coli, a gene encoding a phosphoglycerate. The gene encoding oleate dehydrogenase, the gene encoding phosphoserine/phosphohydroxythreonine aminotransferase and/or the gene encoding phosphoserine phosphatase are incorporated into the genome of the genetically modified pantothenate-producing strain (e.g., Escherichia coli), for example, at the location of one or more of the loci encoding the following genes: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase.

In one embodiment, the genetically engineered pantothenate-producing strain further has reduced activity or inactivated: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase and/or branched-chain amino acid aminotransferase; preferably, the branched-chain amino acid aminotransferase is weakened.

As used herein, the "enzyme with reduced activity or inactivated" means that in the genetically modified pantothenate-producing strain, each of the enzymes independently has reduced activity or is inactivated (i.e., has no detectable activity), that is, one or more enzymes may have reduced activity while others are inactivated, including situations where all have reduced activity or are all inactivated.

As used herein, the "branched-chain amino acid aminotransferase is attenuated" or "attenuated branched-chain amino acid aminotransferase" means that the enzymatic activity of the branched-chain amino acid aminotransferase is reduced compared to the reference activity, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.

In one embodiment, the present invention provides a genetically modified pantoate producing strain, which:

having enhanced (e.g., overexpressed) activity of acetolactate synthase, NADH-dependent acetohydroxyacid reductoisomerase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase, and phosphoserine phosphatase;

have weakened branched-chain amino acid aminotransferase; and

Optionally, there is inactivation (e.g., knockout) of: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase and/or phosphate acetyltransferase.

In one embodiment, the weakened branched-chain amino acid aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has weakened branched-chain amino acid aminotransferase activity.

In one embodiment, the present invention provides a genetically modified pantoate producing strain, which:

Having enhanced: acetolactate synthase from Bacillus subtilis, acetolactate synthase I, acetolactate synthase II and L-valine feedback-resistant acetolactate synthase III from Escherichia coli, NADH-dependent acetohydroxyacid reductoisomerase from Thermacetogenium phaeum, dihydroxyacid dehydratase, 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum and 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (such as aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase from Corynebacterium glutamicum, phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase activity;

Optionally, having inactivated (e.g., knocked out), if present: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, and/or phosphate acetyltransferase; and

Has weakened branched-chain amino acid aminotransferase.

In one embodiment, the genetically engineered pantoate-producing strain:

Expressing a gene encoding a weakened branched-chain amino acid aminotransferase, such as the weakened branched-chain amino acid aminotransferase encoding gene shown in SEQ ID NO: 32; and/or

Overexpressed genes: acetolactate synthase from Bacillus subtilis, acetolactate synthase I from Escherichia coli, acetolactate synthase II from Escherichia coli, acetolactate synthase III from Escherichia coli with L-valine feedback resistance, NADH-dependent acetohydroxyacid reductoisomerase from Thermacetogeniumphaeum strain, dihydroxyacid dehydratase, 3-carboxylic acid synthase from Corynebacterium glutamicum, a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase system, a gene encoding 3-methyl-2-oxobutyrate hydroxymethyltransferase from Escherichia coli, a gene encoding phosphoglycerate dehydrogenase, a gene encoding phosphoserine/phosphohydroxythreonine aminotransferase, and a gene encoding phosphoserine phosphatase, and/or a gene encoding a phosphoserine phosphatase.

The gene encoding one or more, preferably all, of the following enzymes is not present or the endogenous gene encoding one or more, preferably all, of the following enzymes is knocked out: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase and phosphate acetyltransferase.

In one embodiment, the gene encoding the acetolactate synthase from Bacillus subtilis encodes the acetolactate synthase shown in SEQ ID NO: 1, for example, comprises the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter, such as the M1-93 promoter (SEQ ID NO: 169).

In one embodiment, the gene encoding acetolactate synthase I from Escherichia coli encodes the large subunit ilvB of acetolactate synthase I shown in SEQ ID NO:3, for example, comprises the nucleotide sequence shown in SEQ ID NO:4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter such as the M1-93 promoter (SEQ ID NO:169).

In one embodiment, the gene encoding acetolactate synthase II from Escherichia coli encodes the acetolactate synthase II large subunit ilvG shown in SEQ ID NO:5, for example, comprises the nucleotide sequence shown in SEQ ID NO:6 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter such as the M1-93 promoter (SEQ ID NO:169).

In one embodiment, the gene encoding L-valine feedback-resistant acetolactate synthase III from Escherichia coli encodes the L-valine feedback-resistant acetolactate synthase III shown in SEQ ID NO: 7, for example, comprises the nucleotide sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the gene encoding NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO:9, for example, comprises the nucleotide sequence shown in SEQ ID NO:10 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as the RBS5 artificial regulatory element (SEQ ID NO:170).

In one embodiment, the gene encoding the dihydroxy-acid dehydratase encodes the dihydroxy-acid dehydratase shown in SEQ ID NO: 11, for example, comprises the nucleotide sequence shown in SEQ ID NO: 12 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as RBSL1 (SEQ ID NO: 171).

In one embodiment, the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum encodes the 3-methyl-2-oxobutanoate hydroxymethyltransferase shown in SEQ ID NO: 13, for example, comprising the nucleotide sequence shown in SEQ ID NO: 14 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli encodes the amino acid sequence shown in SEQ ID NO: 15, for example, comprises the nucleotide sequence shown in SEQ ID NO: 16 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding 2-dehydropantothenate-2-reductase encodes the 2-dehydropantothenate-2-reductase shown in SEQ ID NO:17, for example, comprises the nucleotide sequence shown in SEQ ID NO:18 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as RBSL2 (SEQ ID NO:172).

In one embodiment, the gene encoding serine hydroxymethyltransferase encodes the serine hydroxymethyltransferase shown in SEQ ID NO: 19, for example, comprises the nucleotide sequence shown in SEQ ID NO: 20 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-46 (SEQ ID NO: 173).

In one embodiment, the gene encoding the aminomethyltransferase encodes the aminomethyltransferase shown in SEQ ID NO:21, for example, comprises the nucleotide sequence shown in SEQ ID NO:22 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding glycine decarboxylase encodes the glycine decarboxylase shown in SEQ ID NO:23, for example, comprises the nucleotide sequence shown in SEQ ID NO:24 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding phosphoglycerate dehydrogenase encodes the phosphoglycerate dehydrogenase shown in SEQ ID NO:25, for example, comprises the nucleotide sequence shown in SEQ ID NO:26 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding phosphoserine/phosphohydroxythreonine aminotransferase encodes the phosphoserine/phosphohydroxythreonine aminotransferase shown in SEQ ID NO: 27, for example, comprises the nucleotide sequence shown in SEQ ID NO: 28 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding the phosphoserine phosphatase encodes the phosphoserine phosphatase shown in SEQ ID NO:29, for example, comprises the nucleotide sequence shown in SEQ ID NO:30 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the genetically engineered pantoate-producing (eg, E. coli) strain:

expressing an enzyme comprising the amino acid sequence shown in SEQ ID NO: 31, for example, expressing a gene comprising the nucleotide sequence shown in SEQ ID NO: 32;

Overexpression of enzymes comprising the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, respectively, such as overexpression of genes comprising the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, respectively; and

Optionally, there is no gene encoding one or more, preferably all of the following enzymes or endogenous genes encoding one or more, preferably all of the following enzymes are knocked out: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase, phosphate acetyltransferase.

In one embodiment, the pantoate-producing strain is selected from the group consisting of Escherichia, Enterobacter, Corynebacterium glutamicum, Bacillus subtilis and yeast, preferably Escherichia coli.

In one embodiment, the genetically modified pantoate-producing strain is Escherichia coli deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC) in Beijing, China, with the deposit number of CGMCC No. 26276.

In one aspect, the present invention provides a method for producing a genetically modified pantoate-producing strain, comprising: imparting or enhancing NADH-dependent acetohydroxyacid reductoisomerase activity in the pantoate-producing strain.

In one embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is derived from a microorganism, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. In a preferred embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity.

In one embodiment, the method comprises expressing or overexpressing a gene encoding the NADH-dependent acetohydroxy acid reductoisomerase in a pantoic acid producing strain. In one embodiment, the gene encoding the NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO: 9, for example, comprising the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably under the control of a strong promoter such as the RBS5 artificial regulatory element (SEQ ID NO: 170).

In one embodiment, the method comprises, in a pantoate-producing strain: expressing or overexpressing a gene encoding the NADH-dependent acetohydroxyacid reductoisomerase.

The pantoate-producing strain may be a strain known in the art or a strain obtainable through technical knowledge in the art.

In addition to conferring or enhancing the NADH-dependent acetohydroxy acid reductoisomerase, the method may also include performing other genetic modifications required for producing D-pantoic acid in the genetically modified pantoic acid-producing strain, such as modifying other enzymes or regulatory molecules involved in the pantoic acid production pathway. For example, other genetic modifications and conferring or enhancing the NADH-dependent acetohydroxy acid reductoisomerase may be performed in a suitable strain (e.g., a strain that does not produce a detectable amount of D-pantoic acid accumulation) to produce a detectable amount of D-pantoic acid accumulation.

For example, in one embodiment, the present invention provides a method for producing a genetically modified pantothenate-producing strain, comprising: conferring or enhancing in the strain: NADH-dependent acetohydroxyacid reductoisomerase, acetolactate synthetase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase activities.

In one embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is derived from a microorganism, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. In a preferred embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity.

In one embodiment, the method comprises expressing or overexpressing a gene encoding the NADH-dependent acetohydroxy acid reductoisomerase in a pantoic acid producing strain. In one embodiment, the gene encoding the NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO: 9, for example, comprising the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably under the control of a strong promoter such as the RBS5 artificial regulatory element (SEQ ID NO: 170).

In one embodiment, the acetolactate synthase comprises an acetolactate synthase from Bacillus subtilis. In particular, the acetolactate synthase comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase activity.

In one embodiment, the acetolactate synthase comprises acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli. In one embodiment, the acetolactate synthase I comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase I activity. In one embodiment, the acetolactate synthase II comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase II activity. In one embodiment, the L-valine feedback-resistant acetolactate synthase III comprises the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having L-valine feedback-resistant acetolactate synthase III activity.

In one embodiment, the method comprises conferring or enhancing the activities of acetolactate synthase from Bacillus subtilis and acetolactate synthase I, acetolactate synthase II and L-valine feedback-resistant acetolactate synthase III from Escherichia coli in the strain.

In one embodiment, the dihydroxyacid dehydratase, 2-dehydropantothenate-2-reductase, glycine lyase system (eg, aminomethyltransferase and/or glycine decarboxylase), phosphoserine/phosphohydroxythreonine aminotransferase, and phosphoserine phosphatase are from Escherichia coli.

In one embodiment, the dihydroxy-acid dehydratase comprises the amino acid sequence of SEQ ID NO:11, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has dihydroxy-acid dehydratase activity.

In one embodiment, the 2-dehydropantothenate-2-reductase comprises the amino acid sequence of SEQ ID NO:17, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has 2-dehydropantothenate-2-reductase activity.

In one embodiment, the serine hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has serine hydroxymethyltransferase activity.

In one embodiment, the aminomethyltransferase comprises the amino acid sequence shown in SEQ ID NO:21, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has aminomethyltransferase activity.

In one embodiment, the glycine decarboxylase comprises the amino acid sequence shown in SEQ ID NO:23, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has glycine decarboxylase activity.

In one embodiment, the phosphoserine/phosphohydroxythreonine aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 27 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine/phosphohydroxythreonine aminotransferase activity.

In one embodiment, the phosphoserine phosphatase comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine phosphatase activity.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase includes 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum and/or Escherichia coli.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase is from Corynebacterium glutamicum. In particular, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homogeneity therewith and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.

In one embodiment, the 3-methyl-2-oxobutanoate hydroxymethyltransferase is from Escherichia coli. In particular, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.

In one embodiment, the phosphoglycerate dehydrogenase is from Corynebacterium glutamicum. Particularly, the phosphoglycerate dehydrogenase comprises the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homogeneity therewith and having phosphoglycerate dehydrogenase activity.

As used herein, the term "conferring or enhancing" means that in the pantoic acid producing strain, one or more of the enzymes have an activity, while the activity of another enzyme is enhanced, including the case where all enzymes have enhanced activity. In a preferred embodiment, all enzymes have enhanced activity.

To confer or enhance one or more enzyme activities, one or more copies of the target gene or its homologous gene can be integrated into the genome (e.g., by homologous recombination), optionally at any site in the genome, (as long as such integration does not significantly negatively affect the growth of the strain and the production of desired compounds such as D-pantoic acid), for example, one copy of any gene in the genome is replaced by one or more copies of the target gene or its homologous gene. Those skilled in the art know how to integrate transgenes and select the locus position of the desired transgene for integration in the strain.

Genes encoding enzymes described herein are expressed or overexpressed to produce or enhance the activity of the enzymes, which can be inserted into the gene loci encoding the strain genome that are not required for strain survival and production of pantoic acid, thereby obtaining genetically modified strains that can produce pantoic acid. These loci include but are not limited to the loci encoding the following enzymes: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase, branched-chain amino acid aminotransferase.

In one embodiment, the method comprises further reducing or inactivating the activity of one or more of the following enzymes: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase, branched-chain amino acid aminotransferase; preferably, the enzymatic activity of branched-chain amino acid aminotransferase is weakened.

As used herein, the "reduction or inactivation" means that in the genetically modified pantoate-producing strain, if the enzyme exists, the activity of the enzyme is reduced or inactivated (i.e., to undetectable activity), that is, one or more enzymes may have reduced activity while others are inactivated, including situations where all have reduced activity or all are inactivated.

In one embodiment, the attenuated branched-chain amino acid aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 31, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has attenuated branched-chain amino acid aminotransferase activity.

In one embodiment, the method comprises:

Expression or overexpression, preferably overexpression: a gene encoding the NADH-dependent acetohydroxyacid reductoisomerase, a gene encoding acetolactate synthase, a gene encoding a dihydroxyacid dehydratase, a gene encoding 3-methyl-2-oxobutyrate hydroxymethyltransferase, a gene encoding 2-dehydropantothenate-2-reductase, a gene encoding a serine hydroxymethyltransferase, a gene encoding a glycine lyase system (e.g., a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase), a gene encoding a phosphoglycerate dehydrogenase, a gene encoding a phosphoserine/phosphohydroxythreonine aminotransferase, and a gene encoding a phosphoserine phosphatase;

Optionally, genes encoding one or more, preferably all, of the following enzymes are knocked out, if present: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase, phosphate acetyltransferase.

In one embodiment, the method of producing a genetically engineered pantoate-producing strain comprises, in the strain:

Expression of a weakened branched-chain amino acid aminotransferase, for example, expression of the protein shown in SEQ ID NO: 31, in particular, expression of a gene comprising the nucleotide sequence shown in SEQ ID NO: 32 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto; and/or

Overexpression: Gene encoding acetolactate synthase from Bacillus subtilis, gene encoding acetolactate synthase I from Escherichia coli, gene encoding acetolactate synthase II from Escherichia coli, gene encoding L-valine feedback-resistant acetolactate synthase III from Escherichia coli, gene encoding acetolactate synthase II from Thermacetogenium phaeum strain, a gene encoding a NADH-dependent acetohydroxyacid reductoisomerase, a gene encoding a dihydroxyacid dehydratase, a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum, a gene encoding a 2-dehydropantothenate-2-reductase, a gene encoding a serine hydroxymethyltransferase, a gene encoding a glycine lyase system (e.g., a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase), a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Escherichia coli, a gene encoding a phosphoglycerate dehydrogenase, a gene encoding a phosphoserine/phosphohydroxythreonine aminotransferase, and a gene encoding a phosphoserine phosphatase, and/or

Optionally, the genes encoding one or more, preferably all, of the following enzymes are knocked out, if present: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase, phosphate acetyltransferase.

In one embodiment, the gene encoding the acetolactate synthase from Bacillus subtilis encodes the acetolactate synthase shown in SEQ ID NO: 1, for example, comprises the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter, such as the M1-93 promoter (SEQ ID NO: 169).

In one embodiment, the gene encoding acetolactate synthase I from Escherichia coli encodes the large subunit ilvB of acetolactate synthase I shown in SEQ ID NO:3, for example, comprises the nucleotide sequence shown in SEQ ID NO:4 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter such as the M1-93 promoter (SEQ ID NO:169).

In one embodiment, the gene encoding acetolactate synthase II from Escherichia coli encodes the acetolactate synthase II large subunit ilvG shown in SEQ ID NO:5, for example, comprises the nucleotide sequence shown in SEQ ID NO:6 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and is preferably placed under the control of a strong promoter such as the M1-93 promoter (SEQ ID NO:169).

In one embodiment, the gene encoding L-valine feedback-resistant acetolactate synthase III from Escherichia coli encodes the L-valine feedback-resistant acetolactate synthase III shown in SEQ ID NO: 7, for example, comprises the nucleotide sequence shown in SEQ ID NO: 8 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the gene encoding NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO:9, for example, comprises the nucleotide sequence shown in SEQ ID NO:10 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as the RBS5 artificial regulatory element (SEQ ID NO:170).

In one embodiment, the gene encoding the dihydroxy-acid dehydratase encodes the dihydroxy-acid dehydratase shown in SEQ ID NO: 11, for example, comprises the nucleotide sequence shown in SEQ ID NO: 12 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as RBSL1 (SEQ ID NO: 171).

In one embodiment, the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum encodes the 3-methyl-2-oxobutanoate hydroxymethyltransferase shown in SEQ ID NO: 13, for example, comprising the nucleotide sequence shown in SEQ ID NO: 14 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli encodes the 3-methyl-2-oxobutanoate hydroxymethyltransferase shown in SEQ ID NO: 15, for example, comprises the nucleotide sequence shown in SEQ ID NO: 16 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding 2-dehydropantothenate-2-reductase encodes the 2-dehydropantothenate-2-reductase shown in SEQ ID NO:17, for example, comprises the nucleotide sequence shown in SEQ ID NO:18 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as RBSL2 (SEQ ID NO:172).

In one embodiment, the gene encoding serine hydroxymethyltransferase encodes the serine hydroxymethyltransferase shown in SEQ ID NO: 19, for example, comprises the nucleotide sequence shown in SEQ ID NO: 20 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-46 (SEQ ID NO: 173).

In one embodiment, the gene encoding the aminomethyltransferase encodes the aminomethyltransferase shown in SEQ ID NO:21, for example, comprises the nucleotide sequence shown in SEQ ID NO:22 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding glycine decarboxylase encodes the glycine decarboxylase shown in SEQ ID NO:23, for example, comprises the nucleotide sequence shown in SEQ ID NO:24 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding phosphoglycerate dehydrogenase encodes the phosphoglycerate dehydrogenase shown in SEQ ID NO:25, for example, comprises the nucleotide sequence shown in SEQ ID NO:26 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the gene encoding phosphoserine/phosphohydroxythreonine aminotransferase encodes the phosphoserine/phosphohydroxythreonine aminotransferase shown in SEQ ID NO: 27, for example, comprises the nucleotide sequence shown in SEQ ID NO: 28 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO: 169).

In one embodiment, the gene encoding the phosphoserine phosphatase encodes the phosphoserine phosphatase shown in SEQ ID NO:29, for example, comprises the nucleotide sequence shown in SEQ ID NO:30 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, preferably under the control of a strong promoter such as M1-93 (SEQ ID NO:169).

In one embodiment, the method comprises in a pantoate producing (eg, E. coli) strain:

expressing an enzyme comprising the amino acid sequence shown in SEQ ID NO: 31, for example, expressing a gene comprising the nucleotide sequence shown in SEQ ID NO: 32;

Overexpression of enzymes comprising the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29, respectively, such as overexpression of genes comprising the nucleotide sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, respectively; and

Optionally, genes encoding the following enzymes are knocked out: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase and/or phosphate acetyltransferase.

In one embodiment, the method is to carry out the steps of imparting or enhancing the enzyme activity and optionally reducing or inactivating the enzyme activity in a strain selected from the genus Escherichia, Enterobacter, Corynebacterium glutamicum, Bacillus subtilis and yeast to obtain the genetically modified pantoate-producing strain.

In one embodiment, the method comprises carrying out the steps of imparting or enhancing enzyme activity and reducing or inactivating enzyme activity in an Escherichia coli strain such as Escherichia coli ATCC 8739 to obtain the genetically modified pantoate-producing strain.

In one embodiment, the method comprises replacing a gene encoding a NADPH-dependent acetohydroxy acid reductoisomerase (e.g., as shown in SEQ ID NO: 167) with a gene encoding a NADH-dependent acetohydroxy acid reductoisomerase in Escherichia coli with a deposit number of CGMCC No. 21699 in Beijing, China, to obtain the genetically modified pantoate-producing strain. Preferably, the NADH-dependent acetohydroxy acid reductoisomerase is from a Thermacetogenium phaeum strain, for example, comprising an amino acid sequence as shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity.

In one embodiment, the gene encoding NADH-dependent acetohydroxyacid reductoisomerase comprises the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the genetically modified pantoate-producing strain obtained by the method is Escherichia coli deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC) in Beijing, China, with a deposit number of CGMCC No. 26276.

In one aspect, the present invention provides a method for producing D-pantoic acid, comprising culturing the genetically modified pantoic acid-producing strain of the present invention or the genetically modified pantoic acid-producing strain prepared according to the method for producing a genetically modified pantoic acid-producing strain of the present invention under conditions suitable for the fermentation production of D-pantoic acid, optionally comprising isolating and purifying the produced D-pantoic acid.

The conditions for fermentation and culturing pantoic acid-producing strains for the production of D-pantoic acid are known in the art, including, for example, but not limited to, pH, temperature, medium components, fermentation time, and the like.

Temperatures for fermentation of D-pantoic acid by pantoic acid producing strains are known in the art, such as about 25-37° C., such as about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C. In one embodiment, the pantoic acid producing yeast strain of the present invention is fermented at 37° C. to produce D-pantoic acid.

The pantoic acid producing strain of the present invention can be fermented at a suitable pH value known in the art, such as pH about 6.5-7.5. In one embodiment, the pantoic acid producing strain of the present invention is fermented at pH about 7.0 to produce D-pantoic acid.

To produce D-pantoic acid, the pantoic acid-producing strain of the present invention can be fermented for a suitable time, for example, about 12-96 hours, such as about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 96, about 120, about 144, about 168 hours.

In one aspect, the present invention provides the use of the genetically modified pantoic acid producing strain of the present invention or the genetically modified pantoic acid producing strain prepared according to the method for producing a genetically modified pantoic acid producing strain of the present invention in producing D-pantoic acid.

In one aspect, the present invention provides a method for improving the production of D-pantoic acid in a pantoic acid-producing strain, comprising: imparting or enhancing the activity of NADH-dependent acetohydroxyacid reductoisomerase in the pantoic acid-producing strain.

In one embodiment, the pantoate-producing strain does not have an NADH-dependent acetohydroxyacid reductoisomerase, for example, has an NADPH-dependent acetohydroxyacid reductoisomerase.

In one embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is derived from a microorganism, such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, etc. In a preferred embodiment, the NADH-dependent acetohydroxy acid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity.

In one embodiment, the method comprises replacing the NADPH-dependent acetohydroxy acid reductoisomerase in a pantoate-producing strain having an NADPH-dependent acetohydroxy acid reductoisomerase with an NADH-dependent acetohydroxy acid reductoisomerase, thereby improving the D-pantoate production capacity of the strain. The replacement can be performed using any suitable method known in the art, such as by homologous recombination, using a gene encoding the NADH-dependent acetohydroxy acid reductoisomerase to replace the gene encoding the NADPH-dependent acetohydroxy acid reductoisomerase in the strain.

The gene encoding the NADH-dependent acetohydroxy acid reductoisomerase introduced into the strain can be placed under the control of an appropriate promoter, such as a strong promoter (e.g., RBS5 artificial regulatory element (SEQ ID NO: 170)), so that the gene is expressed or overexpressed in the strain, preferably overexpressed. In one embodiment, the gene encoding the NADH-dependent acetohydroxy acid reductoisomerase encodes the NADH-dependent acetohydroxy acid reductoisomerase shown in SEQ ID NO: 9, for example, comprising the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto.

In one embodiment, the method includes replacing the NADPH-dependent acetohydroxy acid reductoisomerase encoding gene of Escherichia coli deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee (CGMCC) in Beijing, China with the deposit number CGMCC No. 21699 with a gene encoding NADH-dependent acetohydroxy acid reductoisomerase to improve the pantoate production of the Escherichia coli of CGMCC No. 21699.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. For example, an optionally included step means that the step exists or does not exist.

As used herein, the term "about" refers to a numerical range that includes a specific value that a person skilled in the art would reasonably consider to be similar to the specific value. In some embodiments, the term "about" refers to within the standard error of measurement using commonly accepted measurements in the art. In some embodiments, approximately refers to +/-10% of a specific value.

When "one or more" is mentioned herein, it should be understood that various specific combinations are disclosed accordingly. For example, one or more of A, B, and C should be considered to disclose specific specific combinations such as A, B, C, AB, AC, BC, and ABC.

Ranges disclosed herein should be considered to have specifically disclosed all possible subranges and individual numerical values within the range. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed subranges from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6.

Description of biological material deposit

Classification name: Escherichia coli

Strain ID: Span050

Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

Abbreviation of depository unit: CGMCC

Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

Date of deposit: January 22, 2021

CGMCC Registration Number: CGMCC No.21699

Classification name: Escherichia coli

Strain ID: Span096

Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

Abbreviation of depository unit: CGMCC

Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

Date of deposit: December 26, 2022

CGMCC Registration Number: CGMCC No.26276

Example

The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In combination with this specification and common knowledge in the art, a person of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, a person of ordinary skill in the art can make any modification or change to the technical solution of the present invention, and such modification and change are also included in the scope of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

Table 1: Strains and plasmids used in the present invention

Note: In Table 1, ATCC 8739, M1-93 and M1-46 are all Escherichia coli. Table 2: Primers used in the present invention

Hereinafter, two primers separated by "/" are used to form a corresponding primer pair for amplifying the target fragment.

Example 1: Insertion of the acetolactate synthase gene alsS into the ATCC 8739 strain at the loci of the propionate kinase encoding gene tdcD and the formate acetyltransferase encoding gene tdcE and knockout of the tdcDE operon

Starting from Escherichia coli ATCC 8739, the acetolactate synthase gene alsS from Bacillus subtilis 168 (from ATCC, No. 23857) was inserted into the propionate kinase encoding gene tdcD and formate acetyltransferase encoding gene tdcE on the chromosome using a two-step homologous recombination method. The specific steps are as follows:

In the first step, pXZ-CS (Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844) plasmid DNA was used as a template and primers tdcDE-incs-up/tdcDE-incs-down were used to amplify a 2719 bp DNA fragment I, which contained a 50 bp upstream homologous arm of tdcDE, a 2619 bp cat-sacB fragment of chloramphenicol gene (cat) and fructan sacrose transferase gene (sacB) DNA, and a 50 bp downstream homologous arm of tdcDE gene for the first step of homologous recombination.

The amplification system was: Phusion 5X buffer (New England Biolabs) 10 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phusion High-Fidelity DNA polymerase (2.5 U/μl) 0.5 μl, distilled water 33.5 μl, and the total volume was 50 μl.

The amplification conditions were pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 2 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

The above DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (CGSC Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Escherichia coli ATCC 8739 by electroporation, and then the DNA fragment I was electroporated into Escherichia coli ATCC 8739 carrying pKD46.

The electroporation conditions are as follows: first prepare electroporation competent cells of Escherichia coli ATCC 8739 carrying the pKD46 plasmid (preparation method according to Dower et al., 1988, Nucleic Acids Res 16: 6127-6145); place 50 μl of competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of bacterial solution and apply it on LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select single colonies for PCR verification. The primers used are XZ-tdcDE-up/XZ-tdcDE-down. The amplification product of the correct colony is a 3615 bp fragment, including 845 bp of tdcDE upstream homology arm, 2619 bp of cat-sacB fragment and 151 bp of tdcDE downstream homology arm. Select a correct single colony and name it Span001.

In the second step, wild-type Bacillus subtilis 168 genomic DNA was used as a template and primers tdcDE-alsSin-up/tdcDE-alsSin-down were used to amplify a 1826 bp DNA fragment II, including a 50 bp upstream homology arm of tdcDE, 1716 bp of alsS gene and a total of 10 bp of the restriction site and protective base of sacI, and a 50 bp downstream homology arm of tdcDE. DNA fragment II was used for the second homologous recombination. The amplification conditions and system were the same as those described in the first step. DNA fragment II was electroporated into strain Span001.

The electroporation conditions are as follows: first prepare electroporation competent cells of Span001 with pKD46 plasmid; place 50 μl competent cells on ice, add 50 ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml LB culture medium to the electroporation cup, blow 5 times and then transfer to a test tube, incubate at 75 rpm and 30°C for 4 hours. Transfer the bacterial liquid to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, streak culture on LB solid culture medium without sodium chloride containing 6% sucrose. After PCR verification, the primers used were XZ-tdcDE-up/XZ-tdcDE-down, and the correct colony amplification product was a 2722bp fragment, including 845bp of the upstream homology arm of tdcDE, 1726bp of the alsS gene and sacI restriction site, and 151bp of the downstream homology arm of tdcDE. A correct single colony was selected and named Span002.

Span002 is a recombinant bacterium obtained by integrating the acetolactate synthase gene (alsS gene, the nucleotide sequence is SEQ ID NO: 2 in the sequence list, encoding the alsS protein shown in SEQ ID NO: 1) into the propionate kinase encoding gene tdcD and formate acetyltransferase encoding gene tdcE sites of Escherichia coli ATCC 8739, in which the propionate kinase encoding gene tdcD (the encoded protein sequence is NCBI ACA76259.1, coded_by=CP000946.1:626900..628108) and the formate acetyltransferase encoding gene tdcE (the encoded protein sequence is NCBI ACA76260.1, coded_by=CP000946.1:628142..630436) are simultaneously knocked out.

Example 2: Regulation of the acetolactate synthase gene alsS

Starting from Span002, artificial regulatory elements were used to regulate the expression of the acetolactate synthase encoding gene alsS integrated in the tdcDE site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers tdcDE-incs-up/alsSPro-CS-down were used to amplify a 2719 bp DNA fragment I, including a 50 bp upstream homology arm of tdcDE, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the alsS gene, for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electroporated to Span002.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span002 by electroporation, and then DNA fragment I was electroporated into E. coli Span002 carrying pKD46.

The electroporation conditions and steps were consistent with the first step of the method for integrating alsS at the tdcDE site described in Example 1. 200 μl of bacterial solution was applied to an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, a single colony was selected for PCR verification using primers XZ-tdcDE-up/tdcDE-YZ285-down. The correct PCR product should be 3749 bp, including 845 bp of the upstream homology arm of tdcDE, 2619 bp of the cat-sacB fragment, and 285 bp of the downstream homology arm of alsS. A correct single colony was selected and named Span003.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template, and the primers alsS-Pro-up/alsS-Pro-down were used to amplify the 188 bp DNA fragment II, including 50 bp of the upstream homology arm of tdcDE, 88 bp of the M1-93 promoter, and 50 bp of the downstream homology arm of alsS. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span003.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-tdcDE-up/tdcDE-YZ285-down. The correct colony amplification product was a 1218 bp fragment, including 8454 bp of the upstream homology arm of tdcDE, 88 bp of the M1-93 promoter sequence, and 285 bp of the downstream homology arm of alsS. A correct single colony was selected and named Span004.

Span004 is a recombinant bacterium obtained by integrating M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of alsS gene of Escherichia coli Span002. In the recombinant bacterium, M1-93 promoter drives the expression of alsS gene.

Example 3: Regulation of the acetolactate synthase gene ilvB

The expression of the acetolactate synthase I large subunit gene ilvB was regulated by the artificial regulatory element M1-93 through a two-step homologous recombination method. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ilvB pro-catup/ilvB pro-catdown were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of the upstream homology arm of ilvB, 2619 bp of the cat-sacB fragment, and 50 bp of the downstream homology arm of ilvB. The amplification system and amplification conditions were the same as those described in Example 1.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span004 by electroporation, and then DNA fragment I was electroporated into E. coli Span004 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers ilvB pro-YZup/ilvB pro-YZdown. The correct PCR product should be 2996 bp, including ilvB upstream homology arm 123 bp, cat-sacB fragment 2619 bp and ilvB downstream homology arm 254 bp. Select a correct single colony and name it Span005.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers ilvB pro-up/ilvB pro-down were used to amplify a 188 bp DNA fragment II. DNA fragment II includes 50 bp of the upstream homology arm of ilvB, 88 bp of the M1-93 promoter, and 50 bp of the downstream homology arm of ilvB. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span005.

The electroporation conditions and steps were the same as those described in Example 1 for the second step of alsS integration at the tdcDE site. The clones were verified by colony PCR, and the primers used were ilvB pro-YZup/ilvB pro-YZdown. The correct colony amplification product was a 465 bp fragment, including 123 bp of the upstream homology arm of ilvB, 88 bp of the M1-93 promoter, and 254 bp of the downstream homology arm of ilvB. A correct single colony was selected and named Span006.

Span006 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of the acetolactate synthase gene ilvB (the encoded protein sequence is SEQ ID NO: 3, NCBIACA75715.1, coded_by=CP000946.1:28583..30271) of Escherichia coli Span004. In this recombinant bacterium, the M1-93 promoter can drive the expression of the ilvB gene.

Example 4: Regulation of the acetolactate synthase gene ilvG

The expression of the acetolactate synthase II large subunit gene ilvG was regulated by the artificial regulatory element M1-93 through a two-step homologous recombination method. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ilvG pro-catup/ilvG pro-catdown were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of ilvG upstream homology arm, 2619 bp of cat-sacB fragment and 50 bp of ilvG downstream homology arm. The amplification system and amplification conditions were the same as those described in Example 1.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span006 by electroporation, and then DNA fragment I was electroporated into E. coli Span006 carrying pKD46.

The electroporation conditions and steps were the same as the first step of the method for integration of alsS at the tdcDE site described in Example 1. 200 μl of bacterial solution was applied to an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, a single colony was selected for PCR verification using primers ilvG pro-YZup/ilvG p-YZdown. The correct PCR product should be 2993 bp, including ilvG upstream homology arm 179 bp, cat-sacB fragment 2619 bp and ilvG downstream homology arm 195 bp. A correct single colony was selected and named Span007.

In the second step, the genomic DNA plasmid DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers ilvG pro-up/ilvG pro-down were used to amplify the 188 bp DNA fragment II. The DNA fragment II includes 50 bp of the upstream homology arm of ilvG, 88 bp of the M1-93 promoter and 50 bp of the downstream homology arm of ilvG. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Span007.

The electroporation conditions and steps were the same as the second step method for alsS integration at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were ilvG pro-YZup/ilvG p-YZdown. The correct colony amplification product was a 462 bp fragment, including 179 bp of the upstream homology arm of ilvG, 88 bp of the M1-93 fragment, and 195 bp of the downstream homology arm of ilvG. A correct single colony was selected and named Span008.

Span008 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of the acetolactate synthase gene ilvG (the encoded protein sequence is SEQ ID NO: 5, NCBIACA79830.1, coded_by=CP000946.1:4677780..4679426) of Escherichia coli Span006. In this recombinant bacterium, the M1-93 promoter can drive the expression of the ilvG gene.

Example 5: Mutation of the acetolactate synthase gene ilvH

A two-step homologous recombination method was used to introduce a mutation into the ilvH gene, a regulatory subunit gene of acetolactate synthase III, to relieve the feedback inhibition of L-valine. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ilvH*-cat-up/ilvH*-cat-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of the upstream homology arm of ilvH, 2619 bp of the cat-sacB fragment, and 50 bp of the downstream homology arm of ilvH. The amplification system and amplification conditions were the same as those described in Example 1.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span008 by electroporation, and then DNA fragment I was electroporated into E. coli Span008 carrying pKD46.

The electroporation conditions and steps were the same as the first step of the method for integration of alsS at the tdcDE site described in Example 1. 200 μl of bacterial solution was applied to an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, a single colony was selected for PCR verification using primers ilvH*-mutYZ-up/ilvH*-mut-down. The correct PCR product should be 3165 bp, including 202 bp of the upstream homology arm of ilvH, 2619 bp of the cat-sacB fragment, and 344 bp of the downstream homology arm of ilvH. A correct single colony was selected and named Span009.

In the second step, the DNA of wild-type E. coli ATCC 8739 was used as a template and the primers ilvH*-mut-up/ilvH*-mut-down were used to amplify a 467 bp DNA fragment II. DNA fragment II contained the mutated ilvH gene. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span009.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were ilvH*-mutYZ-up/ilvH*-mut-down. The correct colony amplification product was a 619 bp fragment, including 163 bp upstream of the ilvH gene and 456 bp of the ilvH gene. A correct single colony was selected and named Span010.

Span010 is a recombinant bacterium obtained by mutating the acetolactate synthase gene ilvH of Escherichia coli Span008 into an ilvH* gene (i.e., an ilvH mutant gene). In the recombinant bacterium, the sequence of the ilvH* gene is SEQ ID NO: 8 in the sequence listing, encoding the ilvH* protein shown in SEQ ID NO: 7.

Example 6: Integration of the acetohydroxyacid reductoisomerase encoding gene ilvC into the alcohol dehydrogenase adhE site and knockout of the adhE gene

Starting from Span010, the ilvC gene encoding acetohydroxy acid reductoisomerase from Escherichia coli was integrated into the alcohol dehydrogenase adhE site by a two-step homologous recombination method. The specific steps include:

In the first step, using pXZ-CS plasmid DNA as a template, primers adhE-CS-up/adhE-CS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of adhE, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of adhE. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span010.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the plasmid was purchased from the CGSC Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Escherichia coli Span010 by electroporation, and then DNA fragment I was electroporated into Escherichia coli Span010 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integrating alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-adhE-up/XZ-adhE-down. The correct PCR product should be 3167 bp, including 221 bp of the upstream homology arm of adhE, 2619 bp of the cat-sacB fragment, and 327 bp of the downstream homology arm of adhE. Select a correct single colony and name it Span011.

In the second step, the genomic DNA of wild-type Escherichia coli ATCC 8739 was used as a template and the primers adhE-ilvC-up/adhE-ilvC-down were used to amplify a 1576 bp DNA fragment II. DNA fragment II includes a 50 bp upstream homology arm of adhE, 1476 bp of ilvC gene and 50 bp downstream homology arm of adhE. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span011.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-adhE-up/XZ-adhE-down. The correct colony amplification product was a 2024 bp fragment, including 221 bp of the upstream homology arm of adhE, 1476 bp of the ilvC gene, and 327 bp of the downstream homology arm of adhE. A correct single colony was selected and named Span012.

Span012 is a recombinant bacterium obtained by integrating the acetohydroxyacid reductoisomerase encoding gene ilvC (the encoded protein sequence is SEQ ID NO: 167, NCBI ACA79824.1, coded_by=CP000946.1:4670539..4672014) into the adhE site of Escherichia coli Span010, and the alcohol dehydrogenase gene adhE (the encoded protein sequence is NCBI ACA78022.1, coded_by=CP000946.1:2627307..2629982) in the recombinant bacterium is knocked out at the same time.

Example 7: Regulation of the acetohydroxyacid reductoisomerase encoding gene ilvC

Starting from Span012, an artificial regulatory element was used to regulate the expression of the acetohydroxyacid reductoisomerase encoding gene ilvC integrated in the adhE site of the alcohol dehydrogenase gene. The specific steps are as follows:

In the first step, pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844) DNA was used as a template and primers adhE-cs-up/ilvC-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of adhE, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of ilvC. The amplification system and amplification conditions are the same as those described in Example 1. DNA fragment I was electroporated to Span012.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the plasmid was purchased from the CGSC Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Escherichia coli Span012 by electroporation, and then DNA fragment I was electroporated into Escherichia coli Span012 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integrating alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-adhE-up/ilvC-YZ347-down. The correct PCR product should be 3187 bp, including 221 bp of adhE upstream homology arm, 2619 bp of cat-sacB fragment and 347 bp of ilvC downstream homology arm. Select a correct single colony and name it Span013.

In the second step, the genomic DNA of M1-46 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers ilvC-Pro-up/ilvC-Pro-down were used to amplify the 188 bp DNA fragment II. DNA fragment II includes 50 bp of upstream homology arm of adhE, 88 bp of M1-46 promoter sequence and 50 bp of downstream homology arm of ilvC. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span013.

The electroporation conditions and steps were the same as the second step method for alsS integration at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-adhE-up/ilvC-YZ347-down. The correct colony amplification product was a 656 bp fragment, including 221 bp of the upstream homology arm of adhE, 88 bp of the M1-46 promoter, and 347 bp of the downstream homology arm of ilvC. A correct single colony was selected and named Span014.

Span014 is a recombinant bacterium obtained by integrating the M1-46 promoter (nucleotide sequence is SEQ ID NO: 173 in the sequence list) into the upstream of the ilvC gene of Escherichia coli Span012. In the recombinant bacterium, the M1-46 promoter drives the expression of the ilvC gene.

Example 8: Integration of the dihydroxyacid dehydratase encoding gene ilvD into the pyruvate formate lyase encoding gene pflB site and knockout of the pflB gene

Starting from Span014, the dihydroxyacid dehydratase encoding gene ilvD from Escherichia coli was integrated into the pyruvate formate lyase encoding gene pflB site by a two-step homologous recombination method and the pflB gene was knocked out. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers pflB-CS-up/pflB-CS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of pflB, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of pflB. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span014.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span014 by electroporation, and then DNA fragment I was electroporated into E. coli Span014 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-pflB-up600/XZ-pflB-down. The correct PCR product should be 3675 bp, including 641 bp of pflB upstream homology arm, 2619 bp of cat-sacB fragment and 415 bp of pflB downstream homology arm. Select a correct single colony and name it Span015.

In the second step, the 1951 bp DNA fragment I was amplified from the gene of Escherichia coli MG1655 (from ATCC, No. 700926) using primers pflB-ilvD-up/pflB-ilvD-down. DNA fragment II includes 50 bp of pflB upstream homology arm, 1851 bp of ilvD gene and 50 bp of pflB downstream homology arm. DNA fragment II was used for the second homologous recombination. DNA fragment II was electrotransferred to strain Span015.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-pflB-up600/XZ-pflB-down. The correct colony amplification product was a 2996 bp fragment, including 641 bp of the upstream homology arm of pflB, 1851 bp of the ilvD gene, and 415 bp of the downstream homology arm of pflB. A correct single colony was selected and named Span016.

Span016 is a recombinant bacterium obtained by integrating the dihydroxy-acid dehydratase encoding gene ilvD (SEQ ID NO: 12) (the encoded protein sequence is SEQ ID NO: 11, NCBI QPA17447.1, coded_by=CP032679.1:3943375..3945225) into the pflB site of Escherichia coli Span014, and the pyruvate formate lyase encoding gene pflB (the encoded protein sequence is NCBI ACA78322.1, coded_by=CP000946.1:2956804..2959086) in the recombinant bacterium is simultaneously knocked out.

Example 9: Expression regulation of the dihydroxyacid dehydratase encoding gene ilvD

Starting from Span016, an artificial regulatory element was used to regulate the expression of the dihydroxyacid dehydratase encoding gene ilvD integrated in the pyruvate formate lyase encoding gene pflB site. The specific steps are as follows:

In the first step, pXZ-CS plasmid DNA was used as a template and primers pflB-CS-up/pflB-Pcs-down were used to amplify a 2719 bp DNA fragment I for homologous recombination in the first step. DNA fragment I includes a 50 bp upstream homology arm of pflB, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of ilvD. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span016.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span016 by electroporation, and then DNA fragment I was electroporated into E. coli Span016 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-pflB-up600/ilvD-YZ496-down. The correct PCR product should be 3756 bp, including 641 bp of pflB upstream homology arm, 2619 bp of cat-sacB fragment and 496 bp of ilvD downstream homology arm. Select a correct single colony and name it Span017.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers pflB-Pro-up/ilvD-Pro-down were used to amplify the 189 bp DNA fragment II. The DNA fragment II includes a 50 bp upstream homology arm of pflB, an 89 bp sequence of the artificial regulatory element RBSL1 and a 50 bp downstream homology arm of ilvD. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Span017.

The electroporation conditions and steps were the same as the second step method for alsS integration at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-pflB-up600/ilvD-YZ496-down. The correct colony amplification product was a 1226 bp fragment, including 641 bp of pflB upstream homology arm, 89 bp of RBSL1 sequence, and 496 bp of ilvD downstream homology arm. A correct single colony was selected and named Span018.

Span018 is a recombinant bacterium obtained by integrating the RBSL1 promoter (nucleotide sequence is SEQ ID NO: 171 in the sequence list) into the upstream of the ilvD gene of Escherichia coli Span016. In the recombinant bacterium, the RBSL1 promoter drives the expression of the ilvD gene.

Example 10: Integration of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB into the fumarate reductase encoding gene frd site and knockout of the frd site

Starting from Span018, the 3-methyl-2-oxobutyrate hydroxymethyltransferase encoding gene panB was integrated into the fumarate reductase encoding gene frd site. The specific steps are as follows:

In the first step, pXZ-CS plasmid DNA was used as a template and primers frd-cs-up/frd-cs-down were used to amplify a 2719 bp DNA fragment I for homologous recombination in the first step. DNA fragment I includes a 50 bp upstream homology arm of frd, a 2619 bp cat-sacB fragment and a 50 bp downstream homology arm of frd. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span018.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span018 by electroporation, and then DNA fragment I was electroporated into E. coli Span018 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-frd-up/XZ-frd-down. The correct PCR product should be 3440 bp, including 426 bp of frd upstream homology arm, 2619 bp of cat-sacB fragment and 395 bp of frd downstream homology arm. Select a correct single colony and name it Span019.

In the second step, the genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) was used as a template, and the primers frd-panB-up/frd-panB-down were used to amplify the 895bp DNA fragment II. The DNA fragment II includes 50bp of the upstream homology arm of frd, 795bp of the panB gene, and 50bp of the downstream homology arm of frd. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Span019.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-frd-up/XZ-frd-down. The correct colony amplification product was a 1661 bp fragment, including 426 bp of the upstream homology arm of frd, 795 bp of the panB gene, and 395 bp of the downstream homology arm of frd. A correct single colony was selected and named Span020.

Span020 is a recombinant bacterium obtained by integrating the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB (the encoded protein sequence is SEQ ID NO: 15, NCBI QPA14045.1, coded_by=CP032679.1:148806..149600) into the frd site of Escherichia coli Span018, and the fumarate reductase encoding gene frd (the encoded protein sequence is NCBI ACA79462.1, coded_by=CP000946.1:4217304..4217699) in the recombinant bacterium is knocked out at the same time.

Example 11: Expression regulation of 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB

Starting from Span020, an artificial regulatory element was used to regulate the expression of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB integrated in the frd site of the fumarate reductase encoding gene. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers frd-cs-up/panB-Pcs-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of frd, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of panB. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span020.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span020 by electroporation, and then DNA fragment I was electroporated into E. coli Span020 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-frd-up/panB-YZ130-down. The correct PCR product should be 3175 bp, including 426 bp of frd upstream homology arm, 2619 bp of cat-sacB fragment and 130 bp of panB downstream homology arm. Select a correct single colony and name it Span021.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers panB-Pro-up/panB-Pro-down were used to amplify the 188 bp DNA fragment II. DNA fragment II includes 50 bp of the upstream homology arm of frd, 88 bp of the M1-93 promoter sequence and 50 bp of the downstream homology arm of panB. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span021.

The electroporation conditions and steps were the same as those described in Example 1 for the second step of alsS integration at the tdcDE site. The clones were verified by colony PCR, and the primers used were XZ-frd-up/panB-YZ130-down. The correct colony amplification product was a 644 bp fragment, including 426 bp of frd upstream homology arm, 88 bp of M1-93 promoter sequence, and 130 bp of panB downstream homology arm. A correct single colony was selected and named Span022.

Span022 is a recombinant bacterium obtained by integrating M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of panB gene of Escherichia coli Span020. In the recombinant bacterium, M1-93 promoter drives the expression of panB gene.

Example 12: Integration of the 2-dehydropantothenate-2-reductase gene panE into the lactate dehydrogenase ldhA site and knockout of the ldhA site

Starting from Span022, the 2-dehydropantothenate-2-reductase gene panE was integrated into the lactate dehydrogenase gene ldhA site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ldhA-csin-up/ldhA-csin-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of the upstream homology arm of ldhA, 2619 bp of the cat-sacB fragment, and 50 bp of the downstream homology arm of ldhA. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span022.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span022 by electroporation, and then DNA fragment I was electroporated into E. coli Span022 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-ldhA-up/XZ-ldhA-down. The correct PCR product should be 3415 bp, including ldhA upstream homology arm 380 bp, cat-sacB fragment 2619 bp and ldhA downstream homology arm 416 bp. Select a correct single colony and name it Span023.

In the second step, the genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) was used as a template, and the primers ldhA-panE-up/ldhA-panE-down were used to amplify a 1012bp DNA fragment II. DNA fragment II includes 50bp of the upstream homology arm of ldhA, 912bp of the panE gene, and 50bp of the downstream homology arm of ldhA. DNA fragment II was used for the second homologous recombination. DNA fragment II was electrotransferred to strain Span023.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-ldhA-up/XZ-ldhA-down. The correct colony amplification product was a 1708 bp fragment, including 380 bp of the upstream homology arm of ldhA, 912 bp of the panE gene, and 416 bp of the downstream homology arm of ldhA. A correct single colony was selected and named Span024.

Span024 is a recombinant bacterium obtained by integrating the 2-dehydropantothenate-2-reductase gene panE (the protein sequence encoded is SEQ ID NO: 17, NCBI QPA14304.1, coded_by=CP032679.1:443607..444518) into the ldhA site of Escherichia coli Span022, and the lactate dehydrogenase gene ldhA (the protein sequence encoded is NCBI ACA77911.1, coded_by=CP000946.1:2508048..2509037) in the recombinant bacterium is knocked out at the same time.

Example 13: Expression regulation of 2-dehydropantothenate-2-reductase gene panE

Starting from Span024, an artificial regulatory element was used to regulate the expression of the 2-dehydropantothenate-2-reductase gene panE integrated in the ldhA site of the lactate dehydrogenase gene. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ldhA-csin-up/panE-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of the upstream homology arm of ldhA, 2619 bp of the cat-sacB fragment, and 50 bp of the downstream homology arm of panE. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span024.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span024 by electroporation, and then DNA fragment I was electroporated into E. coli Span024 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-ldhA-up/panE-YZ245-down. The correct PCR product should be 3244 bp, including ldhA upstream homology arm 380 bp, cat-sacB fragment 2619 bp and panE downstream homology arm 245 bp. Select a correct single colony and name it Span025.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers panE-Pro-up/panE-Pro-down were used to amplify the 189 bp DNA fragment II. The DNA fragment II includes 50 bp of the upstream homology arm of ldhA, 89 bp of the artificial promoter RBSL2 sequence and 50 bp of the downstream homology arm of panE. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Span025.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-ldhA-up/panE-YZ245-down. The correct colony amplification product was a 714 bp fragment, including 380 bp of the upstream homology arm of ldhA, 89 bp of the artificial promoter RBSL2 sequence, and 245 bp of the downstream homology arm of panE. A correct single colony was selected and named Span026.

Span026 is a recombinant bacterium obtained by integrating the RBSL2 promoter (nucleotide sequence is SEQ ID NO: 172 in the sequence list) into the upstream of the panE gene of Escherichia coli Span024. In the recombinant bacterium, the RBSL2 promoter drives the expression of the panE gene.

Example 14: Integration of the serine hydroxymethyltransferase gene glyA at the methylglyoxal synthase gene mgsA site and knockout of the mgsA site

Starting from Span026, the serine hydroxymethyltransferase gene glyA was inserted into the methylglyoxal synthase gene mgsA site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers mgsA-cs-up/mgsA-cs-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of mgsA, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of mgsA. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span026.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span026 by electroporation, and then DNA fragment I was electroporated into E. coli Span026 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-mgsA-up/XZ-mgsA-down. The correct PCR product should be 3646 bp, including 516 bp of mgsA upstream homology arm, 2619 bp of cat-sacB fragment and 511 bp of mgsA downstream homology arm. Select a correct single colony and name it Span027.

In the second step, the genomic DNA of wild-type Escherichia coli ATCC8739 was used as a template and the primers mgsA-glyA-up/mgsA-glyA-down were used to amplify a 1354 bp DNA fragment II. DNA fragment II includes a 50 bp upstream homology arm of mgsA, a 1254 bp glyA fragment, and a 50 bp downstream homology arm of mgsA. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span027.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-mgsA-up/XZ-mgsA-down. The correct colony amplification product was a 2281 bp fragment, including a 516 bp upstream homology arm of mgsA, a 1254 bp glyA fragment, and a 511 bp downstream homology arm of mgsA. A correct single colony was selected and named Span028.

Span028 is a recombinant bacterium obtained by integrating the serine hydroxymethyltransferase gene glyA (the protein sequence encoded is SEQ ID NO: 19, NCBI ACA76793.1, coded_by=CP000946.1:1227416..1228669) into the mgsA site of Escherichia coli Span026, and the mgsA gene (the protein sequence encoded is NCBI ACA78263.1, coded_by=CP000946.1:2883345..2883803) in the recombinant bacterium is knocked out at the same time.

Example 15: Regulation of serine hydroxymethyltransferase gene glyA

Starting from Span028, artificial regulatory elements were used to regulate the expression of the serine hydroxymethyltransferase gene glyA integrated at the mgsA site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers mgsA-cs-up/glyA-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of mgsA, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of glyA. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span028.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span028 by electroporation, and then DNA fragment I was electroporated into E. coli Span028 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-mgsA-up/glyA-YZ364-down. The correct PCR product should be 3499 bp, including 516 bp of mgsA upstream homology arm, 2619 bp of cat-sacB fragment and 364 bp of glyA downstream homology arm. Select a correct single colony and name it Span029.

In the second step, the genomic DNA of M1-46 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers glyA-Pro-up/glyA-Pro-down were used to amplify a 188 bp DNA fragment II. DNA fragment II includes 50 bp of the upstream homology arm of mgsA, 88 bp of the M1-46 promoter, and 50 bp of the downstream homology arm of glyA. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span029.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-mgsA-up/glyA-YZ364-down. The correct colony amplification product was a 968 bp fragment, including 516 bp of mgsA upstream homology arm, 88 bp of M1-46 promoter, and 364 bp of glyA downstream homology arm. A correct single colony was selected and named Span030.

Span030 is a recombinant bacterium obtained by integrating the M1-46 promoter (nucleotide sequence is SEQ ID NO: 173 in the sequence list) into the upstream of the glyA gene of Span028. In the recombinant bacterium, the M1-46 promoter drives the expression of the glyA gene.

Example 16: Regulation of the wild-type aminomethyltransferase gene gcvT of Escherichia coli

Starting from Span030, the expression of the wild-type aminomethyltransferase gene gcvT of Escherichia coli was regulated using artificial regulatory elements. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers gcvT-Pcat-up/gcvT-PsacB-down were used to amplify a 2719 bp DNA fragment I for homologous recombination in the first step. DNA fragment I includes a 50 bp upstream homology arm of the gcvT gene, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of gcvT. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span030.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span030 by electroporation, and then DNA fragment I was electroporated into E. coli Span030 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers gcvT-up-500/gcvT-350-down. The correct PCR product should be 3197 bp, including 228 bp of the upstream homology arm of the gcvT gene, 2619 bp of the cat-sacB fragment, and 350 bp of the downstream homology arm of gcvT. Select a correct single colony and name it Span031.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers gcvT-M93-up/gcvT-M93-down were used to amplify the 188 bp DNA fragment II. DNA fragment II includes 50 bp of the upstream homology arm of the gcvT gene, 88 bp of the M1-93 promoter, and 50 bp of the downstream homology arm of gcvT. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span031.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were gcvT-up-500/gcvT-350-down. The correct colony amplification product was a 666bp fragment, including 228bp of the upstream homology arm of the gcvT gene, 88bp of the M1-93 promoter, and 350bp of the downstream homology arm of gcvT. A correct single colony was selected and named Span032.

Span032 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of the aminomethyltransferase gene gcvT (encoded protein sequence is SEQ ID NO: 21, NCBIACA76476.1, coded_by=CP000946.1:862077..863171) of Escherichia coli Span030. In this recombinant bacterium, the M1-93 promoter can drive the expression of the gcvT gene.

Example 17: Regulation of the wild-type glycine decarboxylase gene gcvP in Escherichia coli

Starting from Span032, the expression of the wild-type glycine decarboxylase gene gcvP of Escherichia coli was regulated using artificial regulatory elements. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers gcvP-Pcat-up/gcvP-PsacB-down were used to amplify a 2719 bp DNA fragment I for homologous recombination in the first step. DNA fragment I includes a 50 bp upstream homology arm of the gcvP gene, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of gcvP. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span032.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span032 by electroporation, and then DNA fragment I was electroporated into E. coli Span032 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers gcvH-up/gcvP-390-down. The correct PCR product should be 3399 bp, including 390 bp of the upstream homology arm of the gcvP gene, 2619 bp of the cat-sacB fragment, and 390 bp of the downstream homology arm of gcvP. Select a correct single colony and name it Span033.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers gcvP-M93-up/gcvP-M93-down were used to amplify the 188bp DNA fragment II. DNA fragment II includes 50bp of the upstream homologous arm of the gcvP gene, 88bp of the M1-93 promoter, and 50bp of the downstream homologous arm of gcvP. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span033.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were gcvH-up/gcvP-390-down. The correct colony amplification product was a 868 bp fragment, including 390 bp of the upstream homology arm of the gcvP gene, 88 bp of the M1-93 promoter, and 390 bp of the downstream homology arm of gcvP. A correct single colony was selected and named Span034.

Span034 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of the glycine decarboxylase gene gcvP (the encoded protein sequence is SEQ ID NO: 23, NCBIACA76478.1, coded_by=CP000946.1:863703..866576) of Escherichia coli Span032. In this recombinant bacterium, the M1-93 promoter can drive the expression of the gcvP gene.

Example 18: Integration of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB from Corynebacterium glutamicum at the phosphate acetyltransferase encoding gene pta and the acetate kinase encoding gene ackA site and knockout of the ackA-pta site

Starting from Span034, the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB from Corynebacterium glutamicum was inserted into the phosphate acetyltransferase encoding gene pta and the acetate kinase encoding gene ackA sites. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ackA-cs-up/pta-cs-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of upstream homology arm of ackA-pta gene, 2619 bp of cat-sacB fragment and 50 bp of downstream homology arm of ackA-pta. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span034.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span034 by electroporation, and then DNA fragment I was electroporated into E. coli Span034 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-ackA-up/XZ-pta-down. The correct PCR product should be 3350 bp, including 320 bp of the upstream homology arm of the ackA-pta gene, 2619 bp of the cat-sacB fragment, and 411 bp of the downstream homology arm of the ackA-pta gene. Select a correct single colony and name it Span035.

In the second step, the genomic DNA of Corynebacterium glutamicum ATCC13032 (ATCC product) was used as a template, and a 916bp DNA fragment II was amplified with primers ackA-panBC-up/ackA-panBC-down. DNA fragment II includes 50bp of homology arms upstream of the ackA-pta gene, 816bp of the panB gene from the glutamicum and 50bp of homology arms downstream of the ackA-pta. It was used for the second homologous recombination. DNA fragment II was electrotransferred to strain Span035.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were XZ-ackA-up/XZ-pta-down. The correct colony amplification product was a 1547 bp fragment, including 320 bp of the upstream homology arm of the ackA-pta gene, 816 bp of the panB gene from Gubang, and 411 bp of the downstream homology arm of ackA-pta. A correct single colony was selected and named Span036.

Span036 is a recombinant bacterium obtained by integrating the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB (panB gene, the nucleotide sequence is SEQ ID NO: 14 in the sequence list, encoding the panB protein shown in SEQ ID NO: 13) of Corynebacterium glutamicum into the phosphate acetyltransferase encoding gene pta and the acetate kinase encoding gene ackA sites of Escherichia coli Span034, in which the pta gene (the protein sequence encoded is NCBI ACA77021.1, coded_by=CP000946.1:1484032..1486176) and the ackA gene (the protein sequence encoded is NCBI ACA77022.1, coded_by=CP000946.1:1486251..1487453) are knocked out at the same time.

Example 19: Regulation of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB from Corynebacterium glutamicum

Starting from Span036, an artificial regulatory element was used to regulate the expression of the 3-methyl-2-oxobutyrate hydroxymethyltransferase gene panB integrated at the ackA-pta site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers ackA-cs-up/panBC-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes 50 bp of upstream homology arm of ackA-pta gene, 2619 bp of cat-sacB fragment and 50 bp of downstream homology arm of panB gene from Gubang. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span036.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span036 by electroporation, and then DNA fragment I was electroporated into E. coli Span036 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers XZ-ackA-up/panBC-YZ425-down. The correct PCR product should be 3364 bp, including 320 bp of the upstream homology arm of the ackA-pta gene, 2619 bp of the cat-sacB fragment, and 425 bp of the downstream homology arm of panB. Select a correct single colony and name it Span037.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers panBC-Pro-up/panBC-Pro-down were used to amplify the 188 bp DNA fragment II. DNA fragment II includes 50 bp of the upstream homology arm of the ackA-pta gene, 88 bp of the M1-93 promoter, and 50 bp of the downstream homology arm of panB. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span037.

The electroporation conditions and steps were the same as those described in Example 1 for the second step of alsS integration at the tdcDE site. The clones were verified by colony PCR, and the primers used were XZ-ackA-up/panBC-YZ425-down. The correct colony amplification product was a 833 bp fragment, including 320 bp of the upstream homology arm of the ackA-pta gene, 88 bp of the M1-93 promoter, and 425 bp of the downstream homology arm of panB. A correct single colony was selected and named Span038.

Span038 is a recombinant bacterium obtained by integrating M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of panB gene of Escherichia coli Span036. In the recombinant bacterium, M1-93 promoter drives the expression of panB gene.

Example 20: Weakened expression of branched-chain amino acid aminotransferase gene ilvE

Starting from Span038, the expression of the branched-chain amino acid aminotransferase gene ilvE was weakened. The specific steps are as follows:

In the first step, pXZ-CS plasmid DNA was used as a template and primers ilvE-cat-up/ilvE-sacB-down were used to amplify a 2719 bp DNA fragment I for homologous recombination in the first step. DNA fragment I included a 50 bp upstream homology arm of the ilvE gene, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the ilvE gene. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span038.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span038 by electroporation, and then DNA fragment I was electroporated into E. coli Span038 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers ilvM-up/ilvE-down. The correct PCR product should be 3832 bp, including 283 bp of the upstream homology arm of the ilvE gene, 2619 bp of the cat-sacB fragment, and 930 bp of the downstream homology arm of the ilvE gene. Select a correct single colony and name it Span039.

In the second step, the genomic DNA of wild-type E. coli ATCC 8739 was used as a template and the primers ilvEGTG-up/ilvE-down were used to amplify a 980 bp DNA fragment II. DNA fragment II includes the ilvE gene with the start codon ATG changed to GTG. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span039.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were ilvM-up/ilvE-down. The correct colony amplification product was a 1213 bp fragment, including 283 bp of the upstream homology arm of the ilvE gene and 930 bp of ilvE with the start codon ATG replaced by GTG. A correct single colony was selected and named Span040.

Span040 is a recombinant bacterium obtained by mutating the start codon ATG of ilvE of Span038 to GTG. The mutated gene is recorded as ilvE* gene (its sequence is SEQ ID NO: 32 in the sequence list), and the ilvE* gene encodes the ilvE* protein (its sequence is SEQ ID NO: 31 in the sequence list).

Example 21: Integration of the phosphoglycerate dehydrogenase gene serA from Corynebacterium glutamicum into the ara site of ribokinase and knockout of the ara site

Starting from Span040, the phosphoglycerate dehydrogenase gene serA from Corynebacterium glutamicum was integrated into the ara site of ribokinase. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers araBCD-CS-up/araBCD-CS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of the ara site, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the ara site. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span041.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span041 by electroporation, and then DNA fragment I was electroporated into E. coli Span041 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers araBCD-YZ300-up/araBCD-YZ468-down. The correct PCR product should be 3378 bp, including 291 bp of the upstream homology arm of the ara site, 2619 bp of the cat-sacB fragment, and 468 bp of the downstream homology arm of the ara site. Select a correct single colony and name it Span041.

In the second step, the genomic DNA of Corynebacterium glutamicum ATCC13032 (ATCC product) was used as a template, and a 1102bp DNA fragment II was amplified with primers araBCD-serA197-up/araBCD-serA197-down. DNA fragment II includes 50bp of homology arms upstream of the ara site, 1002bp of serA gene and 50bp of homology arms downstream of the ara site. DNA fragment II is used for the second homologous recombination. DNA fragment II is electrotransferred to bacterial strain Span041.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were araBCD-YZ300-up/araBCD-YZ468-down. The correct colony amplification product was a 1761 bp fragment, including 291 bp of the upstream homology arm of the ara site, 1002 bp of the serA gene, and 468 bp of the downstream homology arm of the ara site. A correct single colony was selected and named Span042.

Span042 is a recombinant bacterium obtained by integrating the phosphoglycerate dehydrogenase gene serA (serA gene, nucleotide sequence is SEQ ID NO: 26, encoding the serA protein shown in SEQ ID NO: 25) of Corynebacterium glutamicum into the ara site of Escherichia coli Span040, and the ara gene (the encoded protein sequence is NCBI ACA79208.1, coded_by=CP000946.1:3929533..3931233, the encoded protein sequence is NCBI ACA79209.1, coded_by=CP000946.1:3931244..3932746) in the recombinant bacterium is knocked out at the same time.

Example 22: Regulation of the phosphoglycerate dehydrogenase gene serA from Corynebacterium glutamicum

Starting from Span042, artificial regulatory elements were used to regulate the expression of the phosphoglycerate dehydrogenase gene serA integrated at the ara site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers araBCD-CS-up/serA197-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of the ara site, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the serA site. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span042.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span042 by electroporation, and then DNA fragment I was electroporated into E. coli Span042 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers araBCD-YZ300-up/SerA197-YZ358-down. The correct PCR product should be 3268 bp, including 291 bp of the upstream homology arm of the ara site, 2619 bp of the cat-sacB fragment, and 358 bp of the downstream homology arm of the serA site. Select a correct single colony and name it Span043.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers serA197-Pro-up/serA197-Pro-down were used to amplify the 188 bp DNA fragment II. The DNA fragment II includes a 50 bp upstream homology arm of the ara site, 88 bp of the M1-93 promoter, and 50 bp of the downstream homology arm of the serA gene. The DNA fragment II was used for the second homologous recombination. The DNA fragment II was electroporated into the strain Span043.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were araBCD-YZ300-up/SerA197-YZ358-down. The correct colony amplification product was a 737 bp fragment, including 291 bp of the upstream homology arm of the ara site, 88 bp of the M1-93 promoter, and 358 bp of the downstream homology arm of the serA site. A correct single colony was selected and named Span044.

Span044 is a recombinant bacterium obtained by integrating the M1-93 promoter (nucleotide sequence is SEQ ID NO: 169 in the sequence list) into the upstream of the serA gene of Escherichia coli Span042. In the recombinant bacterium, the M1-93 promoter drives the expression of the serA gene.

Example 23: Integration of the phosphoserine/phosphohydroxythreonine aminotransferase gene serC and the phosphoserine phosphatase gene serB from Escherichia coli into the valine-pyruvate aminotransferase gene avtA site and knockout of the avtA site

Starting from Span044, the phosphoserine/phosphohydroxythreonine aminotransferase gene serC and the phosphoserine phosphatase gene serB from Escherichia coli were integrated into the valine-pyruvate aminotransferase gene avtA site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers avtA-CS-up/avtA-CS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of the avtA site, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the avtA site. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span044.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span044 by electroporation, and then DNA fragment I was electroporated into E. coli Span044 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of bacterial solution and apply it on LB plates containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers avtA-YZ-up/avtA-YZ-down. The correct PCR product should be 3454 bp, including 416 bp of homology arm upstream of the avtA site, 2619 bp of cat-sacB fragment and 419 bp of homology arm downstream of the avtA site. Select a correct single colony and name it Span045.

In the second step, the genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) was used as a template, and the primers avtA-serCB-up/serC-down were used to amplify the 1181 bp fragment II. The genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) was used as a template, and the primers serB-up/avtA-serCB-down were used to amplify the 1062 bp fragment III. PCR amplification was performed with primers avtA-serCB-up/avtA-serCB-down, and the template was equimolar fragment II and fragment III. The amplification system and conditions were the same as those described in Example 1 to perform fusion PCR to obtain fragment IV. Fragment IV is a 2179 bp DNA fragment, which is used for the second homologous recombination. Fragment IV includes 50 bp of avtA upstream homology arm, 1089 bp of serC gene, 21 bp of RBS sequence for serB gene translation initiation and 969 bp of serB gene, and 50 bp of avtA downstream homology arm. DNA fragment IV was electroporated into strain Span045.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were avtA-YZ-up/avtA-YZ-down. The correct colony amplification product was a 2914 bp fragment, including 416 bp of homology arm upstream of the avtA site, 1089 bp of serC gene, 21 bp of RBS sequence for translation initiation of serB gene and 969 bp of serB gene, and 419 bp of homology arm downstream of the avtA site. A correct single colony was selected and named Span046.

Span046 is a recombinant bacterium obtained by integrating the Escherichia coli phosphoserine/phosphohydroxythreonine aminotransferase gene serC and the phosphoserine phosphatase gene serB (serCB gene cluster, the nucleotide sequence is SEQ ID NO: 174 in the sequence list, the serCB gene cluster encodes the serC protein shown in SEQ ID NO: 27 and the serB protein shown in SEQ ID NO: 29) into the avtA site of Escherichia coli Span044, and the avtA gene (the encoded protein sequence is NCBIACA75824.1, coded_by=CP000946.1:153868..155121) in the recombinant bacterium is knocked out at the same time.

In SEQ ID NO: 174, positions 1-88 are the M1-93 promoter sequence, positions 89-1177 are the serC gene sequence, positions 1178-1198 are the RBS sequence for serB gene translation initiation, and positions 1199-2167 are the sequence of the serB gene.

Example 24: Expression regulation of the serCB gene cluster integrated at the avtA site

Starting from Span046, artificial regulatory elements were used to regulate the expression of the phosphoserine/phosphohydroxythreonine aminotransferase gene serC and the phosphoserine phosphatase gene serB from Escherichia coli integrated at the avtA site. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers avtA-CS-up/serCB-ProCS-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of the avtA site, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the serC gene. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span046.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span046 by electroporation, and then DNA fragment I was electroporated into E. coli Span046 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integration of alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers avtA-YZ-up/serCB-YZ317-down. The correct PCR product should be 3456 bp, including 416 bp of homology arm upstream of the avtA site, 2619 bp of cat-sacB fragment and 421 bp of homology arm downstream of the serC gene. Select a correct single colony and name it Span047.

In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and the primers serCB-Pro-up/serCB-Pro-down were used to amplify the 188 bp DNA fragment II. DNA fragment II includes 50 bp of homology arm upstream of the avtA site, 88 bp of M1-93 promoter sequence and 50 bp of homology arm downstream of the serC gene. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span047.

The electroporation conditions and steps were consistent with the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were avtA-YZ-up/serCB-YZ317-down. The correct colony amplification product was a 925 bp fragment, including 416 bp of the upstream homology arm of the avtA site, 88 bp of the M1-93 promoter sequence, and 421 bp of the downstream homology arm of the serC gene. A correct single colony was selected and named Span048.

Span048 is a recombinant bacterium obtained by integrating the M1-93 promoter into the upstream of the serCB gene cluster of Escherichia coli Span046, and contains the serCB gene cluster expression cassette shown in SEQ ID NO: 174. The M1-93 promoter (nucleotide sequence is positions 1-88 of SEQ ID NO: 174 in the sequence list) in the recombinant bacterium drives the expression of serC and serB genes in the serCB gene cluster.

Example 25: Knockout of L-serine deaminase I gene sdaA

Starting from Span048, the coding gene sdaA of L-serine deaminase I was knocked out. The specific steps are as follows:

In the first step, using pXZ-CS plasmid DNA as a template, primers sdaA-delcat-up/sdaA-delsacB-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. DNA fragment I includes a 50 bp upstream homology arm of the sdaA site, a 2619 bp cat-sacB fragment, and a 50 bp downstream homology arm of the sdaA gene. The amplification system and amplification conditions were the same as those described in Example 1. DNA fragment I was electrotransferred to Span048.

DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Span048 by electroporation, and then DNA fragment I was electroporated into E. coli Span048 carrying pKD46.

The electroporation conditions and steps are consistent with the first step of the method for integrating alsS at the tdcDE site described in Example 1. Take 200 μl of the bacterial solution and apply it on an LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select a single colony for PCR verification using primers sdaA-YZ-up/sdaA-YZ-down. The correct PCR product should be 3428 bp, including 383 bp of the upstream homology arm of the sdaA site, 2619 bp of the cat-sacB fragment, and 426 bp of the downstream homology arm of the sdaA gene. Select a correct single colony and name it Span049.

In the second step, the genomic DNA of E. coli ATCC 8739 was used as a template and the primers sdaA-YZ-up/SdaAdel-down were used to amplify a 433 bp DNA fragment II. DNA fragment II includes a 383 bp upstream homology arm and a 50 bp downstream homology arm of sdaA. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Span049.

The electroporation conditions and steps were the same as the second step method for integration of alsS at the tdcDE site described in Example 1. The clones were verified by colony PCR, and the primers used were sdaA-YZ-up/sdaA-YZ-down. The correct colony amplification product was a 809 bp fragment, including 383 bp of the upstream homology arm of the sdaA site and 426 bp of the downstream homology arm of the sdaA gene. A correct single colony was selected and named Span050.

Span050 is a recombinant bacterium obtained by knocking out the L-serine deaminase I gene sdaA (the encoded protein sequence is NCBI ACA77468.1, coded_by=CP000946.1:2018393..2019757) of Escherichia coli Span048, and the recombinant bacterium does not contain the sdaA gene.

Span050 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 22, 2021, with the deposit number CGMCC No.21699.

Example 26: Production of pantoic acid using Span050

The seed culture medium consists of the following components (the solvent is water):

Macroelements: glucose 20g/L, (NH4)2HPO4 3.5g/L, KH2PO4 3.91g/L, K2HPO4 4.48g/L, MgSO4·7H2O 0.18g/L, betaine-HCl 0.15g/L;

Trace elements: FeCl3·6H2O 1.5μg/L, CoCl2·6H2O 0.1μg/L, CuCl2·2H2O 0.1μg/L, ZnCl2 0.1μg/L, Na2MoO4·2H2O 0.1μg/L, MnCl2·4H2O 0.2μg/L, H3BO3 0.05μg/L.

The fermentation medium is mostly the same as the seed medium, except that the glucose concentration is 50 g/L and the fermentation medium also contains 5 g/L serine.

The fermentation of Span050 includes the following steps:

(1) Seed culture: Fresh clones from the LB plate were inoculated into a test tube containing 4 ml of seed culture medium and cultured overnight at 37°C, 250 rpm with shaking. Then, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed culture medium at an inoculum volume of 2% (V/V) and cultured at 37°C, 250 rpm with shaking for 12 hours to obtain a seed culture solution for inoculation of the fermentation medium.

(2) Fermentation culture: The volume of the fermentation medium in a 250 ml Erlenmeyer flask was 25 ml. The seed culture solution was inoculated into the fermentation medium at an inoculum amount of a final concentration of OD550 = 0.1, and fermented at 37° C., 250 rpm, for 60 hours to obtain a fermentation solution.

Analysis method: Agilent-1260 high performance liquid chromatograph was used to determine the components in the fermentation broth after 3 days of fermentation. The concentrations of glucose and pantoic acid in the fermentation broth were determined using Biorad's Aminex HPX-87H organic acid analysis column.

The results showed that the Span050 strain could produce 1.2 g/L of pantoic acid after 3 days of fermentation. This showed that the pantoic acid synthesis pathway in the Span050 strain had been opened and pantoic acid accumulation could be achieved during the fermentation process.

Example 27: Fermentation of Span050 in a 5L tank

The composition, preparation and analysis method of the seed culture medium are the same as those described in Example 26.

Fermentation medium: glucose 30 g/L, magnesium sulfate 5 g/L, potassium dihydrogen phosphate 10.5 g/L, yeast powder 20 g/L, diammonium hydrogen phosphate 6 g/L, citric acid monohydrate 1.84 g/L, trace elements are the same as the fermentation medium in Example 26, and the solvent is water.

Feed medium: 600 g/L glucose, solvent is water.

Fermentation was carried out in a 5L fermenter (BIOTECH-5BG, Shanghai Baoxing), including the following steps:

(1) Seed culture: 50 mL of seed culture medium was placed in a 500 mL Erlenmeyer flask and sterilized at 115°C for 15 min. After cooling, the recombinant E. coli Span050 was inoculated into the seed culture medium at a rate of 1% (V/V) and cultured at 37°C and 250 rpm for 12 hours to obtain a seed solution for inoculation of the fermentation medium.

(2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, and sterilized at 115°C for 25 min. The seed solution is inoculated into the fermentation medium at a final concentration of OD550 = 0.2, the dissolved oxygen is maintained at 30%, and the pH is maintained at 7.0 using ammonia water as a neutralizer. The glucose concentration in the tank is controlled below 5g/L by feeding the medium, and the culture is carried out at 37°C for 3 days to obtain the fermentation broth. The fermentation broth is all the substances in the fermenter.

The results showed that after 3 days of fermentation, the production of pantoic acid in Span050 reached 22 g/L, which has great potential for industrial application.

Example 28: Construction of Span096 strain

Starting from Span050, an exogenous NADH-dependent acetohydroxy acid reductoisomerase KARI (SEQ ID NO: 9) was used to replace the NADPH-dependent IlvC (SEQ ID NO: 167) of Escherichia coli to obtain a new recombinant Escherichia coli Span096 that produces D-pantoic acid. The strain construction process is as follows:

In the first step, pXZ-CS plasmid (Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844) DNA was used as a template and primers adhE-cs-up/adhE-cs-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.

The amplification system was: NewEngland Biolabs Phusion 5X buffer 10 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phusion High-Fidelity DNA polymerase (2.5 U/μl) 0.5 μl, distilled water 33.5 μl, and the total volume was 50 μl.

The amplification conditions were pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 2 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

The above DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the plasmid was purchased from the CGSC Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Span050 by electroporation, and then the DNA fragment I was electroporated into Span050 carrying pKD46.

The electroporation conditions are as follows: first prepare electroporation competent cells of recombinant Escherichia coli Span050 carrying pKD46 plasmid (Dower et al., 1988, Nucleic Acids Res 16: 6127-6145); place 50 μl competent cells on ice, add 50 ng DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, and the electroporation parameters are 2.5 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm and 30°C for 2 hours. Take 200 μl of bacterial solution and spread it on LB plate containing ampicillin (final concentration of 100 μg/ml) and chloramphenicol (final concentration of 34 μg/ml). After overnight culture at 30°C, select single colonies for PCR verification. The primers used are XZ-adhE-up/XZ-adhE-down. The correct colony amplification product is a fragment of 3167 bp. Select a correct single colony and name it Span050-CS.

The second step was to integrate the acetohydroxyacid reductoisomerase kari gene into the Span050-CS engineered bacteria. The acetohydroxyacid reductoisomerase encoding gene kari was obtained by whole gene synthesis based on the kari sequence from Thermacetogenium phaeum strain reported in the literature (Brinkmann-Chen, S., Cahn, J.K.B. & Arnold, F.H. Uncovering rare NADH-preferring key-acid reductoisomerases. Metab Eng 26, 17-22, doi: 10.1016/j.ymben.2014.08.003 (2014).) and after codon optimization (SEQ ID NO: 10). During the synthesis, the RBS5 artificial regulatory element (SEQ ID NO: 170) was added in front of the kari gene to promote the expression of the kari gene, and the gene was inserted into the pUC57 vector to construct the plasmid pUC57-RBS5-kari (gene synthesis and vector construction were completed by Nanjing GenScript Biotechnology Co., Ltd.). The RBS5 artificial regulatory element and the kari gene were integrated into the Span050-CS engineered bacteria to achieve the expression of the kari gene.

Using pUC57-RBS5-kari plasmid DNA as a template, primers adhE-RBS5-up/adhE-kari-down were used to amplify 1188bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated to Span050-CS. The electroporation conditions were as follows: First, prepare electroporation competent cells of Span050-CS with pKD46 plasmid using the same steps as in the first step. Then, place 50μl competent cells on ice, add 50ng DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5kv. After electroporation, quickly transfer 1ml LB culture medium to the electroporation cup, blow 5 times, transfer to a test tube, 75 rpm, and incubate at 30°C for 4 hours. The bacterial liquid was transferred to LB liquid medium without sodium chloride containing 10% sucrose, and after culturing for 24 hours, it was streaked on LB solid medium without sodium chloride containing 6% sucrose. PCR verification was performed using XZ-adhE-up/XZ-adhE-down. The correct colony amplification product was a 1454 bp fragment. The PCR product was sequenced and a correct single colony was selected and named Span096.

The Span096 strain was deposited in CGMCC with the accession number CGMCC No.26276.

Example 29: Production of pantoate using Span096

The seed culture medium consists of the following components (the solvent is water):

Macroelements: glucose 20g/L, (NH4)2HPO4 3.5g/L, KH2PO4 3.91g/L, K2HPO4 4.48g/L, MgSO4·7H2O 0.18g/L, betaine-HCl 0.15g/L;

Trace elements: FeCl3·6H2O 1.5μg/L, CoCl2·6H2O 0.1μg/L, CuCl2·2H2O 0.1μg/L, ZnCl2 0.1μg/L, Na2MoO4·2H2O 0.1μg/L, MnCl2·4H2O 0.2μg/L, H3BO3 0.05μg/L.

The fermentation medium is mostly the same as the seed medium, except that the glucose concentration is 50 g/L and the fermentation medium also contains 5 g/L serine.

The fermentation of Span096 includes the following steps:

(1) Seed culture: Fresh clones from the LB plate were inoculated into a test tube containing 4 ml of seed culture medium and cultured overnight at 37°C, 250 rpm with shaking. Then, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed culture medium at an inoculum volume of 2% (V/V) and cultured at 37°C, 250 rpm with shaking for 12 hours to obtain a seed culture solution for inoculation of the fermentation medium.

(2) Fermentation culture: The volume of the fermentation medium in a 250 ml Erlenmeyer flask was 25 ml. The seed culture solution was inoculated into the fermentation medium at an inoculum amount of a final concentration of OD550 = 0.1. The fermentation was carried out at 37° C. and 250 rpm for 60 hours to obtain a fermentation solution.

Analysis method: Agilent-1260 high performance liquid chromatograph was used to determine the components in the fermentation broth after 3 days of fermentation. The concentrations of glucose and pantoic acid in the fermentation broth were determined using Biorad's Aminex HPX-87H organic acid analysis column.

The results showed that the Span096 strain could produce 1.8 g/L of pantoic acid after 3 days of fermentation, which was 50% higher than that of the starting strain Span050.

Example 30: Fermentation of Span096 in a 5L tank

The composition, preparation and analysis method of the seed culture medium are the same as those described in Example 29.

Fermentation medium: glucose 30 g/L, magnesium sulfate 5 g/L, potassium dihydrogen phosphate 10.5 g/L, yeast powder 20 g/L, diammonium hydrogen phosphate 6 g/L, citric acid monohydrate 1.84 g/L, trace elements are the same as the fermentation medium in Example 26, and the solvent is water.

Feed medium: 600 g/L glucose, solvent is water.

Fermentation was carried out in a 5L fermenter (BIOTECH-5BG, Shanghai Baoxing), including the following steps:

(1) Seed culture: 50 mL of seed culture medium was placed in a 500 mL Erlenmeyer flask and sterilized at 115°C for 15 min. After cooling, the recombinant E. coli Span096 was inoculated into the seed culture medium at a rate of 1% (V/V), and cultured at 37°C and 250 rpm for 12 hours to obtain a seed solution for inoculation of the fermentation medium.

(2) Fermentation culture: The volume of the fermentation medium in a 5L fermenter is 3L, and sterilized at 115°C for 25 min. The seed solution is inoculated into the fermentation medium at a final concentration of OD550 = 0.2, the dissolved oxygen is maintained at 30%, and the pH is maintained at 7.0 using ammonia water as a neutralizer. The glucose concentration in the tank is controlled below 5g/L by feeding the medium, and the culture is carried out at 37°C for 3 days to obtain the fermentation broth. The fermentation broth is all the substances in the fermenter.

The results showed that after 3 days of fermentation, the production of pantoic acid in Span096 reached 31 g/L, which has great potential for industrial application.

Sequence information:

Bacillus subtilis acetolactate synthase (alsS), amino acid sequence (SEQ ID NO: 1): MLTKATKEQKSLVKNRGAELVVDCLVEQGVTHVFGIPGAKIDAVFDALQDKGPEIIVARHEQNAAFMAQAVGRLTGKPGVVLVTSGPGASNLATGLLTANTEGDPVVALAGNVIRADRLKRTHQSLDNAALFQPITKYSVEVQDVKNIPEAVTNAFRIASAGQAGAAFVSFPQDVVNEVTNTKNVRAVAAPKLGPAADDAISAAIAKIQTAKLPVVLVGMKGGRPEAIKAVRKLLKKVQLPFVETYQAAGTLSRDLEDQYFGRIGLFRNQPGDLLLEQADVV LTIGYDPIEYDPKFWNINGDRTIIHLDEIIADIDHAYQPDLELIGDIPSTINHIEHDAVKVEFAEREQKILSDLKQYMHEGEQVPADWKSDRAHPLEIVKELRNAVDDHVTVTCDIGSHAIWMSRYFRSYEPLTLMISNGMQTLGVALPWAIGASLVKPGEKVVSVSGDGGFLFSAMELETAVRLKAPIVHIVWNDSTYDMVAFQQLKKYNRTSAVDFGN IDIVKYAESFGATGLRVESPDQLADVLRQGMNAEGPVIIDVPVDYSDNINLASDKLPKEFGELMKTKAL

Bacillus subtilis acetolactate synthase (alsS), nucleotide sequence (SEQ ID NO: 2): atgttgacaaaagcaacaaaagaacaaaaatcccttgtgaaaaacagaggggcggagcttgttgttgattgcttagtggagcaaggtgtcacacatgtatttggcattccaggtgcaaaaattgatgcggtatttgacgctttacaagataaaggacctgaaattatcgttgcccggcacgaacaaaacgcagcattcatggcccaagc agtcggccgtttaactggaaaaccgggagtcgtgttagtcacatcaggaccgggtgcctctaacttggcaacaggcctgctgacagcgaacactgaaggagaccctgtcgttgcgcttgctggaaacgtgatccgtgcagatcgtttaaaacggacacatcaatctttggataatgcggcgctattccagcc gattacaaaatacagtgtagaag ttcaagatgtaaaaaatataccggaagctgttacaaatgcatttaggatagcgtcagggcaggcaggctggggccgcttttgtgagctttccgcaagatgttgtgaatgaagtcacaaatacgaaaaacgtgcgtgctgttgcagcgccaaaaactcggtcctgcagcagatgatgcaatcagtg cggccatagcaaaaatccaaacagcaaaactt cctgtcgttttggtcggcatgaaaggcggaagaccggaagcaattaaagcggttcgcaagcttttgaaaaaggttcagcttccatttgttgaaacatatcaagctgccggtaccctttctagagatttagaggatcaatattttggccgtatcggtttgttccgcaaccagcctggcgatttactgctagag caggcagatgttgttctgacgatc ggctatgacccgattgaatatgatccgaaattctggaatatcaatggagaccggacaattatccatttagacgagattatcgctgacattgatcatgcttaccagcctgatcttgaattgatcggtgacattccgtccacgatcaatcatatcgaacacgatgctgtgaaagtggaatttgcagagcgtgagcagaaaatcctttctgattta aa acaatatatgcatgaaggtgagcaggtgcctgcagattggaaatcagacagcgcaccctcttgaaatcgttaaagagttgcgtaatgcagtcgatgatcatgttacagtaacttgcgatatcggttcgcacgccatttggatgtcacgttatttccgcagctacgagccgttaacattaatgatcagtaacggtat gcaaacactcggcgttg cgcttccttgggcaatcggcgcttcattggtgaaaccgggagaaaaagtggtttctgtctctggtgacggcggttcttattctcagcaatggaattagacagcagttcgactaaaagcaccaattgtacacattgtatggaacgacagcacatatgacatggttgcattccagcaattgaaaaaataaccgtacat ctgcggtcgatttc ggaaatatcgatatcgtgaaatatgcggaaagcttcggagcaactggcttgcgcgtagaatcaccagaccagctggcgcgtagaatcaccagaccagctggcagatgttctgcgtcaaggcatgaacgctgaaggtcctgtcatcatcgatgtcccggttgactacagtgataacattaatttagcaagtgacaagcttccgaaagaattcggggaactcatgaaaacgaa agctctctag

Acetolactate synthase I of Escherichia coli, amino acid sequence (SEQ ID NO: 3)

MASSGTTSTRKRFTGAEFIVHFLEQQGIKIVTGIPGGSILPVYDALSQSTQIRHILARHEQGAGFIAQGMARTDGKPAV

CMACSGPGATNLVTAIADARLDSIPLICITGQVPASMIGTDAFQEVDTYGISIPITKHNYLVRHIEELPQVMSDAFRIAQ

SGRPGPVWIDIPKDVQTAVFEIEAQPAVAEKAAAPAFSEESIRDAATMINAAKRPVLYLGGGVINAPARVRELAEKAQ

LPTTMTLMALGMLPKAHPLSLGMLGMHGVRSTNYILQEADLLIVLGARRFDDRAIGKTEQFCPNAKIIHVDIDRAEL

GKIKQPHVAIQADVDDVLAQLIPLVEAQPRAEWHQLVADLQREFPCPIPKACDPLSHYGLINAVAACVDDNAIITTDV

GQHQMWTAQAYPLNRPRQWLTSGGLGTMGFGLPAAIGAALANPDRKVLCFSGDGSLMMNIQEMATASENQLDVK

IILMNNEALGLVHQQQSLFYEQGVFAATYPGKINFMQIAAGFGLETCDLNNEADPQAALQEIINRPGPALIHVRIDAE

EKVYPMVPPGAANTEMVGE

Acetolactate synthase I of Escherichia coli, nucleotide sequence (SEQ ID NO: 4)

atggcaagttcgggcacaacatcgacgcgtaagcgctttaccggcgcagaatttatcgttcatttcctggaacagcagggcattaagattgtgacgggcattccgggcggttctatcct

gcctgtttacgatgccttaagccaaagtacgcaaatccgccatattctggctcgccatgaacagggcgcgggatttatcgctcagggaatggcgcgcaccgacggtaaaccggcggt

ctgtatggcctgtagcggaccgggtgcgactaacctggtgaccgccattgccgatgcgcggctggactccatcccgctgatttgcatcactggtcaggttcccgcctcgatgatcggc

accgacgccttccaggaagtcgacacctacggcatctctatccccatcaccaaacacaactatctggtcagacatatcgaagaactcccgcaggtcatgagcgatgccttccgcattg

cgcaatcaggccgcccaggcccggtgtggatagacattcctaaggatgtgcaaacggcggtttttgagattgaagctcagcccgcggtggcagaaaaagccgctgcacccgccttt

agcgaagaaagcattcgtgacgcagctacaatgattaacgctgccaaacgcccggtgctttatctgggtggtggtgtgatcaatgcgcctgcgcgggtgcgtgaactggcggagaaa

gcgcaactgcctaccaccatgactttaatggcgctgggcatgctgccaaaagcgcatccgttgtcgctgggtatgctggggatgcacggcgtgcgcagcactaactatatcttgcagg

aggcggatttactgattgtgctcggtgcgcgttttgatgaccgggcgattggcaaaaccgagcagttctgtccgaatgccaaaatcattcatgtcgatatcgaccgtgcagagctgggta

aaatcaagcagccgcatgtggcgattcaggcggatgttgatgacgtgctggcgcagttgatcccgctggtggaagcgcaaccgcgtgcagagtggcaccagttggtagcggatttg

cagcgtgagtttccgtgtccaatcccgaaagcgtgcgatccattaagccattacggcctgatcaacgccgttgccgcctgtgtcgatgacaatgcgattatcaccaccgatgtggggca

gcatcagatgtggaccgcgcaagcttatccgctcaatcgcccacgccagtggctgacctccggtgggctgggcacgatgggttttggcctgcctgcggcgattggcgcggcgctgg

cgaacccggatcgcaaagtgttgtgtttctccggcgacggcagcctgatgatgaatattcaggagatggcgaccgccagtgaaaatcagctggatgtcaaaatcattctgatgaacaa

cgaagcgctggggctggtgcatcagcaacagagtctgttctacgagcaaggcgtttttgccgccacctatccgggcaaaatcaactttatgcagattgccgccggattcggcctcgaa

acctgtgatttgaataacgaagccgatccgcaggctgcattgcaggaaatcatcaatcgccctggcccggcgctgatccatgtgcgcattgatgccgaagaaaaagtttacccgatgg

tgccgccaggtgcggcgaatactgaaatggtgggggaataa

Acetolactate synthase II of Escherichia coli, amino acid sequence (SEQ ID NO: 5)

MNGAQWVVHALRAQGVNTVFGYPGGAIMPVYDALYDGGVEHLLCRHEQGAAMAAIGYARATGKTGVCIATSGP

GATNLITGLADALLDSIPVVAITGQVSAPFIGTDAFQEVDVLGLSLACTKHSFLVQSLEELPRIMAEAFDVASSGRPGP

VLVDIPKDIQLASGDLEPWFTTVENEVTFPHAEVEQARQMLAKAQKPMLYVGGGVGMAQAVPALREFLATTKMPA

TCTLKGLGAVEADYPYYLGMLGMMHGTKAANFAVQECDLLIAVGARFDDRVTGKLNTFAPHASVIHMDIDPAEMNK

LRQAHVALQGDLNALLPALQQPLNINDWQQHCAQLRDEHAWRYDHPGDAIYAPLLLKQLSDRKPADCVVTTDVG

QHQMWAAQHIAHTRPENFITTSSGLGTMGFGLPAAVGAQVARPNDTVVCISGDGSFMMNVQELGTVKRKQLPLKIV

LLDNQRLGMVRQWQQLFFQERYSETTLTDNPDFLMLASAFGIPGQHITRKDQVEAALNTMLNSDGPYLLHVSIDEL

ENVWPLVPPGASNSEMLEKLS

Acetolactate synthase II of Escherichia coli, nucleotide sequence (SEQ ID NO: 6)

atgaatggcgcacagtgggtggtacatgcgttgcgggcacagggtgtgaataccgttttcggttatccgggtggcgcaattatgccggtttacgatgcattgtatgacggcggcgtgga

gcacttgctgtgccgacatgaacagggtgcggcaatggcggctatcggttatgcccgtgctactggcaaaactggcgtatgtatcgccacgtctggtccgggcgcaaccaacctgat

aaccgggcttgcggacgcactgttagattccatccccgttgttgccatcaccggtcaagtgtccgcaccgtttatcggcacggacgcatttcaggaagtggatgtcctgggattgtcgct

agcctgtaccaagcacagcttcctggtgcagtcgctggaagagttgccgcgcatcatggctgaagcattcgacgttgccagctcaggtcgtcctggtccggttctggtcgatatcccaa

aagatatccaattagccagcggcgacctggaaccgtggttcaccaccgttgaaaacgaagtgactttcccacatgccgaagtcgagcaagcgcgccagatgctggcaaaagcgca

aaaaccgatgctgtacgttggtggtggcgtgggtatggcgcaggcagttcctgctttacgagaatttctcgctaccacaaaaatgcctgccacctgcacgctgaaagggctgggcgca

gttgaagcagattatccgtactatctgggcatgctgggaatgcatggcaccaaagcggcgaacttcgcggtgcaggagtgcgacttgctgatcgccgtgggtgcacgttttgatgatgacc

gggtgaccggcaaactgaacaccttcgcaccacacgccagtgttatccatatggatatcgacccggcagaaatgaacaagctgcgtcaggcacatgtggcattacaaggtgatttaa

atgctctgttaccagcattacagcagccgttaaatatcaatgactggcagcaacactgcgcgcagctgcgtgatgaacatgcctggcgttacgaccatcccggtgacgctatctacgcg

ccgttgttgttaaaacaactgtcggatcgtaaacctgcggattgcgtcgtgaccacagatgtggggcagcaccagatgtgggctgcgcagcacatcgcccacactcgcccggaaaat

ttcatcacctccagcggcttaggtaccatgggttttggtttaccggcggcggttggcgcacaagtcgcgcgaccgaacgataccgttgtctgtatctccggtgacggctctttcatgatg

aatgtgcaagagctgggcaccgtaaaacgcaagcagttaccgttgaaaatcgtcttactcgataaccaacggttagggatggttcgacaatggcagcaactgttttttcaggaacgata

cagcgaaaccacccttactgataaccccgatttcctcatgttagccagcgccttcggcatccctggccaacacatcacccgtaaagaccaggttgaagcggcactcaacaccatgctg

aacagtgatgggccatacctgcttcatgtctcaatcgacgaacttgagaacgtctggccgctggtgccgccaggtgccagtaattcagaaatgttggagaaattatcatga

L-valine feedback-resistant acetolactate synthase III, amino acid sequence (SEQ ID NO: 7)

MRRILSVLLENESDALFRVIGLFSQRGYNIESLTVAPTDDPTLSRMTIQTVGDEKVLEQIEKQLHKLVDVLRVSELGQ

GAHVEREIMLVKIQASGYGRDEVKRNTEIFRGQIIDVTPSLYTVQLAGTSGKLDAFLASIRDVAKIVEVARSGVVGLS

RGDKIMR

L-valine feedback-resistant acetolactate synthase III, nucleotide sequence (SEQ ID NO: 8)

atgcgccggatattatcagtcttactcgaaaatgaatcagacgcgttattccgcgtgattggccttttttcccagcgtggctacaacattgaaagcctgaccgttgcgccaaccgacgatc

cgacattatcgcgtatgaccatccagaccgtgggcgatgaaaaagtacttgagcagatcgaaaagcaattacacaagctggtcgatgtcttgcgcgtgagtgagttggggcagggcg

cgcatgttgagcgggaaatcatgctggtgaaaattcaggccagcggttacgggcgtgacgaagtgaaacgtaatacggaaatattccgtgggcaaattatcgatgtcacaccctcgct

ttataccgttcaattagcaggcaccagcggtaagcttgatgcatttttagcatcgattcgcgatgtggcgaaaattgtggaggttgctcgctctggtgtggtcggactttcgcgcggcgat

aaaataatgcgttga

NADH-dependent acetohydroxyacid reductoisomerase, amino acid sequence (SEQ ID NO: 9)

MKIYYDQDADLQYLDGKTVAVIGYGSQGHAQSQNLRDSGVKVVVADIPSSENWKKAEEAQFQPLTADEAAREADII

QILVPDEKQAALYRESIAPNLRPGKALVFSHGFNIHFKQIVPPPDVDVFMVAPKGPGHLVRRMYEEGAGVPSLVAVEQ

DYSGQALNLALAYAKGIGATRAGVIQTTFKEETETDLFGEQAVLCGGITELIRAGFDTLVDAGYQPEIAYFECLHEMK

LIVDLIYEGGISTMRYSISDTAEYGDLTRGKRIITEATREEMKKILKEIQDGVFAREWLLENQVGRPVYNALRRKEQN

HLIETVGARLRGMMPWLKKKVI

NADH-dependent acetohydroxyacid reductoisomerase, nucleotide sequence (SEQ ID NO: 10)

atgaaaatctattacgatcaggacgcggatctgcaatatctggatggcaaaaccgtggctgttatcggctacggttcacagggccatgcgcagtcgcaaaatctgcgtgacagcggtgttaaagtggttgtcgcggatattccgagctctgaaaactggaaaaaagctgaagaagcgcagttccaaccgct gacggctgacgaagcagcccgcgaagcggatattatccagattctggtgccggatgaa aaacaagcagctctgtatcgtgaatcaatcgccccgaatctgcgcccgggcaaagcactggtgtttagccacggcttcaacattcactttaaacagatcgtgccgccgccggacgtcgatgtgtttatggtcgcaccgaaaggtccgggtcacctggtgcgtcgcatgtacgaagaaggcgccggtg ttccgtctctggttgcagtcgaacaggactatagtggtcaagccctgaatctggcgctggcct acgcaaaaggcattggtgccacccgtgcaggcgtcatccagaccacgttcaaagaagaaaccgaaaccgacctgtttggtgaacaagccgtcctgtgcggcggtattaccgaactgatccgcgcaggcttcgacaccctggtggatgctggttatcagccggaaattgcgtactttgaatgtctgcatgaaatga aactgattgttgacctgatctatgaaggcggtatttccaccatgcgttatagtat ctccgacaccgctgaatacggcgatctgacgcgtggtaaacgcattatcaccgaagcgacgcgcgaagaaatgaagaaaattctgaaagaaatccaggatggcgtgttcgcccgtgaatggctgctggaaaaccaagtgggtcgcccggtttataatgcccggtttataatgccctgcgtcgcaaagaacagaaccacctgattgaa accgtgggcgcacgtctgcgcggtatgatgccgtggctgaagaaaaaagttatctaa

Dihydroxyacid dehydratase, amino acid sequence (SEQ ID NO: 11)

MPKYRSATTTHGRNMAGARALWRATGMTDADFGKPIIAVVNSFTQFVPGHVHLRDLGKLVAEQIEAAGGVAKEFN

TIAVDDGIAMGHGGGMLYSLPSRELIADSVEYMVNAHCADAMVCISNCDKITPGMLMASLRLNIPVIFVSGGPMEAG

KTKLSDQIIKLDLVDAMIQGADPKVSDSQSDQVERSACPTCGSCSGMFTANSMNCLTEALGLSQPGNGSLLATHAD

RKQLFLNAGKRIVELTKRYYEQNDESALPRNIASKAAFENAMTLDIAMGGSTNTVLHLLAAAQEAEIDFTMSDIDK

LSRKVPQLCKVAPSTQKYHMEDVHRAGGVIGILGELDRAGLLNRDVKNVLGLTLPQTLEQYDVMLTQDDAVKNMF

RAGPAGIRTTQAFSQDCRWDTLDDDRANGCIRSLEHAYSKDGGLAVLYGNFAENGCIVKTAGVDDSILKFTGPAKV

YESQDDAVEAILGGKVVAGDVVVIRYEGPKGPGGMQEMLYPTSFLKSMGLGKACALITDGRFSGGTSGLSIGHVSP

EAASGGSIGLIEDGDLIAIDIPNRGIQLQVSDAELAARREAQDARGDKAWTPKNRERQVSFALRAYASLATSADKGA

VRDKSKLGG

Dihydroxyacid dehydratase, nucleotide sequence (SEQ ID NO: 12)

atgcctaagtaccgttccgccaccaccactcatggtcgtaatatggcgggtgctcgtgcgctgtggcgcgccaccggaatgaccgacgccgatttcggtaagccgattatcgcggttg

tgaactcgttcacccaatttgtaccgggtcacgtccatctgcgcgatctcggtaaactggtcgccgaacaaattgaagcggctggcggcgttgccaaagagttcaacaccattgcggtg

gatgatgggattgccatgggccacggggggatgctttattcactgccatctcgcgaactgatcgctgattccgttgagtatatggtcaacgcccactgcgccgacgccatggtctgcat

ctctaactgcgacaaaatcaccccggggatgctgatggcttccctgcgcctgaatattccggtgatctttgtttccggcggcccgatggaggccgggaaaaccaaactttccgatcaga

tcatcaagctcgatctggttgatgcgatgatccagggcgcagacccgaaagtatctgactcccagagcgatcaggttgaacgttccgcgtgtccgacctgcggttcctgctccggggat

gtttaccgctaactcaatgaactgcctgaccgaagcgctgggcctgtcgcagccgggcaacggctcgctgctggcaacccacgccgaccgtaagcagctgttccttaatgctggtaa

acgcattgttgaattgaccaaacgttattacgagcaaaacgacgaaagtgcactgccgcgtaatatcgccagtaaggcggcgtttgaaaacgccatgacgctggatatcgcgatgggt

ggatcgactaacaccgtacttcacctgctggcggcggcgcaggaagcggaaatcgacttcaccatgagtgatatcgataagctttcccgcaaggttccacagctgtgtaaagttgcgc

cgagcacccagaaataccatatggaagatgttcaccgtgctggtggtgttatcggtattctcggcgaactggatcgcgcggggttatgaaccgtgatgtgaaaaacgtacttggcctg

acgttgccgcaaacgctggaacaatacgacgttatgctgacccaggatgacgcggtaaaaaatatgttccgcgcaggtcctgcaggcattcgtaccacacaggcattctcgcaagatt

gccgttgggatacgctggacgacgatcgcgccaatggctgtatccgctcgctggaacacgcctacagcaaagacggcggcctggcggtgctctacggtaactttgcggaaaacgg

ctgcatcgtgaaaacggcaggcgtcgatgacagcatcctcaaattcaccggcccggcgaaagtgtacgaaagccaggacgatgcggtagaagcgattctcggcggtaaagttgtc

gccggagatgtggtagtaattcgctatgaaggcccgaaaggcggtccggggatgcaggaaatgctctacccaaccagcttcctgaaatcaatgggtctcggcaaagcctgtgcgct

gatcaccgacggtcgtttctctggtggcacctctggtctttccatcggccacgtctcaccggaagcggcaagcggcggcagcattggcctgattgaagatggtgacctgatcgctatc

gacatcccgaaccgtggcattcagttacaggtaagcgatgccgaactggcggcgcgtcgtgaagcgcaggacgctcgaggtgacaaagcctggacgccgaaaaatcgtgaacgt

caggtctcctttgccctgcgtgcttatgccagcctggcaaccagcgccgacaaaggcgcggtgcgcgataaatcgaaactggggggttaa

Corynebacterium glutamicum 3-methyl-2-oxobutanoate hydroxymethyltransferase, amino acid sequence (SEQ ID NO: 13)MPMSGIDAKKIRTRHFREAKVNGQKVSVLTSYDALSARIFDEAGVDMLLVGDSAANVVLGRDTTLSITLDEMIVLAKAVTIATKRALVVVDLPFGTYEVSPNQAVESAIRVMRETGAAAVKIEGGVEIAQTIRRIVDAGIPVVGHIGYTPQSEHSLGGHVVQGRGASSGKLIADARALEQAGAFAVVLEMVPAEAAREVTEDLSITTIGIGAGNGTDGQVLVWQDAFGLNRGKKPRFVREYATLGDSLHDAAQAYIADIHAGTFPGEAESF

Corynebacterium glutamicum 3-methyl-2-oxobutyrate hydroxymethyltransferase, nucleotide sequence (SEQ ID NO: 14) atgcccatgtcaggcattgatgcaaagaaaatccgcacccgtcatttccgcgaagctaaagtaaacggccagaaagtttcggttctcaccagctatgatgcgctttcggcgcgcatttttgatgaggctggcgtcgatatgctccttgttggtgattccgctgccaacgttgtgctgggtcgcgataccaccttgtcga tcaccttggatgagatgattgtgctggccaaggcggtgacgatcgctacgaagcgtgcgcttgtggtggttgatctgccgtttggtacctatgaggtgagcccaaatcaggcggtggagtccgcgatccgggtcatgcgtgaaacgggtgcggctgcggtgaagatcgagggtggcgtgg agatcgcgcagacgattcgacgcatt gttgatgctggaattccggttgtcggccacatcgggtacaccccgcagtccgagcattccttgggcggccacgtggttcagggtcgtggcgcgagttctggaaagctcatcgccgatgcccgcgcgttggagcaggcgggtgcgtttgcggttgtgttggagatggttccagcagagg cagcgcgcgaggttaccgaggatcttt ccatcaccactatcggaatcggtgccggcaatggcacagatgggcaggcaggttttggtgtggcaggatgccttcggcctcaaccgcggcaagaagccacgcttcgtccgcgagtacgccaccttgggcgattccttgcacgacgccgcgcaggcctacatcgccgatatccacgcgggtaccttcccaggcgaagc ggagtccttttaa

Escherichia coli 3-methyl-2-oxobutyrate hydroxymethyltransferase, amino acid sequence (SEQ ID NO: 15)

MKPTTISLLQKYKQEKKRFATITAYDYSFAKLFADEGLNVMLVGDSLGMTVQGHDSTLPVTVADIAYHTAAVRRGA

PNCLLLADLPFMAYATPEQAFENAATVMRAGANMVKIEGGEWLVETVQMLTERAVPVCGHLGLTPQSVNIFGGYK

VQGRGDEAGDQLLSDALALEAAGAQLLVLECPVVELAKRITEALAIPVIGIGAGNVTDGQILVMHDAFGITGGHIPK

FAKNFLAETGDIRAAVRQYMAEVESGVYPGEEHSFH

Escherichia coli 3-methyl-2-oxobutyrate hydroxymethyltransferase, nucleotide sequence (SEQ ID NO: 16)

ttaatggaaactgtgttcttcgcccggataaacgccggactccacttcagccatatactgccgcacagccgcgcggatgtcgcccgtttcggcgaggaaatttttagcgaatttaggaat

gtgaccgccggtaataccaaaggcgtcgtgcatcacgaggatctgcccgtcagtgacgttgcctgcgccaatgccaataaccgggatcgccagtgcttcggtaatacgttttgccagt

tcaaccggcacgcattccagcaccagcagctgtgccccagcagcttctaaggctaatgcatcgctgagcagttgatcgcccgcttcatcgccgcgcccctgaactttgtagccaccga

aaatattcactgactgtggtgttaaacctaagtgaccacatacaggaacggcacgttcggtcagcatttgtacggtttctaccagccactcaccgccttcaattttgaccatgttagcaccg

gcacgcataaccgttgcggcgttttcgaaggcttgttccggcgtggcatacgccataaacggcaggtcagccagcagcagcagcagtttggtgcgccgcgacgtacggcggcagtgtg

gtaggcgatatcggcaacggtaactggcagggtggagtcgtgcccctgaaccgtcatgcccagcgaatcgcccaccagcatgacgttaagcccttcatcagcaaagagtttggcga

agctatagtcataagcggtgatggtcgcgaaacgttttttttcctgtttgtacttctgcagtaaggagatggtggtcggtttcat

2-Dehydropantothenate-2-reductase, amino acid sequence (SEQ ID NO: 17)

MKITVLGCGALGQLWLTALCKQGHEVQGWLRVPQPYCSVNLVETDGSIFNESLTANDPDFLATSDLLLVTLKAWQV

SDAVKSLASTLPVTTPILLIHNGMGTIEELQNIQQPLLMGTTTHAARRDGNVIIHVANGITHIGPARQQDGDYSYLADI

LQTVLPDVAWHNNIRAELWRKLAVNCVINPLTAIWNCPNGELRHHPQEIMQICEEVAAVIEREGHHTSAEDLRDYVM

QVIDATAENISSMLQDIRALRHTEIDYINGFLLRRARAHGIAVPENTRLFEMVKRKESEYERIGTGLPRPW

2-Dehydropantothenate-2-reductase, nucleotide sequence (SEQ ID NO: 18)

atgaaaattaccgtattggggatgcggtgccttagggcaattatggcttacagcactttgcaaacagggtcatgaagttcagggctggctgcgcgtaccgcaaccttattgtagcgtgaat

ctggttgagacagatggttcgatatttaacgaatcgctgaccgccaacgatcccgattttctcgccaccagcgatctgctcctggtgacgctgaaagcatggcaggtttccgatgccgtc

aaaagcctcgcgtccacactgcctgtaactacgccaatactgttaattcacaacggcatgggcaccatcgaagagttgcaaaacattcagcagccattactgatgggcaccacccaccc

atgcagcccgccgcgacggcaatgtcattattcatgtggcaaacggtatcacgcatattggcccggcacggcaacaggacggggattacagttatctggcggatattttgcaaaccgt

gttgcctgacgttgcctggcataacaatattcgcgccgagctgtggcgcaagctggcagtcaactgcgtgattaatccactgactgccatctggaattgcccgaacggtgaattacgtc

atcatccgcaagaaattatgcagatatgcgaagaagtcgcggcggtgatcgaacgcgaagggcatcatacttcagcagaagatttgcgtgattacgtgatgcaggtgattgatgccac

agcggaaaatatctcgtcgatgttgcaggatatccgcgcgctgcgccacactgaaatcgactatatcaatggtttctcttacgccgcgcccgcgcgcatgggattgccgtaccggaaa

acacccgcctgtttgaaatggtaaaaagaaaggagagtgaatatgagcgcatcggcactggtttgcctcgcccctggtag

Serine hydroxymethyltransferase, amino acid sequence (SEQ ID NO: 19)

MLKREMNIADYDAELWQAMEQEKVRQEEHIELIASENYTSPRVMQAQGSQLTNKYAEGYPGKRYYGGCEYVDIVE

QLAIDRAKELFGADYANVQPHSGSQANFAVYTALLEPGDTVLGMNLAHGGHLTHGSPVNFSGKLYNIVPYGIDATG

HIDYADLEKQAKEHKPKMIIGGFSAYSGVVDWAKMREIADSIGAYLFVDMAHVAGLVAAGVYPNPVPHAHVVTTT

THKTLAGPRGGLILAKGGSEELYKKLNSAVFPGGQGGPLMHVIAGKAVALKEAMEPEFKTYQQQVAKNAKAMVEV

FLERGYKVVSGGTDNHLFLVDLVDKNLTGKEADAALGRANITVNKNSVPNDPKSPFVTSGIRVGTPAITRRGFKEAE

AKELAGWMCDVLDSINDEAVIERIKGKVLDICARYPVYA

Serine hydroxymethyltransferase, nucleotide sequence (SEQ ID NO: 20)

atgttaaagcgtgaaatgaacattgccgattatgatgccgaactgtggcaggctatggagcaggaaaaagtacgtcaggaagagcacatcgaactgatcgcctccgaaaactacacc

agcccgcgcgtaatgcaggcgcagggttctcagctgaccaacaaatatgctgaaggttatccgggcaaacgctactacggcggttgcgagtatgttgatatcgttgaacaactggcg

atcgatcgtgcgaaagaactgttcggcgctgactacgctaacgtccagccgcactccggctcccaggctaactttgcggtctacaccgcgctgctggaaccaggtgataccgttctgg

gtatgaacctggcgcatggcggtcacctgactcacggttctccggttaacttctccggtaaactgtacaacatcgttccttacggtatcgatgctaccggtcatatcgactacgccgatct

ggaaaaacaagccaaagaacacaagccgaaaatgattatcggtggtttctctgcatattccggcgtggtggactgggcgaaaatgcgtgaaatcgctgacagcatcggtgcttacctg

ttcgttgatatggcgcacgttgcgggcctggttgctgctggcgtctacccgaacccggttcctcatgctcacgttgttactaccaccactcacaaaaccctggcgggtccgcgcggcgg

cctgatcctggcgaaaggtggtagcgaagagctgtacaaaaaactgaactctgccgttttccctggtggtcagggcggtccgttgatgcacgtaatcgccggtaaagcggttgctctg

aaagaagcgatggagcctgagttcaaaacttaccagcagcaggtcgctaaaaacgctaaagcgatggtagaagtgttcctcgagcgcggctacaaagtggtttccggcggcactga

taaccacctgttcctggttgatctggttgataaaaacctgaccggtaaagaagcagacgccgctctgggccgtgctaacatcaccgtcaacaaaaacagcgtaccgaacgatccgaa

gagcccgtttgtgacctccggtattcgtgtaggtactccggcgattacccgtcgcggctttaaagaagccgaagcgaaagaactggctggctggatgtgtgacgtgctggacagcatc

aatgatgaagccgttatcgagcgcatcaaaggtaaagttctcgacatctgcgcacgttacccggtttacgcataa

Aminomethyltransferase, amino acid sequence (SEQ ID NO: 21)

MAQQTPLYEQHTLCGARMVDFHGWMMPLHYGSQIDEHHAVRTDAGMFDVSHMTIVDLRGSRTREFLRYLLANDV

AKLTKSGKALYSGMLNASGGVIDDLIVYYFTEDFFRLVVNSATREKDLSWITQHAEPFGIEITVRDDLSMIAVQGPNA

QAKAATLFNDAQRQAVEGMKPFFGVQAGDLFIATTGYTGEAGYEIALPNEKAADFWRALVEAGVKPCGLGARDTL

RLEAGMNLYGQEMDETISPLAANMGWTIAWEPADRDFIGREALEVQREHGTEKLVGLVMTEKGVLRNELPVRFTD

AQGNQHEGIITSGTFSPTLGYSIALARVPEGIGETAIVQIRNREMPVKVTKPVFVRNGKAVA

Aminomethyltransferase, nucleotide sequence (SEQ ID NO: 22)

atggcacaacagactcctttgtacgaacaacacacgctttgcggcgctcgcatggtggatttccacggctggatgatgccgttgcattacggttcgcaaatcgacgaacatcatgcggt

acgtaccgatgccggaatgtttgatgtgtcacatatgaccatcgtcgatctccgcggcagccgcacccgggagtttctgcgttatctgctggcgaacgatgtggcgaagctcaccaaa

agcggcaaagccctttactcggggatgttgaatgcctctggcggtgtgatagatgatctcatcgtctactactttactgaagatttcttccgcctcgttgttaactccgccacccgcgaaaa

agacctctcctggattacccaacacgctgaacctttcggcatcgaaattaccgttcgtgatgacctttccatgattgccgtgcaagggccgaatgcgcaggcaaaagctgccacactgtt

taatgacgcccagcgtcaggcggtggaagggatgaaaccgttctttggcgtgcaggcgggcgatctgtttattgccaccactggttataccggtgaagcgggctatgaaattgcgctg

cccaatgaaaaagcggccgatttctggcgtgcgctggtggaagcgggtgttaagccatgtggcttgggcgcgcgtgacacgctgcgtctggaagcgggcatgaatctttatggtcag

gagatggacgaaactatttctcctttagccgccaacatgggctggactatcgcctgggaaccggcagatcgtgactttatcggtcgtgaagccctggaagtgcagcgtgagcatggta

cagaaaaactggttggtctggtgatgaccgaaaaaggcgtgctgcgtaatgaactgccggtacgctttaccgatgcgcagggcaaccagcatgaaggcattatcaccagcggtacttt

ctccccgacgctgggttacagcattgcgctggcgcgcgtgccggaaggtattggcgaaacggcgattgtgcaaattcgcaaccgtgaaatgccggttaaagtgacaaaacctgttttt

gtgcgtaacggcaaagccgtcgcgtga

Glycine decarboxylase, amino acid sequence (SEQ ID NO: 23)

MTQTLSQLENSGAFIERHIGPDAAQQQEMLNAVGAQSLNALTGQIVPKDIQLATPPQVGAPATEYAALAELKAIASR

NKRFTSYIGMGYTAVQLPPVILRNMLENPGWYTAYTPYQPEVSQGRLEALLNFQQVTLDLTGLDMASASLLDEATA

AAEAMAMAKRVSKLKNANRFFVASDVHPQTLDVVRTRAETFGFEVIVDDAQKVLDHQDVFGVLLQQVGTTGEIH

DYTALISELKSRKIVVSVAADIMALVLLTAPGKQGADIVFGSAQRFGVPMGYGGPHAAFFAAKDEYKRSMPGRIIGV

SKDAAGNTALRMAMQTREQHIRREKANSNICTSQVLLANIASLYAVYHGPVGLKRIANRIHRLTDILAAGLQQKGLK

LRHAHYFDTLCVEVADKAGVLTRAEAAEINLRSDILNAVGITLDETTTRENVMQLFSVLLGDNHGLDIDTLDKDVA

HDSRSIQPAMLRDDEILTHPVFNRYHSETEMMRYMHSLERKDLALNQAMIPLGSCTMKLNAAAEMIPITWPEFAEL

HPFCPPEQAEGYQQMIAQLADWLVKLTGYDAVCMQPNSGAQGEYAGLLAIRHYHESCNEGHRDICLIPASAHGTNP

ASAHMAGMQVVVVACDKNGNIDLTDLRAKAEQAGDNLSCIMVTYPSTHGVYEETIREVCEVVHQFGGQVYLDGA

NMNAQVGITSPGFIGADVSHLNLHKTFCIPHGGGGPGMGPIGVKAHLAPFVPGHSVVQIEGMLTRQGAVSAAPFGS

ASILPISWMYIRMMGAEGLKKASQVAILNANYIASRLQDAFPVLYTGRDGRVAHECILDIRPLKEETGISELDIAKRLI

DYGFHAPTMSFPVAGTLMVEPTESESKVELDRFIDAMLAIRAEIDQVKAGVWPLEDNPLVNAPHIQSELVAEWAHPY

SREVAVFPAGVADKYWPTVKRLDDVYGDRNLFCSCVPISEYQ

Glycine decarboxylase, nucleotide sequence (SEQ ID NO: 24)

atgacacagacgttaagccagcttgaaaacagcggcgcttttattgaacgccatatcggaccggacgccgcgcaacagcaagaaatgctgaatgccgttggtgcacaatcgttaaac

gcgctgaccggccagattgtgccgaaagatattcagcttgcgaccaccgcaggttggcgcaccggcgaccgaatacgccgcactggcagaactcaaggctattgccagtcgca

ataaacgcttcacgtcttacatcggcatgggttacaccgccgtgcagctaccgccggttatcctgcgtaacatgctggaaaatccgggctggtataccgcgtacactccgtatcaacct

gaagtctcccagggccgccttgaagcactgctcaacttccagcaggtaacgctggatttgactggactggatatggcctctgcttctcttctggacgaggccaccgctgccgccgaag

caatggcgatggcgaaacgcgtcagcaaactgaaaaatgccaaccgcttcttcgtggcttccgatgtgcatccgcaaacgctggatgtggtccgtactcgtgccgaaacctttggtttt

gaagtgattgtcgatgacgcgcaaaaagtgctcgaccatcaggacgtcttcggcgtgctgttacagcaggtaggcactaccggtgaaattcacgactacactgcgctttattagcgaact

gaaatcacgcaaaattgtggtcagcgttgccgccgatattatggcgctggtgctgttaactgcgccgggtaaacagggcgcggatattgtttttggttcggcgcaacgcttcggcgtgc

cgatgggctacggtggcccacgcggcattctttgcggcgaaagatgaatacaaacgctcaatgccgggccgtattatcggtgtatcgaaagatgcagctggcaacaccgctctgc

gcatggcgatgcagactcgcgagcaacatattcgtcgtgagaaagcgaactccaacatttgtacttcccaggtactgctggcaaacatcgccagcctgtatgccgtttatcacggcccg

gttggcctgaaacgtatcgctaaccgcattcaccgtctgaccgatatcctggcggcaggcctgcaacaaaaaggtctgaagctgcgccatgcgcactatttcgacactctgtgtgtgga

agtggccgataaggcgggcgtactgacgcgtgccgaagcggctgaaatcaacctgcgtagcgatatcctgaacgcggttggggatcacccttgatgaaaccaccacgcgcgaaaac

gtgatgcagcttttcagcgtactgctgggggataaccacggcctggacatcgacacgctggacaaagacgtggctcacgacagccgctctattcaacctgcgatgctgcgcgacga

cgaaatcctcacccatccggtgtttaatcgctaccacagcgaaaccgaaatgatgcgctatatgcactcgctggagcgtaaagatctggcgctgaatcaggcgatgatcccgctgggt

tcctgcaccatgaaactgaacgccgccgccgagatgatcccaatcacctggccggaatttgccgaactgcacccgttctgcccgccggagcaggccgaaggttatcagcagatgatt

gcgcagctggctgactggctggtgaaactgaccggttacgacgccgtttgtatgcagccgaactctggcgcacagggcgaatacgcgggcctgctggcgattcgtcattatcatgaa

agctgcaacgaagggcatcgcgatatctgcctgatcccggcttctgcgcacggaactaaccccgcttctgcacatatggcaggaatgcaggtggtggttgtggcgtgtgataaaaac

ggcaacatcgatctgactgatctgcgcgcgaaagcggaacaggcgggcgataacctctcctgtatcatggtgacttatccttctacccacggcgtgtatgaagaaacgatccgtgaag

tgtgtgaagtcgtgcatcagttcggcggtcaggtttaccttgatggcgcgaacatgaacgcccaggttggcatcacctcgccgggctttattggtgcggacgtttcacaccttaacctac

ataaaactttctgcattccgcacggcggtggtggtccgggtatgggaccgatcggcgtgaaagcgcatttggcaccgtttgtaccgggtcatagcgtggtgcaaatcgaaggcatgtt

aacccgtcagggcgcggtttctgcggcaccgttcggtagcgcctctatcctgccaatcagctggatgtacatccgcatgatgggcgcagaagggctgaaaaaagcaagccaggtgg

caatcctcaacgccaactatattgccagccgcctgcaggatgccttcccggtgctgtataccggtcgcgacggtcgcgtggcgcacgaatgtattctcgatattcgcccgctgaaaga

agaaaccggcatcagcgagctggatattgccaagcgcctgatcgactacggtttccacgcgccgacgatgtcgttcccggtggcgggtacgctgatggttgaaccgactgaatctga

aagcaaagtggaactggatcgctttatcgacgcgatgctggctatccgcgcagaaattgaccaggtgaaagccggtgtctggccgctggaagataacccgctggtgaacgcgccgc

acattcagagcgaactggtcgccgagtgggcgcatccgtacagccgtgaagttgcggtattcccggcaggtgtggcagacaaatactggccgacagtgaaacgtctggatgatgttt

acggcgaccgtaacctgttctgctcctgcgtaccgattagcgaataccagtaa

Phosphoglycerate dehydrogenase, amino acid sequence (SEQ ID NO: 25)

MSQNGRPVVLIADKLAQSTVDALGDAVEVRWVDGPNRPELLDAVKEADALLVRSATTVDAEVIAAAPNLKIVGRA

GVGLDNVDIPAATEAGVMVANAPTSNIHSACEHAISLLLSTARQIPAADATLREGEWKRSSFNGVEIFGKTVGIVGFG

HIGQLFAQRLAAFETTIVAYDPYANPARAAQLNVELVELDELMSRSDFVTIHLPKTKETAGMFDAQLLAKSKKGQIII

NAARGGLVDEQALADAIESGHIRGAGFDVYSTEPCTDSPLFKLPQVVVTPHLGASTEEAQDRAGTDVADSVLKALA

GEFVADAVNVSGGRVGEEVAVWMDLA

Phosphoglycerate dehydrogenase, nucleotide sequence (SEQ ID NO: 26)

atgagccagaatggccgtccggtagtcctcatcgccgataagcttgcgcagtccactgttgacgcgcttggagatgcagtagaagtccgttgggttgacggacctaaccgcccagaa

ctgcttgatgcagttaaggaagcggacgcactgctcgtgcgttctgctaccactgtcgatgctgaagtcatcgccgctgcccctaacttgaagatcgtcggtcgtgccggcgtgggctt

ggacaacgttgacatccctgctgccactgaagctggcgtcatggttgctaacgcaccgacctctaatattcactccgcttgtgagcacgcaatttctttgctgctgtctactgctcgccag

atccctgctgctgatgcgacgctgcgtgagggcgagtggaagcggtcttctttcaacggtgtggaaattttcggaaaaactgtcggtatcgtcggttttggccacattggtcagttgtttg

ctcagcgtcttgctgcgtttgagaccaccattgttgcttacgatccttacgctaaccctgctcgtgcggctcagctgaacgttgagttggttgagttggatgagctgatgagccgttctgac

tttgtcaccattcaccttcctaagaccaaggaaactgctggcatgtttgatgcgcagctccttgctaagtccaagaagggccagatcatcatcaacgctgctcgtggtggccttgttgatg

agcaggctttggctgatgcgattgagtccggtcacattcgtggcgctggtttcgatgtgtactccaccgagccttgcactgattctcctttgttcaagttgcctcaggttgttgtgactcctca

cttgggtgcttctactgaagaggctcaggatcgtgcgggtactgacgttgctgattctgtgctcaaggcgctggctggcgagttcgtggcggatgctgtgaacgtttccggtggtcgcgt

gggcgaagaggttgctgtgtggatggatctggcttaa

Phosphoserine/phosphohydroxythreonine aminotransferase, amino acid sequence (SEQ ID NO: 27)

MAQIFNFSSSGPAMLPAEVLKQAQQELRDWNGLGTSVMEVSHRGKEFIQVAEEAEKDFRDLLNVPSNYKVLFCHGG

GRGQFAAVPLNILGDKTTADYVDAGYWAASAIKEAKKYCTPNVFDAKVTVDGLRAVKPMREWQLSDNAAYMHY

CPNETIDGIAIDETPDFGADVVVAADFSSTILSRPIDVSRYGVIYAGAQKNIGPAGLTIVIVREDLLGKANIACPSILDYS

ILNDNGSMFNTPPTFAWYLSGLVFKWLKANGGVAEMDKINQQKAELLYGVIDNSDFYRNDVAKANRSRMNVPFQL

ADSALDKLFLEESFAAGLHALKGHRVVGGMRASIYNAMPLEGVKALTDFMVEFERRHG

Phosphoserine/phosphohydroxythreonine aminotransferase, nucleotide sequence (SEQ ID NO: 28)

atggctcaaatcttcaattttagttctggtccggcaatgctaccggcagaggtgcttaaacaggctcaacaggaactgcgcgactggaacggtcttggtacgtcggtgatggaagtgag

tcaccgtggcaaagagttcattcaggttgcagaggaagccgagaaggattttcgcgatcttcttaatgtcccctccaactacaaggtattattctgccatggcggtggtcgcggtcagttt

gctgcggtaccgctgaatattctcggtgataaaaccaccgcagattatgttgatgccggttatgggcggcaagtgccattaaagaagcgaaaaaatactgcacgcctaatgtctttgac

gccaaagtgactgttgatggtctgcgcgcggttaagccaatgcgtgaatggcaactctctgataatgctgcttatatgcattattgcccgaatgaaaccatcgatggtatcgccatcgacg

aaacgccagacttcggcgcagatgtggtggtcgccgctgacttctcttcaaccattctttcccgtccgattgacgtcagccgttatggtgtaatttacgctggcgcgcagaaaaatatcgg

cccggctggcctgacaatcgtcatcgttcgtgaagatttgctgggcaaagcgaatatcgcgtgtccgtcgattctggattattccatcctcaacgataacggctccatgtttaacacgccg

ccgacatttgcctggtatctatctggtctggtctttaaatggctgaaagcgaacggcggtgtagctgaaatggataaaatcaatcagcaaaaagcagaactgctatatggggtgattgat

aacagcgatttctaccgcaatgacgtggcgaaagctaaccgttcgcggatgaacgtgccgttccagttggcggacagtgcgcttgacaaattgttccttgaagagtcttttgctgctggc

cttcatgcactgaaaggtcaccgtgtggtcggcggaatgcgcgcttctatttataacgccatgccgctggaaggcgttaaagcgctgacagacttcatggttgagttcgaacgccgtca

cggttaa

Phosphoserine phosphatase, amino acid sequence (SEQ ID NO: 29)

MPNITWCDLPEDVSLWPGLPLSLSGDEVMPLDYHAGRSGWLLYGRGLDKQRLTQYQSKLGAAMVIVAAWCVEDY

QVIRLAGSLTARATRLAHEAQLDVAPLGKIPHLRTPGLLVMDMDSTAIQIECIDEIAKLAGTGEMVAEVTERAMRGEL

DFTASLRSRVATLKGADANILQQVRENLPLMPGLTQLVLKLETLGWKVAIASGGFTFFAEYLRDKLRLTAVVANELEI

MDGKFTGNVIGDIVDAQYKAKTLTRLAQEYEIPLAQTVAIGDGANDLPMIKAAGLGIAYHAKPKVNEKAEVTIRHA

DLMGVFCILSGSLNQK

Phosphoserine phosphatase, nucleotide sequence (SEQ ID NO: 30)

atgcctaacattacctggtgcgacctgcctgaagatgtctctttatggccgggtctgcctctttcattaagtggtgatgaagtgatgccactggattaccacgcaggtcgtagcggctggc

tgctgtatggtcgtgggctggataaacaacgtctgacccaataccagagcaaactgggtgcggcgatggtgattgttgccgcctggtgcgtggaagattatcaggtgattcgtctggca

ggttcactcaccgcacgggctacacgcctggcccacgaagcgcagctggatgtcgccccgctggggaaaatcccgcacctgcgcacgccgggtttgctggtgatggatatggact

ccaccgccatccagattgaatgtattgatgaaattgccaaactggccggaacgggcgagatggtggcggaagtaaccgaacgggcgatgcgcggcgaactcgattttaccgccag

cctgcgcagccgtgtggcgacgctgaaaggcgctgacgccaatattctgcaacaggtgcgtgaaaatctgccgctgatgccaggcttaacgcaactggtgctcaagctggaaacgc

tgggctggaaagtggcgattgcctccggcggctttactttctttgctgaatacctgcgcgacaagctgcgcctgaccgccgtggtagccaatgaactggagatcatggacggtaaattt

accggcaatgtgatcggcgacatcgtagacgcgcagtacaaagcgaaaactctgactcgcctcgcgcaggagtatgaaatcccgctggcgcagaccgtggcgattggcgatggag

ccaatgacctgccgatgatcaaagcggcagggctggggattgcctaccatgccaagccaaaagtgaatgaaaaggcggaagtcaccatccgtcacgctgacctgatgggggtattc

tgcatcctctcaggcagcctgaatcagaagtaa

Branched-chain amino acid aminotransferase, amino acid sequence (SEQ ID NO: 31)

VTTKKADYIWFNGEMVRWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFPVSQ

SIDELMEACRDVIRKNNLTSAYIRPLIFVGDVGMGVNPPAGYSTDVIIAAFPWGAYLGAEALEQGIDAMVSSWNRAA

PNTIPTAAKAGGNYLSSLLVGSEARRHGYQEGIALDVNGYISEGAGENLFEVKDGVLFTPPFTSSALPGITRDAIIKLA

KELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPVTKRIQQAFFGLFTGETEDKWGWLDQ

V N

Branched-chain amino acid aminotransferase, nucleotide sequence (SEQ ID NO: 32)

gtgaccacgaagaaagctgattacatttggttcaatggggagatggttcgctgggaagacgcgaaggtgcatgtgatgtcgcacgcgctgcactatggcacctcggtttttgaaggca

tccgttgctacgactcgcacaaaggaccggttgtattccgccatcgtgagcatatgcagcgtctgcatgactccgccaaaatctatcgctttccggtttcgcagagcattgatgagctgat

ggaagcttgtcgtgacgtgatccgcaaaaacaatctcaccagcgcctatatccgtccgctgatcttcgtcggtgatgttggcatgggcgttaacccgccagcgggatactcaaccgatg

tgattatcgccgctttcccgtggggagcgtatctgggcgcagaagcgctggagcaggggatcgatgcgatggtttcctcctggaaccgcgcagcaccaaacaccatcccaaccgcg

gcaaaagccggtggtaactacctctcttccctgctggtgggtagtgaagcacgccgccacggttatcaggaaggtatcgcgctggatgtgaatggttacatctctgaaggtgcaggcg

aaaacctgtttgaagtgaaagacggcgtgctgttcaccccaccgttcacctcctccgcgctgccgggtattacccgtgatgccatcatcaaactggcaaaagagctgggaattgaagtc

cgtgagcaggtgctgtcgcgcgaatccctgtacctggcggatgaagtgtttatgtccggtactgcggcagaaatcacgccagtgcgcagcgtagatggtattcaggttggtgaaggc

cgttgcggcccggttaccaaacgcatccagcaagccttcttcggcctcttcactggcgaaaccgaagataaatggggctggttagatcaagttaatcaataa

Escherichia coli acetohydroxyacid reductoisomerase, amino acid sequence (SEQ ID NO: 167)

MANYFNTLNLRQQLAQLGKCRFMGRDEFADGASYLQGKKVVIVGCGAQGLNQGLNMRDSGLDISYALRKEAIAE

KRASWRKATENGFKVGTYEELIPQADLVINLTPDKQHSDVVRTVQPLMKDGAALGYSHGFNIVEVGEQIRKDITVV

MVAPKCPGTEVREEYKRGFGVPTLIAVHPENDPKGEGMAIAKAWAAATGGHRAGVLESSFVAEVKSDLMGEQTILC

GMLQAGSLLCFDKLVEEGTDPAYAEKLIQFGWETITEALKQGGITLMMDRLSNPAKLRAYALSEQLKEIMAPLFQKH

MDDIISGEFSSGMMADWANDDKKLLTWREETGKTAFETAPQYEGKIGEQEYFDKGVLMIAMVKAGVELAFETMV

DSGIIEESAYYESLHELPLIANTIARKRLYEMNVVISDTAEYGNYLFSYACVPPLLKPFMAELQPGDLGKAIPEGAVDN

GQLRDVNEAIRSHAIEQVGKKLRGYMTDMKRIAVAG

Escherichia coli acetohydroxyacid reductoisomerase, nucleotide sequence (SEQ ID NO: 168)

atggctaactacttcaatacactgaatctgcgccagcagctggcacagctgggcaaatgtcgctttatgggccgcgatgaattcgccgatggcgcgagctaccttcagggtaaaaaag

tagtcatcgtcggctgtggcgcacagggtctgaaccagggcctgaacatgcgtgattctggtctcgatatctcctacgctctgcgtaaagaagcgattgccgagaagcgcgcgtcctg

gcgtaaagcgaccgaaaatggttttaaagtgggtacttacgaagaactgatcccacaggcggatctggtgattaacctgacgccggacaagcagcactctgatgtagtgcgcaccgt

acagccactgatgaaagacggcgcggcgctgggctactcgcacggtttcaacatcgtcgaagtgggcgagcagatccgtaaagatatcaccgtagtgatggttgcgccgaaatgcc

caggcaccgaagtgcgtgaagagtacaaacgtgggttcggcgtaccgacgctgattgccgttcacccggaaaacgatccgaaaggcgaaggcatggcgattgccaaagcctggg

cggctgcaaccggtggtcaccgtgcgggtgtgctggaatcgtccttcgttgcggaagtgaaatctgacctgatgggcgagcaaaccatcctgtgcggtatgttgcaggctggctctct

gctgtgcttcgacaagctggtggaagaaggtaccgatccagcatacgcagaaaaactgattcagttcggttgggaaaccatcaccgaagcactgaaacagggcggcatcaccctga

tgatggaccgtctctctaacccggcgaaactgcgtgcttatgcgctttctgaacagctgaaagagatcatggcacccctgttccagaaacatatggacgacatctccggcgaattct

cttccggtatgatggcggactgggccaacgatgataagaaaactgctgacctggcgtgaagagaccggcaaaaccgcgtttgaaaccgcgccgcagtatgaaggcaaaatcggcga

gcaggagtacttcgataaaggcgtactgatgatcgcgatggtgaaagcgggcgttgaactggcgttcgaaaccatggtcgattccggcatcattgaagagtctgcatattatgaatcac

tgcacgagctgccgctgattgccaacaccatcgcccgtaagcgtctgtacgaaatgaacgtggttatctctgataccgctgagtacggtaactatctgttctcttacgcttgtgtgccgttg

ctgaaaccgtttatggcagagctgcaaccgggcgacctgggtaaagctattccggaaggcgcggtagataacgggcaactgcgtgatgtgaacgaagcgattcgcagccatgcgat

tgagcaggtaggtaagaaactgcgcggctatatgacagatatgaaacgtattgctgttgcgggttaa

M1-93 promoter (SEQ ID NO: 169)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggaaacagct

RBS5 artificial regulatory element (SEQ ID NO: 170)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggaggactacg

RBSL1 (SEQ ID NO: 171)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggaggtcagca

RBSL2 (SEQ ID NO: 172)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggagggttcga

M1-46 (SEQ ID NO: 173)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcctctcgccccaccaattcggtttaaaccaggaaacagct

serCB gene cluster (SEQ ID NO: 174)

ttatctctggcggtgttgacaagagataacaacgttgatataattgagcccgtattgttagcatgtacgtttaaaccaggaaacagctatggctcaaatcttcaattttagttctggtccggca

atgctaccggcagaggtgcttaaacaggctcaacaggaactgcgcgactggaacggtcttggtacgtcggtgatggaagtgagtcaccgtggcaaagagttcattcaggttgcagag

gaagccgagaaggattttcgcgatcttcttaatgtcccctccaactacaaggtattattctgccatggcggtggtcgcggtcagtttgctgcggtaccgctgaatattctcggtgataaaac

caccgcagattatgttgatgccggttatgggcggcaagtgccattaaagaagcgaaaaaatactgcacgcctaatgtctttgacgccaaagtgactgttgatggtctgcgcgcggtta

agccaatgcgtgaatggcaactctctgataatgctgcttatatgcattattgcccgaatgaaaccatcgatggtatcgccatcgacgaaacgccagacttcggcgcagatgtggtggtc

gccgctgacttctcttcaaccattctttcccgtccgattgacgtcagccgttatggtgtaatttacgctggcgcgcagaaaaatatcggcccggctggcctgacaatcgtcatcgttcgtga

agatttgctgggcaaagcgaatatcgcgtgtccgtcgattctggattattccatcctcaacgataacggctccatgtttaacacgccgccgacatttgcctggtatctatctggtctggtcttt

aaatggctgaaagcgaacggcggtgtagctgaaatggataaaatcaatcagcaaaaagcagaactgctatatggggtgattgataacagcgatttctaccgcaatgacgtggcgaaa

gctaaccgttcgcggatgaacgtgccgttccagttggcggacagtgcgcttgacaaattgttccttgaagagtcttttgctgctggccttcatgcactgaaaggtcaccgtgtggtcggc

ggaatgcgcgcttctatttataacgccatgccgctggaaggcgttaaagcgctgacagacttcatggttgagttcgaacgccgtcacggttaagtcgacaaaaggagaacaaacatgc

ctaacattacctggtgcgacctgcctgaagatgtctctttatggccgggtctgcctctttcattaagtggtgatgaagtgatgccactggattaccacgcaggtcgtagcggctggctgct

gtatggtcgtgggctggataaacaacgtctgacccaataccagagcaaactgggtgcggcgatggtgattgttgccgcctggtgcgtggaagattatcaggtgattcgtctggcaggtt

cactcaccgcacgggctacacgcctggcccacgaagcgcagctggatgtcgccccgctggggaaaatcccgcacctgcgcacgccgggtttgctggtgatggatatggactccac

cgccatccagattgaatgtattgatgaaattgccaaactggccggaacgggcgagatggtggcggaagtaaccgaacgggcgatgcgcggcgaactcgattttaccgccagcctgc

gcagccgtgtggcgacgctgaaaggcgctgacgccaatattctgcaacaggtgcgtgaaaatctgccgctgatgccaggcttaacgcaactggtgctcaagctggaaacgctgggc

tggaaagtggcgattgcctccggcggctttactttctttgctgaatacctgcgcgacaagctgcgcctgaccgccgtggtagccaatgaactggagatcatggacggtaaatttaccgg

caatgtgatcggcgacatcgtagacgcgcagtacaaagcgaaaactctgactcgcctcgcgcaggagtatgaaatcccgctggcgcagaccgtggcgattggcgatggagccaat

gacctgccgatgatcaaagcggcagggctggggattgcctaccatgccaagccaaaagtgaatgaaaaggcggaagtcaccatccgtcacgctgacctgatgggggtattctgcat

cctctcaggcagcctgaatcagaagtaa

Claims (17)

1. A genetically modified pantoate-producing strain having or having enhanced NADH-dependent acetohydroxyacid reductoisomerase, preferably the NADH-dependent acetohydroxyacid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxyacid reductoisomerase activity. 2. The genetically engineered pantoate producing strain of claim 1, wherein: Further having or having enhanced activity of acetolactate synthase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase, Optionally, having reduced activity or inactivated: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase and/or branched-chain amino acid aminotransferase; preferably, wherein the branched-chain amino acid aminotransferase is attenuated, Preferably, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum and/or from Escherichia coli. Preferably, the phosphoglycerate dehydrogenase is from Corynebacterium glutamicum, and/or the dihydroxyacid dehydratase, 2-dehydropantothenate-2-reductase, glycine lyase system (e.g. aminomethyltransferase and/or glycine decarboxylase), phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase are from Escherichia coli, Preferably, the acetolactate synthase includes acetolactate synthase from Bacillus subtilis, and/or acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli. 3. The genetically engineered pantoate producing strain of claim 2, wherein: The acetolactate synthase from Bacillus subtilis comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase activity; and/or The acetolactate synthase I comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase I activity; and/or The acetolactate synthase II comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase II activity; and/or The L-valine feedback-resistant acetolactate synthase III comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having L-valine feedback-resistant acetolactate synthase III activity; and/or The dihydroxy-acid dehydratase comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having dihydroxy-acid dehydratase activity; and/or The 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity; and/or The 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity; and/or The 2-dehydropantothenate-2-reductase comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has 2-dehydropantothenate-2-reductase activity; and/or The serine hydroxymethyltransferase comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has serine hydroxymethyltransferase activity; and/or The aminomethyltransferase comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has aminomethyltransferase activity; and/or The glycine decarboxylase comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has glycine decarboxylase activity; and/or The phosphoglycerate dehydrogenase comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoglycerate dehydrogenase activity; and/or The phosphoserine/phosphohydroxythreonine aminotransferase comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine/phosphohydroxythreonine aminotransferase activity; and/or The phosphoserine phosphatase comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine phosphatase activity; and/or The weakened branched-chain amino acid aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has weakened branched-chain amino acid aminotransferase activity. 4. The genetically modified pantoate producing strain of any one of claims 1 to 3, wherein expressing a gene encoding a weakened branched-chain amino acid aminotransferase; and/or Overexpressed: a gene encoding acetolactate synthase, a gene encoding an NADH-dependent acetohydroxyacid reductoisomerase from a Thermacetogenium phaeum strain, a gene encoding a dihydroxyacid dehydratase, a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum, a gene encoding a 2-dehydropantothenate-2-reductase, a gene encoding a serine hydroxymethyltransferase, a gene encoding a glycine lyase system (e.g., a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase), a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Escherichia coli, a gene encoding a phosphoglycerate dehydrogenase, a gene encoding a phosphoserine/phosphohydroxythreonine aminotransferase, and a gene encoding a phosphoserine phosphatase, and/or Optionally, there is no gene encoding one or more, preferably all of the following enzymes or endogenous genes encoding one or more, preferably all of the following enzymes are knocked out: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase, phosphate acetyltransferase, Preferably, the gene encoding acetolactate synthase includes a gene encoding acetolactate synthase from Bacillus subtilis, and/or a gene encoding acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli. 5. The genetically engineered pantoate producing strain of claim 4, wherein: The gene encoding the acetolactate synthase from Bacillus subtilis comprises the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding acetolactate synthase I comprises the nucleotide sequence shown in SEQ ID NO: 4 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding acetolactate synthase II comprises the nucleotide sequence shown in SEQ ID NO: 6 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding L-valine feedback-resistant acetolactate synthase III comprises the nucleotide sequence shown in SEQ ID NO: 8 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding NADH-dependent acetohydroxyacid reductoisomerase comprises the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding the dihydroxy-acid dehydratase comprises the nucleotide sequence of SEQ ID NO: 12 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum comprises the nucleotide sequence shown in SEQ ID NO: 14 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli comprises the nucleotide sequence shown in SEQ ID NO: 16 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 2-dehydropantothenate-2-reductase comprises the nucleotide sequence shown in SEQ ID NO: 18 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding serine hydroxymethyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 20 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding aminomethyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 22 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding glycine decarboxylase comprises the nucleotide sequence shown in SEQ ID NO: 24 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoglycerate dehydrogenase comprises the nucleotide sequence shown in SEQ ID NO: 26 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoserine/phosphohydroxythreonine aminotransferase comprises the nucleotide sequence shown in SEQ ID NO: 28 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoserine phosphatase comprises the nucleotide sequence shown in SEQ ID NO: 30 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding the weakened branched-chain amino acid aminotransferase comprises the nucleotide sequence shown in SEQ ID NO: 32 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. 6. The genetically modified pantoic acid-producing strain according to any one of claims 1 to 5, wherein: the genetically modified pantoic acid-producing strain belongs to the genus Escherichia, Enterobacter, Corynebacterium glutamicum, Bacillus subtilis and yeast, preferably Escherichia coli, more preferably, Escherichia coli deposited in the General Microbiological Center (CGMCC) of the China National Microbiological Culture Collection Administration in Beijing, China, with a deposit number of CGMCC No. 26276. 7. A method for producing a genetically modified pantoate-producing strain according to any one of claims 1 to 6, comprising conferring or enhancing the activity of NADH-dependent acetohydroxyacid reductoisomerase in the pantoate-producing strain, preferably the NADH-dependent acetohydroxyacid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxyacid reductoisomerase activity. 8. The method of claim 7, further comprising, in the strain: conferring or enhancing the activity of acetolactate synthase, dihydroxyacid dehydratase, 3-methyl-2-oxobutyrate hydroxymethyltransferase, 2-dehydropantothenate-2-reductase, serine hydroxymethyltransferase, glycine lyase system (e.g., aminomethyltransferase and/or glycine decarboxylase), phosphoglycerate dehydrogenase, phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase, Optionally, attenuating or inactivating, if present: the activity of L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate transaminase, phosphate acetyltransferase and/or branched-chain amino acid aminotransferase; preferably, attenuating the activity of branched-chain amino acid aminotransferase, Preferably, the 3-methyl-2-oxobutanoate hydroxymethyltransferase comprises 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum and/or Escherichia coli, Preferably, the phosphoglycerate dehydrogenase is from Corynebacterium glutamicum, and/or the dihydroxyacid dehydratase, 2-dehydropantothenate-2-reductase, glycine lyase system (e.g. aminomethyltransferase and/or glycine decarboxylase), phosphoserine/phosphohydroxythreonine aminotransferase and phosphoserine phosphatase are from Escherichia coli, Preferably, the acetolactate synthase includes acetolactate synthase from Bacillus subtilis, and/or acetolactate synthase I, acetolactate synthase II and/or L-valine feedback-resistant acetolactate synthase III from Escherichia coli. 9. The method of claim 8, wherein: The acetolactate synthase from Bacillus subtilis comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase activity; and/or The acetolactate synthase I comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase I activity; and/or The acetolactate synthase II comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having acetolactate synthase II activity; and/or The L-valine feedback-resistant acetolactate synthase III comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having L-valine feedback-resistant acetolactate synthase III activity; and/or The dihydroxy-acid dehydratase comprises an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having dihydroxy-acid dehydratase activity; and/or The 3-methyl-2-oxobutanoate hydroxymethyltransferase from Corynebacterium glutamicum comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity; and/or The 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli comprises the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity; and/or The 2-dehydropantothenate-2-reductase comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has 2-dehydropantothenate-2-reductase activity; and/or The serine hydroxymethyltransferase comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has serine hydroxymethyltransferase activity; and/or The aminomethyltransferase comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has aminomethyltransferase activity; and/or The glycine decarboxylase comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has glycine decarboxylase activity; and/or The phosphoglycerate dehydrogenase comprises the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoglycerate dehydrogenase activity; and/or The phosphoserine/phosphohydroxythreonine aminotransferase comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine/phosphohydroxythreonine aminotransferase activity; and/or The phosphoserine phosphatase comprises the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has phosphoserine phosphatase activity; and/or The weakened branched-chain amino acid aminotransferase comprises the amino acid sequence shown in SEQ ID NO: 31 or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical thereto and has weakened branched-chain amino acid aminotransferase activity. 10. The method of any one of claims 7 to 9, comprising, in the strain: expressing a gene encoding a weakened branched-chain amino acid aminotransferase; and/or Overexpression: a gene encoding acetolactate synthase from Bacillus subtilis, a gene encoding acetolactate synthase I, a gene encoding acetolactate synthase II, a gene encoding L-valine feedback-resistant acetolactate synthase III, a gene encoding an NADH-dependent acetohydroxyacid reductoisomerase from a Thermacetogenium phaeum strain, a gene encoding a dihydroxyacid dehydratase, a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum, a gene encoding a 2-dehydropantothenate-2-reductase, a gene encoding a serine hydroxymethyltransferase, a gene encoding a glycine lyase system (e.g., a gene encoding an aminomethyltransferase and/or a gene encoding a glycine decarboxylase), a gene encoding a 3-methyl-2-oxobutyrate hydroxymethyltransferase from Escherichia coli, a gene encoding a phosphoglycerate dehydrogenase, a gene encoding a phosphoserine/phosphohydroxythreonine aminotransferase, and a gene encoding a phosphoserine phosphatase, and/or Optionally, endogenous genes encoding one or more, preferably all, of the following enzymes are knocked out, if present: L-serine deaminase I, propionate kinase, formate acetyltransferase, alcohol dehydrogenase, pyruvate formate lyase, fumarate reductase, lactate dehydrogenase, methylglyoxal synthase, acetate kinase, ribokinase, valine-pyruvate aminotransferase and/or phosphate acetyltransferase. 11. The method of claim 10, wherein: The gene encoding the acetolactate synthase from Bacillus subtilis comprises the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding acetolactate synthase I comprises the nucleotide sequence shown in SEQ ID NO: 4 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding acetolactate synthase II comprises the nucleotide sequence shown in SEQ ID NO: 6 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding L-valine feedback-resistant acetolactate synthase III comprises the nucleotide sequence shown in SEQ ID NO: 8 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding NADH-dependent acetohydroxyacid reductoisomerase comprises the nucleotide sequence shown in SEQ ID NO: 10 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding the dihydroxy-acid dehydratase comprises the nucleotide sequence of SEQ ID NO: 12 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 3-methyl-2-oxobutyrate hydroxymethyltransferase from Corynebacterium glutamicum comprises the nucleotide sequence shown in SEQ ID NO: 14 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 3-methyl-2-oxobutanoate hydroxymethyltransferase from Escherichia coli comprises the nucleotide sequence shown in SEQ ID NO: 16 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding 2-dehydropantothenate-2-reductase comprises the nucleotide sequence shown in SEQ ID NO: 18 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding serine hydroxymethyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 20 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding aminomethyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 22 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding glycine decarboxylase comprises the nucleotide sequence shown in SEQ ID NO: 24 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoglycerate dehydrogenase comprises the nucleotide sequence shown in SEQ ID NO: 26 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoserine/phosphohydroxythreonine aminotransferase comprises the nucleotide sequence shown in SEQ ID NO: 28 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding phosphoserine phosphatase comprises the nucleotide sequence shown in SEQ ID NO: 30 or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and/or The gene encoding the weakened branched-chain amino acid aminotransferase comprises the nucleotide sequence shown in SEQ ID NO: 32 or its degenerate sequence, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. 12. The method according to any one of claims 7 to 11, wherein the strain is selected from the group consisting of Escherichia, Enterobacter, Corynebacterium glutamicum, Bacillus subtilis and yeast, preferably Escherichia coli. 13. A method for producing a genetically modified pantoate-producing strain, comprising: replacing a gene encoding an NADPH-dependent acetohydroxy acid reductoisomerase with a gene encoding an NADH-dependent acetohydroxy acid reductoisomerase in Escherichia coli at the General Microbiological Center of China Microorganism Culture Collection (CGMCC) in Beijing, China, with a deposit number of CGMCC No. 21699, Preferably, the NADH-dependent acetohydroxy acid reductoisomerase is from a Thermacetogenium phaeum strain, more preferably, the NADH-dependent acetohydroxy acid reductoisomerase comprises the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity, more preferably, the gene encoding the NADH-dependent acetohydroxy acid reductoisomerase comprises the nucleotide sequence shown in SEQ ID NO: 10, or a degenerate sequence thereof, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto. 14. The method of any one of claims 7 to 13, wherein the genetically modified pantoic acid-producing strain is Escherichia coli deposited in the General Microbiological Center of China Microorganism Culture Collection Committee (CGMCC) in Beijing, China, with a deposit number of CGMCC No. 26276. 15. A method for improving the production of D-pantoic acid in a pantoic acid-producing strain, comprising: conferring or enhancing the activity of NADH-dependent acetohydroxy acid reductoisomerase in the pantoic acid-producing strain, preferably the NADH-dependent acetohydroxy acid reductoisomerase is from Thermacetogenium phaeum, more preferably comprising the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity thereto and having NADH-dependent acetohydroxy acid reductoisomerase activity. 16. A method for producing D-pantoic acid, comprising culturing the genetically modified pantoic acid-producing strain of any one of claims 1 to 6 or the genetically modified pantoic acid-producing strain obtained by the method of any one of claims 7 to 15 under conditions suitable for the fermentative production of D-pantoic acid, optionally comprising isolating and purifying the produced D-pantoic acid. 17. Use of the genetically modified pantoic acid-producing strain according to any one of claims 1 to 6 or the genetically modified pantoic acid-producing strain obtained by the method according to any one of claims 7 to 15 in producing D-pantoic acid.