CN112662605B - YH66_10715 gene-modified L-isoleucine-producing strain and its construction method and application - Google Patents

Abstract

The invention discloses a YH66_10715 gene-modified high-yield L-isoleucine strain and its construction method and application. In the present invention, a point mutation is introduced into the wild-type YH66_10715 gene in Corynebacterium glutamicum, and the nucleotide sequence obtained by mutating the 991st base of SEQ ID NO: 1 from cytosine (C) to thymine (T) . The present invention also provides a recombinant strain obtained by introducing the polynucleotide sequence into L-isoleucine-producing Corynebacterium glutamicum. The recombinant strain includes the YH66_10715 gene containing a point mutation. The obtained strain is the same as the unmodified strain. compared to the strains that were favorable for the production of high concentrations of L-isoleucine.

Description

YH66_10715 genetically modified L-isoleucine strain and its construction method and application

technical field

The invention belongs to the technical field of genetic engineering and microorganisms, and in particular relates to a L-isoleucine-producing strain genetically modified by YH66_10715 and a construction method and application thereof.

Background technique

L-Isoleucine is one of the eight essential amino acids in the human body and one of the three branched-chain amino acids. Because of its special structure and function, it has a particularly important position in the metabolism of human life. Therefore, it has been widely used in industries such as food, animal feed and medicine. The production methods of L-isoleucine mainly include protein hydrolysis, chemical synthesis and biological fermentation. Biological fermentation has become the preferred method for industrial production because of its low cost of raw materials, easy control, energy saving and environmental protection. Currently, L-isoleucine-producing strains are mainly Corynebacterium glutamicum and Escherichia coli. Corynebacterium glutamicum has the characteristics of no endotoxin, less metabolic isoenzymes, which is conducive to the release of feedback inhibition or transcriptional attenuation, and strong anti-feedback inhibition ability of key metabolic enzymes. Therefore, most researchers tend to study Corynebacterium glutamicum and Its subspecies, such as Corynebacterium glutamicum (Brevibacterium flavum) and Brevibacterium lactofermentum (Brevibacterium lactofermentum) and so on.

In Corynebacterium glutamicum, after glucose is absorbed into the cell, oxaloacetate is generated through a series of enzymatic reactions. Oxaloacetic acid is further converted to L-aspartic acid, and then after ten steps of biological reaction, L-isoleucine is produced, and L-lysine, L-methionine, L-serine and L-valine are involved in the middle. Metabolic pathway of other amino acids such as acid. The key enzymes in the L-isoleucine biosynthesis pathway include aspartate kinase (AK), homoserine dehydrogenase (HDH), homoserine kinase (HSK), threonine Dehydratase (Threonine dehydrogenase, TD) and acetohydroxy acid synthase (Acetohydroxy acid synthase, AHAS), and AHAS is the first enzyme that catalyzes the synthesis of L-isoleucine and L-valine at the same time, Therefore, the metabolic regulation of L-isoleucine is relatively complex. The intracellularly synthesized L-isoleucine is transported extracellularly through BrnFE, and the extracellular L-isoleucine is also transported into the cell by BrnQ.

The selection of excellent strains is still the key to high-yield L-isoleucine. With the development of high-throughput screening technology, combined with traditional mutagenesis technology, it is helpful to find high-yield L-isoleucine mutants with unknown traits. . These mutants have the potential to alter the activity of a single point in the L-isoleucine metabolic pathway or to recombine the entire pathway to increase acid production.

Although some strains that can improve L-isoleucine production have been screened, in order to meet the increasing demand, more mutants with high L-isoleucine production still need to be found.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides a polynucleotide and a recombinant strain containing the polynucleotide, and the recombinant strain is used to improve the L-isoleucine production capacity of bacteria. In order to achieve the above object, the inventors of the present invention have found that the YH66_10715 gene (GenBank: AKF27993.1) in the genome of Corynebacterium glutamicum ATCC15168 (GenBank: CP011309.1) with L-isoleucine production ability can be obtained by The gene is modified or its expression is improved to obtain a recombinant strain with improved L-isoleucine production. Compared with the unmodified wild-type strain, the recombinant strain has a stronger L-isoleucine production capacity.

The present invention adopts the following technical scheme to realize:

In a first aspect of the present invention, there is provided an L-isoleucine-producing bacterium having improved expression of a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:3. According to the present invention, the improved expression is enhanced expression of the polynucleotide, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has a point mutation, or the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has point mutated and expression is enhanced.

The amino acid sequence of the SEQ ID NO: 3 is the protein encoded by the gene YH66_10715.

The bacteria have enhanced L-isoleucine production capacity compared to the unmodified strain.

In the present invention, the term "bacteria having L-isoleucine-producing ability" refers to the ability to produce and accumulate the desired L-isoleucine in the culture medium and/or cells of the bacteria to such an extent that when Bacteria with L-isoleucine can be collected when the bacteria are cultured in the medium. The bacterium having L-isoleucine-producing ability may be a bacterium capable of accumulating the desired L-isoleucine in the medium and/or cells of the bacteria in a larger amount than that obtainable by the unmodified strain.

The term "unmodified strain" refers to a control strain that has not been modified in such a way that it has specific characteristics. That is, examples of unmodified strains include wild-type strains and parental strains.

In the present invention, unless otherwise specified, the term "L-isoleucine" refers to L-isoleucine in free form, a salt thereof, or a mixture thereof.

The polynucleotide may encode an amino acid sequence that is about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more identical to the amino acid sequence of SEQ ID NO:3 Amino acid sequences of high, or about 99% or greater sequence homology. As used herein, the term "homology" refers to the percent identity between two polynucleotides or two polypeptide modules. Sequence homology between one module and another can be determined by using methods known in the art. Such sequence homology can be determined, for example, by the BLAST algorithm.

Expression of a polynucleotide can be enhanced by substituting or mutating expression regulatory sequences, introducing mutations to the polynucleotide sequence, by increasing the copy number of the polynucleotide via chromosomal insertion or introduction of a vector, or a combination thereof, and the like.

The expression regulatory sequences of the polynucleotide can be modified. Expression regulatory sequences control the expression of polynucleotides to which they are operably linked, and can include, for example, promoters, terminators, enhancers, silencers, and the like. Polynucleotides can have changes in the initiation codon. Polynucleotides can be incorporated into chromosomes at specific sites, thereby increasing copy number. Herein, a specific site may include, for example, a transposon site or an intergenic site. Additionally, polynucleotides can be incorporated into expression vectors that are introduced into host cells to increase copy number.

In one embodiment of the invention, the copy number is increased by incorporating a polynucleotide or a polynucleotide with a point mutation into a specific site in the chromosome of a microorganism.

In one embodiment of the invention, the overexpression of the said nucleic acid sequence.

In one embodiment of the present invention, a polynucleotide or a polynucleotide having a point mutation is incorporated into an expression vector, and the expression vector is introduced into a host cell to increase the copy number.

In one embodiment of the present invention, a polynucleotide with a promoter sequence or a polynucleotide with a point mutation with a promoter sequence is incorporated into an expression vector, and the expression vector is introduced into a host cell, Thereby the amino acid sequence is overexpressed.

In a specific embodiment of the present invention, the polynucleotide may comprise the nucleotide sequence of SEQ ID NO:1.

In one embodiment of the invention, the polynucleotide encoding the amino acid sequence of SEQ ID NO: 3 has a point mutation such that proline at position 331 of the amino acid sequence of SEQ ID NO: 3 is replaced by a different amino acid .

According to the present invention, the proline at position 331 is preferably replaced by serine.

According to the present invention, the amino acid sequence shown in SEQ ID NO: 3, wherein the amino acid sequence after the 331st position proline is replaced by serine is shown in SEQ ID NO: 4.

In one embodiment of the present invention, the polynucleotide sequence with point mutation is formed by mutating the 991st base of the polynucleotide sequence shown in SEQ ID NO: 1.

According to the present invention, the mutation includes the mutation of the 991st base of the polynucleotide sequence shown in SEQ ID NO: 1 from cytosine (C) to thymine (T).

In one embodiment of the present invention, the polynucleotide sequence with point mutation comprises the polynucleotide sequence shown in SEQ ID NO:2.

As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a polynucleotide sequence, whereby the regulatory sequence controls transcription and/or translation of the polynucleotide sequence. The regulatory sequence can be a strong promoter capable of increasing the expression level of the polynucleotide. Regulatory sequences may be promoters derived from microorganisms belonging to the genus Corynebacterium or may be promoters derived from other microorganisms. For example, the promoter can be a trc promoter, gap promoter, tac promoter, T7 promoter, lac promoter, trp promoter, araBAD promoter or cj7 promoter.

In a specific embodiment of the present invention, the promoter is the promoter of the polynucleotide (YH66_10715) encoding the amino acid sequence of SEQ ID NO:3.

As used herein, the term "vector" refers to a polynucleotide construct that contains a gene's regulatory and gene sequences and is configured to express a target gene in a suitable host cell. Alternatively, a vector may in turn refer to a polynucleotide construct containing sequences useful for homologous recombination such that, due to the vector introduced into the host cell, regulatory sequences of endogenous genes in the genome of the host cell may be altered, or may The expressed target gene is inserted into the host's genome at a specific site. In this regard, the vector used in the present invention may further comprise a selectable marker to determine the introduction of the vector into the host cell or the insertion of the vector into the chromosome of the host cell. Selectable markers can include markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with such selective agents, transformed cells can be selected because only cells expressing the selectable marker can survive or display different phenotypic traits.

In some embodiments of the invention, the vector used is the pK18mobsacB plasmid, the pXMJ19 plasmid.

As used herein, the term "transformation" refers to the introduction of a polynucleotide into a host cell such that the polynucleotide is replicable as an extragenomic element or inserted into the genome of the host cell. The method of transforming the vector used in the present invention may include a method of introducing nucleic acid into cells. In addition, as disclosed in the related art, the electrical pulse method can be implemented according to the host cell.

According to the present invention, the bacteria may be microorganisms belonging to the genus Corynebacterium, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum ), Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, Brevibacterium rose ( Brevibacterium roseum), Brevibacterium thiogenitalis, etc.

In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC15168.

In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YPILE001, which has been deposited in the General Microorganism Center of the China Microorganism Culture Collection Administration Committee on August 17, 2020, and the deposit address: Beijing No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, City, Zip Code: 100101, Abbreviation of the depositary institution: CGMCC, biological deposit number is CGMCC No.20437.

The second aspect of the present invention provides a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector comprising the polynucleotide sequence, and a recombinant strain containing the polynucleotide sequence.

According to the present invention, the polynucleotide sequence includes a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3, the 331st proline of the sequence being substituted by a different amino acid.

According to the present invention, the proline at position 331 is preferably replaced by serine. .

According to the present invention, the amino acid sequence shown in SEQ ID NO: 3, wherein the amino acid sequence after the 331st position proline is replaced by serine is shown in SEQ ID NO: 4.

According to the present invention, preferably, the polynucleotide sequence encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO:3 contains the polynucleotide sequence shown in SEQ ID NO:1.

In one embodiment of the present invention, the polynucleotide sequence is formed by mutating the 991st base of the polynucleotide sequence shown in SEQ ID NO: 1.

According to the present invention, the mutation refers to a change in the base/nucleotide of the site, and the mutation method can be selected from at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination methods. kind. In the present invention, PCR site-directed mutagenesis and/or homologous recombination are preferably employed.

According to the present invention, the mutation includes the mutation of cytosine (C) at position 991 of the polynucleotide sequence shown in SEQ ID NO: 1 to thymine (T).

In one embodiment of the present invention, the polynucleotide sequence includes the polynucleotide sequence shown in SEQ ID NO:2.

According to the present invention, the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:4.

According to the present invention, the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.

In one embodiment of the present invention, the plasmid is the pK18mobsacB plasmid.

In another embodiment of the present invention, the plasmid is the pXMJ19 plasmid.

Specifically, the polynucleotide sequence and the plasmid can be constructed into a recombinant vector through the NEBuider recombination system.

According to the present invention, the recombinant strain contains the polynucleotide sequence.

As an embodiment of the present invention, the starting bacteria of the recombinant strain is Corynebacterium glutamicum YPILE001, and the biological deposit number is CGMCC No.20437.

As an embodiment of the present invention, the starting bacteria of the recombinant strain is ATCC 15168.

The third aspect of the present invention provides the above-mentioned polynucleotide sequence, the amino acid sequence encoded by the polynucleotide sequence, the recombinant vector comprising the polynucleotide sequence, and the recombinant strain containing the polynucleotide sequence in the production of L - Application of isoleucine.

The fourth aspect of the present invention also provides a method for constructing a recombinant strain producing L-isoleucine.

According to the present invention, the construction method comprises the following steps:

The polynucleotide sequence of the wild-type YH66_10715 gene shown in SEQ ID NO: 1 in the host strain was modified, and the 991st base was mutated to obtain a recombinant strain comprising the mutated YH66_10715 encoding gene.

According to the construction method of the present invention, the transformation includes at least one of mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.

According to the construction method of the present invention, the mutation refers to the mutation of the 991st base in SEQ ID NO: 1 from cytosine (C) to thymine (T); specifically, the polynucleotide comprising the gene encoding the mutation YH66_10715 The acid sequence is shown in SEQ ID NO:2.

Further, the construction method comprises the steps:

(1) transform the nucleotide sequence of the wild-type YH66_10715 gene as shown in SEQ ID NO: 1, so that the 991st base is mutated to obtain the mutated YH66_10715 gene polynucleotide sequence;

(2) connecting the mutated polynucleotide sequence with a plasmid to construct a recombinant vector;

(3) introducing the recombinant vector into a host strain to obtain the recombinant strain comprising the gene encoding the mutation YH66_10715.

According to the construction method of the present invention, the step (1) comprises: constructing the YH66_10715 gene of the point mutation: according to the genome sequence of the unmodified strain, synthesizing two pairs of primers P1 and P2 and P3 and P4 for amplifying the YH66_10715 gene fragment, by PCR A point mutation was introduced into the wild-type YH66_10715 gene SEQ ID NO: 1 by site-directed mutagenesis, and the nucleotide sequence of the point-mutated YH66_10715 gene SEQ ID NO: 2 was obtained, denoted as YH66_10715 ^C991T .

In one embodiment of the present invention, the genome of the unmodified strain can be derived from the ATCC15168 strain, whose genome sequence GenBank: CP011309.1 can be obtained from the NCBI website.

In one embodiment of the present invention, in the step (1), the primer is:

P1: 5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAAGTTCTTCGTTCACGATCC 3' (SEQ ID NO: 5)

P2: 5' GTTGATTGGAGCCTCGAAGCCTG A AACCAGACGGTGGTAG 3' (SEQ ID NO: 6)

P3: 5' CTACCACCGTCTGGTT T CAGGCTTCGAGGCTCCAATCAAC 3' (SEQ ID NO: 7)

P4: 5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGGAACGCCAGTTGGAAG3' (SEQ ID NO: 8)

In one embodiment of the present invention, the PCR amplification is performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 40s (30 cycles), overextension at 72°C 10min.

In one embodiment of the present invention, the overlapping PCR amplification is carried out as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 90s (30 cycles), overrun at 72°C Extend for 10min.

According to the construction method of the present invention, the step (2) includes the construction of recombinant plasmids, including: assembling the isolated and purified YH66_10715 ^C991T and pK18mobsacB plasmids through the NEBuider recombination system to obtain the recombinant plasmids.

According to the construction method of the present invention, the step (3) includes the construction of a recombinant strain, and the recombinant plasmid is transformed into a host strain to obtain a recombinant strain.

In one embodiment of the present invention, the conversion of step (3) is an electroconversion method.

In one embodiment of the invention, the host strain is ATCC 15168.

In one embodiment of the present invention, the host strain is Corynebacterium glutamicum YPILE001, and the biological deposit number is CGMCC No.20437.

In one embodiment of the invention, the recombination is achieved by homologous recombination.

The fifth aspect of the present invention also provides a method for constructing a recombinant strain producing L-isoleucine.

According to the present invention, the construction method comprises the following steps:

Amplify the upstream and downstream homology arm fragments of YH66_10715, the YH66_10715 gene coding region and its promoter region sequence, and introduce the YH66_10715 or YH66_10715 ^C991T gene into the genome of the host strain by homologous recombination to achieve the strain overexpressing YH66_10715 or YH66_10715 ^C991T gene.

In one embodiment of the present invention, the primer for amplifying the upstream homology arm fragment is:

P7: 5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTTTGCTTCCAAAGGCGTG3'

P8:5'CGGGGTTTCAAACGCCACTCATTTCAGCGTCAGATG3'

In one embodiment of the present invention, the primers for amplifying the downstream homology arm fragments are:

P11:5'GAATTGTACTTCGACTGCTAATGCCGAAATAAGGCCGG3'

P12: 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCCTTCTAAAACTGCCG 3'

In one embodiment of the present invention, the primers for amplifying the coding region of the gene and the sequence of its promoter region are:

P9: 5'CATCTG ACGCTGAAATGAGTGGCGTTTGAAACCCCG 3' (SEQ ID NO: 13)

P10: 5' CCGGCCTTAT TTCGGCATTAGCAGTCGAAGTACAATTC 3' (SEQ ID NO: 14)

In one embodiment of the present invention, the aforementioned P7/P12 is used as the primer, and the amplified upstream homology arm fragment, the downstream homology arm fragment, the gene coding region and the sequence fragment of the promoter region are mixed as The template is amplified to obtain integrated homology arm fragments.

In one embodiment of the present invention, the PCR system used: 10×Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg ²⁺ (25 mM) 4 μL, primers (10 μM) 2 μL each, Ex Taq (5U/ μL) 0.25 μL, total volume 50 μL; PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 180s (30 cycles), and overextension at 72°C for 10 min.

In one embodiment of the present invention, the NEBuider recombination system is used to assemble the shuttle plasmid PK18mobsacB and the integrated homology arm fragment to obtain an integrated plasmid.

In one embodiment of the present invention, the integrated plasmid is transfected into a host strain, and the YH66_10715 or YH66_10715 ^C991T gene is introduced into the genome of the host strain by means of homologous recombination.

In one embodiment of the present invention, the host strain is Corynebacterium glutamicum YPILE001, the biological deposit number is CGMCC No.20437.

In one embodiment of the invention, the host strain is ATCC 15168.

In one embodiment of the present invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO:2.

The sixth aspect of the present invention also provides a method for constructing a recombinant strain for producing L-isoleucine.

According to the present invention, the construction method comprises the following steps:

Amplify the YH66_10715 gene coding region and the promoter region sequence, or the YH66_10715 ^C991T gene coding region and the promoter region sequence, construct an overexpression plasmid vector, and transfer the vector into the host strain to realize that the strain overexpresses YH66_10715 or YH66_10715 ^C991T gene.

In one embodiment of the present invention, the primers for amplifying the coding region of the gene and the sequence of its promoter region are:

P17: 5' GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGTGGCGTTTGAAACCCCG 3' (SEQ ID NO: 21)

P18: 5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACGAATTGTACTTCGACTGCTAA 3' (SEQ ID NO: 22).

In one embodiment of the present invention, the PCR system: 10×Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg ²⁺ (25 mM) 4 μL, primers (10 μM) 2 μL each, Ex Taq (5U/μL) ) 0.25 μL, and the total volume was 50 μL; the PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 120s (30 cycles), and over-extension at 72°C for 10 min.

In one embodiment of the present invention, the NEBuider recombination system is used to assemble the shuttle plasmid pXMJ19 and the YH66_10715 or YH66_10715 ^C991T fragment with its own promoter to obtain an overexpression plasmid.

In one embodiment of the present invention, the host strain is Corynebacterium glutamicum YPILE001, the biological deposit number is CGMCC No.20437.

In one embodiment of the invention, the host strain is ATCC 15168.

In one embodiment of the present invention, the host strain is a strain carrying the polynucleotide sequence shown in SEQ ID NO:2.

The recombinant strain obtained by the present invention can be used alone in fermentation to produce L-isoleucine, or can be mixed with other L-isoleucine-producing bacteria to produce L-isoleucine by fermentation.

Another aspect of the present invention provides a method of producing L-isoleucine, the method comprising culturing the bacterium; and obtaining L-isoleucine from the culture.

Cultivation of bacteria can be carried out in a suitable medium under culture conditions known in the art. The medium may contain: carbon sources, nitrogen sources, trace elements, and combinations thereof. During cultivation, the pH of the culture can be adjusted. In addition, the culturing may include preventing the generation of air bubbles, for example, by using an antifoaming agent. In addition, culturing can include injecting a gas into the culture. The gas can include any gas capable of maintaining aerobic conditions of the culture. In the culture, the temperature of the culture may be 20 to 45°C. The resulting L-isoleucine can be recovered from the culture by treating the culture with sulfuric acid or hydrochloric acid, etc., followed by a combination of methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.

The beneficial effects of the present invention

In the present invention, by knocking out the YH66_10715 gene, it is found that the product encoded by the gene has an impact on the L-isoleucine production capacity, and the recombinant strain is obtained by introducing point mutations in the coding sequence, or increasing the copy number or overexpression of the gene, The obtained strain is favorable for the production of high concentrations of L-isoleucine compared to the unmodified strain.

Biological Preservation Instructions

Corynebacterium glutamicum (Corynebacterium glutamicum), deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, preservation address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, zip code: 100101, preservation institution abbreviation: CGMCC, preservation The date is August 17, 2020, the biological deposit number is CGMCC No.20437, and the strain name is YPILE001.

Detailed ways

The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the intended protection scope of the present invention.

Unless otherwise stated, the starting materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental method of unreceipted specific conditions in the following examples, usually according to routine conditions such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer the proposed conditions.

Unless otherwise defined or clearly indicated by context, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The basal medium used for culturing the bacterial strains in the following examples has the same composition, and correspondingly required sucrose, kanamycin or chloramphenicol are added to this basal medium composition, and the basal medium composition is as follows:

Table 1 Composition of solid medium

Element formula glucose 5g/L polypeptone 10g/L beef paste 10g/L yeast 5g/L Sodium chloride 55g/L Agar powder 20g/L pH (adjusted with NaOH) 7.0 Culture conditions 31℃

Example 1 Construction of the transformation vector pK18-YH66_10715 ^C991T comprising the coding region of the YH66_10715 gene with a point mutation

According to the genome sequence of Corynebacterium glutamicum ATCC15168 (GenBank: CP011309.1) published by NCBI, two pairs of primers for amplifying the coding region sequence of the YH66_10715 gene (GenBank: AKF27993.1) were designed and synthesized. Strain ATCC15168 and high isoleucine-producing Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437) introduced point mutation, the amino acid sequence corresponding to the encoded protein is SEQ ID NO: 3, the nucleotide sequence of the YH66_10715 gene Cytosine (C) at position 991 is changed to thymine (T) (SEQ ID NO: 2: YH66_10715 ^C991T ), corresponding to the amino acid sequence of the encoded protein at position 331 proline (P) is changed to serine (S) (SEQ ID NO: 2) NO: 4: YH66_10715 ^P331S ). The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

P1: 5' CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAG AAGTTCTTCGTTCACGATCC 3' (SEQ ID NO: 5)

P2: 5' GTTGATTGGAGCCTCGAAGCCTGAAAACCAGACGGTGGTAG 3' (SEQ ID NO: 6)

P3: 5' CTACCACCGTCTGGTTTCAGGCTTCGAGGCTCCAATCAAC 3' (SEQ ID NO: 7)

P4: 5' CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGGAACGCCAGTTGGAAG3' (SEQ ID NO: 8)

Construction method: Take Corynebacterium glutamicum ATCC15168 as the template, and carry out PCR amplification with primers P1 and P2, P3 and P4 respectively.

PCR system: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg ²⁺ (25mM) 4μL, primers (10pM) 2μL each, Ex Taq (5U/μL) 0.25μL, total volume 50μL.

The PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 40s (30 cycles), and over-extension at 72°C for 10min to obtain two sizes of about 771bp and 771bp respectively. 756bp, containing the DNA fragment of the coding region of the YH66_10715 gene (YH66_10715 ^C991T -Up and YH66_10715 ^C991T- Down).

YH66_10715 ^C991T -Up and YH66_10715 ^C991T -Down were separated and purified by agarose gel electrophoresis; the above two DNA fragments were used as templates, and P1 and P4 were used as primers to amplify YH66_10715 ^C991T- Up-Down snippets.

PCR system: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg ²⁺ (25mM) 4μL, primers (10pM) 2μL each, Ex Taq (5U/μL) 0.25μL, total volume 50μL.

The PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 90s (30 cycles), and overextension at 72°C for 10 min.

This DNA fragment causes the cytosine (C) at the 991st position in the coding region of the YH66_10715 gene in the mutagenic strain of ATCC15168 Corynebacterium glutamicum YPILE001 (Biodeposit No. CGMCC No. 20437) to be changed to thymine (T), resulting in The amino acid 331 of the encoded protein was changed from proline (P) to serine (S).

After the pK18mobsacB plasmid (purchased from Addgene) was digested with XbaI, the YH66_10715 ^C991T -Up-Down and linearized pK18mobsacB plasmids were separated and purified by agarose gel electrophoresis, and then assembled by the NEBuider recombination system to obtain the vector pK18-YH66_10715 ^C991T , This plasmid contains a kanamycin resistance marker. The vector pK18-YH66_10715 ^{C991T was} sent to a sequencing company for sequencing identification, and the vector pK18-YH66_10715 C991T containing the correct point mutation (CT) was kept for future ^use .

Example 2 Construction of engineering strain of YH66_10715 ^C991T comprising point mutation

Construction method: The allelic replacement plasmid pK18-YH66_10715 ^C991T was transformed into the high isoleucine-producing Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437) by electroporation; the single colonies produced by the culture were subjected to primer P1 It was identified with the universal primer M13R, and the strains that could amplify a band of about 1527bp were positive strains. The positive strains were cultured on the medium containing 15% sucrose; the single colonies produced by the culture were cultured on the medium containing kanamycin and without kanamycin, respectively, and the medium without kanamycin The strains that did not grow on the kanamycin-containing medium were further identified by PCR using the following primers (synthesized by Shanghai Invitrogen Company):

P5: 5' AAGGACGGC AAGCCACTC 3' (SEQ ID NO: 9)

P6: 5'AGTTCGTAGAGGTCCTTG3' (SEQ ID NO: 10)

The above-mentioned PCR amplification product is subjected to sscp electrophoresis after high temperature denaturation and ice bath (with plasmid pK18-YH66_10715 ^C991T amplified fragment as positive control, ATCC15168 amplified fragment as negative control, and water as blank control), due to different fragment structures, electrophoresis position Therefore, the strains whose electrophoretic positions of the fragments are inconsistent with those of the negative control fragments and are the same as those of the positive control fragments are the strains with successful allelic substitution. Using primers P5 and P6 to amplify the target fragment of the successfully allelic replacement strain again by PCR, and connect it to the PMD19-T vector for sequencing, and determine whether the replacement is successful by comparing the base sequences; The mutant strain of Corynebacterium acidophilus YPILE001 (Biological Deposit No. CGMCC No. 20437) was successfully replaced and named as YPI019.

Table 2 Preparation of PAGE for sscp electrophoresis

Example 3 Construction of engineering strains overexpressing YH66_10715 or YH66_10715 ^C991T gene on the genome

According to the genome sequence of Corynebacterium glutamicum ATCC15168 (GenBank: CP011309.1) published by NCBI, three pairs of primers for amplifying the upstream and downstream homology arm fragments and the coding region and promoter region of the YH66_10715 gene were designed and synthesized. The YH66_10715 or YH66_10715 ^C991T gene was introduced into the strain Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437).

The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

P7: 5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTTTGCTTCCAAAGGCGTG3'

P8: 5'CGGGGGTTTCAAACGCCACTCATTTCAGCGTCAGATG 3'

P9: 5'CATCTGACGCTGAAATGAGTGGCGTTTGAAACCCCG 3'

P10: 5'CCGGCCTTATTTCGGCATTAGCAGTCGAAGTACAATTC 3'

P11: 5'GAATTGTACTTCGACTGCTAATGCCGAAATAAGGCCGG 3'

P12: 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCCTTCTAAAACTGCCG 3'

Construction method: Take Corynebacterium glutamicum ATCC15168 or YPI019 as the template, and use primers P7/P8, P9/P10, P11/P12 to carry out PCR amplification to obtain the upstream homology arm fragment of about 735bp, YH66_10715 or YH66_10715 ^C991T gene fragment About 1470 bp and the downstream homology arm fragment of about 914 bp, and then using P7/P12 as the primer, the three amplified fragments above were mixed as the template for amplification, and the integrated homology arm fragment was obtained. After the PCR reaction, the amplified product was recovered by electrophoresis, and the required DNA fragment of about 3119 bp was recovered using a column-type DNA gel recovery kit. The NEBuider recombination system was used to phase with the shuttle plasmid pk18mobsacB recovered by Xba I digestion. Connect to obtain the integrated plasmid pk18mobsacB-YH66_10715 or pk18mobsacB-YH66_10715 ^C991T , the plasmid contains the kanamycin resistance marker, and the recombinant plasmid integrated into the genome can be obtained by kanamycin screening.

PCR system: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg ²⁺ (25mM) 4μL, primers (10pM) 2μL each, Ex Taq (5U/μL) 0.25μL, total volume 50μL.

The PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 180s (30 cycles), and overextension at 72°C for 10 min.

The 2 integrated plasmids were electrotransformed into Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCCNo.20437), and the single colony produced by the culture was identified by PCR with primers P13/P14, and PCR amplified a fragment containing a size of about 1056bp The positive strains are the positive strains, and the original strains are the ones that fail to amplify the fragments. Positive strains were screened with 15% sucrose and cultured on media containing kanamycin and without kanamycin, respectively. The strains that do not grow on the base are further identified by PCR using the P15/P16 primers, and the amplified bacteria with a size of about 845 bp are YH66_10715 or YH66_10715 ^C991T genes integrated into the genome of strain Corynebacterium glutamicum YPILE001 (Biological Preservation Number is CGMCC No.20437) The strains above were named YPI-020 (without mutation point) and YPI-021 (with mutation point).

P13:5'TGATGGATGGGCTCACTC3'

P14: 5'CGAACTCGACGTTTTTCATC3'

P15: 5'CAGTACAAGTACGACAACG 3'

P16: 5'CGAATTTGTATAGCCTAG3'

Example 4 Engineering strains overexpressing YH66_10715 or YH66_10715 ^C991T genes on plasmids

According to the sequence of Corynebacterium glutamicum ATCC15168 genome (GenBank: CP011309.1) published by NCBI, a pair of primers for amplifying the coding region and promoter region of the YH66_10715 gene were designed and synthesized. The primers were designed as follows (synthesized by Shanghai Invitrogen Company):

P17: 5' GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGTGGCGTTTGAAACCCCG 3'

P18: 5'ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACGAATTGTACTTCGACTGCTAA 3'

Construction method: Using ATCC15168 or YPI019 as the template, and using primers P17/P18 for PCR amplification, the YH66_10715 or YH66_10715 ^C991T gene fragment of about 1510bp was obtained. The amplified product was recovered by electrophoresis and recovered by column DNA gel recovery kit. The required DNA fragment of 1510bp was connected with the shuttle plasmid pXMJ19 digested and recovered by EcoR I using the NEBuider recombination system to obtain the overexpression plasmid pXMJ19-YH66_10715 or pXMJ19- ^{YH66_10715C991T} . The plasmid contains a chloramphenicol resistance marker, and the plasmid can be transformed into the strain by chloramphenicol screening.

PCR system: 10×Ex Taq Buffer 5μL, dNTP Mixture (2.5mM each) 4μL, Mg ²⁺ (25mM) 4μL, primers (10pM) 2μL each, Ex Taq (5U/μL) 0.25μL, total volume 50μL.

The PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 52°C for 30s, extension at 72°C for 120s (30 cycles), and overextension at 72°C for 10 min.

The plasmids were electro-transformed into Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437), and the single colony produced by the culture was identified by M13R (-48) and P18 primers by PCR, and the PCR amplification contained a size of about 920bp. The fragments of the transformed strains were named YPI-022 (without point mutation) and YPI-023 (with point mutation).

Example 5 Construction of the engineering strain that lacks the YH66_10715 gene on the genome

According to the genome sequence of Corynebacterium glutamicum ATCC15168 (GenBank: CP011309.1) published by NCBI, two pairs of primers for amplifying the two ends of the coding region of the YH66_10715 gene were synthesized as upstream and downstream homology arm fragments. The primers are designed as follows (synthesized by Shanghai handsome company):

P19:5'CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAAAATTTGCAACTCTCGC3'

P20:5'CGGCTAGCTAAGTGAATTAGCGGTGACTCCTCATTGAC3'

P21: 5'GTCAATGAGGAGTCACCGCTAATTCACTTAGCTAGCCG 3'

P22: 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAAAATGCTTTTCGACGTCC 3'

Using Corynebacterium glutamicum ATCC15168 as a template and primers P19/P20 and P21/P22, PCR amplification was performed to obtain an upstream homology arm fragment of 894 bp and a downstream homology arm fragment of 690 bp. OVER PCR was performed with primers P19/P22 to obtain the entire homology arm fragment of 1484 bp. After the PCR reaction, the amplified product was recovered by electrophoresis, and the required 1484bp DNA fragment was recovered by a column-type DNA gel recovery kit, and the shuttle plasmid pk18mobsacB plasmid recovered by NEBuider recombination system and Xba I digestion ligated to obtain a knockout plasmid. This plasmid contains a kanamycin resistance marker.

The knockout plasmid was electrotransformed into Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437), and the single colonies produced by the culture were identified by PCR with the following primers (synthesized by Shanghai Yingjun Company):

P23: 5'GCGCTGA CTATGCAGATTC 3'

P24: 5'AAACT CGACC TTCACGTC 3'

The strains that amplified bands of about 1484 bp and about 2940 bp by the above PCR were positive strains, and the strains that only amplified the 2940 bp band were the starting strains. Positive strains were screened on 15% sucrose medium and cultured on kanamycin-containing and kanamycin-free medium, respectively, and grown on kanamycin-free medium, while kanamycin-containing medium was grown on The strains that do not grow on the medium of nutrient-rich medium were further identified by PCR using the P23/P24 primers, and the strain with a 1484bp band amplified was a genetically engineered strain in which the coding region of the YH66_10715 gene was knocked out, and was named as YPI-024.

Example 6L-Isoleucine Fermentation Experiment

The strains constructed in Example 2-5 and the original Corynebacterium glutamicum YPILE001 (Biological Deposit No. CGMCC No. 20437) were placed in the BLBIO-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.) Fermentation experiments were carried out with the medium shown in Table 3 and the fermentation control process shown in Table 4. Each strain was repeated three times and the results are shown in Table 5.

Table 3 Fermentation medium formula

Element Formula (g/L) glucose 90 Ammonium sulfate 12 Magnesium sulfate 0.87 Potassium dihydrogen sulfate 2 acidified corn steep liquor 3mL/L yeast 4 Defoamer (2% foam enemy) 4mL/L VB₁ 0.006 VH 0.003 Biotin 0.088

Table 4 fermentation control process

Table 5L-Isoleucine Fermentation Test Results

The results are shown in Table 5. In the isoleucine-producing engineering bacterium Corynebacterium glutamicum YPILE001 (biodeposit number: CGMCC No. 20437), the point mutation YH66_10715 ^C991T in the coding region of the YH66_10715 gene all contribute to L- Increase of isoleucine production; overexpression of YH66_10715 and YH66_10715 ^C991T helps to increase the production of L-isoleucine, while knockout of YH66_10715 gene is not conducive to the accumulation of L-isoleucine.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

sequence listing

<110> Ningxia Yipin Biotechnology Co., Ltd.

<120> YH66_10715 genetically modified high-yielding L-isoleucine strain and its construction method and application

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1434

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

gtggcgtttg aaaccccgga agaaattgtc aagttcatca aggatgaaaa cgtcgagttc 60

gttgacgttc gattcaccga ccttcccggc accgagcagc acttcagcat cccagctgcc 120

agcttcgatg cagatacagt cgaagaaggt ctcgcattcg acggatcctc gatccgtggc 180

ttcaccacga tcgacgaatc tgacatgaat ctcctgccag acctcggaac ggccaccctt 240

gatccattcc gcaaggcaaa gaccctgaac gttaagttct tcgttcacga tcctttcacc 300

cgcgaggcat tctcccgcga cccacgcaac gtagcacgca aggcagagca gtacctggca 360

tccaccggca ttgcagacac ctgcaacttc ggcgccgagg ctgagttcta cctcttcgac 420

tccgttcgct actccaccga gatgaactcc ggcttctacg aagtagatac cgaagaaggc 480

tggtggaacc gtggcaagga aaccaacctc gacggaaccc caaacctggg cgcaaagaac 540

cgcgtcaagg gtggctactt cccagtagca ccatacgacc aaaccgttga cgtgcgcgat 600

gacatggttc gcaacctcgc agcttccggc ttcgctcttg agcgtttcca ccacgaagtc 660

ggtggcggac agcaggaaat caactaccgc ttcaacacca tgctccacgc ggcagatgat 720

atccagacct tcaagtacat catcaagaac accgctcgcc tccacggcaa ggctgcaacc 780

ttcatgccta agccactggc tggcgacaac ggttccggca tgcacgctca ccagtccctc 840

tggaaggacg gcaagccact cttccacgat gagtccggct acgcaggcct gtccgacatc 900

gcccgctact acatcggcgg catcctgcac cacgcaggcg ctgttctggc gttcaccaac 960

gcaaccctga actcctacca ccgtctggtt ccaggcttcg aggctccaat caacctggtg 1020

tactcacagc gcaaccgttc cgctgctgtc cgtatcccaa tcaccggatc caacccaaag 1080

gcaaagcgca tcgaattccg cgctccagac ccatcaggca acccatacct gggcttcgca 1140

gcgatgatga tggccggcct cgacggcatc aagaaccgca tcgagccaca cgctccagtg 1200

gacaaggacc tctacgaact gccaccagag gaagctgcat ccattccaca ggcaccaacc 1260

tccctggaag catccctgaa ggcactgcag gaagacaccg acttcctcac cgagtctgac 1320

gtcttcaccg aggatctcat cgaggcgtac atccagtaca agtacgacaa cgagatctcc 1380

ccagttcgcc tgcgcccaac cccgcaggaa ttcgaattgt acttcgactg ctaa 1434

<210> 2

<211> 1434

<212> DNA

<213> Corynebacterium glutamicum

<400> 2

gtggcgtttg aaaccccgga agaaattgtc aagttcatca aggatgaaaa cgtcgagttc 60

gttgacgttc gattcaccga ccttcccggc accgagcagc acttcagcat cccagctgcc 120

agcttcgatg cagatacagt cgaagaaggt ctcgcattcg acggatcctc gatccgtggc 180

ttcaccacga tcgacgaatc tgacatgaat ctcctgccag acctcggaac ggccaccctt 240

gatccattcc gcaaggcaaa gaccctgaac gttaagttct tcgttcacga tcctttcacc 300

cgcgaggcat tctcccgcga cccacgcaac gtagcacgca aggcagagca gtacctggca 360

tccaccggca ttgcagacac ctgcaacttc ggcgccgagg ctgagttcta cctcttcgac 420

tccgttcgct actccaccga gatgaactcc ggcttctacg aagtagatac cgaagaaggc 480

tggtggaacc gtggcaagga aaccaacctc gacggaaccc caaacctggg cgcaaagaac 540

cgcgtcaagg gtggctactt cccagtagca ccatacgacc aaaccgttga cgtgcgcgat 600

gacatggttc gcaacctcgc agcttccggc ttcgctcttg agcgtttcca ccacgaagtc 660

ggtggcggac agcaggaaat caactaccgc ttcaacacca tgctccacgc ggcagatgat 720

atccagacct tcaagtacat catcaagaac accgctcgcc tccacggcaa ggctgcaacc 780

ttcatgccta agccactggc tggcgacaac ggttccggca tgcacgctca ccagtccctc 840

tggaaggacg gcaagccact cttccacgat gagtccggct acgcaggcct gtccgacatc 900

gcccgctact acatcggcgg catcctgcac cacgcaggcg ctgttctggc gttcaccaac 960

gcaaccctga actcctacca ccgtctggtt tcaggcttcg aggctccaat caacctggtg 1020

tactcacagc gcaaccgttc cgctgctgtc cgtatcccaa tcaccggatc caacccaaag 1080

gcaaagcgca tcgaattccg cgctccagac ccatcaggca acccatacct gggcttcgca 1140

gcgatgatga tggccggcct cgacggcatc aagaaccgca tcgagccaca cgctccagtg 1200

gacaaggacc tctacgaact gccaccagag gaagctgcat ccattccaca ggcaccaacc 1260

tccctggaag catccctgaa ggcactgcag gaagacaccg acttcctcac cgagtctgac 1320

gtcttcaccg aggatctcat cgaggcgtac atccagtaca agtacgacaa cgagatctcc 1380

ccagttcgcc tgcgcccaac cccgcaggaa ttcgaattgt acttcgactg ctaa 1434

<210> 3

<211> 477

<212> PRT

<213> Corynebacterium glutamicum

<400> 3

Met Ala Phe Glu Thr Pro Glu Glu Ile Val Lys Phe Ile Lys Asp Glu

1 5 10 15

Asn Val Glu Phe Val Asp Val Arg Phe Thr Asp Leu Pro Gly Thr Glu

20 25 30

Gln His Phe Ser Ile Pro Ala Ala Ser Phe Asp Ala Asp Thr Val Glu

35 40 45

Glu Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Thr Thr Ile

50 55 60

Asp Glu Ser Asp Met Asn Leu Leu Pro Asp Leu Gly Thr Ala Thr Leu

65 70 75 80

Asp Pro Phe Arg Lys Ala Lys Thr Leu Asn Val Lys Phe Phe Val His

85 90 95

Asp Pro Phe Thr Arg Glu Ala Phe Ser Arg Asp Pro Arg Asn Val Ala

100 105 110

Arg Lys Ala Glu Gln Tyr Leu Ala Ser Thr Gly Ile Ala Asp Thr Cys

115 120 125

Asn Phe Gly Ala Glu Ala Glu Phe Tyr Leu Phe Asp Ser Val Arg Tyr

130 135 140

Ser Thr Glu Met Asn Ser Gly Phe Tyr Glu Val Asp Thr Glu Glu Gly

145 150 155 160

Trp Trp Asn Arg Gly Lys Glu Thr Asn Leu Asp Gly Thr Pro Asn Leu

165 170 175

Gly Ala Lys Asn Arg Val Lys Gly Gly Tyr Phe Pro Val Ala Pro Tyr

180 185 190

Asp Gln Thr Val Asp Val Arg Asp Asp Met Val Arg Asn Leu Ala Ala

195 200 205

Ser Gly Phe Ala Leu Glu Arg Phe His His Glu Val Gly Gly Gly Gln

210 215 220

Gln Glu Ile Asn Tyr Arg Phe Asn Thr Met Leu His Ala Ala Asp Asp

225 230 235 240

Ile Gln Thr Phe Lys Tyr Ile Ile Lys Asn Thr Ala Arg Leu His Gly

245 250 255

Lys Ala Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser

260 265 270

Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe

275 280 285

His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr

290 295 300

Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn

305 310 315 320

Ala Thr Leu Asn Ser Tyr His Arg Leu Val Pro Gly Phe Glu Ala Pro

325 330 335

Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile

340 345 350

Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala

355 360 365

Pro Asp Pro Ser Gly Asn Pro Tyr Leu Gly Phe Ala Ala Met Met Met

370 375 380

Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val

385 390 395 400

Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro

405 410 415

Gln Ala Pro Thr Ser Leu Glu Ala Ser Leu Lys Ala Leu Gln Glu Asp

420 425 430

Thr Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu

435 440 445

Ala Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Ser Pro Val Arg Leu

450 455 460

Arg Pro Thr Pro Gln Glu Phe Glu Leu Tyr Phe Asp Cys

465 470 475

<210> 4

<211> 477

<212> PRT

<213> Corynebacterium glutamicum

<400> 4

Met Ala Phe Glu Thr Pro Glu Glu Ile Val Lys Phe Ile Lys Asp Glu

1 5 10 15

Asn Val Glu Phe Val Asp Val Arg Phe Thr Asp Leu Pro Gly Thr Glu

20 25 30

Gln His Phe Ser Ile Pro Ala Ala Ser Phe Asp Ala Asp Thr Val Glu

35 40 45

Glu Gly Leu Ala Phe Asp Gly Ser Ser Ile Arg Gly Phe Thr Thr Ile

50 55 60

Asp Glu Ser Asp Met Asn Leu Leu Pro Asp Leu Gly Thr Ala Thr Leu

65 70 75 80

Asp Pro Phe Arg Lys Ala Lys Thr Leu Asn Val Lys Phe Phe Val His

85 90 95

Asp Pro Phe Thr Arg Glu Ala Phe Ser Arg Asp Pro Arg Asn Val Ala

100 105 110

Arg Lys Ala Glu Gln Tyr Leu Ala Ser Thr Gly Ile Ala Asp Thr Cys

115 120 125

Asn Phe Gly Ala Glu Ala Glu Phe Tyr Leu Phe Asp Ser Val Arg Tyr

130 135 140

Ser Thr Glu Met Asn Ser Gly Phe Tyr Glu Val Asp Thr Glu Glu Gly

145 150 155 160

Trp Trp Asn Arg Gly Lys Glu Thr Asn Leu Asp Gly Thr Pro Asn Leu

165 170 175

Gly Ala Lys Asn Arg Val Lys Gly Gly Tyr Phe Pro Val Ala Pro Tyr

180 185 190

Asp Gln Thr Val Asp Val Arg Asp Asp Met Val Arg Asn Leu Ala Ala

195 200 205

Ser Gly Phe Ala Leu Glu Arg Phe His His Glu Val Gly Gly Gly Gln

210 215 220

Gln Glu Ile Asn Tyr Arg Phe Asn Thr Met Leu His Ala Ala Asp Asp

225 230 235 240

Ile Gln Thr Phe Lys Tyr Ile Ile Lys Asn Thr Ala Arg Leu His Gly

245 250 255

Lys Ala Ala Thr Phe Met Pro Lys Pro Leu Ala Gly Asp Asn Gly Ser

260 265 270

Gly Met His Ala His Gln Ser Leu Trp Lys Asp Gly Lys Pro Leu Phe

275 280 285

His Asp Glu Ser Gly Tyr Ala Gly Leu Ser Asp Ile Ala Arg Tyr Tyr

290 295 300

Ile Gly Gly Ile Leu His His Ala Gly Ala Val Leu Ala Phe Thr Asn

305 310 315 320

Ala Thr Leu Asn Ser Tyr His Arg Leu Val Ser Gly Phe Glu Ala Pro

325 330 335

Ile Asn Leu Val Tyr Ser Gln Arg Asn Arg Ser Ala Ala Val Arg Ile

340 345 350

Pro Ile Thr Gly Ser Asn Pro Lys Ala Lys Arg Ile Glu Phe Arg Ala

355 360 365

Pro Asp Pro Ser Gly Asn Pro Tyr Leu Gly Phe Ala Ala Met Met Met

370 375 380

Ala Gly Leu Asp Gly Ile Lys Asn Arg Ile Glu Pro His Ala Pro Val

385 390 395 400

Asp Lys Asp Leu Tyr Glu Leu Pro Pro Glu Glu Ala Ala Ser Ile Pro

405 410 415

Gln Ala Pro Thr Ser Leu Glu Ala Ser Leu Lys Ala Leu Gln Glu Asp

420 425 430

Thr Asp Phe Leu Thr Glu Ser Asp Val Phe Thr Glu Asp Leu Ile Glu

435 440 445

Ala Tyr Ile Gln Tyr Lys Tyr Asp Asn Glu Ile Ser Pro Val Arg Leu

450 455 460

Arg Pro Thr Pro Gln Glu Phe Glu Leu Tyr Phe Asp Cys

465 470 475

<210> 5

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 5

cagtgccaag cttgcatgcc tgcaggtcga ctctagaagt tcttcgttca cgatcc 56

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 6

gttgattgga gcctcgaagc ctgaaaccag acggtggtag 40

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 7

ctaccaccgt ctggtttcag gcttcgaggc tccaatcaac 40

<210> 8

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 8

cagctatgac catgattacg aattcgagct cggtaccctg gaacgccagt tggaag 56

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 9

aaggacggca agccactc 18

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 10

agttcgtaga ggtccttg 18

<210> 11

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 11

cagtgccaag cttgcatgcc tgcaggtcga ctctaggttt gcttccaaag gcgtg 55

<210> 12

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 12

cggggtttca aacgccactc atttcagcgt cagatg 36

<210> 13

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 13

catctgacgc tgaaatgagt ggcgtttgaa accccg 36

<210> 14

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 14

ccggccttat ttcggcatta gcagtcgaag tacaattc 38

<210> 15

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 15

gaattgtact tcgactgcta atgccgaaat aaggccgg 38

<210> 16

<211> 56

<212> DNA

<213> Artificial Sequence

<400> 16

cagctatgac catgattacg aattcgagct cggtaccctg ccttctaaaa ctgccg 56

<210> 17

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 17

tgatggatgg gctcactc 18

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 18

cgaactcgac gttttcatc 19

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 19

cagtacaagt acgacaacg 19

<210> 20

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 20

cgaatttgta tagcctag 18

<210> 21

<211> 54

<212> DNA

<213> Artificial Sequence

<400> 21

gcttgcatgc ctgcaggtcg actctagagg atccccgtgg cgtttgaaac cccg 54

<210> 22

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 22

atcaggctga aaatcttctc tcatccgcca aaacgaattg tacttcgact gctaa 55

<210> 23

<211> 54

<212> DNA

<213> Artificial Sequence

<400> 23

cagtgccaag cttgcatgcc tgcaggtcga ctctagaaaa tttgcaactc tcgc 54

<210> 24

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 24

cggctagcta agtgaattag cggtgactcc tcattgac 38

<210> 25

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 25

gtcaatgagg agtcaccgct aattcactta gctagccg 38

<210> 26

<211> 57

<212> DNA

<213> Artificial Sequence

<400> 26

cagctatgac catgattacg aattcgagct cggtacccaa aatgcttttc gacgtcc 57

<210> 27

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 27

gcgctgacta tgcagattc 19

<210> 28

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 28

aaactcgacc ttcacgtc 18

Claims (10)

1. A bacterium having an improved L-isoleucine producing ability, which is characterized by being the following (a) or (b):

(a) (ii) the polynucleotide encoding the amino acid sequence of SEQ ID No. 3 on its genome is overexpressed;

(b) the polynucleotide encoding the amino acid sequence of SEQ ID NO. 3 is point mutated such that the polynucleotide of SEQ ID NO. 3

The 331 st proline of the amino acid sequence is replaced by serine and is over-expressed;

the starting bacterium of the bacterium is Corynebacterium glutamicum having L-isoleucine-producing ability.

2. The bacterium according to claim 1, wherein the Corynebacterium glutamicum is Corynebacterium glutamicum YPILE001, having a biological deposit number of CGMCC number 20437, or Corynebacterium glutamicum ATCC 15168.

3. The bacterium of claim 1 or 2, wherein the nucleotide sequence of the polynucleotide encoding the amino acid sequence of SEQ ID No. 3 is as shown in SEQ ID No. 1.

4. The bacterium of claim 1 or 2, wherein the point-mutated polynucleotide sequence is mutated at base 991 of the polynucleotide sequence shown in SEQ ID NO. 1.

5. The bacterium of claim 4, wherein the nucleotide sequence of the point mutant polynucleotide is set forth in SEQ ID NO. 2.

6. A method of increasing L-isoleucine production in a bacterium, comprising: culturing the bacterium of any one of claims 1-5, and recovering L-isoleucine from the culture.

7. The application of the polynucleotide in improving the L-isoleucine production of the bacteria is characterized in that the polynucleotide is overexpressed in the bacteria, the starting bacteria of the bacteria is corynebacterium glutamicum with L-isoleucine production capacity, and the polynucleotide is a polynucleotide encoding an amino acid sequence shown as SEQ ID NO. 4.

8. The use of claim 7, wherein the polynucleotide is produced by mutation at base 991 of the polynucleotide sequence shown in SEQ ID NO. 1.

9. The use of claim 8, wherein the polynucleotide has the sequence shown in SEQ ID NO 2.

10. The use according to any one of claims 7 to 9, wherein the corynebacterium glutamicum is corynebacterium glutamicum YPILE001, having the biological collection number CGMCC number 20437, or corynebacterium glutamicum ATCC 15168.