CN115322990B - Polynucleotide having promoter activity and use thereof for producing target compounds - Google Patents

Abstract

The present disclosure relates to a polynucleotide having a promoter activity, a transcription expression cassette, a recombinant expression vector, a recombinant host cell comprising the polynucleotide having a promoter activity, and a method for regulating transcription of a target gene, a method for preparing a protein, and a method for producing a target compound, which belong to the technical fields of biotechnology and genetic engineering. The polynucleotide with promoter activity provided by the present disclosure is a polynucleotide comprising a sequence as set forth in SEQ ID NO:2-4, the promoter activity of the mutant is significantly improved compared to the wild-type promoter. The mutant is applied to the production of target compounds, the conversion rate of the target compounds can be obviously improved, and a strong constitutive promoter with great application potential is provided for the industrial fermentation of the target compounds such as amino acid, organic acid and the like.

Description

Polynucleotides with promoter activity and use thereof in producing target compounds

Technical Field

The present disclosure belongs to the field of biotechnology and genetic engineering technology, and specifically relates to a polynucleotide with promoter activity, a transcription expression cassette comprising the polynucleotide with promoter activity, a recombinant expression vector, a recombinant host cell, a method for regulating the transcription of a target gene, a method for preparing a protein, and a method for producing a target compound.

Background Art

Microbial fermentation can produce a variety of target compounds, such as amino acids, organic acids, bio-based materials, pharmaceutical compounds, etc. These target compounds can be widely used in medicine, health, food, animal feed and cosmetics, and have great economic value. In recent years, with the increasing market demand for amino acids, organic acids, bio-based materials, raw materials, etc., how to increase the yield of target compounds and realize industrial large-scale production of target compounds is an important issue that needs to be solved urgently.

Breeding high-yield fermentation microorganisms is an important means to increase the industrial production of target compounds. Compared with traditional mutagenesis breeding technology, genetic engineering breeding technology has the advantages of strong targeting, high stability and high efficiency. Modifying key genes in microbial metabolic pathways through genetic engineering methods is an important method to increase the fermentation yield of target compounds. Factors affecting gene expression include promoter activity, gene translation efficiency, gene copy number, etc. However, an increase in gene copy number will reduce the stability of the bacterial genome. In contrast, increasing promoter activity to improve gene expression efficiency has become an important means of modifying key genes.

Identification of strong promoters or constitutive promoters can provide efficient gene expression elements for genetic engineering breeding of fermentation microorganisms. Among them, strong promoters have a high affinity for transcriptase and can efficiently initiate the transcription of target genes. Constitutive promoters do not require any inducers, that is, they can sustainably express the target protein. Using strong promoters or constitutive promoters to transform key genes in metabolic pathways can effectively increase the expression level of key genes and increase metabolic flux.

Therefore, developing strong promoters or constitutive promoters with high activity to enhance the expression of key genes in the synthesis pathway of target compounds, increase the yield of target compounds, and enhance the potential for industrial application is an important issue that needs to be urgently addressed in the field of microbial fermentation.

Summary of the invention

Problem that the invention aims to solve

In view of the technical problems existing in the prior art, for example, it is necessary to develop more strong promoters or constitutive promoters with high activity to improve the expression of key genes in the synthetic pathway of the target compound. To this end, the present disclosure provides a polynucleotide with promoter activity, which is a mutant of a polynucleotide comprising a sequence as shown in any one of SEQ ID NOs: 2-4. Compared with the wild-type promoter, the promoter activity of the mutant provided by the present disclosure is significantly improved, and is no longer induced by an inducer, and is a new type of strong constitutive promoter. Operably connecting the mutant to the target gene can effectively improve the expression of the target gene, thereby effectively increasing the yield of the target compound while maintaining genome stability.

Solutions for solving problems

The present disclosure provides a polynucleotide having promoter activity, wherein the polynucleotide is selected from any one of the following groups consisting of (i)-(vi):

(i) a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 2, wherein the mutant has a mutated nucleotide at one or more positions in the sequence of SEQ ID NO: 2 from position 204 to position 211; the activity of the mutant is higher than the promoter activity of the polynucleotide having a sequence as shown in SEQ ID NO: 2, and the nucleotide sequence in the sequence of SEQ ID NO: 2 from position 204 to position 211 is not CCACAATG;

(ii) a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 3, wherein the mutant has a mutated nucleotide at one or more positions in the sequence of SEQ ID NO: 3; the activity of the mutant is higher than the promoter activity of the polynucleotide having a sequence as shown in SEQ ID NO: 3, and the nucleotide sequence in the sequence of SEQ ID NO: 3 at positions 164 to 171 is not CCACAATG;

(iii) a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 4, wherein the mutant has a mutated nucleotide at one or more positions in the sequence of SEQ ID NO: 4, wherein the activity of the mutant is higher than the promoter activity of the polynucleotide having a sequence as shown in SEQ ID NO: 4, and the nucleotide sequence in the sequence of SEQ ID NO: 4, wherein the nucleotide sequence is not CCACAATG;

(iv) a polynucleotide comprising a reverse complementary sequence to the nucleotide sequence shown in any one of (i) to (iii);

(v) a polynucleotide comprising a reverse complementary sequence of a sequence capable of hybridizing to the nucleotide sequence shown in any one of (i) to (iii) under high stringency hybridization conditions or very high stringency hybridization conditions;

(vi) a polynucleotide comprising a sequence having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the nucleotide sequence shown in any one of (i) to (iii).

In some embodiments, according to the polynucleotide having promoter activity described in the present disclosure, the mutant has a promoter activity increased by 5-13 times or more compared to the polynucleotide comprising the sequence shown in SEQ ID NO: 2-4.

In some embodiments, according to the polynucleotide having promoter activity of the present disclosure, the mutant corresponds to nucleotide sequences 204-211 of the sequence shown in SEQ ID NO: 2, or 164-171 of the sequence shown in SEQ ID NO: 3, or 106-113 of the sequence shown in SEQ ID NO: 4, and is selected from any one of the following groups consisting of (p ₁ )-(p ₁₈ ):

(p ₁ )ACTGTAGG,

(p ₂ )TATTATGG,

(p ₃ )AATTGGGG,

(p ₄ )TATGGTTG,

(p ₅ )TAGGGTAG,

(p ₆ )AATGGAAT,

(p ₇ )TAGACTTC,

(p ₈ )AATGGGTA,

(p ₉ )TACCATTA,

(p ₁₀ )ACTGAGGG,

(p ₁₁ )ACTAGAAG,

(p ₁₂ )AATTAGTG,

(p ₁₃ )AATAGGGT,

(p ₁₄ )TAGTATTG,

(p ₁₅ )ACTGGACT,

(p ₁₆ )TAACATGG,

(p ₁₇ )ACTAGGGG,

(p ₁₈ )TATAAGTT.

In some embodiments, according to the polynucleotide having promoter activity described in the present disclosure, the nucleotide sequence of the mutant is selected from the sequence shown in any one of SEQ ID NOs: 5-22.

The present disclosure provides a transcription expression cassette, wherein the transcription expression cassette comprises the polynucleotide with promoter activity according to the present disclosure; optionally, the transcription expression cassette further contains a target gene, and the target gene is operably linked to the polynucleotide with promoter activity; preferably, the target gene is a protein coding gene.

The present disclosure provides a recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide having promoter activity according to the present disclosure, or the transcription expression cassette according to the present disclosure.

The present disclosure provides a recombinant host cell, wherein the recombinant host cell comprises the transcription expression cassette according to the present disclosure, or the recombinant expression vector according to the present disclosure.

In some embodiments, according to the recombinant host cell described in the present disclosure, the host cell is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the host cell is Corynebacterium glutamicum or Escherichia coli; more preferably, the host cell is Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067.

The present disclosure provides a use of the polynucleotide having promoter activity according to the present disclosure, the transcription expression cassette according to the present disclosure, the recombinant expression vector according to the present disclosure, and the recombinant host cell according to the present disclosure in at least one of the following:

(a) regulating the transcription level of a gene, or preparing a reagent or kit for regulating the transcription level of a gene;

(b) preparing a protein, or preparing a reagent or kit for preparing a protein;

(c) producing a target compound, or preparing a reagent or kit for producing a target compound.

In some embodiments, according to the use described in the present disclosure, the protein is selected from a gene expression regulatory protein or a protein related to the synthesis of a target compound.

In some embodiments, according to the use described in the present disclosure, the target compound includes at least one of an amino acid and an organic acid; optionally, the amino acid includes at least one of proline, lysine, glutamic acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, and tryptophan, and the organic acid includes at least one of citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, and malic acid.

The present disclosure provides a method for regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide having promoter activity according to the present disclosure to the target gene.

The present disclosure provides a method for preparing a protein, wherein the method comprises the step of expressing the protein using the transcription expression cassette according to the present disclosure, the recombinant expression vector according to the present disclosure, or the recombinant host cell according to the present disclosure; optionally, the protein is a protein related to the synthesis of a target compound or a gene expression regulatory protein;

Optionally, the method further comprises the step of isolating or purifying the protein.

The present disclosure provides a method for producing a target compound, wherein the method comprises the steps of using the transcription expression cassette according to the present disclosure, the recombinant expression vector according to the present disclosure, or the recombinant host cell according to the present disclosure to express a protein or a gene expression regulatory protein related to the synthesis of the target compound, and producing the target compound in an environment where the protein or the gene expression regulatory protein related to the synthesis of the target compound exists;

Optionally, the target compound includes at least one of an amino acid and an organic acid; optionally, the amino acid includes at least one of lysine, glutamic acid, threonine, proline, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, and tryptophan, and the organic acid includes at least one of citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, and malic acid;

Optionally, the protein related to the synthesis of the target compound is a protein related to the synthesis of L-amino acids; optionally, the proteins related to the synthesis of L-amino acids include pyruvate carboxylase, phosphoenolpyruvate carboxylase, γ-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transport protein, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconate repressor protein, glucose dehydrogenase, One or a combination of two or more of aspartate kinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthetase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase;

Optionally, the method further comprises the step of isolating or purifying the target compound.

Effects of the Invention

In some embodiments, the polynucleotide with promoter activity provided by the present disclosure is a mutant of a polynucleotide comprising a sequence as shown in any one of SEQ ID NOs: 2-4, and the promoter activity of the mutant is significantly improved compared with the wild-type promoter comprising a sequence as shown in SEQ ID NOs: 2-4. After the mutant is operably linked to the target gene, the expression efficiency of the target gene can be significantly improved, and the stable and efficient expression of the target gene can be achieved without any induction conditions, providing an expression element with great application potential for the transformation of key genes in the synthetic pathway of the target compound. The mutant is applied to the production of the target compound, which can significantly improve the conversion rate of the target compound, and provides a strong constitutive promoter with great application potential for the industrial fermentation of target compounds such as amino acids and organic acids.

In some embodiments, the polynucleotide with promoter activity provided by the present disclosure has a promoter activity that is 5-13 times higher than that of the wild-type promoter, and the promoter activity of the mutant in the present disclosure is higher than the promoter activity of the endogenous strong promoter tuf of Corynebacterium glutamicum.

In some more specific embodiments, the polynucleotides with promoter activity provided by the present disclosure have promoter activity that is 5.89-12.61 times higher than that of the wild-type promoter.

In some embodiments, the present disclosure provides a transcription expression cassette, a recombinant expression vector, and a recombinant host cell, comprising the above-mentioned polynucleotide with promoter activity. In the transcription expression cassette, the recombinant expression vector, and the recombinant host cell, the polynucleotide with promoter activity is operably linked to the target gene, and the efficient expression of the key gene in the synthesis pathway of the target compound can be achieved.

In some embodiments, the present disclosure provides a method for preparing proteins, which can increase the expression level of proteins related to the synthesis of amino acids, organic acids, etc. or gene expression regulatory proteins, thereby achieving efficient production of target compounds.

In some embodiments, the present disclosure provides a method for producing a target compound. By utilizing the above-mentioned polynucleotide with promoter activity, the expression efficiency of proteins related to the synthesis of the target compound can be improved, thereby stably and efficiently producing the target compound and realizing large-scale industrial production of the target compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the results of comparing the activities of NCgl1418 promoters of different lengths.

DETAILED DESCRIPTION

When used in conjunction with the term "comprising" in the claims and/or the specification, the word "a" or "an" can mean "one", but can also mean "one or more", "at least one" and "one or more than one".

As used in the claims and description, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this application, the term "about" indicates that a value includes the standard deviation of error for the device or method being employed to determine the value.

Although the disclosure supports a definition of the term "or" as only alternatives as well as "and/or," the term "or" in the claims means "and/or" unless explicitly stated as only alternatives or the alternatives are mutually exclusive.

When used in the claims or specification, a selected/optional/preferred "numerical range" includes both the numerical endpoints at both ends of the range and all natural numbers covered between the numerical endpoints relative to the aforementioned numerical endpoints.

As used in the present disclosure, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and recovered by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

As used in the present disclosure, the term "wild-type" refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism, can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. As used in the present disclosure, "naturally occurring" and "wild-type" are synonyms. In some embodiments, the wild-type promoter in the present disclosure refers to the promoter of the wild-type NCgl1418 gene, that is, the polynucleotide of the sequence shown in SEQ ID NO: 2.

As used in the present disclosure, the term "mutant" refers to a polynucleotide comprising an alteration (i.e., substitution, insertion and/or deletion) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein a substitution refers to replacing a nucleotide occupying a position with a different nucleotide. A deletion refers to the removal of a nucleotide occupying a position. An insertion refers to the addition of a nucleotide adjacent to and immediately following the nucleotide occupying the position.

In some embodiments, the "mutation" disclosed herein is a "substitution", which is a mutation caused by the replacement of a base in one or more nucleotides by another different base, also known as a base substitution mutation or a point mutation.

Specifically, the sequence shown in SEQ ID NO: 1 is the core region sequence of the NCgl1418 gene promoter, including the main sequences of the -35 region and the -10 region. The mutant in the present disclosure is a nucleotide with a mutation introduced at the -10 region position, and it is found that after the mutation is introduced at the above position, the promoter activity of the mutant is significantly enhanced, and a new type of strong constitutive promoter is obtained, and the promoter activity is higher than the promoter activity of the endogenous strong promoter tuf of Corynebacterium glutamicum.

In some embodiments, a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 2 refers to a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 2, wherein the mutant has a mutated nucleotide at one or more positions of positions 204-211 of the sequence shown in SEQ ID NO: 2, and does not comprise a polynucleotide having positions 204-211 of the sequence shown in SEQ ID NO: 2 mutated to CCACAATG. Compared to a polynucleotide having a sequence as shown in SEQ ID NO: 2, the mutant has an improved promoter activity.

In some embodiments, a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 3 refers to a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 3, wherein the mutant has a mutated nucleotide at one or more positions of positions 164-171 of the sequence shown in SEQ ID NO: 3, and does not comprise a polynucleotide having positions 164-171 of the sequence shown in SEQ ID NO: 3 mutated to CCACAATG. Compared to a polynucleotide having a sequence as shown in SEQ ID NO: 3, the mutant has an improved promoter activity.

In some embodiments, a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 4 refers to a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 4, wherein the mutant has a mutated nucleotide at one or more positions of positions 106-113 of the sequence shown in SEQ ID NO: 4, and does not comprise a polynucleotide having positions 106-113 of the sequence shown in SEQ ID NO: 4 mutated to CCACAATG. Compared to a polynucleotide having a sequence as shown in SEQ ID NO: 4, the mutant has an improved promoter activity.

In some embodiments, a mutant of the polynucleotide comprising the sequence shown in SEQ ID NO: 2 in the present disclosure has a promoter activity increased by 5-13 times or more compared to the polynucleotide comprising the sequence shown in SEQ ID NO: 2.

Furthermore, the mutants have 8.31, 12.18, 8.93, 8.07, 7.63, 10.31, 5.89, 5.92, 6.49, 7.66, 8.63, 8.41, 10.21, 9.52, 9.91, 8.52, 12.61, and 9.16 times enhanced promoter activity compared to the polynucleotide comprising the sequence shown in SEQ ID NO:2.

As used in the present disclosure, the term "promoter" refers to a nucleic acid molecule that is usually located upstream of the target gene coding sequence, provides a recognition site for RNA polymerase, and is located upstream of the 5' direction of the mRNA transcription start site. It is a nucleic acid sequence that is not translated, and RNA polymerase binds to this nucleic acid sequence to initiate transcription of the target gene. In the synthesis of ribonucleic acid (RNA), the promoter can interact with transcription factors that regulate gene transcription, control the start time and degree of gene expression (transcription), and contains a core promoter region and a regulatory region, just like a "switch", which determines the activity of the gene and then controls which protein the cell starts to produce.

As used in the present disclosure, the term "promoter core region" refers to a nucleic acid sequence located in the promoter region of a prokaryotic organism, which is a core sequence region that exerts a promoter function, and mainly includes the -35 region, the -10 region, the region between the -35 region and the -10 region, and the transcription start site, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase. In some embodiments, the polynucleotide with promoter activity disclosed in the present disclosure is a mutant comprising the promoter core region of the NCgl1418 gene, and a mutant with a mutation introduced into the -10 region of the promoter core region to obtain a promoter activity significantly improved compared to the promoter of the NCgl1418 gene.

As used in the present disclosure, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). Sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

In some embodiments, two or more sequences or subsequences have a "sequence identity" or "percent identity" of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotides when compared and aligned for maximum correspondence using a sequence comparison algorithm or as measured by visual inspection. In certain embodiments, the sequences are substantially identical over the entire length of either or both of the compared biopolymers (e.g., polynucleotides).

As used in this disclosure, the term "complementary" refers to hybridization or base pairing between nucleotides or nucleotides, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleotide being sequenced or amplified.

As used in this disclosure, the term "high stringency conditions" refers to, for probes of at least 100 nucleotides in length, prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide following standard Southern blotting procedures. Finally, the carrier material is washed three times at 65° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

As used in this disclosure, the term "very high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization for 12 to 24 hours at 42° C. in 5X SSPE (saline sodium phosphate EDTA), 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide. The carrier material is finally washed three times at 70° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

In some specific embodiments, the polynucleotides with promoter activity in the present disclosure can be used to initiate the expression of protein coding genes. In other embodiments, the polynucleotides with promoter activity in the present disclosure can be used to initiate the expression of non-coding genes.

As used in this disclosure, the term "expression" includes any step involved in RNA production and protein production including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

As used in the present disclosure, the term "transcription expression cassette" is a recombinant expression element comprising a polynucleotide having promoter activity. In some embodiments, the polynucleotide having promoter activity is a mutant of a polynucleotide comprising nucleotides 204-211 of the sequence shown in SEQ ID NO: 2. In some embodiments, the polynucleotide having promoter activity is a mutant of a polynucleotide comprising nucleotides 164-171 of the sequence shown in SEQ ID NO: 3. In some embodiments, the polynucleotide having promoter activity is a mutant of a polynucleotide comprising nucleotides 106-113 of the sequence shown in SEQ ID NO: 4. In some more specific embodiments, the transcription expression cassette includes a target gene operably linked to the mutant, and the mutant with improved promoter activity in the present disclosure is used to regulate the expression of the target gene. In some embodiments, the transcriptional regulatory element that regulates the target gene may include enhancers, silencers, insulators and other elements in addition to the mutant having promoter activity. In some embodiments, the target gene in the present disclosure is specifically a protein-coding gene. The target gene is "operably linked" to a polynucleotide with promoter activity, which means that the polynucleotide with promoter activity is functionally linked to the target gene to initiate and mediate the transcription of the target gene. The operably linked method can be any method described by those skilled in the art.

As used in the present disclosure, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence to express a target gene in a suitable host. "Recombinant expression vector" refers to a DNA structure used to express, for example, a polynucleotide encoding a desired polypeptide. A recombinant expression vector may include, for example, a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) a transcription subunit that is suitable for transcription and translation initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector may be used, including plasmids, viruses, phages, and transposons. Possible vectors for use in the present disclosure include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, phage DNA, yeast plasmids, and vectors derived from a combination of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies. In the present disclosure, "recombinant expression vector" and "recombinant vector" may be used interchangeably.

As used in the present disclosure, the term "target gene" refers to any gene to which the polynucleotide having promoter activity in the present disclosure is linked to regulate its transcription level.

In some embodiments, the target gene refers to a gene encoding a target protein in a microorganism. Exemplarily, the target gene is a gene encoding an enzyme associated with the biosynthesis of the target compound, a gene encoding an enzyme associated with reducing power, a gene encoding an enzyme associated with glycolysis or TCA cycle, or a gene encoding an enzyme associated with the release of the target compound, etc.

As used in the present disclosure, the term "target compound" may be selected from amino acids, organic acids, or other types of compounds that may be obtained through biosynthesis in the art.

In some embodiments, the target compound is an "amino acid" or "L-amino acid". "Amino acid" or "L-amino acid" generally refers to a basic building block of a protein in which an amino group and a carboxyl group are bound to the same carbon atom. Exemplarily, the amino acid is selected from one or a combination of two or more of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and proline, or other types of amino acids in the art.

In some embodiments, the target compound is an organic acid. The organic acid can be an organic compound with acidity, for example, those compounds including carboxyl and sulfonic acid groups. Exemplary, the organic acid includes one or a combination of two or more of lactic acid, acetic acid, succinic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, citric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, malic acid, or other types of organic acids in the art.

The term "protein coding gene" in this disclosure refers to a DNA molecule that can guide the synthesis of proteins through certain rules. The process of protein coding gene guiding protein synthesis generally includes a transcription process with double-stranded DNA as a template and a translation process with mRNA as a template. Protein coding genes contain CDS sequences (Coding Sequences) that can guide the production of mRNA encoding proteins.

Exemplarily, protein encoding genes include but are not limited to proteins encoding proteins related to the synthesis of target compounds. In some embodiments, protein encoding genes involve proteins encoding proteins related to the synthesis of L-amino acids. Exemplarily, proteins related to the synthesis of L-amino acids include but are not limited to pyruvate carboxylase, phosphoenolpyruvate carboxylase, γ-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylic acid reductase, amino acid transport protein, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconate repressor protein, glucose dehydrogenase, one or a combination of two or more thereof. In some embodiments, the protein related to the synthesis of L-amino acids is a protein related to the synthesis of L-lysine, and the protein related to the synthesis of L-lysine includes aspartate kinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydropicolinate synthetase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydropicolinate ester succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transport protein, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase. One or a combination of more than two.

In some embodiments, the protein coding gene is used to encode a protein related to the synthesis of organic acids, and illustratively, the protein coding gene is used to encode a protein related to the synthesis of citric acid, or is used to encode a protein related to the synthesis of succinic acid. In some embodiments, the protein coding gene is related to a protein related to gene editing, such as Cpf1 protein.

The term "gene expression regulatory protein" disclosed in the present invention includes but is not limited to exogenous gene expression regulatory tool proteins, such as dCas9 protein and dCpf1 protein required for CRISPRi regulation, Hfq protein required for sRNA regulation, etc., as well as endogenous or exogenous transcription regulatory factors, thereby regulating the expression of key genes in metabolic pathways.

The term "host cell" in the present disclosure means any cell type that is susceptible to transformation, transfection, transduction, etc. with a transcription initiation element or an expression vector comprising a polynucleotide of the present disclosure. The term "recombinant host cell" encompasses a host cell that is different from the parent cell after the introduction of a transcription initiation element or a recombinant expression vector, and the recombinant host cell is specifically achieved by transformation.

The term "transformation" in the present disclosure has a meaning generally understood by those skilled in the art, i.e., the process of introducing exogenous DNA into a host. The transformation method includes any method of introducing nucleic acid into a cell, including but not limited to electroporation, calcium phosphate precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method and lithium acetate-DMSO method.

Host cell of the present disclosure can be prokaryotic cell or eukaryotic cell, as long as can import the cell of the polynucleotide with promoter activity of the present disclosure.In some embodiments, host cell refers to prokaryotic cell, and particularly, host cell derives from the microorganism that is suitable for fermentation production amino acid, organic acid, for example Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia.As preferably, host cell is the Corynebacterium glutamicum that derives from Corynebacterium.Wherein, Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067 etc.

The host cells disclosed herein can be cultured according to conventional methods in the art, including but not limited to well plate culture, shake flask culture, batch culture, continuous culture and fed-batch culture, etc., and various culture conditions such as temperature, time and pH value of the culture medium can be appropriately adjusted according to actual conditions.

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

Mutants containing the promoter core region of the NCgl1418 gene

The present invention utilizes the promoter core region sequence of the NCgl1418 gene to introduce a mutation in the promoter -10 region of the NCgl1418 gene, thereby obtaining a mutant of the promoter core region of the NCgl1418 gene containing a mutation in the -10 region.

The polynucleotide with promoter activity in the present invention is obtained by mutating the promoter core region of the NCgl1418 gene, specifically introducing a mutation in the -10 region (CCACAATG) of the promoter core region of the NCgl1418 gene. Compared with the wild-type promoter comprising the promoter core region of the NCgl1418 gene, the mutant in the present invention has significantly improved promoter activity and is a new type of strong constitutive promoter. When applied to the fermentation of the target compound, the mutant shows a higher conversion rate of the target compound than the wild-type promoter.

In addition, by truncating the promoter to different lengths, NCgl1418 promoter fragments of 203 bp (SEQ ID NO: 3) and 145 bp (SEQ ID NO: 4) were obtained, respectively. Both fragments have the core region of the NCgl1418 promoter and can also show significantly enhanced promoter activity in an environment with increased salt concentration and osmotic pressure. Then, by using the promoter modification method in the above embodiment, that is, mutating one or more positions in the 164-171 position of the sequence shown in SEQ ID NO: 3, or mutating one or more positions in the 106-113 position of the sequence shown in SEQ ID NO: 4, a strong constitutive promoter mutant will be obtained.

In some embodiments, the mutant disclosed herein refers to a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2, and refers to a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2, wherein the mutant has a mutated nucleotide at one or more positions of positions 204-211 of the sequence shown in SEQ ID NO: 2 and does not include a polynucleotide in which positions 204-211 of the sequence shown in SEQ ID NO: 2 are mutated to CCACAATG. Compared to the polynucleotide comprising the sequence shown in SEQ ID NO: 2, the mutant has an improved promoter activity.

In some embodiments, the mutant has a mutated nucleotide at 1, 2, 3, 4, 5, 6, 7 or 8 positions corresponding to positions 204-211 of the sequence shown in SEQ ID NO:2.

In some embodiments, a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 3 refers to a mutant comprising a polynucleotide having a sequence as shown in SEQ ID NO: 3, wherein the mutant has a mutated nucleotide at one or more positions of positions 164-171 of the sequence shown in SEQ ID NO: 3 and does not comprise a polynucleotide having positions 164-171 of the sequence shown in SEQ ID NO: 3 mutated to CCACAATG. Compared to a polynucleotide having a sequence as shown in SEQ ID NO: 3, the mutant has an improved promoter activity.

In some embodiments, the mutant has a mutated nucleotide at 1, 2, 3, 4, 5, 6, 7 or 8 positions corresponding to positions 164-171 of the sequence shown in SEQ ID NO:3.

In some embodiments, a mutant comprising a polynucleotide of the sequence shown in SEQ ID NO: 4 refers to a mutant comprising a polynucleotide of the sequence shown in SEQ ID NO: 4, wherein the mutant has a mutated nucleotide at one or more positions of positions 106-113 of the sequence shown in SEQ ID NO: 4 and does not contain a polynucleotide in which positions 106-113 of the sequence shown in SEQ ID NO: 4 are mutated to ACACCGAGTG. Compared with the polynucleotide comprising the sequence shown in SEQ ID NO: 4, the mutant has an increased promoter activity. In some embodiments, the mutant has a mutated nucleotide at 1, 2, 3, 4, 5, 6, 7 or 8 positions corresponding to positions 106-113 of the sequence shown in SEQ ID NO: 4.

In some embodiments, the polynucleotide having promoter activity in the present disclosure further includes a polynucleotide that is complementary to the nucleotide sequence of the mutant shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

In some embodiments, the polynucleotides having promoter activity in the present disclosure further include polynucleotides that are reverse complementary to the mutants or hybridized sequences shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 under high stringency hybridization conditions or very high stringency hybridization conditions. And the nucleotide sequence of the polynucleotide corresponding to positions 204-211 of the sequence shown in SEQ ID NO: 2 is not CCACAATG, the nucleotide sequence corresponding to positions 164-171 of the sequence shown in SEQ ID NO: 3 is not CCACAATG, and the nucleotide sequence corresponding to positions 106-113 of the sequence shown in SEQ ID NO: 4 is not CCACAATG.

In some embodiments, the polynucleotide with promoter activity in the present disclosure is a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity (including all ranges and percentages between these values) with the above-mentioned polynucleotide sequence. And the nucleotide sequence of the polynucleotide in the 204th to 211th positions corresponding to the sequence shown in SEQ ID NO: 2 is not CCACAATG, the nucleotide sequence in the 164th to 171th positions corresponding to the sequence shown in SEQ ID NO: 3 is not CCACAATG, and the nucleotide sequence in the 106th to 113th positions corresponding to the sequence shown in SEQ ID NO: 4 is not CCACAATG.

In some specific embodiments, the mutant corresponds to the nucleotide sequence at positions 204-211 of the sequence shown in SEQ ID NO: 2, or the nucleotide sequence at positions 164-171 of the sequence shown in SEQ ID NO: 3, or the nucleotide sequence at positions 106-113 of the sequence shown in SEQ ID NO: 4 is selected from any one of the following groups consisting of (p ₁ )-(p ₁₈ ): (p ₁ ) ACTGTAGG, (p ₂ ) TATTATGG, (p ₃ ) AATTGGGG, (p ₄ ) TATGGTTG, (p ₅ ) TAGGGTAG, (p ₆ ) AATGGAAT, (p ₇ ) TAGACTTC, (p ₈ ) AATGGGTA, (p ₉ ) TACCATTA, (p ₁₀ ) ACTGAGGG, (p ₁₁ ) ACTAGAAG, (p ₁₂ ) AATTGGGG, (p ₁₃ ) )AATAGGGT, (p ₁₄ )TAGTATTG, (p ₁₅ )ACTGGACT, (p ₁₆ )TAACATGG, (p ₁₇ )ACTAGGGGG, (p ₁₈ )TATAAGTT.

In some specific embodiments, the nucleotide sequence of the mutant is selected from the sequence shown in any one of SEQ ID NOs: 5-22.

In some embodiments, the polynucleotides with promoter activity in the present disclosure have promoter activity increased by 5-13 times or more compared to the polynucleotides of the sequence shown in SEQ ID NO: 2. Further, compared to the polynucleotides comprising the sequence shown in SEQ ID NO: 2, the promoter activity is increased by 8.31, 12.18, 8.93, 8.07, 7.63, 10.31, 5.89, 5.92, 6.49, 7.66, 8.63, 8.41, 10.21, 9.52, 9.91, 8.52, 12.61, 9.16 times.

Recombinant expression vector and recombinant host cell

In some embodiments, the present invention uses the ATCC13032 genome (Corynebacterium glutamicumATCC 13032, NC_003450.3)) as a template, 1418-F and 1418-R as primers, to amplify the DNA fragment of the NCgl1418 gene promoter; uses the pXM-gfp plasmid as a template, pGFP-F and pGFP-R primers to amplify the vector fragment removing the lacI gene and tac promoter; and recombines and connects the above fragments to obtain the recombinant expression vector pXM-P _NCgl1418 -gfp.

In some embodiments, the present invention uses pXM-P _NCgl1418 -gfp as a template and performs reverse PCR amplification on pXM-P _NCgl1418 -gfp with 1418mutant-F and 1418mutant-R primers to obtain a linearized plasmid fragment; the linearized plasmid fragment is phosphorylated and ligated, and resistant clones are collected to obtain a promoter mutant library of the NCgl1418 gene.

In some embodiments, the present disclosure transforms Corynebacterium glutamicum ATCC13032 with promoter mutant library of NCgl1418 gene and pXM-Con and pXM-P _NCgl1418 -gfp respectively to obtain recombinant host cells. The fluorescence intensity of recombinant host cells is screened after plate culture to screen mutants with improved promoter strength.

In some specific embodiments, the present disclosure uses ATCC 13032 genome as a template, uses primers 1418-LF and 1418-LR, and primers lysE-F and lysE-R, respectively, to obtain the promoter sequence of NCgl1418 gene and the DNA sequence of lysE gene by PCR amplification. Using pEC-XK99E as a template, using primers pEC-F and pEC-R, a vector fragment is obtained by PCR amplification, and the above three fragments are recovered and recombined to obtain the recombinant expression vector pEC-P _NCgl1418 -lysE.

In some specific embodiments, the present disclosure uses pXM-P _tuf -gfp as a template, and uses primers tuf-lysE-F and tuf-lysE-F to amplify the promoter sequence of the tuf gene containing the RBS region of the NCgl1418 gene. At the same time, using pXM-P _NCgl1418 -lysE as a template, using primers tuf-pEC-F and tuf-pEC-R, a vector fragment containing the lysE gene is obtained by PCR amplification. The above two fragments are recovered and recombined to obtain the recombinant expression vector pEC-P _tuf -lysE.

In some specific embodiments, the present invention uses pXM-P _NCgl1418 -gfp as a template, uses primers 10P2-pEC-F and 10P2-pEC-R, obtains a vector fragment with a mutant promoter 10P2 and containing the lysE gene by PCR amplification, then uses T4 PNK to phosphorylate the vector fragment, and obtains pEC-P _10P2 -lysE by self-cyclization.

In some specific embodiments, the present invention uses pXM-P _NCgl1418 -gfp as a template, uses primers 10P17-pEC-F and 10P2-pEC-R, and obtains a vector fragment with a mutant promoter 10P17 and containing the lysE gene through PCR amplification, then phosphorylates the linearized vector fragment, and obtains pEC-P _10P17 -lysE through self-cyclization.

In other embodiments, the present disclosure can also construct the desired recombinant vector using the promoter sequences of 10P1, 10P3, 10P4, 10P5, 10P6, 10P7, 10P8, 10P9, 10P10, 10P11, 10P12, 10P13, 10P14, 10P15, 10P16, and 10P18 according to specific cloning needs.

In some embodiments, the SCgL30 strain of Corynebacterium glutamicum disclosed herein mutates the threonine at position 311 of aspartate kinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 to isoleucine, thereby constructing a strain SCgL30 having a certain lysine synthesis ability.

In some embodiments, the present disclosure transforms pEC-P _10P2 -lysE into SCgL30 recombinant strain to obtain a recombinant host cell. In some embodiments, the present disclosure transforms pEC-P _10P17 -lysE into SCgL30 recombinant strain to obtain a recombinant host cell. In other embodiments, the present disclosure can also transform the recombinant vector containing the promoter sequence of 10P1, 10P3, 10P4, 10P5, 10P6, 10P7, 10P8, 10P9, 10P10, 10P11, 10P12, 10P13, 10P14, 10P15, 10P16, 10P18 into SCgL30 recombinant strain to obtain a recombinant host cell.

Production process of target compound

(1) A polynucleotide having promoter activity is operably linked to a gene encoding a protein related to the synthesis of a target compound or a gene encoding a gene regulating a gene expression to obtain a recombinant expression vector capable of producing a protein related to the synthesis of a target compound or a gene regulating a gene expression, and a host cell is transformed with the recombinant expression vector to obtain a recombinant host cell.

(2) Fermenting the recombinant host cells, collecting the target compound from the recombinant host cells or the culture fluid of the recombinant host cells, and completing the production process of the target compound.

In the above production process, since the polynucleotide has improved promoter activity, in the recombinant host cell, the transcription activity of the gene encoding the protein related to the synthesis of the target compound or the gene expression regulatory protein is increased, and the expression level of the protein related to the synthesis of the target compound or the gene expression regulatory protein is increased, thereby significantly improving the yield of the target compound.

In some specific embodiments, no inducer is added in the steps of the method for preparing amino acids used in the present disclosure. In a specific embodiment, no IPTG is added in the steps of the method for preparing amino acids used in the present disclosure.

In some embodiments, the target compound is an amino acid, and the protein coding gene associated with the synthesis of the target compound refers to a protein coding gene associated with the synthesis of amino acids. In some embodiments, the target compound is an L-amino acid, and the protein coding gene associated with the synthesis of amino acids refers to a protein coding gene associated with the synthesis of L-amino acids. In some specific embodiments, the L-amino acid is L-lysine, and the protein associated with amino acid synthesis is the lysine transporter LysE. Increasing the expression of LysE with a polynucleotide having promoter activity can promote the extracellular emission and accumulation of lysine.

In some specific embodiments, the host cell is Corynebacterium glutamicum, which is an important strain for producing L-lysine. After Corynebacterium glutamicum is transformed with a polynucleotide, a transcription expression cassette or a recombinant expression vector having strong constitutive promoter activity, the expression level of proteins related to lysine synthesis in Corynebacterium glutamicum is significantly increased, thereby greatly improving the ability of Corynebacterium glutamicum to accumulate L-lysine by long-term fermentation.

In some specific embodiments, the host cell is a modified Corynebacterium glutamicum as follows: the threonine at position 311 of aspartate kinase (encoded by lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 is mutated to isoleucine.

In some specific embodiments, the culture conditions of the recombinant host cells are as follows: the recombinant host cells are inoculated with TSB medium containing corresponding antibiotics, cultured overnight at 30°C, 220r/min, and the fermentation medium is transferred according to the initial OD 0.3. The culture system is a 24-well plate with 1mL of liquid, and the fermentation is terminated after culture at 30°C, 800r/min for 24h, and the residual glucose content, OD ₆₀₀ and lysine yield are detected.

For the lysine fermentation medium, the formula is: glucose 80 g/L, yeast powder 8 g/L, urea 9 g/L, K ₂ HPO ₄ 1.5 g/L, MOPS 42 g/L, FeSO ₄ 0.01 g/L, MnSO ₄ 0.01 g/L, MgSO ₄ 0.6 g/L, chloramphenicol final concentration of 5 μg/mL, and/or kanamycin final concentration of 25 μg/mL.

In some specific embodiments, the target compound can be recovered from the recombinant host cell or the culture medium of the recombinant cell by methods commonly used in the art, including but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.

In the art, methods for manipulating microorganisms are known, as explained in publications such as Current Methods in Molecular Biology (Online ISBN: 9780471142720, John Wiley and Sons, Inc.), Microbial Metabolic Engineering: Methods and Procedures (Qiong Cheng Ed., Springer), and Systems Metabolic Engineering: Methods and Procedures (Hal S. Alper Ed., Springer).

Example

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples (although representing specific embodiments of the present disclosure) are given for illustrative purposes only, because after reading the detailed description, various changes and modifications made within the spirit and scope of the present disclosure will become apparent to those skilled in the art.

The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

The primer sequences used in the plasmid construction in the embodiment of Table 1 are as follows:

Example 1. Characterization of plasmid containing NCgl1418 gene promoter sequence

We first cultured the Corynebacterium glutamicum ATCC13032 strain using CGXII medium with or without 0.6M NaCl or lysine sulfate, and determined that the promoter of the NCgl1418 gene was a high-salt hyperosmotic inducible promoter through transcriptome sequencing analysis. In addition, by truncating the promoter at different lengths, we obtained NCgl1418 promoter fragments of 203bp (SEQ ID NO: 3), 145bp (SEQ ID NO: 4), and 94bp, respectively, and compared the strength of the NCgl1418 promoter of different lengths under high salt (addition of 0.6M sodium sulfate) and normal medium conditions. The results are shown in Figure 1. The data show that although the 94bp NCgl1418 promoter contains the core sequence (-35 region and -10 region), it has basically lost the normal function of the promoter; although the induction intensity of the 145bp promoter decreased under high salt conditions, it can still reach more than 74% of the activity of the 243bp promoter; the 203bp promoter basically maintains the activity of the 243bp promoter under high salt osmotic pressure conditions, which is 94% of the activity of the 243bp promoter; the above results show that the promoter activity of the NCgl1418 promoter and its activity under high salt osmotic pressure conditions need to include at least the 145bp DNA sequence shown in SEQ ID NO:3.

Primers 1418-F (SEQ ID NO: 24)/1418-R (SEQ ID NO: 25) were designed based on the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI (NC_003450.3). The promoter sequence of the NCgl1418 gene (SEQ ID NO: 2) was obtained by PCR amplification using the ATCC 13032 genome as a template. At the same time, the vector fragment with the lacI gene and tac promoter removed was obtained by PCR amplification using primers pGFP-F (SEQ ID NO: 26) and pGFP-R (SEQ ID NO: 27) using the pXM-gfp reported in the literature as a ^template [1]. After the two fragments were recovered, they were recombined and ligated using the Vazyme Clon Express Multies one-step recombination kit to obtain the recombinant vector pXM-P _NCgl1418 -gfp. At the same time, the vector fragment was phosphorylated by T4 PNK, and the control vector pXM-con was constructed by self-cyclization. The above recombinant vector was transformed into Corynebacterium glutamicum ATCC 13032 to obtain a recombinant strain.

Example 2. Construction of a control plasmid containing the tuf promoter sequence

At present, it is known that the commonly used strong constitutive promoter endogenous to Corynebacterium glutamicum is P _tuf , therefore, the present disclosure uses this promoter as a control to construct a plasmid to characterize the relative strength of the NCgl1418 mutant promoter.

According to the genome sequence (NC_003450.3) of Corynebacterium glutamicum ATCC 13032 published by NCBI, primers tuf-F (SEQ ID NO:28) and tuf-R (SEQ ID NO:29) were designed. The promoter sequence (SEQ ID NO:23) with tuf gene was obtained by PCR amplification using ATCC13032 genome as template. At the same time, using pXM-P _NCgl1418 -gfp as template, primers tuf-pGFP-F (SEQ ID NO:30) and tuf-pGFP-R (SEQ ID NO:31) were used to obtain a vector fragment containing the RBS region of NCgl1418 gene by PCR amplification. After the above fragments were recovered, they were recombined using the VazymeClon Express Multies recombination kit, and the ligation products were transformed into Trans T1 competent cells, coated with chloramphenicol resistance plates for overnight culture, positive clones were selected for colony PCR verification, and the correct transformants were sequenced and confirmed. The obtained recombinant vector was named pXM-P _tuf -gfp. The recombinant vector was transformed into Corynebacterium glutamicum ATCC13032 to obtain a recombinant strain.

Example 3. Construction of NCgl1418 gene promoter mutation library

Considering that the promoter -10 region may have an important regulatory effect on the strength and properties of the promoter, in this example, the -10 region of the core region of the NCgl1418 gene promoter (SEQ ID NO: 1) was randomly mutated, and the underlined parts are the main sequences of the promoter -35 region and -10 region:

TATTAAAGATCACACCGAGTG GTGGAA TTTCCTCAAGTGATTTAC CCACAATG GACTTTG;

The specific mutation sequence is:

TATTAAAGATCACACCGAGTG GTGGAA TTTCCTCAAGTGATTTAC NNNNNNNN GACTTTG.

pXM-P _NCgl1418 -gfp was amplified by reverse PCR using 1418mutant-F/R primers (SEQ ID NO: 38 and SEQ ID NO: 39), and the linearized plasmid fragments obtained were phosphorylated and ligated, and transformed into Escherichia coli T1 competent cells to obtain resistant clones. All clones were collected and plasmids were extracted to obtain a mutant library of the NCgl1418 gene promoter.

Example 4. Screening of NCgl1418 gene promoter mutation library and characterization of mutant promoters

The promoter mutant library was transformed into Corynebacterium glutamicum ATCC13032, and strains ATCC13032 (pXM-Con) and ATCC13032 (pXM-P _NCgl1418 -gfp) were used as empty vectors and wild-type controls. The strains were inoculated in TSB medium containing 5 μg/mL chloramphenicol, and cultured at 30°C and 220 r/min for 8 to 10 hours. Then, according to the initial OD1, they were transferred to CGXIIY medium supplemented with 0.6 M Na ₂ SO _4. The culture system was 24-well plates with 1 mL of liquid, and cultured at 30°C and 800 r/min for 6 hours. The resulting bacterial solution was diluted 50 times with PBS buffer, and then ultrasonically treated for 6 minutes, and then fluorescent sorting (top 0.01%) was performed by flow cytometry.

The strains obtained by the above sorting, the wild-type NCgl1418 promoter, the tuf promoter and the promoter-free control strain were inoculated into TSB medium containing 5 μg/mL chloramphenicol, and cultured overnight at 30°C and 220 r/min. The components of TSB liquid medium are (g/L): glucose, 5g/L; yeast powder, 5g/L; soy peptone, 9g/L; urea, 3g/L; succinic acid, 0.5g/L; K ₂ HPO ₄ ·3H ₂ O, 1g/L; MgSO ₄ ·7H ₂ O, 0.1g/L; biotin, 0.01mg/L; vitamin B1, 0.1mg/L; MOPS, 20g/L.

According to the initial OD 0.5, the cells were transferred to CGXIIY medium with or without 0.6 M Na ₂ SO _4. The culture system was 1 mL of liquid in a 24-well plate. The cells were cultured at 30°C and 800 r/min for 24 h. The GFP fluorescence intensity and OD ₆₀₀ of different strains were detected. The fluorescence intensity per unit cell (minus the fluorescence intensity per unit cell of the control strain under the same conditions) was used to characterize the relative strength of the mutant promoter under different conditions. The formula of CGXIIY medium is: glucose 50g/L, _NH4Cl 16.5g/L, urea 5g/L, _{KH2PO4 1g} /L, _{K2HPO4 1g} /L, MOPS 42g/L, MgSO4 0.25g/L, _FeSO4 · _7H2O 0.01g/L, _MnSO4 ·H2O0.01g/L, _ZnSO4 ·7H2O 0.001g/L, CuSO4 0.2mg/L, NiCl· _{6H2O 0.02mg} /L, CaCl2 _0.01g / _L , protocatechuic acid _0.03g /L, biotin 0.2mg/L, vitamin B1 0.1mg/L, and the final concentration of chloramphenicol is 5μg/ _mL . According to the fluorescence intensity obtained by detection, 18 strong constitutive mutant promoters were screened and obtained, the intensity of which was higher than that of the endogenous strong constitutive promoter P _tuf of Corynebacterium glutamicum, and was basically no longer induced by high salt and hyperosmotic conditions (Table 2).

Table 2

^a Fluorescence intensity of each promoter/fluorescence intensity of wild-type NCgl1418 gene promoter (without adding Na ₂ SO ₄ )

^b Fluorescence intensity with Na ₂ SO ₄ added/fluorescence intensity without Na ₂ SO ₄ added

Example 5. Application of mutant promoter to regulate LysE expression in lysine synthesis

According to the genome sequence of Corynebacterium glutamicum ATCC 13032 (NC_003450.3) published by NCBI, primers 1418-LF (SEQ ID NO: 32) and 1418-LR (SEQ ID NO: 33), lysE-F (SEQ ID NO: 34) and lysE-R (SEQ ID NO: 35) were designed respectively. The promoter sequence of the NCgl1418 gene and the DNA sequence of the lysE gene were obtained by PCR amplification using the ATCC 13032 genome as a template. At the same time, the vector fragment was obtained by PCR amplification using pEC-XK99E reported in the literature as a template ^[2] and primers pEC-F (SEQ ID NO: 36) and pEC-R (SEQ ID NO: 37). After the three fragments were recovered, they were recombined and ligated using the Vazyme Clon Express Multies one-step recombination kit, and the ligation products were transformed into Trans T1 competent cells and coated on kanamycin-resistant plates for overnight culture. Positive clones were selected for colony PCR verification, and the correct transformants were sequenced for confirmation. The obtained recombinant vector was named pEC-P _NCgl1418 -lysE.

Using pXM-P _tuf -gfp as a template, primers tuf-lysE-F (SEQ ID NO: 40) and tuf-lysE-R (SEQID NO: 41) were used to amplify the promoter sequence (SEQ ID NO: 47) of the tuf gene in the RBS region of the NCgl1418 gene. At the same time, using pXM-P _NCgl1418 -lysE as a template, primers tuf-pEC-F (SEQ ID NO: 42) and tuf-pEC-R (SEQID NO: 43) were used to obtain a vector fragment containing the lysE gene by PCR amplification. After the above two fragments were recovered, they were recombined and connected using the Vazyme Clon Express II recombination kit, and the connection products were transformed into Trans T1 competent cells, coated with kanamycin resistance plates for overnight culture, positive clones were selected for colony PCR verification, and the correct transformants were sequenced and confirmed, and the obtained recombinant vector was named pEC-P _tuf -lysE.

Using pXM-P _NCgl1418 -lysE as a template, primers 10P2-pEC-F (SEQ ID NO: 44), 10P17-pEC-F (SEQ ID NO: 45) and 10P2-pEC-R (SEQ ID NO: 46) were used to obtain a vector fragment containing mutant promoters 10P2 and 10P17 and lysE gene by PCR amplification. Then, the vector fragment was phosphorylated by T4 PNK, and pEC-P _10P2 -lysE and pEC-P _10P17 -lysE were obtained by self-cyclization.

The above recombinant vectors pEC-P _NCgl1418 -lysE, pEC-P _tuf -lysE, pEC-P _10P2 -lysE, pEC-P _10P17 -lysE and pEC-XK99E were transformed into Corynebacterium glutamicum ScgL30 to obtain recombinant strains and control strains. The above strains were inoculated with TSB medium containing 25 μg/mL kanamycin, cultured at 30°C, 220r/min overnight, and transferred to fermentation medium according to the initial OD 0.3. The culture system was 24-well plates with 1 mL of liquid, cultured at 30°C, 800r/min for 24 hours, and then the fermentation was terminated, and the residual glucose content, OD ₆₀₀ and lysine yield were detected. The formula of lysine fermentation medium is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, _{K2HPO4 1.5g} /L, MOPS 42g/L, FeSO4 _0.01g /L, _MnSO4 0.01g/L, MgSO4 0.6g/L, _and the final concentration of kanamycin is 25μg/mL. The test results are shown in Table 3. The data show that when P _tuf was used to overexpress LysE, the lysine yield and glucose conversion rate were increased by 31% and 32% respectively compared with the control strain, while when the wild-type NCgl1418 gene promoter was used to overexpress LysE, the lysine yield and glucose conversion rate were only increased by 16% and 15% due to the weak promoter strength under hypotonic conditions. When LysE was overexpressed using the strong constitutive promoters 10P2 and 10P17 obtained by mutation screening, the lysine yield was increased by 38% and 69% respectively, and the glucose conversion rate was increased by 45% and 88% respectively compared with the control strain, which were much higher than the wild-type NCgl1418 promoter and the endogenous strong promoter P _tuf , showing good application prospects.

Table 3 Effect of constitutive strong promoter on regulating LysE expression in lysine synthesis

In addition, FIG. 1 of the present disclosure proves that a promoter with a length of 145 bp can reach more than 74% of the activity of a promoter with a length of 243 bp under high salt osmotic pressure conditions; a promoter with a length of 203 bp basically maintains the activity of a promoter with a length of 243 bp under high salt osmotic pressure conditions, which is 94% of the activity of a promoter with a length of 243 bp; it is explained that since the promoter fragments of SEQ ID NO: 3 and SEQ ID NO: 4 contain the core region of the promoter of the NCgl1418 gene, the promoter fragments of SEQ ID NO: 3 and SEQ ID NO: 4 can also show significantly enhanced promoter activity under an environment with increased salt concentration and osmotic pressure. Therefore, by adopting the method for modifying the core region of the promoter in the above embodiment, i.e., mutating one or more positions in the 164-171 position of the sequence shown in SEQ ID NO: 3, or mutating one or more positions in the 106-113 position of the sequence shown in SEQ ID NO: 4, a strong constitutive promoter mutant can also be obtained.

References:

[1]Sun DH et al.,Journal of Industrial Microbiology&Biotechnology2019,46(2):203-208.

[2]O Kirchner, et al. Journal of Biotechnology, 2003, 104: 287-299.

All technical features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can also be replaced by other features with the same, equal or similar functions. Therefore, unless otherwise specified, each feature disclosed is only an example of a series of equal or similar features.

In addition, from the above description, those skilled in the art can easily understand the key features of the present disclosure from this disclosure, and many modifications can be made to the invention to adapt to various different purposes and conditions of use without departing from the spirit and scope of the present disclosure. Therefore, such modifications are also intended to fall within the scope of the appended claims.

Sequence Listing

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> Polynucleotides having promoter activity and their use in producing target compounds

<130> 6A17-2133227I

<160> 47

<170> SIPOSequenceListing 1.0

<210> 1

<211> 60

<212> DNA

<213> Corynebacterium glutamicum

<400> 1

tattaaagat cacaccgagt ggtggaattt cctcaagtga tttacccaca atggactttg 60

<210> 2

<211> 243

<212> DNA

<213> Corynebacterium glutamicum

<400> 2

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacccacaat ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 3

<211> 203

<212> DNA

<213> Corynebacterium glutamicum

<400> 3

gacacctgtg agtttcaaac tccccattat cgccttagtc aggcggtagt ggggagtttt 60

tgtttatgca ggtggcgcga ttcttagatt tcataagggt aacagatctg tttctatgta 120

ttaaagatca caccgagtgg tggaatttcc tcaagtgatt tacccacaat ggactttgtt 180

gatacccaat tcgagaaagg cca 203

<210> 4

<211> 145

<212> DNA

<213> Corynebacterium glutamicum

<400> 4

tttgtttatg caggtggcgc gattcttaga tttcataagg gtaacagatc tgtttctatg 60

tattaaagat cacaccgagt ggtggaattt cctcaagtga tttacccaca atggactttg 120

ttgataccca attcgagaaa ggcca 145

<210> 5

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P1

<400> 5

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacactgtag ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 6

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P2

<400> 6

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactattatg ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 7

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P3

<400> 7

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacaattggg ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 8

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P4

<400> 8

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactatggtt ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 9

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P5

<400> 9

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactagggta ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 10

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P6

<400> 10

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacaatggaa tgactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 11

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P7

<400> 11

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactagactt cgactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 12

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P8

<400> 12

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacaatgggt agactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 13

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P9

<400> 13

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactaccatt agactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 14

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P10

<400> 14

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacactgagg ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 15

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P11

<400> 15

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacactagaa ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 16

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P12

<400> 16

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacaattagt ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 17

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P13

<400> 17

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacaataggg tgactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 18

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P14

<400> 18

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactagtatt ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 19

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P15

<400> 19

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacactggac tgactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 20

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P16

<400> 20

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactaacatg ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 21

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P17

<400> 21

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tacactaggg ggactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 22

<211> 243

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P18

<400> 22

taaaactcgc gatgaagtag aaaaacaacg caacacttaa gacacctgtg agtttcaaac 60

tccccattat cgccttagtc aggcggtagt ggggagtttt tgtttatgca ggtggcgcga 120

ttcttagatt tcataagggt aacagatctg tttctatgta ttaaagatca caccgagtgg 180

tggaatttcc tcaagtgatt tactataagt tgactttgtt gatacccaat tcgagaaagg 240

cca 243

<210> 23

<211> 349

<212> DNA

<213> Artificial Sequence

<220>

<223> Ptuf

<400> 23

agatcgttta gatccgaagg aaaacgtcga aaagcaattt gcttttcgac gccccaccccc 60

gcgcgtttta gcgtgtcagt aggcgcgtag ggtaagtggg gtagcggctt gttagatatc 120

ttgaaatcgg ctttcaacag cattgatttc gatgtattta gctggccgtt accctgcgaa 180

tgtccacagg gtagctggta gtttgaaaat caacgccgtt gcccttagga ttcagtaact 240

ggcacatttt gtaatgcgct agatctgtgt gctcagtctt ccaggctgct tatcacagtg 300

aaagcaaaac caattcgtgg ctgcgaaagt cgtagccacc acgaagtcc 349

<210> 24

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418-F

<400> 24

cttttcacca gtgagacggg taaaactcgc gatgaagtag 40

<210> 25

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418-R

<400> 25

gttcttctcc tttactcatc attggccttt ctcgaattgg g 41

<210> 26

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> pGFP-F

<400> 26

atgagtaaag gagaagaact tttcac 26

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> pGFP-R

<400> 27

cccgtctcac tggtgaaaag 20

<210> 28

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-F

<400> 28

caccagtgag acgggagatc gtttagatcc gaaggaaaa 39

<210> 29

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-R

<400> 29

ctcgaattgg gtatcaacgg acttcgtggt ggctacga 38

<210> 30

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-pGFP-F

<400> 30

gttgataccc aattcgagaa agg 23

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-pGFP-R

<400> 31

cccgtctcac tggtgaaaag 20

<210> 32

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418-L-F

<400> 32

agcggcatgc atttacgttt aaaactcgcg atgaagtag 39

<210> 33

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418-L-R

<400> 33

agatttccat gatcaccatc attggccttt ctcgaattgg g 41

<210> 34

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> lysE-F

<400> 34

atggtgatca tggaaatctt cattac 26

<210> 35

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> lysE-R

<400> 35

gtctgtttcc tgtgtgaaac taacccatca acatcagttt gatg 44

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> pEC-F

<400> 36

tttcacacag gaaacagacc atg 23

<210> 37

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> pEC-R

<400> 37

aacgtaaatg catgccgctt c 21

<210> 38

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418mutant-F

<400> 38

nnnnnnnnga ctttgttgat acccaattcg ag 32

<210> 39

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> 1418mutant-R

<400> 39

gtaaatcact tgaggaaatt ccacc 25

<210> 40

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-lysE-F

<400> 40

agcggcatgc atttacgtta gatcgtttag atccgaagga aaacg 45

<210> 41

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-lysE-R

<400> 41

agatttccat gatcaccatc attggccttt ctcgaattgg g 41

<210> 42

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-pEC-F

<400> 42

atggtgatca tggaaatctt cattac 26

<210> 43

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> tuf-pEC-R

<400> 43

aacgtaaatg catgccgctt c 21

<210> 44

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P2-pEC-F

<400> 44

tattatggga ctttgttgat acccaattcg ag 32

<210> 45

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P17-pEC-F

<400> 45

actaggggga ctttgttgat acccaattcg ag 32

<210> 46

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> 10P2-pEC-R

<400> 46

gtaaatcact tgaggaaatt ccacc 25

<210> 47

<211> 375

<212> DNA

<213> Artificial Sequence

<220>

<223> Promoter sequence of tuf gene containing RBS region

<400> 47

agatcgttta gatccgaagg aaaacgtcga aaagcaattt gcttttcgac gccccaccccc 60

gcgcgtttta gcgtgtcagt aggcgcgtag ggtaagtggg gtagcggctt gttagatatc 120

ttgaaatcgg ctttcaacag cattgatttc gatgtattta gctggccgtt accctgcgaa 180

tgtccacagg gtagctggta gtttgaaaat caacgccgtt gcccttagga ttcagtaact 240

ggcacatttt gtaatgcgct agatctgtgt gctcagtctt ccaggctgct tatcacagtg 300

aaagcaaaac caattcgtgg ctgcgaaagt cgtagccacc acgaagtccg ttgataccca 360

attcgagaaa ggcca 375

Claims (21)

1. A polynucleotide having promoter activity, wherein the nucleotide sequence of the polynucleotide is selected from the group consisting of SEQ ID NOs: any one of 5 to 22 the sequence shown.

2. A transcriptional expression cassette, wherein the transcriptional expression cassette comprises the polynucleotide having promoter activity of claim 1.

3. The transcriptional expression cassette of claim 2, wherein the transcriptional expression cassette further comprises a gene of interest operably linked to the polynucleotide having promoter activity.

4. The transcriptional expression cassette of claim 3, wherein the gene of interest is a protein-encoding gene.

5. A recombinant expression vector, wherein the recombinant expression vector comprises the polynucleotide of claim 1 having promoter activity, or the transcriptional expression cassette of any one of claims 2-4.

6. A recombinant host cell, wherein the recombinant host cell comprises the transcriptional expression cassette of any one of claims 2-4, or the recombinant expression vector of claim 5; the host cell is Corynebacterium glutamicum.

7. The recombinant host cell of claim 6, wherein the host cell is corynebacterium glutamicum ATCC 13032, corynebacterium glutamicum ATCC 13869, or corynebacterium glutamicum ATCC 14067.

8. Use of a polynucleotide having promoter activity according to claim 1, a transcriptional expression cassette according to any one of claims 2-4, a recombinant expression vector according to claim 5, a recombinant host cell according to claim 6 or 7 in at least one of:

(a) Regulating the transcription level of the gene, or preparing a reagent or kit for regulating the transcription level of the gene;

(b) Preparing a protein, or preparing a reagent or kit for preparing a protein;

(c) Producing the compound of interest, or preparing a reagent or kit for producing the compound of interest.

9. The use according to claim 8, wherein the protein is selected from a gene expression regulatory protein or a protein associated with synthesis of a compound of interest.

10. The use according to claim 8 or 9, wherein the target compound comprises at least one of an amino acid, an organic acid.

11. The use of claim 10, wherein the amino acid comprises at least one of proline, lysine, glutamic acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and the organic acid comprises at least one of citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, malic acid.

12. A method of regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide having promoter activity of claim 1 to the target gene.

13. A method of producing a protein, wherein the method comprises the step of expressing the protein using the transcriptional expression cassette of any one of claims 2-4, the recombinant expression vector of claim 5, or the recombinant host cell of claim 6 or 7.

14. The method according to claim 13, wherein the protein is a protein involved in synthesis of a compound of interest or a gene expression regulatory protein.

15. The method of claim 13, wherein the method further comprises the step of isolating or purifying the protein.

16. A method for producing a compound of interest, wherein the method comprises the step of producing the compound of interest in the presence of a protein involved in synthesis of the compound of interest or a gene expression regulatory protein, using the transcriptional expression cassette of any one of claims 2 to 4, the recombinant expression vector of claim 5, or the recombinant host cell of claim 6 or 7.

17. The method of claim 16, wherein the target compound comprises at least one of an amino acid, an organic acid.

18. The method of claim 17, wherein the amino acid comprises at least one of lysine, glutamic acid, threonine, proline, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan, and the organic acid comprises at least one of citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, malic acid.

19. The method of claim 16, wherein the protein associated with synthesis of the compound of interest is a protein associated with synthesis of L-amino acids.

20. The method of claim 19, wherein the protein associated with L-amino acid synthesis comprises one or more of pyruvate carboxylase, phosphoenolpyruvate carboxylase, gamma-glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transporter, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconic acid repressor, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydropyridine dicarboxylic acid synthase, dihydropyridine formate reductase, succinyldiaminopimelate aminotransferase, tetrahydropyridine dicarboxylate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, diaminopimelate dehydrogenase, and pyruvate carboxylase.

21. The method of claim 16, wherein the method further comprises the step of isolating or purifying the compound of interest.