CN113278620A - Mutant hypertonic inducible promoter Pprox and application thereof - Google Patents

Abstract

The present invention provides a polynucleotide mutant having promoter activity, which has enhanced promoter activity in an environment of increased salt concentration and osmotic pressure compared to a wild-type promoter. The polynucleotide is operably connected with the target gene, so that the expression strength of the target gene in a high-salt and high-osmotic-pressure stress environment can be remarkably improved, a downstream product can be stably and efficiently produced, and the problems that high inducers such as IPTG (isopropyl-beta-thiogalactoside) and the like are added at present and toxicity is caused to a strain are effectively solved.

Description

Mutant hypertonic inducible promoter Pprox and application thereof

Technical Field

The invention belongs to the field of genetic engineering and molecular biology, and particularly relates to a polynucleotide mutant with promoter activity, a transcription expression cassette containing a promoter, a recombinant expression vector, a recombinant host cell, a method for regulating and controlling the transcription of a target gene, and a method for producing a target compound.

Background

Promoters are important means for regulating the expression of target genes and can be divided into constitutive promoters and inducible promoters. Constitutive promoters are generally not affected by external factors and express the target gene at a substantially constant level, as a series of constitutive promoters have been reported in the literature (Rytter, J. V., et al., Synthetic promoter libraries for)Corynebacterium glutamicumAppl. Microbiol. Biotechnol., 2014, 98, 2617-2623.). However, for the synthesis of some toxic proteins or metabolites, the expression of constitutive promoters often causes a large metabolic burden to the strain, and has a large limitation in practical application.

Inducible promoters are promoters that can substantially increase the transcription level of a target gene when stimulated by certain physical or chemical signals. Such promoters may control the timing of transcription initiation and thus may be more favorable for metabolic flux regulation and redistribution by the strain. Inducible promoters widely used in the biological field include tac, trc, etc., however, the promoters need to be additionally added with expensive inducers, such as IPTG, and the addition of the inducers also causes certain toxicity to the strains or causes great interference to the fermentation system.

During the fermentation of industrial strains, the high salt hyperosmotic conditions are almost all environmental inducers that industrial strains will face in the late stages of fermentation (Varela C et al, Metabolic flux redistribution in)Corynebacterium glutamicumin response to osmotic stress, appl. Microbiol. Biotechnol., 2003, 60 (5): 547-555), however, there are currently few reports of high-salt hyperosmotic inducible promoters in industrial strains, especially C.glutamicum. Therefore, high-activity high salts have been developedThe hypertonic inducible promoter not only can provide a universal self-inducible element for the development of industrial strains, but also can increase the expression of genes related to a target compound at the later stage of industrial fermentation, thereby increasing the synthesis of the target compound, and is a problem to be solved by genetic modification of the current industrial strains.

Disclosure of Invention

In view of the technical problems of the prior art, the present invention first identifies a hypertonic inducible promoter P_proPAnd obtaining two mutant polynucleotides with improved promoter activity by mutating 5' -UTR and promoter core region thereof, wherein the mutant polynucleotides show higher salt concentration and osmotic pressure than wild type P under the environment with higher salt concentration and osmotic pressure_proPEnhanced promoter activity of the promoter. The promoter mutant polynucleotide is operably connected with a protein coding gene related to amino acid synthesis or a coding gene of a gene expression regulatory protein, so that the high-efficiency expression of the protein coding gene in a high-salt and high-osmotic-pressure environment can be realized, and the problems that an expensive inducer needs to be added to the current inducible promoter and the inducer causes toxicity to a strain are effectively solved.

Accordingly, the present invention provides a polynucleotide having promoter activity, wherein the polynucleotide is selected from any one of the group consisting of:

(i) comprises the amino acid sequence shown as SEQ ID NO: 2-3, or a nucleotide sequence shown in any sequence;

(ii) comprises the amino acid sequence shown as SEQ ID NO: 2-3, the reverse complement of the nucleotide sequence shown in any sequence;

(iii) (iii) a reverse complement of a sequence that is capable of hybridizing to the nucleotide sequence set forth in (i) or (ii) under high stringency hybridization conditions or very high stringency hybridization conditions;

(iv) (iii) a sequence having at least 80%, optionally at least 90%, preferably at least 95%, more preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity to the nucleotide sequence set forth in (i) or (ii).

Wherein said polynucleotide having promoter activity has increased promoter activity relative to a wild-type promoter in an environment of increased salt concentration or osmotic pressure.

In a second aspect, the present invention provides a transcription expression cassette comprising said polynucleotide having promoter activity; optionally, the transcription expression cassette further contains a gene encoding a protein or a gene encoding a gene expression regulatory protein, which is operably linked to the polynucleotide having promoter activity.

In a third aspect, the present invention provides a recombinant expression vector comprising said polynucleotide having promoter activity, or said transcriptional expression cassette.

In a fourth aspect, the present invention provides a recombinant host cell, wherein said recombinant host cell comprises said transcription expression cassette, or said recombinant expression vector.

Preferably, 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 ATCC13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067 or a derivative strain of Corynebacterium glutamicum.

In a fifth aspect, the invention provides the use of a polynucleotide having said promoter activity in at least one of:

(a) regulating the transcription level of a gene, or preparing a reagent or a 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 compound of interest, or preparing a reagent or kit for producing a compound of interest. Preferably, the promoter is operably linked to a gene encoding a gene expressing a regulatory protein, a gene encoding a protein involved in the synthesis of a target compound, or a gene encoding a protein involved in membrane transport. In particular embodiments, the target compounds are amino acids and organic acids. Preferably, the amino acid is one or a combination of two or more of the following: proline, hydroxyproline, lysine, glutamic acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, or a derivative of any of the foregoing amino acids;

preferably, the organic acid is one or a combination of two or more of the following: citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, propionic acid, hexenoic acid, decanoic acid, octanoic acid, pentanoic acid, malic acid or derivatives of any of the above organic acids.

In a sixth aspect, the present invention provides a method for regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide having promoter activity to a target RNA or a target gene; optionally, the target RNA is at least one of tRNA, sRNA, and the target gene is at least one of a gene encoding a protein involved in synthesis of a target compound, a gene encoding a gene expression regulatory protein, and a gene encoding a protein involved in membrane transport;

optionally, the target gene is at least one of: pyruvate carboxylase gene, phosphoenolpyruvate carboxylase gene, gamma-glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene, pyrroline-5-carboxylate reductase gene, amino acid transporter gene, ptsG system-related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, glucorepressor gene, glucose dehydrogenase gene, aspartokinase gene, aspartate semialdehyde dehydrogenase gene, aspartate ammonia lyase gene, dihydrodipicolinate synthase gene, dihydropicolinate reductase gene, succinyldiaminopimelate aminotransferase gene, tetrahydropyridinedicarboxylate succinylase gene, succinyldiaminopimelate deacylase gene, diaminopimelate epimerase gene, diaminopimelate deacylase gene, gamma-glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene, pyrroline-5-carboxylate reductase gene, amino acid transporter gene, ptsG system-related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, glucorepressor gene, glucose dehydrogenase gene, aspartokinase gene, dihydrodipicolinate reductase gene, dihydropicolinate aminotransferase gene, dihydropicolinate reductase gene, succinyldiaminopimelate epimerase gene, diaminopimelate gene, dihydrodipicolinate gene, dihydropicolinate deacylase gene, beta-type reductase gene, beta-glucosidase gene, beta-glucosidase, beta-glucosidase, beta-glucosidase, beta-glucosidase, beta-glucosidase, and the like, Glyceraldehyde-3-phosphate dehydrogenase gene, transketolase gene, diaminopimelate dehydrogenase gene.

In a seventh aspect, the present invention provides a method for producing a protein, comprising the step of expressing said protein using said transcription expression cassette, said recombinant expression vector, or said recombinant host cell; optionally, the protein is a protein associated with synthesis of a target compound, a protein associated with membrane transport, or a gene expression regulatory protein; optionally, the method further comprises the step of isolating or purifying the protein.

In an eighth aspect, the present invention provides a method for producing a target compound, comprising the step of expressing a protein involved in synthesis of a target compound, a protein involved in membrane transport or a gene expression regulatory protein using the transcription expression cassette, the recombinant expression vector or the recombinant host cell, and producing a target compound in the presence of the protein involved in synthesis of a target compound, the protein involved in membrane transport or the gene expression regulatory protein; optionally, the target compound comprises at least one of an amino acid and an organic acid;

optionally, the amino acid is one or a combination of two or more of: proline, hydroxyproline, lysine, glutamic acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, or a derivative of any of the foregoing amino acids;

optionally, the organic acid is one or a combination of two or more of the following: citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, propionic acid, hexenoic acid, decanoic acid, octanoic acid, pentanoic acid, malic acid or derivatives of any of the above organic acids.

Optionally, the protein involved in the synthesis of the target compound is a protein involved in the synthesis of an L-amino acid; optionally, the protein involved in L-amino acid synthesis is 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, glucorepressor, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydropyridinedicarboxylate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, glucuronyl-glutamate decarboxylase, glucuronyl-glutamate dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, dihydropicolinate reductase, glucuronyl-3-phosphate dehydrogenase, glucuronyl-pyruvate-decarboxylase, glucuronyl-dehydrogenase, glucuronyl-3-phosphate dehydrogenase, glucuronyl-phosphate-dehydrogenase, glucuronyl-phosphate-reductase, glucuronyl-phosphate-dehydrogenase, dihydropicolinate synthase, dihydropicolinate-reductase, dihydropicolinate, and a salt, a salt, One or more of transketolase and diaminopimelate dehydrogenase;

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

Finally, the invention also provides the polynucleotide with promoter activity, the transcription expression cassette, the recombinant expression vector and the application of the recombinant host cell in at least one of the following methods:

(a) regulating the transcription level of a gene, or preparing a reagent or a 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 compound of interest, or preparing a reagent or kit for producing a compound of interest.

The present invention provides a polynucleotide mutant having promoter activity, which has enhanced promoter activity in an environment of increased salt concentration and osmotic pressure compared to a wild-type promoter. The polynucleotide is operably connected with the target gene, so that the expression strength of the target gene in a high-salt and high-osmotic-pressure stress environment can be remarkably improved, a downstream product can be stably and efficiently produced, and the problems that high inducers such as IPTG (isopropyl-beta-thiogalactoside) and the like are added at present and toxicity is caused to a strain are effectively solved. For example, the expression of the protein related to amino acid synthesis in a stress environment can be improved by producing the amino acid by using the polynucleotide with promoter activity, and the expression of other pathway proteins is weakened, so that metabolic flow is enriched towards the direction of amino acid synthesis more, the amino acid is stably and efficiently produced, and the aim of excessive accumulation of the amino acid is fulfilled. Specifically, for example, when L-lysine is produced by the above-mentioned method, L-lysine can be produced stably and efficiently in a high-salt and high-osmotic pressure environment.

Drawings

FIG. 1 shows a schematic view of aproPGene promoter and mutant promoter strength and hyperosmotic induction activity.

Detailed Description

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification 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 specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the summary supports the definition of the term "or" as being merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless it is explicitly stated that only alternatives or mutual exclusions between alternatives are mutually exclusive.

When used in the claims or specification, the term "range of values" is selected/preferred to include both the end points of the range and all natural numbers subsumed within the middle of the end points of the range with respect to the aforementioned end points of values.

The 'high-salt environment' of the invention can be high-concentration Na in the culture medium₂SO₄、NaCl、K₂SO₄Inorganic salt ions such as KCl, or the like, or the concentration thereof is increased by accumulation of a product such as lysine or some intermediate metabolite in the fermentation liquid (e.g., lysine sulfate or the like) or by substrate feeding (e.g., ammonium sulfate or the like)Substrate) or any other salt concentration that may be present in the fermentation broth. In some specific embodiments, a "high salt environment" relates to a salt concentration above 0.2M; in some more specific embodiments, a "high salt environment" relates to a salt concentration in the range of 0.2 to 0.8M. For example, the salt concentration is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M.

In the present invention, "high osmotic pressure environment" refers to an increased osmotic pressure in response to an increased salt concentration. In some preferred embodiments, the nucleic acid sequence of SEQ ID NO: 1-2 shows higher promoter activity or higher transformation efficiency under a high-salt environment formed by sulfate; in some preferred embodiments, the sulfate salt is Na₂SO₄Or K₂SO₄。

The term "polynucleotide" in the present invention refers to a polymer composed of nucleotides. Polynucleotides may be in the form of individual fragments, or may be a component of a larger nucleotide sequence structure, derived from nucleotide sequences that have been isolated at least once in quantity or concentration, and which are capable of being recognized, manipulated, and recovered in sequence, and their component nucleotide sequences, by standard molecular biology methods (e.g., using cloning vectors). 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, a "polynucleotide" refers to a polymer of nucleotides removed from other nucleotides (either individual fragments or whole fragments), or may be an integral part or component of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences.

The terms "sequence identity" and "percent identity" in the context of the present invention refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions in the aligned polynucleotides or polypeptides containing the same nucleotide or amino acid residue is scored and compared to the number of positions in the aligned polynucleotides or polypeptides containing different nucleotide or amino acid residues. Polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or deleted nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or both polynucleotides). Polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or deleting amino acids (i.e., amino acid insertions or amino acid deletions in one or both 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 same 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 specific embodiments, the polynucleotide having promoter activity comprises a nucleotide sequence identical to SEQ ID NO: 2-3, and the polynucleotide retains the high-salt and high-osmotic-pressure-inducible promoter activity.

In some embodiments, the polynucleotide having promoter activity comprises a sequence that hybridizes under high stringency hybridization conditions or very high stringency hybridization conditions to SEQ ID NO: 2-3 or the reverse complement thereof, and the polynucleotide retains the high-salt and high-osmotic-pressure-inducible promoter activity.

In some specific embodiments, the polynucleotide having promoter activity comprises 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%, 100% sequence identity to the nucleotide sequence of any of the above, and the polynucleotide retains high-salt, high-osmolarity-inducible promoter activity.

As used herein, the term "high stringency conditions" means prehybridization and hybridization at 42 ℃ in 5 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 for probes of at least 100 nucleotides in length, for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 65 ℃.

As used herein, the term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 ℃ in 5 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 for 12 to 24 hours. Finally, the carrier material is washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 70 ℃.

The term "complementary" herein refers to hybridization or base pairing between nucleotides or nucleotides, e.g., between 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, etc.

The term "promoter" in the present invention refers to a nucleic acid molecule, which is usually located upstream of the coding sequence of a target gene, provides a recognition site for RNA polymerase, and is located upstream in the 5' direction of the transcription initiation site of mRNA. 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 ribonucleic acid (RNA) synthesis, a promoter may interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription), including the core promoter region and regulatory regions, like a "switch," which determines the activity of the gene and, in turn, which protein the cell begins to produce.

The term "promoter core region" in the present invention refers to a nucleic acid sequence located in a promoter region of a prokaryote, which is a core sequence region functioning as a promoter and mainly includes a region between-35 region, -10 region, -35 region and-10 region and a transcription initiation site, -35 region is a recognition site for RNA polymerase, and-10 region is a binding site for RNA polymerase.

In some embodiments, the polynucleotides of the invention having promoter activity can be used to initiate expression of a gene encoding a protein. In other embodiments, the polynucleotides of the invention having promoter activity can be used to initiate expression of a non-coding gene.

The term "expression" in the present invention includes any step involving RNA production and protein production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The term "target RNA" of the present invention includes functional RNA that plays a role in genetic coding, translation, regulation, gene expression, and the like. In the present disclosure, the target RNA linked to the polynucleotide having promoter activity may be any functional RNA in the art. In some embodiments, the target RNA is tRNA or sRNA. In other embodiments, the target RNA may also be sgRNA, crRNA, tracrRNA, miRNA, siRNA, or other species of RNA.

The term "target gene" in the present invention relates to any gene linked to a polynucleotide having promoter activity in the present invention to regulate the transcription level thereof. In some embodiments, the target gene is a gene encoding a protein involved in the synthesis of the target compound. In some embodiments, the target gene is a gene encoding a gene expression regulatory protein. In some embodiments, the target gene is a gene encoding a protein associated with membrane transport. Illustratively, the target gene is a gene encoding an enzyme involved in biosynthesis of the target compound, a gene encoding an enzyme involved in reducing power, a gene encoding an enzyme involved in glycolysis or TCA cycle, or a gene encoding an enzyme involved in release of the target compound, or the like. Illustratively, the target gene includes at least one of the following genes: pyruvate carboxylase gene, phosphoenolpyruvate carboxylase gene, gamma-glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene, pyrroline-5-carboxylate reductase gene, amino acid transporter gene, ptsG system-related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, glucorepressor gene, glucose dehydrogenase gene, aspartokinase gene, aspartate semialdehyde dehydrogenase gene, aspartate ammonia lyase gene, dihydrodipicolinate synthase gene, dihydropicolinate reductase gene, succinyldiaminopimelate aminotransferase gene, tetrahydropyridinedicarboxylate succinylase gene, succinyldiaminopimelate deacylase gene, diaminopimelate epimerase gene, diaminopimelate deacylase gene, gamma-glutamyl kinase gene, glutamate semialdehyde dehydrogenase gene, pyrroline-5-carboxylate reductase gene, amino acid transporter gene, ptsG system-related gene, pyruvate dehydrogenase gene, homoserine dehydrogenase gene, oxaloacetate decarboxylase gene, glucorepressor gene, glucose dehydrogenase gene, aspartokinase gene, dihydrodipicolinate reductase gene, dihydropicolinate aminotransferase gene, dihydropicolinate reductase gene, succinyldiaminopimelate epimerase gene, diaminopimelate gene, dihydrodipicolinate gene, dihydropicolinate deacylase gene, beta-type reductase gene, beta-glucosidase gene, beta-glucosidase, beta-glucosidase, beta-glucosidase, beta-glucosidase, beta-glucosidase, and the like, Glyceraldehyde-3-phosphate dehydrogenase gene, transketolase gene, diaminopimelate dehydrogenase gene.

The term "target compound" in the present invention may be selected from at least one of amino acids and organic acids, and may be selected from other kinds of compounds that may be biosynthetically derived in the art.

In some embodiments, the compound of interest is an "amino acid" or an "L-amino acid". "amino acid" or "L-amino acid" generally refers to the basic building block of a protein in which an amino group and a carboxyl group are bound to the same carbon atom. Illustratively, 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, proline, or other types of amino acids known in the art.

In some embodiments, the target compound is an organic acid. The organic acid may be an organic compound having acidity, for example, those including a carboxyl group and a sulfonic acid group. Exemplary organic acids include 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, decanoic acid, octanoic acid, valeric acid, citric acid, or other types of organic acids known in the art.

The term "protein-encoding gene" in the present invention refers to a synthetic DNA molecule capable of directing a protein through a certain rule, and the process of directing protein synthesis by a protein-encoding gene generally includes a transcription process using double-stranded DNA as a template and a translation process using mRNA as a template. The protein-encoding gene contains a CDS Sequence (Coding Sequence) that directs the production of mRNA encoding the protein.

Illustratively, protein-encoding genes include, but are not limited to, genes encoding proteins involved in the synthesis of the target compound, and in some embodiments, genes encoding proteins involved in the synthesis of L-lysine. For proteins involved in the synthesis of L-lysine, one or a combination of two or more of aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate reductase, succinyl diaminopimelate aminotransferase, tetrahydropyridinedicarboxylate succinylase, succinyl diaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase is included. In some embodiments, the protein-encoding gene is directed to encoding a protein associated with synthetic organic acids, illustratively, the protein-encoding gene is directed to encoding a protein associated with synthetic citric acid, or is directed to encoding a protein associated with synthetic succinic acid. The polynucleotide with promoter activity can be suitable for improving the expression of target genes in the high-salt and high-osmotic-pressure stress environment and realizing the high-efficiency production of target products.

The term "gene expression regulatory protein" of the present invention includes, but is not limited to, exogenous gene expression regulatory tool proteins, such as dCas9 protein, dCpf1 protein, Hfq protein required for sRNA regulation, and the like, which are required for CRISPRi regulation, and endogenous or exogenous transcriptional regulatory factors, which in turn regulate the expression of key genes in metabolic pathways.

The term "transcription expression cassette" in the present invention refers to an expression element comprising a transcription regulatory element and a target gene, wherein the transcription regulatory element regulates the expression of the target gene. In the present invention, the transcription regulatory element includes a promoter, and may further include an enhancer, a silencer, an insulator, and the like. In the present invention, the target gene is specifically a protein-encoding gene. A gene of interest is "operably linked" to a polynucleotide, meaning that a polynucleotide having promoter activity is functionally linked to the gene of interest to initiate and mediate transcription of the gene of interest, in any manner described by one skilled in the art.

The term "vector" in the present invention refers to a DNA construct comprising a DNA sequence operably linked to suitable control sequences for the expression of a gene of interest in a suitable host. "recombinant expression vector" refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide. Recombinant expression vectors can include, for example, a collection comprising i) genetic elements that have a regulatory effect on gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into protein; and iii) transcriptional subunits of appropriate transcriptional and translational initiation and termination sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not critical and any vector may be used, including plasmids, viruses, phages and transposons. Possible vectors for use in the present invention 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 combinations of plasmids and phage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40 and pseudorabies.

The term "host cell" in the present invention means any cell type that is easily transformed, transfected, transduced, or the like with a transcription initiation element or expression vector comprising a polynucleotide of the present invention. The term "recombinant host cell" encompasses host cells which differ from the parent cell after introduction of a transcription initiation element or a recombinant expression vector, which is effected in particular by transformation.

The term "transformation" in the present invention has a meaning generally understood by those skilled in the art, i.e., a process of introducing exogenous DNA into a host. The method of transformation includes any method of introducing nucleic acid into a cell including, but not limited to, electroporation, calcium phosphate (CaPO)₄) Precipitation method, calcium chloride (CaCl)₂) Precipitation, microinjection, polyethylene glycol (PEG), DEAE-dextran, cationic liposome, and lithium acetate-DMSO.

The host cell of the present invention may be a prokaryotic cell or a eukaryotic cell, as long as the host cell can be introduced with the polynucleotide having promoter activity of the present invention. In one embodiment, the host cell refers to a prokaryotic cell, in particular, the host cell is derived from a microorganism suitable for the fermentative production of amino acids, such as Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia. Preferably, the host cell is Corynebacterium glutamicum derived from the genus Corynebacterium. Among them, the Corynebacterium glutamicum may be Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 13869, Corynebacterium glutamicum ATCC 14067, etc., and a mutant strain producing an amino acid, particularly lysine, or a derivative strain of Corynebacterium glutamicum prepared from the above strains. In some embodiments, the host cell of the present invention may be any type of strain having an amino acid-producing ability, including wild-type strains and recombinant strains.

Illustratively, the host cell is a lysine-producing host cell. In some embodiments, for lysine-producing host cells, can be in the Corynebacterium glutamicum ATCC13032 based on the expression of feedback inhibition of aspartate kinase strain. In addition, lysine-producing host cells may also be other kinds of strains having lysine-producing ability.

In some embodiments, one or more genes selected from the group consisting of:

a. encoding alcohol dehydrogenaseadhEA gene;

b. encoding acetate kinaseackAA gene;

c. encoding a phosphate acetyltransferaseptaA gene;

d. encoding lactate dehydrogenaseldhAA gene;

e. encoding formate transportersfocAA gene;

f. encoding pyruvate formate lyasepflBA gene;

g. process for coding pyruvate oxidasepoxBA gene;

h. encoding an aspartokinase I/homoserine dehydrogenase I bifunctional enzymethrAA gene;

i. encoding homoserine kinasethrBA gene;

j. encoding lysine decarboxylaseldcCA gene; and

h. encoding lysine decarboxylasecadAA gene.

In some embodiments, one or more genes selected from the group consisting of:

a. encoding dihydrodipyridine synthetase for relieving lysine feedback inhibitiondapAA gene;

b. encoding dihydrodipicolinate reductasedapBA gene;

c. encoding diaminopimelate dehydrogenaseddhA gene;

d. encoding tetrahydropyriddicarboxylic acid succinylasesdapDAnd encoding succinyldiaminopimelate deacylasedapE；

e. Encoding aspartate-semialdehyde dehydrogenaseasdA gene;

f. encoding phosphoenolpyruvate carboxylaseppcA gene;

g. encoding nicotinamide adenine dinucleotide transhydrogenasepntABA gene;

i. transport proteins encoding lysinelysEA gene.

Illustratively, the host cell is a threonine producing host cell. In some embodiments, the threonine producing host cell is a strain that expresses the feedback-released aspartate kinase LysC on the basis of corynebacterium glutamicum ATCC 13032. In other embodiments, the threonine-producing host cell can also be other species of strain that have threonine-producing ability.

In some embodiments, one or more genes selected from the group consisting of:

a. encoding the threonine operonthrABCA gene;

b. encoding feedback inhibition-relieved homoserine dehydrogenasehomA gene;

c. encoding glyceraldehyde-3-phosphate dehydrogenasegapA gene;

d. encoding pyruvate carboxylasepycA gene;

e. encoding malic acid: process for preparing quinone oxidoreductasesmqoA gene;

f. encoding transketolasetktA gene;

g. encoding 6-phosphogluconate dehydrogenasegndA gene;

h. encoding threonine exportthrEA gene;

i. encoding enolaseenoA gene.

Illustratively, the host cell is a host cell that produces isoleucine. In some embodiments, the host cell that produces isoleucine is produced by substituting alanine for L-threonine dehydrataseilvAA strain in which the amino acid at position 323 of the gene is used to produce L-isoleucine. In other embodiments, the host cell producing isoleucine may also be another species of strain having isoleucine-producing ability.

Illustratively, the host cell is a host cell producing O-acetylhomoserine. In some embodiments, the host cell producing O-acetylhomoserine is a strain that produces O-acetylhomoserine by inactivation of O-acetylhomoserine (thiol) -lyase. In other embodiments, the host cell producing O-acetylhomoserine can also be other kinds of strains having O-acetylhomoserine producing ability.

Illustratively, the host cell is a host cell that produces methionine. In some embodiments, the methionine producing host cell is a strain that produces methionine by inactivating the transcriptional regulators of methionine and cysteine. In other embodiments, the methionine-producing host cell may be a strain of another species having methionine-producing ability.

The cultivation of the host cell of the present invention may be performed according to a conventional method in the art, including, but not limited to, a well plate culture, a shake flask culture, a batch culture, a continuous culture, a fed-batch culture, and the like, and various culture conditions such as temperature, time, pH of a medium, and the like may be appropriately adjusted according to actual circumstances.

In some specific embodiments, the recombinant host cell is cultured under conditions selected from the group consisting of: inoculating recombinant host cells into a TSB culture medium containing corresponding antibiotics, carrying out overnight culture at 30 ℃ at 220 r/min, respectively switching over lysine fermentation culture media with or without 0.6M sodium sulfate (simulating a high-salt hyperosmotic environment caused by accumulation of high-concentration products at the later stage of fermentation) according to the initial OD 0.3, wherein the culture system is a 24-pore plate containing 1 mL of liquid, carrying out culture at 30 ℃ at 800 r/min for 24 h, terminating fermentation, detecting the content and OD of residual glucose, and detecting the content and OD₆₀₀And lysine production.

For the lysine fermentation medium, the formula is as follows: 80 g/L glucose, 8 g/L yeast powder and 9 g/L, K urea₂HPO₄1.5 g/L、MOPS 42 g/L、FeSO₄ 0.01 g/L、MnSO₄ 0.01 g/L、MgSO₄0.6 g/L, the final concentration of chloramphenicol is 5 mug/mL, and/or the final concentration of kanamycin is 25 mug/mL.

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

Examples

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

The experimental techniques and experimental procedures used in this example are, unless otherwise specified, conventional techniques, e.g., those in the following examples, in which specific conditions are not specified, and generally according to conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The materials, reagents and the like used in the examples are commercially available from normal sources unless otherwise specified.

The sequences of the primers used for plasmid construction in the examples of Table 1 are shown below:

example 1 Inclusion of endogenous sourcesproPPlasmid construction of Gene promoter sequences

ProP is proline-uptake protein, is induced to express under the condition of hypertonic condition, and enhances the tolerance of the strain to the hypertonic environment by improving the uptake of compatible substance proline. The literature reports that the expression of ProP is obviously up-regulated under hypertonic conditions, which indicates that the promoter of the gene may also be a hypertonic inducible promoter (Franzel, B., et al., Adaptation ofCorynebacterium glutamicumto salt-stress conditions, Proteomics, 2010, 10(3): 445-. Corynebacterium glutamicum (C.glutamicum) according to the NCBI publicationCorynebacterium glutamicum) The genomic sequence of ATCC13032 (NC-003450.3), the primers proP-F (SEQ ID NO: 4) and proP-R (SEQ ID NO: 5) were designed. The gene of ATCC13032 was used as a template to obtain a gene vector by PCR amplificationproPPromoter of Gene (P) _proP ) Sequence (SEQ ID NO: 1). At the same time, reported in literature pXM-gfpAs a template (Sun DH et al, Journal of Industrial Microbiology&Biotechnology, 2019, 46 (2): 203-lacIGenes andtacvector fragments of promoters. The fragments were recovered and recombined and ligated using a Vazyme Clon Express Multies recombination kit, and the ligation products were transformed into Trans T1Culturing the state cells on a chloramphenicol resistant plate overnight, selecting positive clones for colony PCR verification, sequencing the correct transformants to confirm, and naming the obtained recombinant vector as pXM-P _proP -gfp. Meanwhile, the vector fragment was phosphorylated using T4 PNK, and the control vector pXM-con was constructed by self-cyclization. The recombinant vector was transformed into Corynebacterium glutamicum ATCC13032 to obtain a recombinant strain.

Example 2 high salt coupleproPInduction of Gene promoters

The strains with the different promoter recombinant vectors are respectively inoculated into a TSB culture medium containing 5 mug/mL chloramphenicol and cultured overnight at 30 ℃ at 220 r/min. Wherein the TSB liquid culture medium comprises the following components in percentage by weight (g/L): glucose, 5 g/L; 5 g/L of yeast powder; soybean peptone, 9 g/L; 3 g/L of urea; succinic acid, 0.5 g/L; k₂HPO₄•3H₂O，1 g/L；MgSO₄•7H₂O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L. 0.6M Na was added or not added separately in accordance with the original OD 0.5₂SO₄The culture system of the CGXIIY culture medium is 1 mL of 24-pore plate liquid, the GFP fluorescence intensity and OD of different strains are detected after culturing for 18 h at 30 ℃ at 800 r/min₆₀₀The relative intensities of the different promoters under different conditions were characterized by the fluorescence intensity of the unit cell (minus the fluorescence intensity of the unit cell of the control strain under the same conditions). Wherein the CGXIIY culture medium formula is as follows: 50 g/L glucose and 2 g/L, NH yeast powder₄Cl 16.5 g/L and urea 5 g/L, KH₂PO₄ 1 g/L、K₂HPO₄1 g/L、MOPS 42 g/L、MgSO₄ 0.25 g/L、FeSO₄·2H₂O 0.01 g/L、MnSO₄·H₂O 0.01 g/L、ZnSO₄·7H₂O 0.001 g/L、CuSO₄ 0.2 mg/L、NiCl·6H₂O 0.02 mg/L、CaCl₂0.01 g/L, 0.03 g/L protocatechuic acid, 0.2 mg/L biotin and 10.1 mg/L vitamin B, and the final concentration of chloramphenicol is 5 mug/mL. The detection result is shown in figure 1, and data are displayedproPThe gene promoter is induced by high salt, and the induction activity reaches 8.9 times.

Example 3.proPPromoter modification and characterization plasmid construction

In order to further improve the promoter strength and retain higher high-salt induction activity, the invention respectively carries out sequence modification and replacement on 5 '-UTR, promoter-35 region and promoter-10 region core region and 5' -UTR under the condition of retaining possible regulatory region to obtain P _proP-1 (SEQ ID NO: 2) and P _proP-2 (SEQ ID NO: 3) two mutant promoters.

With pXM-P _proP -gfpAs a template, the substitution was carried out by PCR amplification using the primers Prop-1-F (SEQ ID NO: 8) and Prop-1-R (SEQ ID NO: 9)proPRecovering the PCR fragment from the 5' -UTR region behind the promoter, phosphorylating the vector fragment with T4 PNK, and constructing pXM-P by self-cyclization _proP-1 -gfp. At the same time, using pXM-P _proP -gfpAs template, the substitution was obtained by PCR amplification using the primers Prop-2-F (SEQ ID NO: 10) and Prop-2-R (SEQ ID NO: 11)proPRecovering the 5' -UTR region and the-35 region and the-10 region core region after the promoter, phosphorylating the vector fragment with T4 PNK, and constructing pXM-P by self-cyclization _proP-2 -gfp. The recombinant vector was transformed into Corynebacterium glutamicum ATCC13032 to obtain a recombinant strain.

Example 4 Induction of high salt on hybrid promoters

The strength and inducible activity of the mutant promoter was compared to those of the high salt or normal medium conditions using a similar method as in example 3. The results are shown in FIG. 1, and the data show that the strength of the two modified promoters is higher than that of the wild type under normal culture medium and high-salt conditionsproPPromoter, P _proP-1 The inducing activity is basically maintained in wild typeproPPerformance of the promoter (8.7 fold), but P _proP-2 The induction activity is reduced by 3.2 times due to the higher background expression level.

Example 5 Regulation of dCpf1 expression Using mutant promoters for promotion of lysine

With pXM-P _proP -gfpFor the template, primers proP-D-F (SEQ ID NO:18) and proP-D-R (SEQ ID NO: 19), P was obtained by PCR amplification _proP A promoter fragment. Meanwhile, the pXM-07 reported in the literature is taken as a template^[5]Firstly, using primers pXM07-F1 (SEQ ID NO: 12) and pXM07-R1 (SEQ ID NO: 13) to obtain a vector fragment I with dCpf1 by PCR amplification; then, using primers pXM07-F2 (SEQ ID NO: 14) and pXM07-R2 (SEQ ID NO: 15), a second vector fragment with an origin of replication was obtained by PCR amplification. Targeting was obtained by PCR amplification using pEC-26 as a template (Li MY et al, Frontiers in Bioengineering and Biotechnology, 2020, 8: 357) using primers pEC26-F (SEQ ID NO: 16) and pEC26-R (SEQ ID NO: 17)gltA、pgi、homAndpckcrRNA array fragment of gene. Recovering the four fragments, and performing recombinant ligation by using a Vazyme Clon Express multiple one-step recombinant kit to obtain a recombinant vector pXM-P _proP dCpf 1. Similarly, as pXM-P _proP-1 -gfpPrimers, proP-D-F (SEQ ID NO: 18) and proP1-D-R (SEQ ID NO: 20), were designed as templates and P was obtained by PCR amplification _proP-1 A promoter fragment. Recombining the promoter fragment, the vector fragment I, the vector fragment II and the crRNA array fragment to obtain a recombinant vector pXM-P _proP-1 dCpf 1. At the same time, using pXM-P _proP Using dCpf1 as template, using primers pXM07-F1 (SEQ ID NO: 12) and pXM07-R2 (SEQ ID NO: 15) to obtain vector fragment III by PCR amplification, recovering the above fragments, and mixing with P _proP And P _proP-1 The promoter sequence fragment is recombined and connected by a Vazyme Clon Express multiple one-step recombination kit to obtain a corresponding control vector pXM-P _proP -con and pXM-P _proP-1 -con。

According to the construction method of lysine strains disclosed in the literature (Becker, J., et al., Metab. Eng., 2011, 13, 159. sup. 168.), the homologous recombination technology based on pK18mobsacB is utilized to carry out the aspartokinase on the genome of the Corynebacterium glutamicum ATCC13032 (lysCGene coding) the 311 st threonine is mutated into isoleucine, and a strain with certain lysine synthesis energy is constructedForce strain SCgL 30. The recombinant vector pXM-P is _proP dCpf1 and pXM-P _proP-1 dCpf1, and the respective control plasmid pXM-P _proP -con and pXM-P _proP-1 -con transformation of the SCgL30 strain to obtain recombinant and control strains, respectively. Respectively inoculating the strains to a TSB culture medium containing 5 mug/mL of chloramphenicol, carrying out overnight culture at 30 ℃ and 220 r/min, respectively switching a lysine fermentation culture medium with or without 0.6M sodium sulfate (simulating a high-salt hyperosmotic environment caused by accumulation of high-concentration products at the later stage of fermentation) according to an initial OD 0.3, wherein a culture system is a 24-hole plate containing 1 mL of liquid, terminating fermentation after culturing for 24 h at 30 ℃ and 800 r/min, and detecting the content of residual glucose, OD600 and the yield of lysine. Wherein the lysine fermentation medium formula is as follows: 80 g/L glucose, 8 g/L yeast powder and 9 g/L, K urea₂HPO₄ 1.5 g/L、MOPS 42 g/L、FeSO₄ 0.01 g/L、MnSO₄ 0.01 g/L、MgSO₄0.6 g/L, and the final concentration of chloramphenicol is 5 mug/mL. The results are shown in Table 2, and show that the wild type gene is weak in promoter strengthproPThe promoter-regulated dCpf1 weakening system has very limited effect on lysine yield and transformation rate, and the modified mutant promoter has no obvious effect under normal culture conditions. However, under the condition of hypertonic induction, the dCpf1 system regulated and controlled by the mutant promoter shows good application effect, and the yield and the conversion rate of lysine are both obviously improved.

TABLE 2 application effect of hybrid promoter for controlling dCpf1 expression in lysine synthesis

Example 6 use of mutant promoters to regulate LysE expression in lysine Synthesis

With pXM-P _proP -gfpAs a template, P was obtained by PCR amplification using the primers proP-lysE-F (SEQ ID NO: 25) and proP-lysE-R (SEQ ID NO: 26) _proP-1 A promoter sequence fragment; with pXM-P _proP-1 -gfpAs template, use was made of the primer proP-lysE-F (SE)Q ID NO: 25) and proP1-lysE-R (SEQ ID NO: 27) by PCR amplification to give P _proP Promoter and sequence fragment. Obtained by PCR amplification using ATCC13032 genome as template and primers lysE-F (SEQ ID NO: 21) and lysE-R (SEQ ID NO: 22)lysEA gene fragment. Meanwhile, a vector fragment was obtained by PCR amplification using pXM-XK99E as a template and pEC-F (SEQ ID NO: 23) and pEC-R (SEQ ID NO: 24) as primers. The above promoter fragments are respectively reacted withlysERecombining and connecting the fragment and the vector fragment, transforming the connection product into Trans T1 competent cells, coating kanamycin resistant plates for overnight culture, selecting positive clones for colony PCR verification, sequencing and confirming correct transformants, and naming the obtained recombinant vector as pEC-P _proP -lysEAnd pEC-P _proP-1 -lysE。

The recombinant vector pEC-P is prepared _proP -lysE、pEC-P _proP-1 -lysEAnd pEC-XK99E were transformed into C.glutamicum ScgL30, respectively, to obtain a recombinant strain and a control strain. The application effect of LysE expression strains regulated by different promoters in lysine synthesis was verified by the method as in example 4 (antibiotic was replaced with kanamycin at a final concentration of 25. mu.g/mL), and the results are shown in Table 3. Data show, wild typeproPThe lysine yield and the conversion rate of the promoter-regulated LysE overexpression can be improved a little under a hyperosmotic condition, and the lysine yield and the conversion rate of the modified hybrid promoter are obviously improved under a hyperosmotic induction condition, so that a good application effect is shown.

TABLE 3 application effect of different promoters in regulation of LysE expression in lysine synthesis

All the technical features of the invention described in the present specification may be combined in any combination. Each feature of the invention described in this specification may be replaced by an alternative feature serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

Furthermore, from the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes to the invention to adapt it to various usages and conditions, and therefore such changes are intended to fall within the scope of the appended claims.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> mutant hypertonic inducible promoter Pprox and application thereof

<160>27

<170> PatentIn Version 3.1

<210>1

<211> 384

<212>DNA

<213> Corynebacterium glutamicum

<400> 1

gcaccgaaaa cagtaacttt cccaagaaaa atataagaaa acttccccac acaggccgtg 60

aagagcctga atttattgat ttttcagaca gatctggaaa tgtgaccaat ttgtaaccca 120

cccccgctca cctgcatgag tgtggggtct ttttgcattc ttccagctcc cagacttgaa 180

aacgatctga cttttcaccc cgaaccttac taaggtcgat tcatgttgaa aagagaggtg 240

gtgttttcac ttccctttta taggcaaagc tttaaggagt cttacaggaa gaagttaaca 300

ccgcccaggg gtgcgttgga tgatgatcat ctacaaacaa acattccgtt atgcactcat 360

aagatatgac gagaggtttt actc 384

<210>2

<211> 381

<212>DNA

<213> Artificial sequence

<400> 2

gcaccgaaaa cagtaacttt cccaagaaaa atataagaaa acttccccac acaggccgtg 60

aagagcctga atttattgat ttttcagaca gatctggaaa tgtgaccaat ttgtaaccca 120

cccccgctca cctgcatgag tgtggggtct ttttgcattc ttccagctcc cagacttgaa 180

aacgatctga cttttcaccc cgaaccttac taaggtcgat tcatgttgaa aagagaggtg 240

gtgttttcac ttccctttta taggcaaagc tttaaggagt cttacaggaa gaagttaaca 300

ccgcccaggg gtgcgttgga tgatgatcat ctacaaacaa acattccgtt atgcacttga 360

tacccaattc gagaaaggcc a 381

<210>3

<211> 250

<212>DNA

<213> Artificial sequence

<400> 3

gcaccgaaaa cagtaacttt cccaagaaaa atataagaaa acttccccac acaggccgtg 60

aagagcctga atttattgat ttttcagaca gatctggaaa tgtgaccaat ttgtaaccca 120

cccccgctca cctgcatgag tgtggggtct ttttgcattc ttccatatta aagatcacac 180

cgagtggtgg aatttcctca agtgatttac ccacaatgga ctttgttgat acccaattcg 240

agaaaggcca 250

<210>4

<211> 40

<212>DNA

<213> Artificial sequence

<400> 4

tttcaccagt gagacggggc accgaaaaca gtaactttcc 40

<210>5

<211> 47

<212>DNA

<213> Artificial sequence

<400> 5

ttcttctcct ttactcatga gtaaaacctc tcgtcatatc ttatgag 47

<210>6

<211> 26

<212>DNA

<213> Artificial sequence

<400>6

atgagtaaag gagaagaact tttcac 26

<210>7

<211> 20

<212>DNA

<213> Artificial sequence

<400>7

cccgtctcac tggtgaaaag 20

<210>8

<211>41

<212>DNA

<213> Artificial sequence

<400>8

cgagaaaggc caatgatgag taaaggagaa gaacttttca c 41

<210>9

<211> 36

<212>DNA

<213> Artificial sequence

<400>9

aattgggtat caagtgcata acggaatgtt tgtttg 36

<210>10

<211> 67

<212>DNA

<213> Artificial sequence

<400>10

acaatggact ttgttgatac ccaattcgag aaaggccaat gatgagtaaa ggagaagaac 60

ttttcac 67

<210>11

<211>68

<212>DNA

<213> Artificial sequence

<400> 11

gggtaaatca cttgaggaaa ttccaccact cggtgtgatc tttaatatgg aagaatgcaa 60

aaagaccc 68

<210>12

<211> 30

<212>DNA

<213> Artificial sequence

<400>12

gtgtcaattt atcaagaatt tgttaataaa 30

<210>13

<211> 26

<212>DNA

<213> Artificial sequence

<400>13

gcggatacat atttgaatgt atttag 26

<210>14

<211> 46

<212>DNA

<213> Artificial sequence

<400>14

acacgcgtct gagcagtatt catgagacaa taaccctgat aaatgc 46

<210>15

<211> 20

<212>DNA

<213> Artificial sequence

<400>15

cccgtctcac tggtgaaaag 20

<210>16

<211> 40

<212>DNA

<213> Artificial sequence

<400>16

cattcaaata tgtatccgcg agagtcaatt cagggtggtg 40

<210>17

<211> 21

<212>DNA

<213> Artificial sequence

<400>17

aatactgctc agacgcgtgt c 21

<210>18

<211> 41

<212>DNA

<213> Artificial sequence

<400>18

ttttcaccag tgagacgggg caccgaaaac agtaactttc c 41

<210>19

<211> 49

<212>DNA

<213> Artificial sequence

<400>19

aattcttgat aaattgacac gagtaaaacc tctcgtcata tcttatgag 49

<210>20

<211> 40

<212>DNA

<213> Artificial sequence

<400>20

attcttgata aattgacacc attggccttt ctcgaattgg 40

<210>21

<211> 26

<212>DNA

<213> Artificial sequence

<400>21

atggtgatca tggaaatctt cattac 26

<210>22

<211> 44

<212>DNA

<213> Artificial sequence

<400>22

gtctgtttcc tgtgtgaaac taacccatca acatcagttt gatg 44

<210>23

<211> 23

<212>DNA

<213> Artificial sequence

<400> 23

tttcacacag gaaacagacc atg 23

<210>24

<211> 21

<212>DNA

<213> Artificial sequence

<400>24

aacgtaaatg catgccgctt c 21

<210>25

<211> 40

<212>DNA

<213> Artificial sequence

<400>25

gcggcatgca tttacgttgc accgaaaaca gtaactttcc 40

<210>26

<211>49

<212>DNA

<213> Artificial sequence

<400>26

aagatttcca tgatcaccat gagtaaaacc tctcgtcata tcttatgag 49

<210>27

<211> 40

<212>DNA

<213> Artificial sequence

<400>27

agatttccat gatcaccatc attggccttt ctcgaattgg 40

Claims (18)

1. A polynucleotide having promoter activity, wherein the polynucleotide is selected from any one of the group consisting of:

i. comprises the amino acid sequence shown as SEQ ID NO: 2-3, or a nucleotide sequence shown in any sequence;

comprising the amino acid sequence set forth in SEQ ID NO: 2-3, the reverse complement of the nucleotide sequence shown in any sequence;

a sequence that is the reverse complement of a sequence that is capable of hybridizing to a nucleotide sequence set forth in i or ii under high stringency hybridization conditions or very high stringency hybridization conditions, and has increased promoter activity in an environment of elevated salt concentration or osmolality;

iv a sequence having at least 99% sequence identity to the nucleotide sequence set forth in i or ii and having increased promoter activity in an environment of increased salt concentration or osmotic pressure.

2. A transcription expression cassette comprising the polynucleotide having promoter activity according to claim 1, and operably linked to a target gene.

3. A recombinant expression vector comprising the polynucleotide having promoter activity of claim 1, or the transcription expression cassette of claim 2.

4. A recombinant host cell comprising the transcription expression cassette of claim 2, or the recombinant expression vector of claim 3.

5. The recombinant host cell of claim 4, wherein the host cell belongs to the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium, or Escherichia.

6. The recombinant host cell of claim 5, wherein the host cell is Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC 13869, or Corynebacterium glutamicum ATCC 14067, or a derivative strain of Corynebacterium glutamicum.

7. A method for controlling transcription of a target gene, comprising the step of operably linking the polynucleotide having promoter activity according to claim 1 to a target RNA or a target gene.

8. The method of claim 7, wherein the target RNA is at least one of tRNA, sRNA; the target gene includes at least one of a gene encoding a protein involved in synthesis of the target compound, a gene encoding a gene expression regulatory protein, and a gene encoding a protein involved in membrane transport.

9. A method for producing a protein, comprising the step of expressing a protein of interest using the transcription expression cassette of claim 2, the recombinant expression vector of claim 3, or the recombinant host cell of any one of claims 4 to 6.

10. The method of claim 9, wherein the target protein is a protein associated with synthesis of a target compound, a protein associated with membrane transport, or a gene expression regulatory protein.

11. The method of claim 9 or 10, further comprising the step of isolating or purifying the protein of interest.

12. A method for producing a target compound, which comprises the step of expressing a protein involved in synthesis of the target compound, a protein involved in membrane transport or a gene expression regulatory protein using the transcription expression cassette according to claim 2, the recombinant expression vector according to claim 3 or the recombinant host cell according to any one of claims 4 to 6, and producing the target compound in the presence of the protein involved in synthesis of the target compound, the protein involved in membrane transport or the gene expression regulatory protein.

13. The method of claim 12, wherein the target compound is at least one of an amino acid and an organic acid.

14. The method of claim 13, wherein the amino acid is one or a combination of two or more of: proline, hydroxyproline, lysine, glutamic acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, or a derivative of any of the above amino acids.

15. The method of claim 12 or 13, wherein the protein involved in the synthesis of the target compound is pyruvate carboxylase, phosphoenolpyruvate carboxylase, γ -glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transporter, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, glucorepressor, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate deacylase, dihydrodipicolinate deacylase, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, etc, One or more of glyceraldehyde-3-phosphate dehydrogenase, transketolase and diaminopimelate dehydrogenase.

16. The method of claim 13, wherein the organic acid is one or a combination of two or more of: citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, oxaloacetic acid, tartaric acid, propionic acid, hexenoic acid, decanoic acid, octanoic acid, pentanoic acid, malic acid or derivatives of any of the above organic acids.

17. The method of any one of claims 12 to 16, further comprising the step of isolating or purifying the target compound.

18. The polynucleotide having promoter activity according to claim 1, the transcription expression cassette according to claim 2, the recombinant expression vector according to claim 3, the use of the recombinant host cell according to any one of claims 4 to 6 for at least one of:

a. regulating the transcription level of a gene, or preparing a reagent or a 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 compound of interest, or preparing a reagent or kit for producing a compound of interest.

Images