WO2022152036A1 - 具有启动子活性的多核苷酸及其在生产目标化合物中的用途 - Google Patents

具有启动子活性的多核苷酸及其在生产目标化合物中的用途 Download PDF

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WO2022152036A1
WO2022152036A1 PCT/CN2022/070499 CN2022070499W WO2022152036A1 WO 2022152036 A1 WO2022152036 A1 WO 2022152036A1 CN 2022070499 W CN2022070499 W CN 2022070499W WO 2022152036 A1 WO2022152036 A1 WO 2022152036A1
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acid
seq
polynucleotide
promoter
protein
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PCT/CN2022/070499
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English (en)
French (fr)
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郑平
陈久洲
黄婧文
孙际宾
周文娟
刘娇
王钰
石拓
马延和
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中国科学院天津工业生物技术研究所
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Priority claimed from CN202110036121.1A external-priority patent/CN113201538B/zh
Priority claimed from CN202110504946.1A external-priority patent/CN115322989B/zh
Priority claimed from CN202110841507.XA external-priority patent/CN113278620B/zh
Application filed by 中国科学院天津工业生物技术研究所 filed Critical 中国科学院天津工业生物技术研究所
Priority to EP22738917.8A priority Critical patent/EP4293115A1/en
Publication of WO2022152036A1 publication Critical patent/WO2022152036A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the present disclosure belongs to the technical field of biotechnology and genetic engineering, and in particular relates to a polynucleotide with promoter activity, a transcription expression cassette, a recombinant expression vector, a recombinant host cell comprising the polynucleotide with promoter activity, and a control target gene Methods of transcription, methods of preparing proteins, and methods of producing target compounds.
  • Corynebacterium especially the non-pathogenic Corynebacterium glutamicum, is one of the most commonly used strains in the fermentation industry due to its strong amino acid synthesis ability. It is widely used in the industrial production of proteins, amino acids, organic acids and other chemicals.
  • the expression regulation and optimization of the target gene is the key to improve the synthesis of protein or product, and the promoter element is an important tool for regulating gene expression.
  • a series of strong promoters [1-2] or constitutive promoters [3] have been identified or developed in Corynebacterium glutamicum, which are used to regulate the expression of key genes in metabolic pathways.
  • promoter elements can finely regulate the expression of target genes, however, for the synthesis of some toxic proteins or metabolites, the use of constitutive promoters is still relatively limited. In addition, expression from strong promoters or constitutive promoters often imposes a large metabolic burden on engineered strains.
  • Inducible promoters can control the timing of transcription initiation and are therefore more conducive to strain regulation and redistribution of metabolic flux.
  • inducible promoters such as tac and trc are widely used in the metabolic regulation of C. glutamicum.
  • the above promoters often require additional expensive inducers, such as IPTG, and the addition of these inducers will also cause certain toxicity to the strain, or cause greater interference to the fermentation system. Therefore, the development of an autoinduction system under industrial fermentation conditions is crucial for the construction of industrial strains.
  • some studies have reported auto-inducible promoters in C. glutamicum, such as lysine-inducible promoters [4] , growth process-regulated promoters [5-6] and so on.
  • the number of the above-mentioned auto-inducible promoters is still relatively small, and the response conditions are relatively narrow, so they cannot be widely used in more fermentation systems and product synthesis.
  • identifying high-salt-hypertonic inducible promoters and developing and constructing an auto-inducible system for high-salt-hypertonic conditions in the late fermentation stage can not only increase the available auto-inducible systems, but also provide universal auto-induction elements for the development of all industrial strains , which has also become a key problem that needs to be solved urgently in the development of industrial strains of Corynebacterium glutamicum.
  • the use of inducible promoters such as tac and trc in the process of industrial fermentation needs to add expensive inducers such as IPTG, and the addition of the inducer causes the problem of toxicity to strains, for this reason,
  • the present disclosure provides a polynucleotide with promoter activity, and the aforementioned polynucleotide exhibits enhanced promoter activity in an environment with elevated salt concentration and osmotic pressure.
  • the operative connection of the polynucleotide and the protein-coding gene related to amino acid synthesis can realize the high-efficiency expression of the protein-coding gene in a high-salt and high-osmotic pressure environment, which effectively solves the problem that the current inducible promoter needs to add a high-inducing agent, and Inducers pose a problem of toxicity to the strain.
  • the polynucleotide with promoter activity provided by the present disclosure is a mutant obtained on the basis of the polynucleotide of the sequence shown in any one of SEQ ID NOs: 1-3, and the mutation in the present disclosure
  • the body exhibits enhanced promoter activity, and operably linking it with the target gene can make the target gene highly expressed in the environment of high salt and high osmotic pressure, avoiding the use of IPTG It provides a novel high-salt or hypertonic inducible promoter for the efficient production of target compounds.
  • the promoter activity of the mutant is significantly higher than that of the wild-type promoter, which has important industrial application value.
  • a polynucleotide with promoter activity wherein the polynucleotide is selected from any one of the following groups (i)-(iv):
  • the nucleotide sequence shown in (i) or (ii) has 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 sequence.
  • transcription expression cassette wherein the transcription expression cassette comprises the polynucleotide with promoter activity according to (1) or (2); optionally, the transcription expression cassette further contains a protein encoding A gene, the protein-coding gene is operably linked to the polynucleotide having promoter activity.
  • a recombinant expression vector wherein the recombinant expression vector comprises the polynucleotide having promoter activity according to (1) or (2), or the transcription expression cassette according to (3).
  • a recombinant host cell wherein the recombinant host cell comprises the transcription expression cassette according to (3), or the recombinant expression vector according to (4).
  • the recombinant host cell according to (5) wherein the host cell is derived from the genus Corynebacterium, Brevibacterium, Arthrobacter, Microbacterium or Escherichia; preferably, the host The 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.
  • a polynucleotide according to (1) or (2), a transcription expression cassette according to (3), a recombinant expression vector according to (4), and according to (5) or (6) Use of the recombinant host cell in at least one of the following:
  • the target compound includes at least one of amino acids and organic acids; alternatively, the amino acids include lysine, glutamic acid and threonine At least one of amino acids, and the organic acid includes at least one of citric acid and succinic acid.
  • a method for regulating transcription of a target gene comprising the step of operably linking the polynucleotide with promoter activity according to any one of (1) to (2) and the target gene.
  • a method for preparing a protein comprising using the transcription expression cassette according to (3), the recombinant expression vector according to (4), or any one of (5)-(6) The step of expressing the protein in the recombinant host cell; optionally, the protein is a protein related to the synthesis of the target compound or a gene expression regulatory protein;
  • the method further comprises the step of isolating or purifying the protein.
  • a method for producing a target compound comprising using the transcription expression cassette described in (3), the recombinant expression vector described in (4), or the recombinant expression described in any one of (5)-(6).
  • the host cell expresses a protein related to the synthesis of the target compound or a gene expression regulatory protein, and the step of producing the target compound in the presence of the protein related to the synthesis of the target compound or the gene expression regulatory protein;
  • the target compound includes at least one of amino acids and organic acids; optionally, the amino acids include at least one of lysine, glutamic acid and threonine, and the organic acid includes lemon at least one of acid and succinic acid;
  • the protein is a protein related to lysine synthesis; optionally, the protein related to lysine synthesis includes aspartate kinase, aspartate semialdehyde dehydrogenase, aspartate Acid ammonia lyase, dihydrodipicolinate synthase, dihydropicolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate Acid deacylase, diaminopimelate epimerase, diaminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate One or more combinations of dehydrogenase and pyruvate carboxylase;
  • the method further comprises the step of isolating or purifying the target compound.
  • polynucleotide with promoter activity wherein the polynucleotide is selected from any one of the following groups (v)-(x):
  • the nucleotide sequence in position 179 is not ACACCGAGTG;
  • the nucleotide sequence in position 139 is not ACACCGAGTG;
  • (x) comprises at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% of the nucleotide sequence shown in any one of (v) to (vii). Sequence identity of a sequence of polynucleotides.
  • the mutant has a 1- to 8-fold increased promoter activity in an environment with increased salt concentration or osmotic pressure compared to a polynucleotide comprising the sequence shown in SEQ ID NO: 2.
  • a transcription expression cassette wherein the transcription expression cassette comprises the polynucleotide with promoter activity according to any one of (13)-(16); optionally, the transcription expression cassette further It 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.
  • a recombinant expression vector wherein the recombinant expression vector comprises the polynucleotide with promoter activity described in any one of (13) to (16), or the transcription expression cassette described in (17).
  • a recombinant host cell wherein the recombinant host cell comprises the transcription expression cassette described in (17), or the recombinant expression vector described in (18).
  • the target compound includes at least one of amino acids and organic acids; optionally, the amino acids include proline, lysine, glutamic acid Amino acid, threonine, glycine, alanine, valine, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine , at least one of arginine, histidine, phenylalanine, tyrosine, tryptophan, and the organic acids include citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, At least one of tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, valeric acid, and malic acid.
  • the amino acids include proline, lysine, glutamic acid Amino acid, threonine, glycine, alanine, valine, leucine, isole
  • (24) A method for regulating transcription of a target gene, wherein the method comprises the step of operably linking the polynucleotide with promoter activity according to any one of (13) to (16) and the target gene.
  • a method for preparing a protein comprising using the transcription expression cassette described in (17), the recombinant expression vector described in (18), or any one of (19)-(20).
  • the method further comprises the step of isolating or purifying the protein.
  • a method for producing a target compound comprising using the transcription expression cassette described in (17), the recombinant expression vector described in (18), or any one of (19)-(20).
  • the recombinant host cell expresses a protein related to the synthesis of the target compound or a gene expression regulatory protein, and the step of producing the target compound in the presence of the protein related to the synthesis of the target compound or the gene expression regulatory protein;
  • the target compound includes at least one of amino acids and organic acids; alternatively, the amino acids include lysine, glutamic acid, threonine, proline, glycine, alanine, valine amino acid, leucine, isoleucine, serine, cysteine, glutamine, methionine, aspartic acid, asparagine, arginine, histidine, phenylalanine, At least one of tyrosine and tryptophan, the organic acids include citric acid, succinic acid, lactic acid, acetic acid, butyric acid, palmitic acid, oxalic acid, tartaric acid, propionic acid, hexenoic acid, capric acid, caprylic acid, At least one of valeric acid and malic acid;
  • the amino acids include lysine, glutamic acid, threonine, proline, glycine, alanine, valine amino acid, leucine, isoleucine, serine, cyst
  • the protein related to the synthesis of the target compound is a protein related to the synthesis of L-amino acid; optionally, the protein related to the synthesis of L-amino acid includes pyruvate carboxylase, phosphoenolpyruvate carboxylate Amylase, ⁇ -glutamyl kinase, glutamate semialdehyde dehydrogenase, pyrroline-5-carboxylate reductase, amino acid transporter, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxalyl Acetate decarboxylase, gluconate repressor protein, glucose dehydrogenase, aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrogenase Picolinate reductase, succinyldia
  • the method further comprises the step of isolating or purifying the target compound.
  • polynucleotide with promoter activity wherein the polynucleotide is selected from any one of the following groups (xi)-(xiv):
  • (xiii) the reverse complement of the sequence capable of hybridizing to the nucleotide sequence shown in (xi) or (xii) under high stringency hybridization conditions or very high stringency hybridization conditions, and in salt concentration or osmotic pressure Increased promoter activity in elevated environments;
  • (xiv) A sequence having at least 99% sequence identity with the nucleotide sequence shown in (xi) or (xii), and having enhanced promoter activity in an environment of elevated salt concentration or osmotic pressure.
  • a transcription expression cassette comprising the polynucleotide having promoter activity as described in (27), operably linked to a target gene.
  • a recombinant expression vector comprising the polynucleotide having promoter activity as described in (27), or the transcription expression cassette as described in (28).
  • a recombinant host cell comprising the transcription expression cassette as described in (28), or the recombinant expression vector as described in (29).
  • a method for regulating transcription of a target gene comprising the step of operably linking the polynucleotide with promoter activity described in (27) to a target RNA or a target gene.
  • the target RNA is at least one of tRNA and sRNA;
  • the target gene includes a gene encoding a protein related to the synthesis of the target compound, a gene expression regulatory protein At least one of the encoding genes of , and the encoding genes of proteins related to membrane transport.
  • a method for preparing a protein comprising using the transcription expression cassette described in (28), the recombinant expression vector described in (29), or the recombinant host cell expression described in any one of (30)-(32). Steps for target protein.
  • the target protein is a protein related to the synthesis of the target compound, a protein related to membrane transport, or a gene expression regulatory protein.
  • a method for producing a target compound comprising using the transcription expression cassette described in (28), the recombinant expression vector described in (29), or the recombinant expression described in any one of (30)-(32).
  • the host cell expresses a protein related to the synthesis of the target compound, a protein related to membrane transport, or a gene expression regulatory protein, in the environment where the protein related to the synthesis of the target compound, the protein related to membrane transport, or the gene expression regulatory protein exists
  • the steps for producing the target compound are as follows.
  • 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 derivatives of any of the above amino acids.
  • organic acid is a combination of one 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, capric acid, caprylic acid, valeric acid, malic acid or derivatives of any of the above organic acids.
  • the present disclosure provides a polynucleotide having promoter activity that is a high-salt, high-osmolarity-inducible promoter with enhanced initiation in an environment of elevated salt concentration and osmotic pressure subactivity.
  • the polynucleotide is operably linked to the target gene, which can significantly improve the expression intensity of the target gene under the stress environment of high salt and high osmotic pressure, and then produce the downstream products stably and efficiently, which effectively solves the problem of adding IPTG and other expensive inducers. , and the problem of toxicity to the strain.
  • the present disclosure provides transcriptional expression cassettes, recombinant expression vectors, and recombinant host cells comprising the above-described polynucleotides having promoter activity.
  • the polynucleotide with promoter activity is operably linked to the protein-coding gene, which can improve the expression strength of the protein-coding gene under the stress environment of high salt and high osmotic pressure. .
  • the present disclosure provides a method for producing amino acids, using the above-mentioned polynucleotides with promoter activity, which can improve the expression of proteins related to amino acid synthesis under stress conditions, while attenuating the expression of other pathway proteins,
  • the metabolic flow is more enriched in the direction of amino acid synthesis, so as to produce amino acids stably and efficiently, and achieve the purpose of excessive accumulation of amino acids.
  • L-Lysine when L-Lysine is produced by the above method, L-Lysine can be produced stably and efficiently in an environment of high salt and high osmotic pressure.
  • the polynucleotides with promoter activity provided by the present disclosure are mutants obtained on the basis of the polynucleotides of the sequences shown in any one of SEQ ID NOs: 1-3.
  • the mutant has enhanced promoter activity in the environment of elevated salt concentration or osmotic pressure, and is a high-salt, high osmotic pressure-inducible promoter.
  • inducible promoters such as tac and trc
  • the mutants in the present disclosure do not require IPTG, which is expensive and has certain toxicity, as an inducer.
  • the promoter activity of the mutant in the present disclosure under high salt and high osmotic pressure environment is further improved than that of the wild-type promoter.
  • the expression efficiency of the target gene can be improved, and the target compound can be produced stably and efficiently.
  • the conversion rate of mutants used in the production of target compounds is higher than that of wild-type promoters, which provides a highly efficient method for industrial fermentation of target compounds such as amino acids and organic acids. Potential inducible promoters for use.
  • the present disclosure provides a mutant of a polynucleotide of the sequence shown in any one of SEQ ID NOs: 1-3, the promoter activity of which is higher in salt concentration or osmolarity compared to the wild-type promoter 1-8 times higher promoter activity in elevated pressure environment.
  • the present disclosure provides a mutant of the polynucleotide of the sequence shown in any one of SEQ ID NO: 1-3, the promoter activity of which is compared with the wild-type promoter, in 1.85-7.62-fold increased promoter activity in environments with elevated salt concentration or osmotic pressure.
  • the present disclosure provides transcriptional expression cassettes, recombinant expression vectors, and recombinant host cells, comprising the above-described polynucleotides having promoter activity.
  • the polynucleotide with promoter activity is operably linked to the target gene, which can improve the ability of key genes in the synthesis pathway of the target compound to survive under the stress environment of high salt and high osmotic pressure. expression strength.
  • the present disclosure provides methods for preparing proteins, which can increase the expression of proteins related to the synthesis of amino acids, organic acids, etc. or gene expression regulatory proteins, thereby achieving efficient production of target compounds.
  • the present disclosure provides a method for producing a target compound.
  • the expression of proteins related to the synthesis of the target compound can be improved under stress conditions, so as to stably and efficiently produce the target compound. compound, to achieve the purpose of improving the metabolic flow of the target compound and accumulating excessively the target compound.
  • Figure 1 shows the induction of NCgl1418 promoter by hyperosmolarity
  • Fig. 2 shows the induction effect of different concentrations of sodium sulfate on NCgl1418 promoter
  • Figure 3 shows the induction of NCgl1418 promoter by high concentrations of sugar
  • Fig. 4 shows the promoter strength comparison result of P NCgl1418 and P tuf ;
  • Figure 5 shows the activity comparison results of NCgl1418 promoters of different lengths
  • Figure 7 shows the results of comparing the activities of the proP gene promoter and the NCgl1418 promoter.
  • a selected/alternative/preferred “numerical range” includes both the numerical endpoints at both ends of the range and, with respect to the aforementioned numerical endpoints, all natural numbers covered in the middle of the numerical endpoints.
  • high-salt environment can be inorganic salt ions such as high-concentration Na 2 SO 4 , NaCl, K 2 SO 4 , KCl, etc. in the medium, or products such as lysine in the fermentation broth as the fermentation time prolongs, or Increased concentrations due to accumulation of certain intermediate metabolites (e.g., lysine sulfate, etc.), or due to substrate influx (e.g., substrates such as ammonium sulfate), or other Arbitrary salt concentration.
  • a “high salt environment” refers to a salt concentration above 0.2M; in some more specific embodiments, a “high salt environment” refers to a salt concentration between 0.2-0.8M.
  • the salt concentration is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M.
  • hypothalmotic environment refers to increased osmotic pressure in response to increased salt concentration.
  • polynucleotide in this disclosure refers to a polymer composed of nucleotides.
  • Polynucleotides may be in the form of individual fragments or part of a larger nucleotide sequence structure derived from a nucleotide sequence that has been isolated at least once in quantity or concentration, and can be obtained by standard Molecular biological methods (eg, using cloning vectors) identify, manipulate, and recover sequences and their component nucleotide sequences.
  • a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C)
  • this also includes an RNA sequence (ie A, U, G, C) where "U" replaces "T”.
  • polynucleotide refers to a polymer of nucleotides removed from other nucleotides (individual fragments or entire fragments), or may be a component or component of a larger nucleotide structure, such as an expression vector or polycistronic sequence.
  • Polynucleotides include DNA, RNA and cDNA sequences.
  • sequence identity and “percent identity” in this disclosure refer to the percentage of nucleotides or amino acids that are identical (ie, identical) between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be determined by aligning the nucleotide or amino acid sequences of the polynucleotides or polypeptides and The number of positions containing the same nucleotide or amino acid residue is scored and compared to the number of positions containing different nucleotide or amino acid residues in the aligned polynucleotides or polypeptides.
  • Polynucleotides can differ at a position, eg, by containing different nucleotides (ie, substitutions or mutations) or deletions of nucleotides (ie, insertions of nucleotides or deletions of nucleotides in one or both polynucleotides).
  • Polypeptides can differ at one position, for example, by containing different amino acids (ie, substitutions or mutations) or missing amino acids (ie, 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 a 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.
  • the polynucleotide having promoter activity comprises the reverse complement of the nucleotide sequence shown in any of SEQ ID NOs: 1-3, 57-58, and the polynucleotide maintains High-salt, high-osmolarity-inducible promoter activity.
  • the polynucleotide with promoter activity is included under high stringency hybridization conditions or very high stringency hybridization conditions, and SEQ ID NO: 1-3, 57-58 shown in any of the sequences The reverse complement of a nucleotide sequence or a sequence to which its reverse complement hybridizes, and the polynucleotide retains a high salt, high osmotic pressure-inducible promoter activity.
  • the polynucleotide having promoter activity comprises 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, and multinucleated
  • the glucosinolate maintains a high-salt, high-osmolarity-inducible promoter activity.
  • wild-type refers to objects that can be found in nature.
  • 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.
  • wild-type refers to the promoter of the wild-type NCgl1418 gene, that is, the polynucleotide of the sequence shown in SEQ ID NO: 1.
  • mutant refers to a "wild-type", or “comparative" polynucleotide or polypeptide that comprises a change at one or more (eg, several) positions (i.e. , the polynucleotide of replacement, insertion and/or lack, wherein, replacement refers to replacing the nucleotide occupying a position with different nucleotides.Deletion refers to removing the nucleotide occupying a certain position. Insertion refers to in Nucleotides are added adjacent to and immediately following the nucleotide occupying the position.
  • a “mutation” in the present disclosure is a “substitution”, which is a mutation caused by the substitution of a base in one or more nucleotides with another different base, also known as a base substitution mutation (substitution) or point mutation (point mutation).
  • sequence shown in SEQ ID NO: 36 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 mutated nucleotide introduced at a position near the -35 region, and it was found that the promoter activity of the mutant under high salt or high osmotic pressure was significantly enhanced after introducing the mutation at the above-mentioned position.
  • a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 1 refers to a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 1, and the mutant is described in SEQ ID NO: 1 ID NO: 1 has mutated nucleotides at one or more positions in positions 170-179 of the sequence shown in SEQ ID NO: 1 and does not contain a polynucleate that is mutated to ACACCGAGTG at positions 170-179 of the sequence shown in SEQ ID NO: 1 Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO: 1.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence shown in SEQ ID NO: 1.
  • a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2 refers to a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2, and the mutant is described in SEQ ID NO: 2 ID NO: 2 has mutated nucleotides at one or more positions in positions 130-139 of the sequence shown in SEQ ID NO: 2, and does not contain a multinucleate that is mutated to ACACCGAGTG at positions 130-139 of the sequence shown in SEQ ID NO: 2 Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO: 2.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence set forth in SEQ ID NO: 2.
  • a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 3 refers to a mutant of the polynucleotide comprising the sequence shown in SEQ ID NO: 3, and the mutant is described in SEQ ID NO: 3 ID NO: 3 has mutated nucleotides at one or more positions in positions 72-81 of the sequence shown in SEQ ID NO: 3 and does not contain a polynucleate in which positions 72-81 of the sequence shown in SEQ ID NO: 3 are mutated to ACACCGAGTG Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO:3.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence set forth in SEQ ID NO: 3.
  • the mutants of the present disclosure have a 1- to 8-fold increase in priming in an environment of elevated salt concentration or osmotic pressure compared to a polynucleotide of the sequence set forth in SEQ ID NO: 1 subactivity.
  • the mutant compared with the polynucleotide of the sequence shown in SEQ ID NO: 1, the mutant has 2.11, 1.85, 3.23, 3.07, 3.57, 2.78, 3.90, 3.28, 2.62 under the environment of increased salt concentration or osmotic pressure , 2.70, 4.47, 7.62-fold increased promoter activity.
  • high stringency conditions refers to 5X SSPE (saline sodium phosphate EDTA) at 42°C for probes of at least 100 nucleotides in length following standard Southern blotting procedures , 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and 50% formamide prehybridized and hybridized for 12 to 24 hours. Finally the carrier material was washed three times for 15 min each at 65°C using 2X SSC, 0.2% SDS.
  • 5X SSPE saline sodium phosphate EDTA
  • very high stringency conditions refers to 5X SSPE (saline sodium phosphate EDTA) at 42°C for probes of at least 100 nucleotides in length following standard Southern blotting procedures ), 0.3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and 50% formamide and prehybridized for 12 to 24 hours. Finally the carrier material was washed three times for 15 min each at 70°C using 2X SSC, 0.2% SDS.
  • 5X SSPE saline sodium phosphate EDTA
  • complementary refers to hybridization or base pairing between nucleotides or nucleotides, such as between two strands of a double-stranded DNA molecule or an oligonucleotide primer and a sequenced or amplified between primer binding sites on single-stranded nucleotides, etc.
  • promoter in the present disclosure refers to a nucleic acid molecule that is generally located upstream of the coding sequence of a gene of interest, provides a recognition site for RNA polymerase, and is located 5' upstream of the mRNA transcription initiation 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.
  • promoters can interact with transcription factors that regulate gene transcription, controlling the initiation time and extent of gene expression (transcription), including core promoter regions and regulatory regions, like "Switches” that determine the activity of genes, which in turn control which proteins cells start producing.
  • promoter core region in the present disclosure refers to a nucleic acid sequence located in the prokaryotic promoter region, which is the core sequence region that exerts the function of the promoter, mainly including the -35 region, the -10 region, the -35 region and the -10 region.
  • the region between the regions and the transcription initiation site, the -35 region is the recognition site of RNA polymerase, and the -10 region is the binding site of RNA polymerase.
  • the polynucleotides having promoter activity of the present disclosure can be used to initiate the expression of protein-encoding genes. In other embodiments, the polynucleotides of the present disclosure having promoter activity can be used to initiate the expression of non-coding genes.
  • RNA production and protein production 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.
  • target gene in the present disclosure refers to any gene that is linked to the polynucleotide having promoter activity in the present disclosure to regulate its transcription level.
  • a gene of interest refers to a gene encoding a protein of interest in a microorganism.
  • the target gene is a gene encoding an enzyme related to the biosynthesis of the target compound, a gene encoding an enzyme related to reducing power, a gene encoding an enzyme related to glycolysis or the TCA cycle, or a gene encoding an enzyme related to the target compound. Genes that release related enzymes, etc.
  • target compound in the present disclosure can be selected from at least one of amino acids and organic acids, and can also be selected from other kinds of compounds that may be obtained by biosynthesis in the art.
  • the compound of interest is an "amino acid” or "L-amino acid.”
  • Amino acid or “L-amino acid” generally refers to the basic building blocks of proteins in which the amino and carboxyl groups are bound to the same carbon atom.
  • the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, glutamine, methionine, aspartic acid , one or both of asparagine, glutamic acid, lysine, arginine, histidine, phenylalanine, tyrosine, tryptophan, 5-aminolevulinic acid, proline A combination of more than one species, or other types of amino acids in the art.
  • the target compound is an organic acid.
  • the organic acid may be an organic compound having an acidity, for example, those compounds including carboxyl groups and sulfonic acid groups.
  • organic acids include 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, oxaloacetic acid, malic acid.
  • organic acids include 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, oxaloacetic acid, malic acid.
  • protein-coding gene in the present disclosure refers to a synthetic DNA molecule that can guide proteins through certain rules.
  • the process of protein-coding genes instructing protein synthesis generally includes transcription using double-stranded DNA as a template and translation using mRNA as a template. process.
  • Protein-coding genes contain CDS sequences (Coding Sequences) that can direct the production of protein-coding mRNAs.
  • protein-coding genes include, but are not limited to, proteins encoding proteins involved in the synthesis of target compounds, and in some embodiments, protein-encoding genes are involved in encoding proteins involved in the synthesis of L-amino acids.
  • proteins involved in the synthesis of L-amino acids include, but are not limited to, pyruvate carboxylase, phosphoenolpyruvate carboxylase, gamma-glutamyl kinase, glutamic semialdehyde dehydrogenase, pyrrole One or both of lino-5-carboxylic acid reductase, amino acid transport protein, ptsG system, pyruvate dehydrogenase, homoserine dehydrogenase, oxaloacetate decarboxylase, gluconate repressor, and glucose dehydrogenase combination of the above.
  • the protein-coding gene relates to encoding a protein associated with the synthesis of L-lysine.
  • proteins related to the synthesis of L-lysine including aspartokinase, aspartate semialdehyde dehydrogenase, aspartate ammonia lyase, dihydrodipicolinate synthase, dihydrogenase Picolinate reductase, succinyldiaminopimelate aminotransferase, tetrahydrodipicolinate succinylase, succinyldiaminopimelate deacylase, diaminopimelate epimerase, diaminopimelate One or more of aminopimelate deacylase, glyceraldehyde-3-phosphate dehydrogenase, lysine transporter, transketolase, diaminopimelate dehydrogenase and pyruvate carboxylase The combination.
  • a protein-coding gene is involved in encoding a protein associated with the synthesis of organic acids, exemplary, a protein-coding gene for encoding a protein associated with the synthesis of citrate, or for encoding a protein associated with the synthesis of succinate .
  • the protein-coding gene involves a protein associated with gene editing, such as the Cpf1 protein.
  • the polynucleotide with promoter activity of the present disclosure can be suitable for improving the expression of the target gene under the stress environment of high salt and high osmotic pressure, so as to realize the efficient production of the target product.
  • gene expression regulatory protein in the present disclosure includes, but is not limited to, exogenous gene expression regulatory tool proteins, such as dCas9 protein, dCpf1 protein required for CRISPRi regulation, Hfq protein required for sRNA regulation, etc., as well as endogenous or exogenous transcriptional regulation factors, which in turn regulate the expression of key genes in metabolic pathways.
  • exogenous gene expression regulatory tool proteins such as dCas9 protein, dCpf1 protein required for CRISPRi regulation, Hfq protein required for sRNA regulation, etc.
  • endogenous or exogenous transcriptional regulation factors which in turn regulate the expression of key genes in metabolic pathways.
  • transcriptional expression cassette in the present disclosure refers to a type of expression element that includes a transcriptional regulatory element and a target gene, and utilizes the transcriptional regulatory element to regulate the expression of the target gene.
  • transcriptional regulatory elements include promoters, and on this basis, elements such as enhancers, silencers, and insulators may also be included.
  • the target gene is specifically a protein-coding gene.
  • the target gene and the polynucleotide are "operably linked", 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, and the operably linked manner can be Use any means described by those skilled in the art.
  • vector in this disclosure refers to a DNA construct that contains DNA sequences operably linked to appropriate control sequences to express a gene of interest in a suitable host.
  • a "recombinant expression vector” refers to a DNA construct used to express, for example, a polynucleotide encoding a desired polypeptide.
  • Recombinant expression vectors may include, for example, i) collections of genetic elements that have regulatory effects on gene expression, such as promoters and enhancers; ii) structural or coding sequences that are transcribed into mRNA and translated into protein; and iii) appropriate transcription and transcriptional subunits of translation initiation and termination sequences.
  • Recombinant expression vectors are constructed in any suitable manner.
  • vectors are not critical and any vector can 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 combinations of plasmids and phage DNA, such as from vaccinia, adenovirus, chicken DNA from viruses such as pox, baculovirus, SV40, and pseudorabies.
  • host cell in the present disclosure means any cell type that is amenable to transformation, transfection, transduction, etc. with a transcription initiation element or expression vector comprising a polynucleotide of the present disclosure.
  • recombinant host cell encompasses a host cell that differs from the parental cell after introduction of a transcription initiation element or recombinant expression vector, in particular by transformation.
  • transformation in the present disclosure has the meaning commonly understood by those skilled in the art, that is, the process of introducing exogenous DNA into a host.
  • the method of transformation includes any method of introducing nucleic acid into cells, 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.
  • the host cell of the present disclosure may be a prokaryotic cell or a eukaryotic cell, as long as it is a cell capable of introducing the polynucleotide having promoter activity of the present disclosure.
  • 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.
  • the host cell is Corynebacterium glutamicum derived from the genus Corynebacterium.
  • Corynebacterium glutamicum can be Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC 13869 or Corynebacterium glutamicum ATCC 14067, etc., and the mutant strains or glutamic acid producing amino acids prepared by the above-mentioned strains Derivative strains of Corynebacterium.
  • the host cells in the present disclosure can be any type of strain with amino acid production ability, including wild-type strains and recombinant strains.
  • the host cell is a lysine-producing host cell.
  • the host cell for lysine production may be a strain expressing aspartokinase released from feedback inhibition based on Corynebacterium glutamicum ATCC 13032.
  • the lysine-producing host cells may also be other kinds of strains having lysine-producing ability.
  • thrA gene encoding aspartate kinase I/homoserine dehydrogenase I bifunctional enzyme
  • the cadA gene encoding lysine decarboxylase.
  • one or more genes selected from the following are enhanced or overexpressed in the lysine-producing host cell:
  • the dapA gene encoding the dihydrodipyridine synthase that relieves the feedback inhibition of lysine
  • dapB gene encoding dihydrodipicolinate reductase
  • dapD encoding tetrahydrodipicolinate succinylase and dapE encoding succinyldiaminopimelate deacylase
  • pntAB gene encoding niacinamide adenine dinucleotide transhydrogenase
  • lysE gene encoding the lysine transporter protein.
  • the host cell is a threonine-producing host cell.
  • the threonine-producing host cell is a strain expressing the feedback-relieved aspartokinase LysC based on Corynebacterium glutamicum ATCC 13032.
  • the threonine-producing host cells can also be other strains of threonine-producing ability.
  • one or more genes selected from the following are enhanced or overexpressed in the threonine-producing host cell:
  • thrABC gene encoding the threonine operon
  • the host cell is an isoleucine-producing host cell.
  • the isoleucine-producing host cell is a strain that produces L-isoleucine by substituting alanine for the amino acid at position 323 of the L-threonine dehydratase ilvA gene.
  • the isoleucine-producing host cells may also be other species of strains capable of isoleucine production.
  • the host cell is a host cell that produces O-acetyl homoserine.
  • the O-acetylhomoserine-producing host cell is a strain that produces O-acetylhomoserine by inactivating O-acetylhomoserine (thiol)-lyase.
  • the host cell that produces O-acetylhomoserine can also be other species of strains that have the ability to produce O-acetylhomoserine.
  • the host cell is a methionine-producing host cell.
  • the methionine-producing host cell is a strain that produces methionine by inactivating transcriptional regulators of methionine and cysteine.
  • the methionine-producing host cells can also be other strains of methionine-producing ability.
  • the host cells of the present disclosure 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 can be appropriately adjusted according to actual conditions Various culture conditions such as temperature, time and pH of the medium, etc.
  • the polynucleotide with promoter activity in the present disclosure can show enhanced promoter activity in the environment of high salt and high osmotic pressure, thereby avoiding the need to add the present invention in the fermentation environment.
  • High-priced and toxic inducers such as IPTG commonly used in the field.
  • Corynebacterium glutamicum ATCC13032 strain was cultivated in CGXII medium with and without the addition of 0.6M NaCl or lysine sulfate, cells were harvested in mid-logarithmic growth, total RNA was extracted and analyzed by transcriptome sequencing.
  • the promoter of the NCgl1418 gene whose transcription level was significantly increased under hypertonic conditions was selected as the most candidate high-salt-hypertonic inducible promoter.
  • the NCgl1418 promoter is selected from any one of the following group consisting of (i)-(iv):
  • nucleotide sequence shown in (i) or (ii) has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, Sequences of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the NCgl1418 promoter has increased promoter activity in a high salt environment. In some specific embodiments, the NCgl1418 promoter has increased promoter activity in a hyperosmotic environment.
  • high-salt environment can be inorganic salt ions such as high-concentration Na 2 SO 4 , NaCl, K 2 SO 4 , KCl, etc. in the medium, or products such as lysine in the fermentation broth as the fermentation time prolongs, or Increased concentrations due to accumulation of certain intermediate metabolites (e.g., lysine sulfate, etc.), or due to substrate influx (e.g., substrates such as ammonium sulfate), or other Arbitrary salt concentration.
  • a “high salt environment” refers to a salt concentration above 0.2M; in some more specific embodiments, a “high salt environment” refers to a salt concentration between 0.2-0.8M.
  • the salt concentration is 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M.
  • hypothalmotic environment refers to increased osmotic pressure in response to increased salt concentration.
  • the NCgl1418 promoter exhibits higher promoter activity or higher transformation efficiency in a "high salt environment" formed with sulfate and hydrochloride; in some preferred embodiments , the sulfate is Na 2 SO 4 or K 2 SO 4 , lysine sulfate, and the hydrochloride is lysine hydrochloride, sodium chloride or potassium chloride.
  • the promoter of the nucleotide sequence shown in any of SEQ ID NOs: 57-58 exhibits higher promoter activity in a "high salt environment" formed with sulfate or Higher conversion efficiency; in some preferred embodiments, the sulfate is Na2SO4 or K2SO4 .
  • the polynucleotide with promoter activity comprises the reverse complement of the nucleotide sequence shown in any of SEQ ID NOs: 57-58, and the polynucleotide maintains high salt, high Osmotic pressure-inducible promoter activity.
  • the polynucleotide having promoter activity comprises the nucleotide sequence set forth in any of SEQ ID NOs: 57-58 under high stringency hybridization conditions or very high stringency hybridization conditions or the reverse complement of the sequence to which its reverse complement hybridizes, and the polynucleotide maintains a high salt, high osmotic pressure-inducible promoter activity.
  • the polynucleotide having promoter activity comprises 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, and multinucleated
  • the glucosinolate maintains a high-salt, high-osmolarity-inducible promoter activity.
  • the present disclosure utilizes the sequence of the promoter core region of the NCgl1418 gene to introduce a mutation in the upstream -35 region of the promoter of the NCgl1418 gene to obtain a mutant of the promoter core region of the NCgl1418 gene containing the upstream mutation of the -35 region.
  • a mutation was introduced into the ACACCGAGTG upstream of the -35 region of the promoter core region of the NCgl1418 gene to obtain a mutant with further improved promoter activity in a high-salt or high-osmotic pressure environment, which is used for genetic engineering breeding of fermenting microorganisms and targets Efficient production of compounds provides novel inducible promoters.
  • the mutants in the present disclosure exhibit increased promoter activity in environments with elevated salt concentration or osmotic pressure; when applied to the fermentation of target compounds, the mutants are comparable to the wild-type promoters. Compared with the promoter, it showed a higher conversion rate of the target compound.
  • NCgl1418 promoter fragments with 203bp (SEQ ID NO:2) and 145bp (SEQ ID NO:3) were obtained, both of which have NCgl1418 promoter fragments.
  • the core region can also show significantly enhanced promoter activity in the environment of elevated salt concentration and osmotic pressure. Therefore, adopt the promoter transformation method in the above-mentioned embodiment, namely mutate one or more positions in the 130th-139th position of the sequence shown in SEQ ID NO:2, or mutate one or more positions of the sequence shown in SEQ ID NO:3 Mutation at one or more of positions 72-81 results in promoter mutants that also have enhanced inducible activity.
  • high-salt environment can be inorganic salt ions such as high-concentration Na 2 SO 4 , NaCl, K 2 SO 4 , KCl, etc. in the medium, or products such as lysine in the fermentation broth as the fermentation time prolongs, or Increased concentrations due to accumulation of certain intermediate metabolites (e.g., lysine sulfate, etc.), or due to substrate influx (e.g., substrates such as ammonium sulfate), or other Arbitrary salt concentration.
  • inorganic salt ions such as high-concentration Na 2 SO 4 , NaCl, K 2 SO 4 , KCl, etc. in the medium, or products such as lysine in the fermentation broth as the fermentation time prolongs, or Increased concentrations due to accumulation of certain intermediate metabolites (e.g., lysine sulfate, etc.), or due to substrate influx (e.g., substrates such as ammonium sulfate), or other Arbitrary salt concentration.
  • a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 1 refers to a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 1, and the mutant is described in SEQ ID NO: 1 ID NO: 1 has mutated nucleotides at one or more positions in positions 170-179 of the sequence shown in SEQ ID NO: 1 and does not contain a polynucleate that is mutated to ACACCGAGTG at positions 170-179 of the sequence shown in SEQ ID NO: 1 Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO: 1.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence set forth in SEQ ID NO: 1.
  • the mutants correspond to 1, 2, 3, 4, 5, 6, 7, 8, 1, 2, 3, 4, 5, 6, 7, 8, Nucleotides with mutations at 9 or 10 positions.
  • a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2 refers to a mutant of a polynucleotide comprising a sequence as shown in SEQ ID NO: 2, and the mutant is described in SEQ ID NO: 2 ID NO: 2 has mutated nucleotides at one or more positions in positions 130-139 of the sequence shown in SEQ ID NO: 2, and does not contain a multinucleate that is mutated to ACACCGAGTG at positions 130-139 of the sequence shown in SEQ ID NO: 2 Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO: 2.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence set forth in SEQ ID NO: 2.
  • the mutants correspond to 1, 2, 3, 4, 5, 6, 7, 8, 1, 2, 3, 4, 5, 6, 7, 8, Nucleotides with mutations at 9 or 10 positions.
  • a mutant of a polynucleotide comprising the sequence shown in SEQ ID NO: 3 refers to a mutant of the polynucleotide comprising the sequence shown in SEQ ID NO: 3, and the mutant is described in SEQ ID NO: 3 ID NO: 3 has mutated nucleotides at one or more positions in positions 72-81 of the sequence shown in SEQ ID NO: 3 and does not contain a polynucleate in which positions 72-81 of the sequence shown in SEQ ID NO: 3 are mutated to ACACCGAGTG Glycosides.
  • the mutants have increased promoter activity compared to polynucleotides comprising the sequence shown in SEQ ID NO: 3.
  • the mutant has increased promoter activity in an environment of elevated salt concentration and osmotic pressure compared to a polynucleotide comprising the sequence set forth in SEQ ID NO: 3.
  • the mutants correspond to 1, 2, 3, 4, 5, 6, 7, 8, Nucleotides with mutations at 9 or 10 positions.
  • the polynucleotides having promoter activity in the present disclosure further include polynucleotides complementary in the direction of the nucleotide sequence of the mutants of the promoter of the NCgl1418 gene.
  • the polynucleotides having promoter activity in the present disclosure also include sequences that hybridize to mutants or hybrids comprising the promoter of the NCgl1418 gene under high stringency hybridization conditions or very high stringency hybridization conditions the reverse complement of the polynucleotide.
  • the nucleotide sequence of the polynucleotide in the 170-179th position corresponding to the sequence shown in SEQ ID NO:1 is not ACACCGAGTG
  • the nucleotide sequence corresponding to the 130-139th position of the sequence shown in SEQ ID NO:2 is not ACACCGAGTG.
  • the nucleotide sequence is not ACACCGAGTG
  • the nucleotide sequence at positions 72-81 corresponding to the sequence shown in SEQ ID NO: 3 is not ACACCGAGTG.
  • the polynucleotide with promoter activity in the present disclosure is 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 percentiles).
  • the nucleotide sequence of the polynucleotide corresponding to the 170-179th position of the sequence shown in SEQ ID NO:1 is not ACACCGAGTG
  • the nucleotide sequence is not ACACCGAGTG
  • the nucleotide sequence at positions 72-81 corresponding to the sequence shown in SEQ ID NO: 3 is not ACACCGAGTG.
  • the mutant corresponds to the nucleotide sequence of positions 170-179 of the sequence shown in SEQ ID NO: 1, and the nucleotide sequence corresponding to positions 130-139 of the sequence shown in SEQ ID NO: 2
  • Nucleotide sequence, the nucleotide sequence at positions 72-81 corresponding to the sequence shown in SEQ ID NO: 3 is selected from any one of the following group consisting of (p 1 )-(p 12 ): (p 1 ) TACTTGCAGA , ( p2 ) AGTGCTGAAA , (p3) GCACGAAAGG , (p4) TATCTAGAGG , (p5) AGGCTTGTCG , (p6)CGCTTCTTTC, ( p7 )TAACTCTTGG, (p8) CCAAGTTCCA , (p9)CGGTGCCACA , (p 10 ) AGCAGTTAGG, (p 11 ) AGATAAATAA, (p 12 ) ATCGATCTAG.
  • the nucleotide sequence of the mutant is selected from the sequence set forth in any one of SEQ ID Nos: 37-48.
  • the polynucleotides with promoter activity in the present disclosure have 1-8 times higher promoter activity than the polynucleotides of the sequence shown in SEQ ID NO: 1. Further, compared with the polynucleotide of the sequence shown in SEQ ID NO: 1, it has 2.11, 1.85, 3.23, 3.07, 3.57, 2.78, 3.90, 3.28, 2.62, 2.70 under the environment of increased salt concentration or osmotic pressure , 4.47, 7.62 times the enhanced promoter activity.
  • the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-F and 1418-R to amplify the promoter sequence (SEQ ID NO: 1) of the NCgl1418 gene by PCR; pXM-gfp As a template, primers pGFP-F and pGFP-R were used to amplify pXM-gfp to obtain a vector fragment with the lacI gene and tac promoter removed; the above fragments were recombined to obtain a recombinant expression vector pXM-P NCgl1418 -gfp.
  • the present disclosure uses pXM-P NCgl1418 -gfp as a template, using primer pairs 1418-203-F/R, 1418-145-F/R and 1418-94-F/R, respectively, by PCR Amplify to obtain NCgl1418 promoter fragments with 203bp, 145bp and 94bp; after the above three fragments are recovered, use T4PNK to phosphorylate the vector fragment, and construct a new vector by self-circularization, which is named as pXM-P 203- gfp, pXM-P 145 -gfp and pXM-P 94 -gfp.
  • the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-DF and 1418-DR to amplify the promoter sequence of the NCgl1418 gene by PCR.
  • primers 1418-DF and 1418-DR to amplify the promoter sequence of the NCgl1418 gene by PCR.
  • pXM-07 as a template, firstly, using primers pXM07-F1 and pXM07-R1, a vector fragment with dCpf1 was obtained by PCR amplification; The vector fragment 2 of the starting point; using pEC-26 as a template, using primers pEC26-F and pEC26-R, the crRNA array fragments targeting gltA, pgi, hom and pck genes are obtained by PCR amplification; after the above-mentioned fragments are recovered, carry out Recombinant ligation to obtain recombinant expression vector pXM-P NCgl1418 -d
  • the present disclosure uses the ATCC 13032 genome as a template and uses primers 1418-LF and 1418-LR to amplify the promoter sequence of the NCgl1418 gene and the DNA sequence of the lysE gene by PCR.
  • primers pEC-F and pEC-R were used to amplify a vector fragment by PCR to obtain a vector fragment. The above three fragments were recovered and then recombined to obtain a recombinant expression vector pEC-P NCgl1418 -lysE.
  • Corynebacterium glutamicum ATCC 13032 Corynebacterium glutamicum ATCC 13032 (Corynebacterium glutamicum ATCC 13032, genome sequence of ATCC 13032: NC_003450.3).
  • the present disclosure uses pXM-P NCgl1418 -gfp as a template, uses 1418mutant-F and 1418mutant-R primers to perform inverse PCR amplification on pXM-P NCgl1418 -gfp, and obtains a linearized plasmid fragment; The plasmid fragments were phosphorylated and ligated, and resistant clones were collected to obtain a library of promoter mutants of the NCgl1418 gene.
  • the present disclosure transforms Corynebacterium glutamicum ATCC13032 with the NCgl1418 gene promoter mutant library and pXM-Con, pXM-P NCgl1418 -gfp, respectively, to obtain recombinant host cells. Screening of mutants with increased promoter strength is performed by screening the fluorescence intensity of recombinant host cells after plating.
  • the present disclosure uses pXM-P NCgl1418 -dCpf1 as a template, uses primers 35P7-F and pXM-R, and primers pXM-F and 35P7-R1 to perform PCR amplification, respectively, to obtain two vector fragments , the above fragments were recombined to obtain the recombinant vector pXM-P 35P7 -dCpf1.
  • the present disclosure takes pEC-P NCgl1418 -lysE as a template, uses primers 35P7-F and 35P7-R2, and obtains a vector fragment with a mutated promoter 35P7 and comprising the lysE gene by PCR amplification, and then The linearized vector fragment was phosphorylated and constructed by self-cyclization to obtain pEC-P 35P7 -lysE.
  • the present disclosure can also use the promoter sequences of 35P1, 35P2, 35P3, 35P4, 35P5, 35P6, 35P8, 35P9, 35P10, 35P11, and 35P12 to construct a desired recombinant vector according to specific cloning needs.
  • the present disclosure uses the ATCC 13032 genome as a template and uses primers proP-F and proP-R to amplify the promoter sequence of the proP gene (SEQ ID NO: 56) by PCR; pXM-gfp As a template, primers pGFP-F and pGFP-R were used to amplify pXM-gfp to obtain a vector fragment with the lacI gene and tac promoter removed; the above fragments were recombined to obtain a recombinant expression vector pXM-P proP -gfp.
  • the present disclosure uses pXM-P proP -gfp as a template, uses primers proP-1-F and proP-1-R, amplifies the obtained fragment by PCR, and uses T4PNK to phosphorylate the vector fragment, And pXM-P proP-1 -gfp was obtained by self-cyclization construction.
  • pXM-P proP -gfp as a template, using primers proP-2-F and proP-2-R, the obtained fragment was amplified by PCR, the vector fragment was phosphorylated by T4PNK, and pXM was obtained by self-circularization construction -P proP-2 -gfp.
  • the present disclosure uses the ATCC 13032 genome as a template and uses primers proP-DF and proP-DR to amplify the P proP promoter sequence by PCR.
  • pXM-07 as a template, firstly, using primers pXM07-F1 and pXM07-R2, a vector fragment with dCpf1 was obtained by PCR amplification; The vector fragment 2 of the starting point; using pEC-26 as a template, using primers pEC26-F and pEC26-R, the crRNA array fragments targeting gltA, pgi, hom and pck genes are obtained by PCR amplification; after the above-mentioned fragments are recovered, carry out Recombinant ligation to obtain recombinant expression vector pXM-P proP -dCpf1.
  • the present disclosure uses pXM-P proP-1- gfp as a template, designs primers proP-DF and proP1-DR, and amplifies the P proP-1 promoter fragment by PCR.
  • pXM-07 as a template, firstly, using primers pXM07-F1 and pXM07-R2, a vector fragment with dCpf1 was obtained by PCR amplification; The vector fragment 2 of the starting point; using pEC-26 as a template, using primers pEC26-F and pEC26-R, the crRNA array fragments targeting gltA, pgi, hom and pck genes are obtained by PCR amplification; after the above-mentioned fragments are recovered, carry out Recombinant ligation to obtain recombinant expression vector pXM-P proP-1 -dCpf1.
  • the present disclosure uses pXM-P proP -gfp as a template, and uses primers proP-lysE-F and proP-lysE-R to obtain a P proP-1 promoter sequence fragment by PCR amplification; P proP-1- gfp was used as a template, and primers proP-lysE-F and proP1-lysE-R were used to amplify the P proP promoter and sequence fragments by PCR.
  • the lysE gene fragment was obtained by PCR amplification with primers lysE-F and lysE-R.
  • the vector fragment was obtained by PCR amplification.
  • the above promoter fragment was recombined with the lysE fragment and the vector fragment respectively, and the ligation product was transformed into Trans T1 competent cells, coated with kanamycin-resistant plates for overnight culture, and positive clones were picked for colony PCR verification, and the The correct transformants were confirmed by sequencing, and the obtained recombinant vectors were named pEC-P proP -lysE and pEC-P proP-1- lysE.
  • the Corynebacterium glutamicum SCgL30 strain of the present disclosure mutates the threonine at position 311 of aspartokinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 to isoleucine, A strain SCgL30 with certain lysine synthesis ability was constructed and obtained.
  • the present disclosure transforms pEC-P 35P7 -lysE into a SCgL30 recombinant strain to obtain a recombinant host cell.
  • the present disclosure transforms pXM-P 35P7 -dCpf1 into a SCgL30 recombinant strain to obtain a recombinant host cell.
  • the present disclosure can also transform SCgL30 recombinant strains with recombinant vectors comprising promoter sequences of 35P1, 35P2, 35P3, 35P4, 35P5, 35P6, 35P8, 35P9, 35P10, 35P11, and 35P12, respectively, to obtain a recombinant host cell.
  • the polynucleotide with promoter activity is operably linked with the protein encoding gene or gene expression regulatory protein encoding gene related to the synthesis of the target compound to obtain the recombination of the protein or gene expression regulatory protein related to the synthesis of the target compound
  • the expression vector is used to transform the host cell with the recombinant expression vector to obtain the recombinant host cell.
  • the polynucleotide has improved promoter activity
  • the transcriptional activity of the protein or gene expression control protein related to the synthesis of the target compound is improved, and the protein or the gene related to the synthesis of the target compound is improved.
  • the expression of gene expression regulatory proteins is increased, thereby significantly increasing the yield of target compounds.
  • no inducer is added in the steps of the method for preparing amino acids employed in the present disclosure.
  • IPTG is not added in the steps of the method for preparing amino acids employed in the present disclosure.
  • the target compound is an amino acid
  • the protein-coding gene related to the synthesis of the target compound refers to a protein-coding gene related to the synthesis of amino acids.
  • the target compound is an L-amino acid
  • the protein-coding gene associated with synthetic amino acid refers to a protein-coding gene associated with synthetic L-amino acid.
  • the L-amino acid is L-lysine
  • the protein related to amino acid synthesis is the lysine transporter LysE
  • increasing the expression of LysE with a polynucleotide with promoter activity can promote lysine Extracellular Emission and Extracellular Accumulation of Acids.
  • the gene expression regulatory protein is dCpf1, and dCpf1 can target and regulate target genes such as gltA, pgi, hom or pck; polynucleotides with promoter activity can increase the expression of dCpf1 under high-salt environment conditions, and improve the The weakening degree of the target gene further promotes the synthesis and substrate utilization of lysine.
  • the host cell is Corynebacterium glutamicum, which is an important strain for the production of L-lysine, a high-salt, high-osmolarity-inducible polynucleoside
  • Corynebacterium glutamicum is an important strain for the production of L-lysine, a high-salt, high-osmolarity-inducible polynucleoside
  • the expression of proteins related to lysine synthesis in Corynebacterium glutamicum is significantly increased, specifically in the environment of high salt and high osmotic pressure
  • the expression level of C. glutamicum was significantly increased, which greatly improved the ability of C. glutamicum to accumulate L-lysine through long-term fermentation.
  • the host cell is Corynebacterium glutamicum modified as follows: the threonine at position 311 of aspartokinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 is mutated to isoleucine acid.
  • 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. at 220 r/min, with or without addition of 0.6 according to the initial OD of 0.3.
  • M sodium sulfate (to simulate the high-salt and hypertonic environment caused by the accumulation of high-concentration products in the late fermentation period) lysine fermentation medium, the culture system is 24-well plate with 1 mL of liquid, 30 ° C, 800 r/min After culturing for 36 hours, the fermentation was terminated, and the detection Residual glucose content, OD 600 and lysine production.
  • the formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K 2 HPO 4 1.5g/L, MOPS 42g/L, FeSO 4 0.01g/L, MnSO 4 0.01 g/L, MgSO 4 0.6 g/L, chloramphenicol at a final concentration of 5 ⁇ g/mL, and/or kanamycin at a final concentration of 25 ⁇ g/mL.
  • the target compound can be recovered from the culture medium of recombinant host cells or recombinant cells by methods commonly used in the art, including but not limited to: filtration, anion exchange chromatography, crystallization or HPLC.
  • the experimental techniques and experimental methods used in the present embodiment are conventional technical methods, such as the experimental methods that do not specify specific conditions in the following examples, usually according to conventional conditions such as people such as Sambrook, molecular cloning: experiment The conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer. Materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • Corynebacterium glutamicum ATCC13032 strain was cultivated in CGXII medium with and without the addition of 0.6 M NaCl or lysine sulfate, cells were harvested in mid-logarithmic growth phase, and total RNA was extracted and analyzed by transcriptome sequencing.
  • the promoter of the NCgl1418 gene whose transcription level was significantly increased under hypertonic conditions was selected as the most candidate high-salt-hypertonic inducible promoter.
  • Primers 1418-F (SEQ ID NO: 6) and 1418-R (SEQ ID NO: 7) were designed according to the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI (NC_003450.3).
  • the promoter sequence (SEQ ID NO: 1) of the NCgl1418 gene was obtained by PCR amplification using the ATCC 13032 genome as a template.
  • the PCR amplification parameters were: 95°C for 5 min, 95°C for 30s, 65-55°C for 30s, 72°C for 1 min, cycle 10 times, 95°C for 30s, 55°C for 30s, 72°C for 1 min, cycle 25 times, and 72°C for 10min extension.
  • the vector fragment with the lacI gene and the tac promoter removed was obtained by PCR amplification.
  • the PCR amplification parameters were: 95 °C 10min , 95°C for 30s, 65-55°C for 30s, 72°C for 3min, cycle 10 times, 95°C for 30s, 55°C for 30s, 72°C for 3min, cycle 25 times, and 72°C for 10min.
  • the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification. , and the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P NCgl1418 -gfp.
  • the vector fragment was phosphorylated by T4 PNK, and the control vector pXM-con was obtained by self-circularization construction.
  • the above-mentioned recombinant vector pXM-P NCgl1418 -gfp and control vector pXM-con were transformed into Corynebacterium glutamicum ATCC 13032 to obtain recombinant strain and control strain.
  • the above strains were respectively inoculated into TSB medium containing 5 ⁇ g/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, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the CGXIIY medium with or without the addition of 0.6M different salts was respectively transferred, and the culture system was 1 mL of 24-well plate, and the GFP fluorescence intensity and OD 600 of different strains were detected after culturing for 18 h at 30 °C and 800 r/min.
  • the relative intensity of the NCgl1418 promoter under different conditions was characterized by the fluorescence intensity of the unit cell (subtracting the fluorescence intensity of the control strain under the same conditions).
  • CGXIIY medium glucose 50g/L, NH 4 Cl 16.5g/L, urea 5g/L, KH 2 PO 4 1g/L, K 2 HPO 4 1g/L, MOPS 42g/L, MgSO 4 0.25g /L, FeSO 4 ⁇ 2H 2 O 0.01g/L, MnSO 4 ⁇ H 2 O 0.01g/L, ZnSO 4 ⁇ 7H 2 O 0.001g/L, CuSO 4 0.2mg/L, NiCl ⁇ 6H 2 O 0.02mg /L, CaCl 2 0.01g/L, protocatechuic acid 0.03g/L, biotin 0.2mg/L, vitamin B1 0.1mg/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • test results are shown in Figure 1.
  • the data show that the addition of 0.6M different salts can induce the conversion rate of the NCgl1418 promoter and the expression of the reporter gene (4.1-7.7 times), so it is determined that the promoter is induced by high salt.
  • Example 3 Using the same method as Example 3, the induction effects of different concentrations of sodium sulfate on the NCgl1418 promoter were detected. The results are shown in Figure 2. The data show that the strength of the NCgl1418 promoter increases with the increase of the sodium sulfate concentration, and it exhibits an obvious gradient-induced activity within a certain range.
  • the PCR amplification parameters were: 95°C 5min, 95°C 30s, 55°C 30s, 72°C 1min, cycle 30 times, 72 °C extension 10min. Meanwhile, using pXM-P NCgl1418 -gfp as a template, using primers tuf-pGFP-F (SEQ ID NO: 12) and tuf-pGFP-R (SEQ ID NO: 13), the RBS containing P NCgl1418 was obtained by PCR amplification
  • the PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 3min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 3min, cycle 25 times, 72°C Extend for 10min.
  • the Vazyme Clon Express Multies recombination kit was used for recombination ligation, and the ligation products were transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification, and the correct results were obtained.
  • the transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P tuf -gfp.
  • the recombinant vector was transformed into Corynebacterium glutamicum ATCC 13032 to obtain a recombinant strain.
  • the strength of the NCgl1418 promoter was compared with that of Ptuf under high salt or normal medium conditions.
  • the results are shown in Figure 4.
  • the data show that the promoter strength of NCgl1418 is lower than that of P tuf under normal osmotic pressure, but under the condition of high osmotic pressure (adding 0.6M sodium sulfate), its transcriptional activity is basically the same as that of P tuf , indicating that in high salt Under osmotic pressure conditions, the promoter has a high strength and can be used for the efficient inducible expression of target genes.
  • primer pairs 1418-203-F/R SEQ ID NO: 14, 15
  • 1418-145-F/R SEQ ID NO: 16, 17
  • PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 3min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 3min, cycle 25 times, Extend for 10 min at 72°C.
  • the vector fragment is phosphorylated by T4PNK, and a new vector is obtained by self-cyclization construction, which is named as pXM-P 203 -gfp, pXM-P 145 -gfp and pXM-P 94 -gfp respectively. .
  • the data show that although the 94bp NCgl1418 promoter contains the core sequence (-35 region and -10 region), it basically loses the normal function of the promoter; The induction strength has decreased, but it can still reach more than 74% of the activity of the 243bp promoter; the promoter with a length of 203bp basically maintains the activity of the 243bp promoter under high salt osmotic pressure, which is 94% of the activity of the 243bp promoter; above The results indicated that the promoter activity of the NCgl1418 promoter and the activity under high salt osmotic pressure conditions at least required a DNA sequence with a length of 145 bp shown in SEQ ID NO: 3.
  • Primers 1418-DF SEQ ID NO: 20
  • 1418-DR SEQ ID NO: 21
  • the PCR amplification parameters were: 95 °C for 10 min, 95 °C for 30 s, 65-55 °C for 30 s, 72 °C for 1 min, cycle 10 times, 95 °C for 30 s, 55°C for 30s, 72°C for 1min, cycle 25 times, and 72°C for 10min extension.
  • the primers pXM07-F1 (SEQ ID NO: 22) and pXM07-R1 (SEQ ID NO: 23) were used to obtain a vector with dCpf1 by PCR amplification.
  • PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 3min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 3min, cycle 25 times, 72°C extension 10min; then use primers pXM07-F2 (SEQ ID NO: 24) and pXM07-R2 (SEQ ID NO: 25) to obtain a vector fragment 2 with an origin of replication by PCR amplification, and the PCR amplification parameters are: 95° C.
  • PCR amplification parameters are: 95°C 10min, 95°C 30s, 65-55°C 30s, 72°C 1min, cycle 10 times, 95°C 30s, 55°C 30s, 72°C 1min, cycle 25 times , 72 °C extension 10min.
  • the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR. After verification, the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P NCgl1418 -dCpf1.
  • the vector fragment three was obtained by PCR amplification, and the PCR amplification parameters were: 95°C 10min, 95°C for 30s, 65-55°C for 30s, 72°C for 3min, cycle 10 times, 95°C for 30s, 55°C for 30s, 72°C for 3min, cycle 30 times, and extend at 72°C for 10min; the above-mentioned fragments are recovered with the obtained NCgl1418
  • the promoter sequence fragment of the gene was recombined and ligated by the Vazyme Clon Express Multies one-step recombination kit, and the ligation product was transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification. The correct transformants were confirmed by sequencing to obtain
  • the threonine at position 311 of the aspartokinase (encoded by the lysC gene) on the genome of Corynebacterium glutamicum ATCC13032 was mutated by homologous recombination technology based on pK18mobsacB to Isoleucine was constructed to obtain a strain SCgL30 with certain lysine synthesis ability.
  • the above recombinant vectors pXM-P NCgl1418 -dCpf1 and pXM-dCpf1-con were transformed into SCgL30 strains to obtain recombinant strains and control strains.
  • the above strains were respectively inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and cultured overnight at 30°C at 220 r/min. According to the initial OD 0.3, 0.6M sodium sulfate was added or not added respectively (simulating the accumulation of high-concentration products in the later stage of fermentation caused by The lysine fermentation medium in a high-salt and hypertonic environment), the culture system was 1 mL of 24-well plate, 30 °C, 800 r/min for 24 h, and then the fermentation was terminated, and the remaining glucose content, OD 600 and lysine production were detected.
  • the lysine fermentation medium formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K 2 HPO 4 1.5g/L, MOPS 42g/L, FeSO 4 0.01g/L, MnSO 4 0.01 g/L, MgSO 4 0.6g/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • the test results are shown in Table 2. The data show that without the addition of sodium sulfate, the lysine production and glucose conversion rate of the target gene weakened strain were increased by 23% and 25%, respectively, compared with the control strain, indicating that the NCgl1418 promoter can regulate the expression of the target gene dCpf1.
  • the lysine production and the glucose conversion rate of the target gene-attenuated strain were increased by 49% and 40%, respectively, compared with the control strain, indicating that the expression intensity of dCpf1 was higher under high-salt conditions.
  • the weakening degree of the target gene is improved, and the synthesis and substrate utilization of lysine are further promoted.
  • NC_003450.3 the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI
  • primers 1418-LF SEQ ID NO: 30
  • 1418-LR SEQ ID NO: 31
  • lysE -F SEQ ID NO:32
  • lysE-R SEQ ID NO:33
  • the PCR amplification parameters were: 95°C for 5 min, 95°C for 30s, 65-55°C for 30s, 72°C for 1 min, cycle 10 times, 95°C for 30s, 55°C for 30s, 72°C for 1 min, cycle 25 times, and 72°C for 10min extension.
  • the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation, and the ligation products were transformed into Trans T1 competent cells, coated with kanamycin-resistant plates and cultured overnight, and positive clones were picked for colonization. PCR was verified, and the correct transformants were confirmed by sequencing, and the obtained recombinant vector was named pEC-P NCgl1418 -lysE.
  • sequence before and after the promoter-35 region may have an important regulatory effect on the strength of the promoter
  • sequence before and after the -35 region of the core region of the promoter of the NCgl1418 gene was randomly mutated.
  • the sequence of the core region of the NCgl1418 gene promoter is:
  • the specific mutation sequence is:
  • the pXM-P NCgl1418 -gfp obtained in Example 2 was amplified by inverse PCR, and the linearized plasmid obtained by Fragments were phosphorylated and ligated to transform E. coli T1 competent cells to obtain resistant clones. Cells were collected from all the obtained clones and plasmids were extracted to obtain two mutant libraries of the NCgl1418 gene promoter.
  • the above promoter mutant library was transformed into Corynebacterium glutamicum ATCC13032, using the strain ATCC13032 (pXM-Con) and ATCC13032 (pXM-P NCgl1418 -gfp) in Example 3 as empty vector and wild-type control, the mutant library was subjected to Three positive and negative screening.
  • the above strains were inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and after culturing for 8-10 h at 30 °C and 220 r/min, respectively, transferred to CGXIIY medium supplemented with 0.6 M Na 2 SO 4 according to the initial OD 1, and cultured.
  • the system was 1 mL of 24-well plate solution, incubated at 30°C for 6 hours at 800 r/min, and the resulting bacterial solution was diluted 50 times with PBS buffer, then sonicated for 6 minutes, and subjected to fluorescence sorting by flow cytometry (forward screening , top 0.01%).
  • the sorted mixed strains were inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and cultured overnight at 30°C at 220 r/min for the next screening.
  • the mixed strain, empty vector and wild-type control strain were inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and after culturing for 8-10 h at 30 °C and 220 r/min, respectively transferred to CGXIIY medium according to the initial OD 0.5, and cultured.
  • the system is 1 mL of 24-well plate solution. After culturing for 6 hours at 30 °C and 800 r/min, the obtained bacterial solution was diluted 50 times with PBS buffer, and then sonicated for 6 min, and subjected to fluorescence sorting by flow cytometry (reverse screening). , bottom 1%). Finally, repeat the process of the first forward screening.
  • strains obtained from the above three screenings, the wild-type NCgl1418 promoter and the control strain without promoter were respectively inoculated into TSB medium containing 5 ⁇ g/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, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the relative intensity of the mutant promoter under different conditions is characterized by the fluorescence intensity of the unit cell (subtracting the fluorescence intensity of the unit cell of the control strain under the same conditions).
  • CGXIIY medium glucose 50g/L, NH 4 Cl 16.5g/L, urea 5g/L, KH 2 PO 4 1g/L, K 2 HPO 4 1g/L, MOPS 42g/L, MgSO 4 0.25g /L, FeSO 4 ⁇ 2H 2 O 0.01g/L, MnSO 4 ⁇ H 2 O 0.01g/L, ZnSO 4 ⁇ 7H 2 O 0.001g/L, CuSO 4 0.2mg/L, NiCl ⁇ 6H 2 O 0.02mg /L, CaCl 2 0.01g/L, protocatechuic acid 0.03g/L, biotin 0.2mg/L, vitamin B1 0.1mg/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • NC_003450.3 the genome sequence of Corynebacterium glutamicum ATCC 13032 published by NCBI
  • primers 1418-DF SEQ ID NO: 21
  • 1418-DR SEQ ID NO: 22
  • the 13032 genome was used as the template to obtain the promoter sequence of the NCgl1418 gene by PCR amplification.
  • the primers pXM07-F1 SEQ ID NO: 23
  • pXM07-R1 SEQ ID NO: 24
  • the recombination ligation was carried out using the Vazyme Clon Express Multies one-step recombination kit to obtain the recombinant vector pXM-P NCgl1418 -dCpf1.
  • pXM-07 as a template, using primers pXM07-F1 (SEQ ID NO: 23) and pGFP-R (SEQ ID NO: 20), vector fragment three was obtained by PCR amplification; The promoter sequence fragment of NCgl1418 gene was recombined by Vazyme Clon Express Multies one-step recombination kit to obtain the control vector pXM-dCpf1-con.
  • primers 35P7-F (SEQ ID NO: 51) and pXM-R (SEQ ID NO: 55), pXM-F (SEQ ID NO: 54) and 35P7-R1 ( SEQ ID NO: 52), two vector fragments (containing the 35P7 promoter) were obtained by PCR amplification, and the Vazyme Clon Express Multies one-step recombination kit was used for recombination ligation to obtain the recombinant vector pXM-P 35P7 -dCpf1.
  • the corresponding control vector pXM-dCpf1-P 35P7 con was constructed and obtained using the same primers and methods described above.
  • the above recombinant vectors pXM-P 35P7 -dCpf1, pXM-P NCgl1418 -dCpf1, pXM-dCpf1-P 35P7 con and pXM-dCpf1-con were transformed into SCgL30 strains to obtain recombinant strains and control strains.
  • the above strains were respectively inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and cultured overnight at 30°C at 220 r/min. According to the initial OD 0.3, 0.6M sodium sulfate was added or not added respectively (simulating the accumulation of high-concentration products in the later stage of fermentation caused by The lysine fermentation medium in the high-salt and hypertonic environment), the culture system was 1 mL of 24-well plate, 30 ° C, 800 r/min culture for 36 h, the fermentation was terminated, the residual glucose content, OD 600 and lysine production were detected.
  • the lysine fermentation medium formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K 2 HPO 4 1.5g/L, MOPS 42g/L, FeSO 4 0.01g/L, MnSO 4 0.01 g/L, MgSO 4 0.6g/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • the test results are shown in Table 6 and Table 7. The data show that without the addition of sodium sulfate, the lysine production and glucose conversion rate of the target gene weakened strain were increased by 28% and 31%, respectively, compared with the control strain, which were higher than those of the wild-type NCgl1418 promoter. The boosts are (23% and 25%).
  • the lysine production and glucose conversion rate of the target gene-attenuated strain were increased by 52% and 55%, respectively, compared with the control strain, which was significantly higher than that of the wild-type NCgl1418 promoter (49 % and 40%).
  • Example 13 Use of mutant promoter to regulate LysE expression in lysine synthesis
  • NCBI Corynebacterium glutamicum ATCC 13032 published by NCBI (NC_003450.3)
  • primers 1418-LF SEQ ID NO: 28
  • 1418-LR SEQ ID NO: 29
  • lysE- F SEQ ID NO:30
  • lysE-R SEQ ID NO:31
  • the promoter sequence of NCgl1418 gene and the DNA sequence of lysE gene were obtained by PCR amplification using ATCC 13032 genome as template.
  • a 35P7 mutant with a mutated promoter and comprising the lysE gene was obtained by PCR amplification. vector fragment. The vector fragment was then phosphorylated using T4PNK and constructed by self-circularization to obtain pEC-P 35P7 -lysE.
  • a promoter with a length of 145bp can reach more than 74% of the activity of a 243bp promoter under high salt osmotic pressure conditions; a promoter with a length of 203bp basically maintains the 243bp promoter under high salt osmotic pressure conditions.
  • the activity of the 243bp promoter is 94% of the activity of the 243bp promoter; it shows that since the promoter fragments of SEQ ID NO: 2 and SEQ ID NO: 3 contain the core region of the promoter of the NCgl1418 gene, SEQ ID NO: 2 and SEQ ID NO: The promoter fragment of 3 can also show significantly enhanced promoter activity in the environment of elevated salt concentration and osmotic pressure.
  • the Ncgl1418 promoter mutant obtained above is obtained by mutating the partial sequence of the core region of the wild-type Ncgl1418 promoter (TATTAAAGATCACACCGA GTGGT GGAATTTCCTCAAGTGATTTAC CCACAAT GGACTTTG, the underlined place is the main sequence of the promoter-35 region and the-10 region respectively), and is obtained by mutation.
  • the promoter activity was significantly enhanced in the environment with increased salt concentration and osmotic pressure, which means that the inducible activity of Ncgl1418 can be further improved by modification. Therefore, the present disclosure intends to further replace the entire core region sequence, 5'-UTR sequence, and other region sequences of the promoter to further improve the inductive activity of the promoter.
  • ProP is a proline uptake protein that induces expression under hypertonic conditions, and enhances the tolerance of strains to hypertonic environments by increasing the uptake of proline, a compatible substance.
  • proP-F SEQ ID NO: 59
  • proP-R SEQ ID NO: 60
  • the promoter (P proP ) sequence SEQ ID NO: 56
  • the proP gene was obtained by PCR amplification using the ATCC 13032 genome as a template.
  • the Vazyme Clon Express Multies recombination kit was used for recombination ligation, and the ligation products were transformed into Trans T1 competent cells, coated with chloramphenicol-resistant plates for overnight culture, and positive clones were picked for colony PCR verification, and the correct results were obtained.
  • the transformants were confirmed by sequencing, and the obtained recombinant vector was named pXM-P proP -gfp.
  • the vector fragment was phosphorylated by T4PNK, and the control vector pXM-con was obtained by self-circularization construction.
  • the above recombinant vector was transformed into Corynebacterium glutamicum ATCC 13032 to obtain a recombinant strain.
  • TSB liquid medium containing 5 ⁇ g/mL chloramphenicol, and cultured overnight at 30°C and 220r/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, 1 g/L; MgSO 4 ⁇ 7H 2 O, 0.1 g/L; biotin, 0.01 mg/L; vitamin B1, 0.1 mg/L; MOPS, 20 g/L.
  • the CGXIIY medium with or without the addition of 0.6M Na 2 SO 4 was respectively transferred, and the culture system was 1 mL of 24-well plate, and the GFP fluorescence intensity and OD of different strains were detected after culturing for 18 h at 30 °C and 800 r/min. 600 , the relative intensities of different promoters under different conditions are characterized by the fluorescence intensity of the unit cell (subtracting the fluorescence intensity of the unit cell of the control strain under the same conditions).
  • CGXIIY medium glucose 50g/L, yeast powder 2g/L, NH 4 Cl 16.5g/L, urea 5g/L, KH 2 PO 4 1g/L, K 2 HPO 4 1g/L, MOPS 42g/ L, MgSO 4 0.25g/L, FeSO 4 ⁇ 2H 2 O 0.01g/L, MnSO 4 ⁇ H 2 O 0.01g/L, ZnSO 4 ⁇ 7H 2 O 0.001g/L, CuSO 4 0.2mg/L, NiCl ⁇ 6H 2 O 0.02 mg/L, CaCl 2 0.01 g/L, protocatechuic acid 0.03 g/L, biotin 0.2 mg/L, vitamin B1 0.1 mg/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • the present invention further verifies the effect of the hybrid transformation of the Ncgl1418 and proP promoters.
  • proP-1-F SEQ ID NO:61
  • proP-1-R SEQ ID NO:62
  • proP-2-F SEQ ID NO: 63
  • proP-2-R SEQ ID NO: 64
  • the proP promoter was replaced by PCR amplification
  • the 5'-UTR region and the core regions of the -35 and -10 regions were recovered, and then the vector fragment was phosphorylated by T4PNK and constructed by self-circularization to obtain pXM-P proP-2- gfp.
  • the above recombinant vector was transformed into Corynebacterium glutamicum ATCC 13032 to obtain a recombinant strain.
  • proP-DF SEQ ID NO: 65
  • proP-DR SEQ ID NO: 66
  • the primers proP-DF (SEQ ID NO: 65) and proP1-DR (SEQ ID NO: 67) were designed, and the P proP-1 promoter was obtained by PCR amplification Fragment.
  • the above promoter fragment is recombined with the vector fragment 1, the vector fragment 2 and the crRNA array fragment to obtain the recombinant vector pXM-P proP-1- dCpf1.
  • the vector fragment three was obtained by PCR amplification, and after the above-mentioned fragment was recovered Recombinantly connect with P proP and P proP-1 promoter sequence fragments by Vazyme Clon Express Multies one-step recombination kit to obtain corresponding control vectors pXM-P proP -con and pXM-P proP-1- con.
  • the above recombinant vectors pXM-P proP -dCpf1 and pXM-P proP-1- dCpf1, and the respective control plasmids pXM-P proP -con and pXM-P proP-1- con were transformed into SCgL30 strains to obtain recombinant strains and controls strains.
  • the above strains were respectively inoculated into TSB medium containing 5 ⁇ g/mL chloramphenicol, and cultured overnight at 30°C at 220 r/min.
  • the culture system was 1 mL of 24-well plate, 30 °C, 800 r/min for 24 h, and the fermentation was terminated, and the remaining glucose content, OD600 and lysine production were detected.
  • the lysine fermentation medium formula is: glucose 80g/L, yeast powder 8g/L, urea 9g/L, K 2 HPO 4 1.5g/L, MOPS 42g/L, FeSO 4 0.01g/L, MnSO 4 0.01 g/L, MgSO 4 0.6g/L, and the final concentration of chloramphenicol was 5 ⁇ g/mL.
  • the test results are shown in Table 10. The results show that due to the weak promoter strength, the dCpf1 weakening system regulated by the wild-type proP promoter has very limited effect on lysine production and conversion rate, while the modified mutant promoter has the same effect under normal culture conditions. No apparent effect. However, under hyperosmotic induction conditions, the dCpf1 system regulated by the mutant promoter showed a good application effect, and the lysine production and conversion rate were significantly improved.
  • Example 19 Use of mutant promoter to regulate LysE expression in lysine synthesis
  • the lysE gene fragment was obtained by PCR amplification with primers lysE-F (SEQ ID NO: 32) and lysE-R (SEQ ID NO: 33) using the ATCC 13032 genome as a template. Meanwhile, using pXM-XK99E as a template and using primers pEC-F (SEQ ID NO:34) and pEC-R (SEQ ID NO:35), a vector fragment was obtained by PCR amplification. The above promoter fragments were recombined with the lysE fragment and the vector fragment, and the ligated products were transformed into Trans T1 competent cells, coated with kanamycin-resistant plates and cultured overnight, and positive clones were picked for colony PCR verification. The correct transformants were confirmed by sequencing, and the obtained recombinant vectors were named pEC-P proP -lysE and pEC-P proP-1- lysE.

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Abstract

具有启动子活性的多核苷酸及其在生产目标化合物中的用途，具体来说，提供了具有启动子活性的多核苷酸，包含具有启动子活性的多核苷酸的转录表达盒、重组表达载体、重组宿主细胞，以及调控目标基因转录的方法、制备蛋白的方法和生产目标化合物的方法。提供的具有启动子活性的多核苷酸是一种高盐、高渗透压诱导型的启动子，在盐浓度、渗透压升高的环境下具有增强的启动子活性。多核苷酸与目标基因可操作地连接，可以显著提高目标基因在高盐、高渗透压的胁迫环境下的表达强度，进而稳定、高效的生产下游产物，有效解决了当前添加IPTG等高昂诱导剂、且对菌株造成毒性的问题。

Description

具有启动子活性的多核苷酸及其在生产目标化合物中的用途
本公开要求如下专利申请的优先权：于2021年01月12日提交中国专利局、申请号为202110036121.1、发明名称为“具有启动子活性的多核苷酸及其在生产目标化合物中的用途”的中国专利申请，于2021年05月10日提交中国专利局、申请号为202110504946.1、发明名称为“具有启动子活性的多核苷酸及其用途和生产目标化合物的方法”的中国专利申请，以及于2021年07月26日提交中国专利局、申请号为202110841507.X、发明名称为“一种突变的高渗诱导型启动子PproP及其应用”的中国专利申请；上述专利申请的全部内容通过引用结合在本公开中。
技术领域
本公开属于生物技术和基因工程技术领域，具体涉及一种具有启动子活性的多核苷酸，包含具有启动子活性的多核苷酸的转录表达盒、重组表达载体、重组宿主细胞，以及调控目标基因转录的方法、制备蛋白的方法和生产目标化合物的方法。
背景技术
棒状杆菌，尤其是非致病的谷氨酸棒状杆菌(Corynebacterium glutamicum)，由于具有较强的氨基酸合成能力，是美国FDA认定的食品安全菌株，是目前发酵工业中最常使用的菌种之一，广泛用于蛋白、氨基酸、有机酸等化学品的工业化生产。目标基因的表达调控和优化是提高蛋白或产物合成的关键，而启动子元件则是调控基因表达的重要工具。目前，谷氨酸棒状杆菌中已经鉴定或开发了一系列的强启动子 [1-2]或组成型启动子 [3]，用于调控代谢途径中关键基因的表达。尽管上述启动子元件可以精细调控目标基因的表达，然而，对于一些毒性蛋白或代谢产物的合成，组成型启动子的使用还存在较大的限制。此外，强启动子或组成型启动子的表达往往对工程菌株也会造成较大的代谢负担。
诱导型启动子可以控制转录起始的时间，因此更有利于菌株的代谢流调控和重新分配。目前tac、trc等诱导型启动子在谷氨酸棒状杆菌的代谢调控中被广泛使用。然而上述启动子往往需要额外添加昂贵的诱导剂，例如IPTG，这些诱导剂的添加也会对菌株造成一定的毒性，或对发酵体系造成较大的干扰。因此开发工业发酵条件下的自诱导系统对于工业菌株的构建至关重要。现阶段，已有研究报道了部分谷氨酸棒状杆菌中的自诱导启动子，例如赖氨酸诱导型启动子 [4]、生长过程调控型启动子 [5-6]等。但上述自诱导启动子的数量还相对较少，响应的条件也相对较窄，无法在更多的发酵体系和产物合成中普及应用。
在发酵罐中pH和溶氧严格控制的条件下，利用谷氨酸棒状杆菌生产谷氨酸、赖氨酸等大宗化学品的产量能够达到100g/L甚至200g/L以上的水平 [7-8]，因此，在发酵后期高浓度产物或中间代谢物的积累以及底物的不断流加势必引发高盐、高渗的压力。同时，高盐高渗条件也几乎是所有工业菌株在发酵后期都将面临的环境诱导因素 [9]，目前，在谷氨酸棒状杆菌中，尚无高盐高渗诱导型启动子调控目标基因表达的应用先例。
因此，鉴定高盐高渗诱导型启动子，开发构建针对发酵后期高盐高渗条件的自诱导系统，不仅可以增加可用的自诱导系统，而且可以为所有工业菌株的开发提供通用的自诱导元件，这也成为当前谷氨酸棒状杆菌工业菌株开发亟需解决的关键问题。
引用文献：
[1]CN101605890A.
[2]Becker,J.,et al.,Metab.Eng.,2011,13,159-168.
[3]Rytter,J.V.,et al.,Appl.Microbiol.Biotechnol.,2014,98,2617-2623.
[4]CN101087881A.
[5]Kim,M.J.,et al.,Appl.Microbiol.Biotechnol.,2016,100,4473-4483.
[6]Ma,Y.C.,et al.,Microb.Cell Fact.,2018,17.
[7]Xu,J.Z.,et al.,Microb Cell Fact,2020,19,39.
[8]户红通等,中国酿造,2018,37(10),51-56.
[9]Varela C et al.Applied Microbiology And Biotechnology 2003,60(5):547-555.
发明内容
发明要解决的问题
鉴于现有技术中存在的技术问题，例如，在工业发酵的过程中使用tac、trc等诱导型启动子需添加IPTG等昂贵诱导剂、且诱导剂的添加对菌株造成毒性的问题，为此，本公开提供了一种具有启动子活性的多核苷酸，前述多核苷酸在盐浓度、渗透压升高的环境下表现出增强的启动子活性。将多核苷酸与氨基酸合成相关的蛋白编码基因可操作的连接，可实现蛋白编码基因在高盐、高渗透压环境下的高效表达，有效解决了当前诱导型启动子需要添加高昂诱导剂、且诱导剂对菌株造成毒性的问题。
在一些实施方式中，本公开提供的具有启动子活性的多核苷酸，为在SEQ ID NO：1-3任一项所示序列的多核苷酸基础上获得的突变体，本公开中的突变体在盐浓度、渗透压升高的环境下表现出增强的启动子活性，将其与目标基因可操作地连接，可以使目标基因在高盐、高渗透压的环境下高效表达，避免使用IPTG等高昂的诱导剂，为目标化合物的高效生产提供了一种新型的高盐或高渗诱导型的启动子。并且，突变体的启动子活性较野生型启动子活性显著提高，具有重要的工业应用价值。
用于解决问题的方案
(1)一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(i)-(iv)中组成的组中的任一项：
(i)包含如SEQ ID NO：1-3任一序列所示的核苷酸序列；
(ii)包含如SEQ ID NO：1-3任一序列所示的核苷酸序列的反向互补序列；
(iii)在高严格性杂交条件或非常高严格性杂交条件下，能够与(i)或(ii)所示的核苷酸序列杂交的序列的反向互补序列；
(iv)与(i)或(ii)所示的核苷酸序列具有至少80％，可选至少90％，优选至少95％，更优选至少97％，更优选至少98％，最优选至少99％的序列同一性的序列。
(2)根据(1)所述的具有启动子活性的多核苷酸，其中，所述多核苷酸在盐浓度或渗透压升高的环境中具有提高的启动子活性。
(3)一种转录表达盒，其中，所述转录表达盒包含根据(1)或(2)所述的具有启动子活性的多核苷酸；可选地，所述转录表达盒还含有蛋白编码基因，所述蛋白编码基因与所述具有启动子活性的多核苷酸可操作地连接。
(4)一种重组表达载体，其中，所述重组表达载体包含根据(1)或(2)所述的具有启动子活性的多核苷酸，或根据(3)所述的转录表达盒。
(5)一种重组宿主细胞，其中，所述重组宿主细胞包含根据(3)所述的转录表达盒，或根据(4)所述的重组表达载体。
(6)根据(5)所述的重组宿主细胞，其中，所述宿主细胞来源于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属；优选地，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌；更优选地，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067。
(7)一种根据(1)或(2)所述的多核苷酸，根据(3)所述的转录表达盒，根据(4)所述的重组表达载体，根据(5)或(6)所述的重组宿主细胞在如下至少一种中的用途：
(a)调控基因的转录水平，或制备用于调控基因的转录水平的试剂或试剂盒；
(b)制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
(c)生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
(8)根据(7)所述的用途，其中，所述蛋白选自基因表达调控蛋白或与目标化合物合成相关的蛋白。
(9)根据(7)或(8)所述的用途，其中，所述目标化合物包括氨基酸和有机酸中的至少一种；可选地，所述氨基酸包括赖氨酸、谷氨酸和苏氨酸中的至少一种，所述有机酸包括柠檬酸和琥珀酸中的至少一种。
(10)一种调控目标基因转录的方法，其中，所述方法包括将(1)-(2)任一项所述的具有启动子活性的多核苷酸与目标基因可操作地连接的步骤。
(11)一种制备蛋白的方法，其特征在于，包括利用根据(3)所述的转录表达盒，根据(4)所述的重组表达载体，或根据(5)-(6)任一项所述的重组宿主细胞表达所述蛋白的步骤；可选地，所述蛋白为与目标化合物合成相关的蛋白或基因表达调控蛋白；
任选地，所述方法还包括分离或纯化所述蛋白的步骤。
(12)一种生产目标化合物的方法，其中，包括利用(3)所述的转录表达盒，(4)所述的重组表达载体，或(5)-(6)任一项所述的重组宿主细胞表达与目标化合物合成相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤；
可选地，所述目标化合物包括氨基酸和有机酸中的至少一种；可选地，所述氨基酸包括赖氨酸、谷氨酸和苏氨酸中的至少一种，所述有机酸包括柠檬酸和琥珀酸中的至少一种；
可选地，所述蛋白为与赖氨酸合成相关的蛋白；可选地，所与赖氨酸合成相关的蛋白包括天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、赖氨酸运输蛋白、转酮酶、二氨基庚二酸脱氢酶和丙酮酸羧化酶中的一种或两种以上的组合；
任选地，所述方法还包括分离或纯化所述目标化合物的步骤。
(13)一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(v)-(x)组成的组中的任一项：
(v)包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第170-179位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高于包含如SEQ ID NO：1所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：1所示序列的第170-179位中的核苷酸序列不为ACACCGAGTG；
(vi)包含如SEQ ID NO：2所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：2所示序列的第130-139位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高于包含如SEQ ID NO：2所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：2所示序列的第130-139位中的核苷酸序列不为ACACCGAGTG；
(vii)包含如SEQ ID NO：3所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：3所示序列的第72-81位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高 于包含如SEQ ID NO：3所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：3所示序列的第72-81位中的核苷酸序列不为ACACCGAGTG；
(viii)包含与(v)至(vii)中任一项所示的核苷酸序列的反向互补序列的多核苷酸；
(ix)包含在高严格性杂交条件或非常高严格性杂交条件下，能够与(v)至(vii)中任一项所示的核苷酸序列杂交的序列的反向互补序列的多核苷酸；
(x)包含与(v)至(vii)中任一项所示的核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％序列同一性的序列的多核苷酸。
(14)根据(13)所述的具有启动子活性的多核苷酸，其中，所述突变体在盐浓度或渗透压升高的环境中具有提高的启动子活性；
优选地，所述突变体与包含如SEQ ID NO：2所示序列的多核苷酸相比，在盐浓度或渗透压升高的环境中具有1-8倍以上提高的启动子活性。
(15)根据(13)-(14)任一项所述具有启动子活性的多核苷酸，其中，所述突变体对应SEQ ID NO：2所示序列的第170-179位，或对应SEQ ID NO：3所示序列的第130-139位，或对应SEQ ID NO：4所示序列的第72-81位的核苷酸序列选自如下(p 1)-(p 12)组成的组中的任一项：
(p 1)TACTTGCAGA，
(p 2)AGTGCTGAAA，
(p 3)GCACGAAAGG，
(p 4)TATCTAGAGG，
(p 5)AGGCTTGTCG，
(p 6)CGCTTCTTTC，
(p 7)TAACTCTTGG，
(p 8)CCAAGTTCCA，
(p 9)CGGTGCCACA，
(p 10)AGCAGTTAGG，
(p 11)AGATAAATAA，
(p 12)ATCGATCTAG。
(16)根据(13)-(15)任一项所述的具有启动子活性的多核苷酸，其中，所述突变体的核苷酸序列选自如SEQ ID NO：37-48任一项所示的序列。
(17)一种转录表达盒，其中，所述转录表达盒包含根据(13)-(16)任一项所述的具有启动子活性的多核苷酸；可选地，所述转录表达盒还含有目标基因，所述目标基因与所述具有启动子活性的多核苷酸可操作地连接；优选地，所述目标基因为蛋白编码基因。
(18)一种重组表达载体，其中，所述重组表达载体包含(13)-(16)任一项所述的具有启动子活性的多核苷酸，或(17)所述的转录表达盒。
(19)一种重组宿主细胞，其中，所述重组宿主细胞包含(17)所述的转录表达盒，或(18)所述的重组表达载体。
(20)根据(19)所述的重组宿主细胞，其中，所述宿主细胞来源于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属；优选地，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌；更优选地，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067。
(21)一种根据(13)-(16)任一项所述的具有启动子活性的多核苷酸，根据(17)所述的转录表达盒，根据(18)所述的重组表达载体，根据(19)或(20)所述的重组宿主细胞在如下至少一种中的用途：
(a)调控基因的转录水平，或制备用于调控基因的转录水平的试剂或试剂盒；
(b)制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
(c)生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
(22)根据(21)所述的用途，其中，所述蛋白选自基因表达调控蛋白或与目标化合物合成相关的蛋白。
(23)根据(21)或(22)所述的用途，其中，所述目标化合物包括氨基酸、有机酸中的至少一种；可选地，所述氨基酸包括脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸中的至少一种，所述有机酸包括柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸中的至少一种。
(24)一种调控目标基因转录的方法，其中，所述方法包括将(13)-(16)任一项所述的具有启动子活性的多核苷酸与目标基因可操作地连接的步骤。
(25)一种制备蛋白的方法，其中，所述方法包括利用(17)所述的转录表达盒，(18)所述的重组表达载体，或(19)-(20)任一项所述的重组宿主细胞表达所述蛋白的步骤；可选地，所述蛋白为与目标化合物合成相关的蛋白或基因表达调控蛋白；
任选地，所述方法还包括分离或纯化所述蛋白的步骤。
(26)一种生产目标化合物的方法，其中，所述方法包括利用(17)所述的转录表达盒，(18)所述的重组表达载体，或(19)-(20)任一项所述的重组宿主细胞表达与目标化合物合成相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤；
可选地，所述目标化合物包括氨基酸、有机酸中的至少一种；可选地，所述氨基酸包括赖氨酸、谷氨酸、苏氨酸、脯氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸中的至少一种，所述有机酸包括柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸中的至少一种；
可选地，所述与目标化合物合成相关的蛋白为与L-氨基酸合成相关的蛋白；可选地，所与L-氨基酸合成相关的蛋白包括丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、转酮酶、二氨基庚二酸脱氢酶和丙酮酸羧化酶中的一种或两种以上的组合；
任选地，所述方法还包括分离或纯化所述目标化合物的步骤。
(27)一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(xi)-(xiv)中组成的组中的任一项：
(xi)包含如SEQ ID NO：57-58任一序列所示的核苷酸序列；
(xii)包含如SEQ ID NO：57-58任一序列所示的核苷酸序列的反向互补序列；
(xiii)在高严格性杂交条件或非常高严格性杂交条件下，能够与(xi)或(xii)所示的核苷酸序列杂交的序列的反向互补序列，且在盐浓度或渗透压升高的环境中具有提高的启动子活性；
(xiv)与(xi)或(xii)所示的核苷酸序列具有至少99％的序列同一性的序列，且在盐浓度或渗透压升高的环境中具有提高的启动子活性。
(28)一种转录表达盒，其包含如(27)所述的具有启动子活性的多核苷酸，并与目标基因可操作连接。
(29)一种重组表达载体，其包含如(27)所述的具有启动子活性的多核苷酸，或如(28)所述的转录表达盒。
(30)一种重组宿主细胞，其包含如(28)所述的转录表达盒，或如(29)所述的重组表达载体。
(31)如(30)所述的重组宿主细胞，所述宿主细胞属于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属。
(32)如(30)所述的重组宿主细胞，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067或谷氨酸棒杆菌的衍生菌株。
(33)一种调控目标基因转录的方法，其特征在于，包括将(27)所述的具有启动子活性的多核苷酸与目标RNA或目标基因可操作地连接的步骤。
(34)如(33)所述的方法，其特征在于，所述目标RNA是tRNA、sRNA中的至少一种；所述目标基因包括与目标化合物合成相关的蛋白的编码基因、基因表达调控蛋白的编码基因、与膜转运相关的蛋白的编码基因中的至少一种。
(35)一种制备蛋白的方法，包括利用(28)所述的转录表达盒，(29)所述的重组表达载体，或(30)-(32)任一项所述的重组宿主细胞表达目标蛋白的步骤。
(36)如(35)所述的方法，所述目标蛋白为与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白。
(37)如(35)或(36)所述的方法，还包括分离或纯化所述目标蛋白的步骤。
(38)一种生产目标化合物的方法，其中，包括利用(28)所述的转录表达盒，(29)所述的重组表达载体，或(30)-(32)任一项所述的重组宿主细胞表达与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白、与膜转运相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤。
(39)如(38)所述的方法，所述目标化合物是氨基酸、有机酸中的至少一种。
(40)如(39)所述的方法，所述氨基酸是如下的一种或两种以上的组合：脯氨酸、羟脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物。
(41)如(38)或(39)所述的方法，所述与目标化合物合成相关的蛋白是丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶二羧酸还原酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、转酮酶、二氨基庚二酸脱氢酶中的一种或两种以上的组合。
(42)如(39)所述的方法，所述有机酸是如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述任一种的有机酸的衍生物。
(43)如(38)至(42)任一项所述的方法，还包括分离或纯化所述目标化合物的步骤。
(44)如(27)所述的具有启动子活性的多核苷酸，(28)所述的转录表达盒，(29)所述的重组表达载体，(30)-(32)任一项所述的重组宿主细胞在如下至少一种中的用途：
a.调控基因的转录水平，或制备用于调控基因的转录水平的试剂或试剂盒；
b.制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
c.生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
发明的效果
在一个实施方案中，本公开提供了具有启动子活性的多核苷酸，其是一种高盐、高渗透压诱导型的启动子，在盐浓度、渗透压升高的环境下具有增强的启动子活性。多核苷酸与目标基因可操作地连接，可以显著提高目标基因在高盐、高渗透压的胁迫环境下的表达强度，进而稳定、高效的生产下游产物，有效解决了当前添加IPTG等高昂诱导剂、且对菌株造成毒性的问题。
在另一个实施方案中，本公开提供了转录表达盒、重组表达载体、重组宿主细胞，包含上述具有启动子活性的多核苷酸。在转录表达盒、重组表达载体、重组宿主细胞中，具有启动子活性的多核苷酸与蛋白编码基因可操作地连接，可以提高蛋白编码基因在高盐、高渗透压的胁迫环境下的表达强度。
在另一个实施方案中，本公开提供了生产氨基酸的方法，利用上述具有启动子活性的多核苷酸，能够提高与氨基酸合成相关的蛋白在胁迫环境下的表达，同时弱化其他途径蛋白的表达，使得代谢流更多地向氨基酸合成方向富集，从而稳定、高效的生产氨基酸，达到过量积累氨基酸的目的。
在另一个实施方案中，当以上述的方法生产L-赖氨酸时，能够在高盐、高渗透压的环境下稳定、高效的生产L-赖氨酸。
在一些实施方式中，本公开提供的具有启动子活性的多核苷酸，为在如SEQ ID NO：1-3任一项所示序列的多核苷酸基础上获得的突变体，本公开中的突变体在盐浓度或渗透压升高的环境下具有增强的启动子活性，是一种高盐、高渗透压诱导型的启动子。与tac、trc等诱导型启动子相比，本公开中的突变体不需要价格高昂并具有一定毒性的IPTG为诱导剂。与野生型启动子相比，本公开中的突变体在高盐、高渗透压环境下的启动子活性较野生型启动子进一步提高。将突变体与目标基因可操作地连接，可以提高目标基因的表达效率，进而稳定、高效的生产目标化合物。并且，在高盐或高渗透压诱导的环境下，将突变体应用于目标化合物生产的转化率高于野生型的启动子，为氨基酸、有机酸等目标化合物的工业发酵提供了一种极具应用潜力的诱导型启动子。在一些实施方式中，本公开提供的如SEQ ID NO：1-3任一项所示序列的多核苷酸的突变体，其启动子活性与野生型的启动子相比，在盐浓度或渗透压升高的环境中具有1-8倍以上提高的启动子活性。
在一些更为具体的实施方式中，本公开提供的如SEQ ID NO：1-3任一项所示序列的多核苷酸的突变体，其启动子活性与野生型的启动子相比，在盐浓度或渗透压升高的环境中具有1.85-7.62倍提高的启动子活性。
在一些实施方式中，本公开提供了转录表达盒、重组表达载体、重组宿主细胞，包含上述具有启动子活性的多核苷酸。在转录表达盒、重组表达载体、重组宿主细胞中，具有启动子活性的多核苷酸与目标基因可操作地连接，能够提高目标化合物合成途径中关键基因在高盐、高渗透压的胁迫环境下的表达强度。
在一些实施方式中，本公开提供了制备蛋白的方法，能够提高与氨基酸、有机酸等合成相关的蛋白或基因表达调控蛋白的表达量，进而实现目标化合物的高效生产。
在一些实施方式中，本公开提供了生产目标化合物的方法，利用上述具有启动子活性的多核苷酸，能够提高与目标化合物合成相关的蛋白在胁迫环境下的表达，从而稳定、高效的生产目标化合物，达到提高目标化合物的代谢流、过量积累目标化合物的目的。
附图说明
图1示出了高渗透压对NCgl1418启动子的诱导作用；
图2示出了不同浓度硫酸钠对NCgl1418启动子的诱导作用；
图3示出了高浓度糖对NCgl1418启动子的诱导作用；
图4示出了P NCgl1418与P tuf的启动子强度对比结果；
图5示出了不同长度NCgl1418启动子的活性比较结果；
图6proP基因启动子及突变启动子强度及高渗诱导活性；
图7示出了proP基因启动子与NCgl1418启动子的活性比较结果。
具体实施方式
当在权利要求和/或说明书中与术语“包含”联用时，词语“一(a)”或“一(an)”可以指“一个”，但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的，词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中，术语“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
当用于权利要求书或说明书时，选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
在本公开中，“高盐环境”可以是培养基中高浓度Na 2SO 4、NaCl、K 2SO 4、KCl等无机盐离子，或是随发酵时间延长、发酵液中赖氨酸等产物或某些中间代谢物积累而增加的浓度(例如，赖氨酸硫酸盐等)，或是由于底物流加而增加的浓度(例如，硫酸铵等底物)，或是发酵液中可能出现的其他任意盐的浓度。在一些具体的实施方案中，“高盐环境”涉及在0.2M以上的盐浓度；在一些更为具体的实施方案中，“高盐环境”涉及在0.2-0.8M的盐浓度。例如，盐浓度为0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M。
在本公开中，“高渗透压环境”是指响应提高的盐浓度而升高的渗透压。
本公开中的术语“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式，也可以是更大的核苷酸序列结构的一个组成部分，其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的，能够通过标准分子生物学方法(例如，使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时，这也包括一个RNA序列(即A、U、G、C)，其中“U”取代“T”。换句话说，“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合 物，或者可以是一个较大核苷酸结构的组成部分或成分，如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。
本公开中的术语“序列同一性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
在一些具体的实施方案中，具有启动子活性的多核苷酸包含与SEQ ID NO：1-3、57-58任一序列所示的核苷酸序列的反向互补序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
在一些具体的实施方式中，具有启动子活性的多核苷酸包含在高严格性杂交条件或非常高严格性杂交条件下，与SEQ ID NO：1-3、57-58任一序列所示的核苷酸序列或其反向互补序列杂交的序列的反向互补序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
在一些具体的实施方案中，具有启动子活性的多核苷酸包含与上述任一种的核苷酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％的序列同一性的序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
如本公开所使用的，术语“野生型的”指在自然界中可以找到的对象。例如，一种存在于生物体中，可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。如本公开所用的，“天然存在的”和“野生型的”是同义词。在一些实施方式中，本公开中野生型的启动子是指野生型NCgl1418基因的启动子，也即如SEQ ID NO：1所示序列的多核苷酸。
如本公开所使用的，术语“突变体”是指相对于“野生型”，或者“相比较的”多核苷酸或多肽，在一个或多个(例如，若干个)位置处包含改变(即，取代、插入和/或缺的多核苷酸，其中，取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。插入是指在邻接并且紧随占据位置的核苷酸之后添加核苷酸。
在一些实施方式中，本公开的“突变”为“取代”，是由一个或多个核苷酸中的碱基被另一个不同的碱基取代所引起的突变，也称为碱基置换突变(subsititution)或点突变(point mutation)。
具体来说，SEQ ID NO：36所示的序列是NCgl1418基因启动子的核心区序列，包括-35区和-10区主要序列。本公开中的突变体是在-35区附近位置处引入的突变的核苷酸，并且发现在上述位置处引入突变后，突变体在高盐或高渗透压下的启动子活性明显增强。
在一些实施方式中，包含如SEQ ID NO：1所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第170-179位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：1所示序列的第170-179位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：1所示序列的多核苷酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID  NO：1所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，包含如SEQ ID NO：2所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：2所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：2所示序列的第130-139位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：2所示序列的第130-139位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：2所示序列的多核苷酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID NO：2所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，包含如SEQ ID NO：3所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：3所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：3所示序列的第72-81位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：3所示序列的第72-81位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：3所示序列的多核苷酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID NO：3所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，本公开中的突变体，与如SEQ ID NO：1所示序列的多核苷酸相比，在盐浓度或渗透压升高的环境下具有1-8倍以上提高的启动子活性。
进一步的，突变体与SEQ ID NO：1所示序列的多核苷酸相比，在盐浓度或渗透压升高的环境下具有2.11、1.85、3.23、3.07、3.57、2.78、3.90、3.28、2.62、2.70、4.47、7.62倍的提高的启动子活性。
如本公开所使用的，术语“高严格条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在65℃处使用2X SSC、0.2％SDS将载体材料洗涤三次，每次15分钟。
如本公开所使用的，术语“非常高严格条件”是指，对于长度为至少100个核苷酸的探针而言，遵循标准DNA印迹程序，在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3％SDS、200微克/ml剪切并变性的鲑精DNA和50％甲酰胺中预杂交和杂交12至24小时。最后在70℃处使用2X SSC、0.2％SDS将载体材料洗涤三次，每次15分钟。
本文中的术语“互补的”是指在核苷酸或核苷酸之间的杂交或碱基配对，例如双链DNA分子的两条链之间或者寡核苷酸引物与被测序或扩增的单链核苷酸上的引物结合位点之间等。
本公开中的术语“启动子”是指一种核酸分子，通常位于目标基因编码序列的上游，为RNA聚合酶提供识别位点，并位于mRNA转录起始位点的5’方向的上游。它是不被翻译的核酸序列，RNA聚合酶与这一核酸序列结合后启动目标基因的转录。在核糖核酸(RNA)的合成中，启动子可以和调控基因转录的转录因子产生相互作用，控制基因表达(转录)的起始时间和表达的程度，包含核心启动子区域和调控区域，就像“开关”，决定基因的活动，继而控制细胞开始生产哪一种蛋白质。
本公开中的术语“启动子核心区”是指位于原核生物启动子区的一段核酸序列，是发挥启动子功能的核心序列区，主要包括-35区、-10区、-35区和-10区之间的区域以及转录起始位点，-35区是RNA聚合酶的识别位点，-10区是RNA聚合酶的结合位点。
在一些具体的实施方案中，本公开中的具有启动子活性的多核苷酸能够用于起始蛋白编码基因的表达。在另外一些实施方案中，本公开中的具有启动子活性的多核苷酸能够用于起始非编码基因的表达。
本公开中的术语“表达”包括涉及RNA产生及蛋白产生的任何步骤，包括但不限于：转录、转录后修饰、翻译、翻译后修饰和分泌。
本公开中的术语“目标基因”涉及与本公开中具有启动子活性的多核苷酸连接，以对其转录水平进行调控的任一种的基因。
在一些实施方案中，目标基因是指编码微生物中目标蛋白质的基因。示例性的，目标基因是编码与目标化合物的生物合成相关的酶的基因、编码与还原力相关的酶的基因，编码与糖酵解或TCA循环相关的酶的基因，或编码与目标化合物的释放相关的酶的基因等等。
本公开中的术语“目标化合物”可以选自氨基酸和有机酸中的至少一种，也可以选自本领域中可能通过生物合成得到的其他种类的化合物。
在一些实施方案中，目标化合物为“氨基酸”或“L-氨基酸”。“氨基酸”或“L-氨基酸”通常是指其中氨基和羧基结合至相同碳原子的蛋白质的基本构成单元。示例性的，氨基酸选自甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苏氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、谷氨酸、赖氨酸、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸、脯氨酸中的一种或两种以上的组合，或者是本领域中其他种类的氨基酸。
在一些实施方案中，目标化合物为有机酸。有机酸可以是具有酸性的有机化合物，例如，其中包括羧基和磺酸基的那些化合物。示例性的，有机酸包括乳酸、醋酸、琥珀酸、丁酸、棕榈酸、草酸、酒石酸、柠檬酸、丙酸、己烯酸、癸酸、辛酸、戊酸、草酰乙酸、苹果酸中的一种或两种以上的组合，或者是本领域中其他种类的有机酸。
本公开中的术语“蛋白编码基因”是指能够通过一定的规则指导蛋白的合成DNA分子，蛋白编码基因指导蛋白合成的过程一般包括以双链DNA为模板的转录过程和以mRNA为模板的翻译过程。蛋白编码基因含有CDS序列(Coding Sequence)，能够指导编码蛋白质的mRNA的产生。
示例性的，蛋白编码基因包括但不限于用于编码与目标化合物合成相关的蛋白，在一些实施方式中，蛋白编码基因涉及用于编码与合成L-氨基酸的相关的蛋白。示例性的，与合成L-氨基酸的相关的蛋白包括但不限于丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶中的一种或两种以上的组合。在一些实施方案中，蛋白编码基因涉及用于编码与合成L-赖氨酸的相关的蛋白。对于与合成L-赖氨酸的相关的蛋白，包括天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、赖氨酸运输蛋白、转酮酶、二氨基庚二酸脱氢酶和丙酮酸羧化酶中的一种或两种以上的组合。
在一些实施方案中，蛋白编码基因涉及用于编码与合成有机酸相关的蛋白，示例性的，蛋白编码基因用于编码与合成柠檬酸有关的蛋白，或用于编码与合成琥珀酸有关的蛋白。
在另外一些实施方案中，蛋白编码基因涉及与基因编辑相关的蛋白，例如Cpf1蛋白。本公开的具有启动子活性的多核苷酸，可适于提高目标基因在高盐、高渗透压的胁迫环境下的表达，实现目标产物的高效生产。
本公开的术语“基因表达调控蛋白”包括不限于外源的基因表达调控工具蛋白，例如CRISPRi调控需要的dCas9蛋白、dCpf1蛋白，sRNA调控需要的Hfq蛋白等，以及内源或外源的转录调控因子，进而调控代谢通路中关键基因的表达。
本公开中的术语“转录表达盒”指的包含转录调控元件与目标基因，利用转录调控元件对目标基因的表达进行调控的一类表达元件。在本公开中，转录调控元件包含启动子，在此基础上，还可以包含增强子、沉默子、绝缘子等元件。在本公开中，目标基因具体为蛋白编码基因。目标基因与多核苷酸“可操作地连接”，是指将具有启动子活性的多核苷酸与目标基因功能性连接，以启动和介导目标基因的转录，所述可操作地连接的方式可以采用本领域技术人员所述的任何方式。
本公开中的术语“载体”指的是DNA构建体，其含有与合适的控制序列可操作地连接的DNA序列，从而在合适的宿主中表达目标基因。“重组表达载体”指用于表达例如编码所需多肽的多核苷酸的DNA结构。重组表达载体可包括，例如包含i)对基因表达具有调控作用的遗传元素的集合，例如启动子和增强子；ii)转录成mRNA并翻译成蛋白质的结构或编码序列；以及iii)适当的转录和翻译起始和终止序列的转录亚单位。重组表达载体以任何合适的方式构建。载体的性质并不重要，并可以使用任何载体，包括质粒、病毒、噬菌体和转座子。用于本公开的可能载体包括但不限于染色体、非染色体和合成DNA序列，例如细菌质粒、噬菌体DNA、酵母质粒以及从质粒和噬菌体DNA的组合中衍生的载体，来自如牛痘、腺病毒、鸡痘、杆状病毒、SV40和伪狂犬病等病毒的DNA。
本公开中的术语“宿主细胞”意指易于用包含本公开的多核苷酸的转录起始元件或表达载体转化、转染、转导等的任何细胞类型。术语“重组宿主细胞”涵盖导入转录起始元件或重组表达载体后不同于亲本细胞的宿主细胞，重组宿主细胞具体通过转化来实现。
本公开中的术语“转化”具有本领域技术人员普遍理解的意思，即将外源性的DNA导入宿主的过程。所述转化的方法包括任何将核酸导入细胞的方法，这些方法包括但不限于电穿孔法、磷酸钙沉淀法、氯化钙(CaCl 2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
本公开的宿主细胞可以是原核细胞或真核细胞，只要是能够导入本公开的具有启动子活性的多核苷酸的细胞即可。在一个实施方案中，宿主细胞指原核细胞，具体地，宿主细胞来源于适合发酵生产氨基酸的微生物，例如棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属。作为优选地，宿主细胞是来源于棒状杆菌属的谷氨酸棒杆菌。其中，谷氨酸棒杆菌可以是谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067等，以及由上述菌株制备的产生氨基酸的突变体菌株或谷氨酸棒杆菌的衍生菌株。在一些实施方式中，本公开中的宿主细胞可以是具有氨基酸生产能力的任意类型的菌株，其包括野生型菌株和重组菌株。
示例地，宿主细胞为生产赖氨酸的宿主细胞。在一些实施方式中，对于生产赖氨酸的宿主细胞，可以是在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶的菌株。此外，生产赖氨酸的宿主细胞也可以是具有赖氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产赖氨酸的宿主细胞中选自以下的一个或多个基因被弱化或表达降低：
a.编码乙醇脱氢酶的adhE基因；
b.编码乙酸激酶的ackA基因；
c.编码磷酸乙酰转移酶的pta基因；
d.编码乳酸脱氢酶的ldhA基因；
e.编码甲酸转运蛋白的focA基因；
f.编码丙酮酸甲酸裂解酶的pflB基因；
g.编码丙酮酸氧化酶的poxB基因；
h.编码天冬氨酸激酶I/高丝氨酸脱氢酶I双功能酶的thrA基因；
i.编码高丝氨酸激酶的thrB基因；
j.编码赖氨酸脱羧酶的ldcC基因；和
h.编码赖氨酸脱羧酶的cadA基因。
在一些实施方式中，所述生产赖氨酸的宿主细胞中选自以下的一个或多个基因被增强或过表达：
a.编码解除赖氨酸反馈抑制的二氢二吡啶合成酶的dapA基因；
b.编码二氢二吡啶二羧酸还原酶的dapB基因；
c.编码二氨基庚二酸脱氢酶的ddh基因；
d.编码四氢吡啶二羧酸琥珀酰酶的dapD和编码琥珀酰二氨基庚二酸脱酰酶的dapE；
e.编码天冬氨酸-半醛脱氢酶的asd基因；
f.编码磷酸烯醇丙酮酸羧化酶的ppc基因；
g.编码烟酸胺腺嘌呤二核苷酸转氢酶的pntAB基因；
i.编码赖氨酸的运输蛋白lysE基因。
示例地，宿主细胞为生产苏氨酸的宿主细胞。在一些实施方式中，生产苏氨酸的宿主细胞为在谷氨酸棒杆菌ATCC 13032基础上表达解除反馈抑制的天冬氨酸激酶LysC的菌株。在另外一些实施方式中，生产苏氨酸的宿主细胞也可以是具有苏氨酸生产能力的其他种类的菌株。
在一些实施方式中，所述生产苏氨酸的宿主细胞中选自以下的一个或多个基因被增强或过表达：
a.编码苏氨酸操纵子的thrABC基因；
b.编码解除反馈抑制的高丝氨酸脱氢酶的hom基因；
c.编码甘油醛-3-磷酸脱氢酶的gap基因；
d.编码丙酮酸羧化酶的pyc基因；
e.编码苹果酸：醌氧化还原酶的mqo基因；
f.编码转酮酶的tkt基因；
g.编码6-磷酸葡糖酸脱氢酶的gnd基因；
h.编码苏氨酸输出的thrE基因；
i.编码烯醇酶的eno基因。
示例地，宿主细胞为生产异亮氨酸的宿主细胞。在一些实施方式中，生产异亮氨酸的宿主细胞是通过用丙氨酸取代L-苏氨酸脱水酶ilvA基因第323位的氨基酸而产生L-异亮氨酸的菌株。在另外一些实施方式中，生产异亮氨酸的宿主细胞也可以是具有异亮氨酸生产能力的其他种类的菌株。
示例地，宿主细胞为生产O-乙酰高丝氨酸的宿主细胞。在一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞是通过使O-乙酰高丝氨酸(硫醇)-裂解酶失活而产生O-乙酰高丝氨酸的菌株。在另外一些实施方式中，生产O-乙酰高丝氨酸的宿主细胞也可以是具有O-乙酰高丝氨酸生产能力的其他种类的菌株。
示例地，宿主细胞为生产蛋氨酸的宿主细胞。在一些实施方式中，生产蛋氨酸的宿主细胞是通过使甲硫氨酸和半胱氨酸的转录调节因子失活而产生蛋氨酸的菌株。在另外一些实施方式中，生产蛋氨酸的宿主细胞也可以是具有蛋氨酸生产能力的其他种类的菌株。
本公开的宿主细胞的培养可以根据本领域的常规方法进行，包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
本公开中的具有启动子活性的多核苷酸作为一种自诱导型的启动子，可在高盐、高渗透压的环境下表现出增强的启动子活性，从而避免了在发酵环境中添加本领域中常用的IPTG等价格高且具有毒性的诱导剂。
除非在本公开中另外定义或由背景清楚指示，否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
高盐、高渗透压诱导型的启动子
在本公开中，利用添加和不添加0.6M NaCl或赖氨酸硫酸盐的CGXII培养基培养谷氨酸棒状杆菌ATCC13032菌株，在对数生长中期收集细胞，提取总RNA并进行转录组测序分析。选择在高渗条件下转录水平明显上升的NCgl1418基因的启动子最为候选高盐高渗诱导型启动子。
在一些具体的实施方案中，NCgl1418启动子选自如下(i)-(iv)中组成的组中的任一项：
(i)包含如SEQ ID NO：1-3任一序列所示的核苷酸序列。
(ii)包含如SEQ ID NO：1-3任一序列所示的核苷酸序列的反向互补序列。
(iii)在高严格性杂交条件或非常高严格性杂交条件下，能够与(i)或(ii)所示的核苷酸序列杂交的序列的反向互补序列；
(iv)与(i)或(ii)所示的核苷酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性的序列。
在一些具体的实施方案中，NCgl1418启动子在高盐环境下具有提高的启动子活性。在一些具体的实施方案中，NCgl1418启动子在高渗透压环境下具有提高的启动子活性。
在本公开中，“高盐环境”可以是培养基中高浓度Na 2SO 4、NaCl、K 2SO 4、KCl等无机盐离子，或是随发酵时间延长、发酵液中赖氨酸等产物或某些中间代谢物积累而增加的浓度(例如，赖氨酸硫酸盐等)，或是由于底物流加而增加的浓度(例如，硫酸铵等底物)，或是发酵液中可能出现的其他任意盐的浓度。在一些具体的实施方案中，“高盐环境”涉及在0.2M以上的盐浓度；在一些更为具体的实施方案中，“高盐环境”涉及在0.2-0.8M的盐浓度。例如，盐浓度为0.2M、0.3M、0.4M、0.5M、0.6M、0.7M、0.8M。
在本公开中，“高渗透压环境”是指响应提高的盐浓度而升高的渗透压。
在一些优选的实施方案中，NCgl1418启动子在以硫酸盐、盐酸盐所形成的“高盐环境”下表现出更高的启动子活性或更高的转化效率；在一些优选的实施方案中，硫酸盐为Na 2SO 4或K 2SO 4、赖氨酸硫酸盐，盐酸盐为赖氨酸盐酸盐、氯化钠或氯化钾。
在一些优选的实施方案中，SEQ ID NO：57-58任一序列所示的核苷酸序列的启动子在以硫酸盐所形成的“高盐环境”下表现出更高的启动子活性或更高的转化效率；在一些优选的实施方案中，硫酸盐为Na 2SO 4或K 2SO 4。
在一些具体的实施方案中，具有启动子活性的多核苷酸包含与SEQ ID NO：57-58任一序列所示的核苷酸序列的反向互补序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
在一些具体的实施方式中，具有启动子活性的多核苷酸包含在高严格性杂交条件或非常高严格性杂交条件下，与SEQ ID NO：57-58任一序列所示的核苷酸序列或其反向互补序列杂交的序列的反向互补序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
在一些具体的实施方案中，具有启动子活性的多核苷酸包含与上述任一种的核苷酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、 92％、93％、94％、95％、96％、97％、98％、99％、100％的序列同一性的序列，且多核苷酸保持高盐、高渗透压诱导型的启动子活性。
包含NCgl1418基因的启动子核心区的突变体
本公开利用NCgl1418基因的启动子核心区序列，在NCgl1418基因的启动子-35区上游引入突变，得到包含-35区上游突变的NCgl1418基因的启动子核心区的突变体。
具体地是在NCgl1418基因的启动子核心区的-35区上游的ACACCGAGTG引入突变，获得了在高盐或高渗透压环境下启动子活性进一步提高的突变体，为发酵微生物的基因工程育种以及目标化合物的高效生产提供了新型的诱导型启动子。与NCgl1418基因的野生型启动子相比，本公开中的突变体在盐浓度或渗透压升高的环境下表现出提高的启动子活性；在应用于目标化合物的发酵时，突变体与野生型启动子相比，表现出更高的目标化合物的转化率。
此外，通过对启动子进行不同长度的截短，分别获得了具有203bp(SEQ ID NO:2)、145bp(SEQ ID NO:3)的NCgl1418启动子片段，这两个片段都具备NCgl1418启动子的核心区，在盐浓度、渗透压升高的环境下也同样能够表现出明显增强的启动子活性。因此，采用上述实施例中的启动子改造方法，即对SEQ ID NO：2所示序列的第130-139位中的一个或多个位置进行突变，或者对SEQ ID NO：3所示序列的第72-81位中的一个或多个位置进行突变，会获得同样诱导活性增强的启动子突变体。
在本公开中，“高盐环境”可以是培养基中高浓度Na 2SO 4、NaCl、K 2SO 4、KCl等无机盐离子，或是随发酵时间延长、发酵液中赖氨酸等产物或某些中间代谢物积累而增加的浓度(例如，赖氨酸硫酸盐等)，或是由于底物流加而增加的浓度(例如，硫酸铵等底物)，或是发酵液中可能出现的其他任意盐的浓度。
在一些实施方式中，包含如SEQ ID NO：1所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第170-179位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：1所示序列的第170-179位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：1所示序列的多核苷酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID NO：1所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，突变体在对应SEQ ID NO：1所示序列的第170-179位中的1个、2个、3个、4个、5个、6个、7个、8个、9个或10个位置处具有突变的核苷酸。
在一些实施方式中，包含如SEQ ID NO：2所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：2所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：2所示序列的第130-139位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：2所示序列的第130-139位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：2所示序列的多核苷酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID NO：2所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，突变体在对应SEQ ID NO：2所示序列的第130-139位中的1个、2个、3个、4个、5个、6个、7个、8个、9个或10个位置处具有突变的核苷酸。
在一些实施方式中，包含如SEQ ID NO：3所示序列的多核苷酸的突变体，是指包含如SEQ ID NO：3所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：3所示序列的第72-81位中的一个或多个位置处具有突变的核苷酸，且不包含SEQ ID NO：3所示序列的第72-81位突变为ACACCGAGTG的多核苷酸。与包含SEQ ID NO：3所示序列的多核苷 酸相比，突变体具有提高的启动子活性。在一些更具体地实施方式中，与包含SEQ ID NO：3所示序列的多核苷酸相比，突变体在盐浓度、渗透压升高的环境下具有提高的启动子活性。
在一些实施方式中，突变体在对应SEQ ID NO：3所示序列的第72-81位中的1个、2个、3个、4个、5个、6个、7个、8个、9个或10个位置处具有突变的核苷酸。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，还包括与NCgl1418基因的启动子的突变体的核苷酸序列方向互补的多核苷酸。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，还包括在高严格性杂交条件或非常高严格性杂交条件下，与包含NCgl1418基因的启动子的突变体或杂交的序列的反向互补的多核苷酸。并且所述多核苷酸在对应SEQ ID NO：1所示序列的第170-179位中的核苷酸序列不为ACACCGAGTG，在对应SEQ ID NO：2所示序列的第130-139位的核苷酸序列不为ACACCGAGTG，在对应SEQ ID NO：3所示序列的第72-81位的核苷酸序列不为ACACCGAGTG。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，为与上述的多核苷酸序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性(包括这些数值之间所有范围和百分数)的序列。并且所述多核苷酸在对应SEQ ID NO：1所示序列的第170-179位中的核苷酸序列不为ACACCGAGTG，在对应SEQ ID NO：2所示序列的第130-139位的核苷酸序列不为ACACCGAGTG，在对应SEQ ID NO：3所示序列的第72-81位的核苷酸序列不为ACACCGAGTG。
在一些具体的实施方式中，所述突变体对应SEQ ID NO：1所示序列的第170-179位的核苷酸序列，在对应SEQ ID NO：2所示序列的第130-139位的核苷酸序列，在对应SEQ ID NO：3所示序列的第72-81位的核苷酸序列选自如下(p 1)-(p 12)组成的组中的任一项：(p 1)TACTTGCAGA，(p 2)AGTGCTGAAA，(p 3)GCACGAAAGG，(p 4)TATCTAGAGG，(p 5)AGGCTTGTCG，(p 6)CGCTTCTTTC，(p 7)TAACTCTTGG，(p 8)CCAAGTTCCA，(p 9)CGGTGCCACA，(p 10)AGCAGTTAGG，(p 11)AGATAAATAA，(p 12)ATCGATCTAG。
在一些具体的实施方式中，所述突变体的核苷酸序列选自如SEQ ID NO：37-48任一项所示的序列。
在一些实施方式中，本公开中的具有启动子活性的多核苷酸，与SEQ ID NO：1所示序列的多核苷酸相比，具有1-8倍以上提高的启动子活性。进一步的，与SEQ ID NO：1所示序列的多核苷酸相比，在盐浓度或渗透压升高的环境下具有2.11、1.85、3.23、3.07、3.57、2.78、3.90、3.28、2.62、2.70、4.47、7.62倍的增强启动子活性。
重组表达载体的构建和重组宿主细胞
在一些具体的实施方案中，本公开以ATCC 13032基因组为模板，利用引物1418-F和1418-R，通过PCR扩增得到NCgl1418基因的启动子序列(SEQ ID NO:1)；以pXM-gfp为模板，利用引物pGFP-F和pGFP-R对pXM-gfp进行扩增获得去除lacI基因和tac启动子的载体片段；将上述片段重组连接，得到重组表达载体pXM-P NCgl1418-gfp。
在一些具体的实施方案中，本公开以pXM-P NCgl1418-gfp为模板，分别利用引物对1418-203-F/R、1418-145-F/R和1418-94-F/R，通过PCR扩增获得具有203bp、145bp和94bp的NCgl1418启动子片段；上述三个片段回收后，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得新载体，将其分别命名为pXM-P 203-gfp、pXM-P 145-gfp和pXM-P 94-gfp。
在一些具体的实施方案中，本公开以ATCC 13032基因组为模板，利用引物1418-D-F和1418-D-R，通过PCR扩增得到NCgl1418基因的启动子序列。以pXM-07为模板，先利用 引物pXM07-F1和pXM07-R1，通过PCR扩增获得带有dCpf1的载体片段一；然后利用引物pXM07-F2和pXM07-R2，通过PCR扩增获得带有复制起点的载体片段二；以pEC-26为模板，利用引物pEC26-F和pEC26-R，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段；将上述片段回收后，进行重组连接，得到重组表达载体pXM-P NCgl1418-dCpf1。
在一些具体的实施方案中，本公开以ATCC 13032基因组为模板,利用引物1418-L-F和1418-L-R，通过PCR扩增得到NCgl1418基因的启动子序列和lysE基因的DNA序列。以pEC-XK99E为模板，利用引物pEC-F和pEC-R通过PCR扩增获得载体片段，将上述三个片段回收后行重组连接，得到重组表达载体pEC-P NCgl1418-lysE。
谷氨酸棒杆菌的具体来源为谷氨酸棒杆菌ATCC 13032(Corynebacterium glutamicum ATCC 13032，ATCC 13032的基因组序列：NC_003450.3)。在一些实施方式中，本公开以pXM-P NCgl1418-gfp为模板，以1418mutant-F和1418mutant-R引物对pXM-P NCgl1418-gfp进行反向PCR扩增，得到的线性化质粒片段；将线性化质粒片段进行磷酸化和连接，收集抗性克隆，得到NCgl1418基因的启动子突变体文库。
在一些实施方式中，本公开以NCgl1418基因的启动子突变体文库和pXM-Con、pXM-P NCgl1418-gfp分别转化谷氨酸棒杆菌ATCC13032，得到重组宿主细胞。通过平板培养后筛选重组宿主细胞的荧光强度，进行启动子强度提高的突变体的筛选。
在一些具体的实施方式中，本公开以pXM-P NCgl1418-dCpf1为模板，分别利用引物35P7-F和pXM-R，以及引物pXM-F和35P7-R1进行PCR扩增，获得两个载体片段，将上述片段进行重组连接，得到重组载体pXM-P 35P7-dCpf1。
在一些具体的实施方案中，本公开以pEC-P NCgl1418-lysE为模板，利用引物35P7-F和35P7-R2，通过PCR扩增得到带有突变启动子35P7且包含lysE基因的载体片段，然后将线性化的载体片段磷酸化，并通过自身环化构建获得pEC-P 35P7-lysE。
在另外一些实施方式中，本公开还可以根据具体的克隆需要，利用35P1、35P2、35P3、35P4、35P5、35P6、35P8、35P9、35P10、35P11、35P12的启动子序列构建所需的重组载体。
在一些具体的实施方案中，本公开以ATCC 13032基因组为模板，利用引物proP-F和proP-R，通过PCR扩增得到proP基因的启动子序列(SEQ ID NO:56)；以pXM-gfp为模板，利用引物pGFP-F和pGFP-R对pXM-gfp进行扩增获得去除lacI基因和tac启动子的载体片段；将上述片段重组连接，得到重组表达载体pXM-P proP-gfp。
在一些具体的实施方案中，本公开以pXM-P proP-gfp为模板，利用引物proP-1-F和proP-1-R，通过PCR扩增获得的片段，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得pXM-P proP-1-gfp。同时，以pXM-P proP-gfp为模板，利用引物proP-2-F和proP-2-R，通过PCR扩增获得的片段，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得pXM-P proP-2-gfp。
在一些具体的实施方案中，本公开以ATCC 13032基因组为模板，利用引物proP-D-F和proP-D-R，通过PCR扩增得到P proP启动子序列。以pXM-07为模板，先利用引物pXM07-F1和pXM07-R2，通过PCR扩增获得带有dCpf1的载体片段一；然后利用引物pXM07-F2和pXM07-R2，通过PCR扩增获得带有复制起点的载体片段二；以pEC-26为模板，利用引物pEC26-F和pEC26-R，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段；将上述片段回收后，进行重组连接，得到重组表达载体pXM-P proP-dCpf1。
在一些具体的实施方案中，本公开以以pXM-P proP-1-gfp为模板，设计引物proP-D-F和proP1-D-R，通过PCR扩增得到P proP-1启动子片段。以pXM-07为模板，先利用引物pXM07-F1和pXM07-R2，通过PCR扩增获得带有dCpf1的载体片段一；然后利用引物pXM07-F2和 pXM07-R2，通过PCR扩增获得带有复制起点的载体片段二；以pEC-26为模板，利用引物pEC26-F和pEC26-R，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段；将上述片段回收后，进行重组连接，得到重组表达载体pXM-P proP-1-dCpf1。
在一些具体的实施方案中，本公开以pXM-P proP-gfp为模板，利用引物proP-lysE-F和proP-lysE-R通过PCR扩增得到P proP-1启动子序列片段；以pXM-P proP-1-gfp为模板，利用引物proP-lysE-F和proP1-lysE-R，通过PCR扩增得到P proP启动子且序列片段。以ATCC 13032基因组为模板，利用引物lysE-F和lysE-R通过PCR扩增得到lysE基因片段。同时，以pXM-XK99E为模板，利用引物pEC-F和pEC-R，通过PCR扩增获得载体片段。将上述启动子片段分别与lysE片段和载体片段进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布卡那霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pEC-P proP-lysE和pEC-P proP-1-lysE。在一些实施方式中，本公开的谷氨酸棒杆菌SCgL30菌株，是将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸，构建获得一株具有一定赖氨酸合成能力的菌株SCgL30。
在一些实施方式中，本公开将pEC-P 35P7-lysE转化SCgL30重组菌株，获得重组宿主细胞。在一些实施方式中，本公开将pXM-P 35P7-dCpf1转化SCgL30重组菌株，获得重组宿主细胞。在另外一些实施方式中，本公开还可以分别将包含35P1、35P2、35P3、35P4、35P5、35P6、35P8、35P9、35P10、35P11、35P12的启动子序列的重组载体转化SCgL30重组菌株，获得重组宿主细胞。
目标化合物的生产过程
(1)将具有启动子活性的多核苷酸，与目标化合物合成相关的蛋白编码基因或基因表达调控蛋白编码基因可操作的连接，得到能够与目标化合物合成相关的蛋白或基因表达调控蛋白的重组表达载体，利用重组表达载体转化宿主细胞，获得重组宿主细胞。
(2)对重组宿主细胞进行发酵培养，从重组宿主细胞或重组宿主细胞的培养液中收集目标化合物，完成目标化合物的生产过程。
上述生产过程中，由于多核苷酸具有改进的启动子活性，在重组宿主细胞中，与目标化合物合成相关的蛋白或基因表达调控蛋白的编码基因的转录活性提高，与目标化合物合成相关的蛋白或基因表达调控蛋白的表达量提高，进而使目标化合物的产量显著提升。
在一些具体的实施方案中，本公开采用的制备氨基酸的方法的步骤中，不添加诱导剂。在一个具体的实施方案中，本公开采用的制备氨基酸的方法的步骤中，不添加IPTG。
在一些实施方式中，目标化合物为氨基酸，与目标化合物合成相关的蛋白编码基因是指与合成氨基酸相关的蛋白编码基因。在一些实施方式中，目标化合物为L-氨基酸，与合成氨基酸相关的蛋白编码基因是指与合成L-氨基酸相关的蛋白编码基因。在一些具体的实施方案中，L-氨基酸为L-赖氨酸，与氨基酸合成相关的蛋白为赖氨酸转运蛋白LysE，以具有启动子活性的多核苷酸增加LysE的表达，可促进赖氨酸的胞外排放和胞外积累。在一些实施方案中，基因表达调控蛋白为dCpf1，dCpf1可以靶向调控gltA、pgi、hom或pck等目标基因；具有启动子活性的多核苷酸可以增加高盐环境条件下dCpf1的表达，提高了靶基因的弱化程度，进一步促进了赖氨酸的合成和底物利用。
在一些具体的实施方案中，宿主细胞为谷氨酸棒杆菌(Corynebacterium glutamicum)，谷氨酸棒杆菌是用于生产L-赖氨酸的重要菌株，高盐、高渗透压诱导型的多核苷酸、转录表达盒或重组表达载体对谷氨酸棒杆菌进行改造后，谷氨酸棒杆菌内与赖氨酸合成相 关的蛋白的表达量显著提高，具体为在高盐、高渗透压的环境下的表达量显著提高，使谷氨酸棒杆菌长时间发酵积累L-赖氨酸的能力大大提高。
在一些具体的实施方案中，宿主细胞是经过如下改良的谷氨酸棒杆菌：谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸。
在一些具体的实施方案中，重组宿主细胞的培养条件为：将重组宿主细胞接种含有相应抗生素的TSB培养基，30℃，220r/min过夜培养，按照初始OD 0.3分别转接添加或不添加0.6M硫酸钠(模拟发酵后期高浓度产物积累造成的高盐高渗环境)的赖氨酸发酵培养基，培养体系为24孔板装液1mL，30℃，800r/min培养36h后终止发酵，检测剩余葡萄糖含量、OD 600和赖氨酸产量。
对于赖氨酸发酵培养基，配方为：葡萄糖80g/L、酵母粉8g/L、尿素9g/L、K 2HPO 4 1.5g/L、MOPS 42g/L、FeSO 4 0.01g/L、MnSO 4 0.01g/L、MgSO 4 0.6g/L，氯霉素终浓度为5μg/mL，和/或卡那霉素终浓度为25μg/mL。
在一些具体的实施方案中，对于重组宿主细胞或重组细胞的培养液回收目标化合物，可通过本领域常用方法，包括但不限于：过滤、阴离子交换色谱、结晶或HPLC。
在本领域，用于操纵微生物的方法是已知的，如《分子生物学现代方法》(Online ISBN：9780471142720,John Wiley and Sons,Inc.)、《微生物代谢工程：方法和规程》(Qiong Cheng Ed.,Springer)和《系统代谢工程：方法和规程》(Hal S.Alper Ed.,Springer)等出版物中被解释。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是，应当理解的是，详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出，因为在阅读该详细说明后，在本公开的精神和范围内所作出的各种改变和修饰，对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法，如无特殊说明均为常规技术方法，例如下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等，如无特殊说明，均可通过正规商业渠道获得。
实施例中质粒构建所使用的引物序列如下表1所示：
Figure PCTCN2022070499-appb-000001
Figure PCTCN2022070499-appb-000002
Figure PCTCN2022070499-appb-000003
实施例1.诱导型启动子的筛选
利用添加和不添加0.6M NaCl或赖氨酸硫酸盐的CGXII培养基培养谷氨酸棒状杆菌ATCC13032菌株，在对数生长中期收集细胞，提取总RNA并进行转录组测序分析。选择在高渗条件下转录水平明显上升的NCgl1418基因的启动子最为候选高盐高渗诱导型启动子。
实施例2.包含启动子序列的重组载体构建
根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，设计引物1418-F(SEQ ID NO:6)和1418-R(SEQ ID NO:7)。以ATCC 13032基因组为模板通过PCR扩增得到NCgl1418基因的启动子序列(SEQ ID NO:1)，PCR扩增参数为：95℃ 5min，95℃ 30s，65-55℃ 30s，72℃ 1min，循环10次，95℃ 30s，55℃ 30s，72℃ 1min，循环25次，72℃延伸10min。同时，以文献报道的pXM-gfp为模板 [10]，利用引物pGFP-F和pGFP-R，通过PCR扩增获得去除lacI基因和tac启动子的载体片段，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min。上述两个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布氯霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pXM-P NCgl1418-gfp。同时，利用T4 PNK将载体片段磷酸化，并通过自身环化构建获得对照载体pXM-con。
实施例3.高盐对NCgl1418启动子的诱导作用
将上述重组载体pXM-P NCgl1418-gfp和对照载体pXM-con分别转化谷氨酸棒杆菌ATCC 13032，获得重组菌株和对照菌株。将上述菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养。其中，TSB液体培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，5g/L；大豆蛋白胨，9g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。按照初始OD 0.5分别转接添加或不添加0.6M不同盐的CGXIIY培养基，培养体系为24孔板装液1mL，30℃，800r/min培养18h后检测不同菌株的GFP荧光强度及OD 600，利用单位菌体的荧光强度(扣除相同条件下对照菌株的单位菌体荧光强度)表征不同条件下NCgl1418启动子的相对强度。其中CGXIIY培养基配方为：葡萄糖50g/L、NH 4Cl 16.5g/L、尿素5g/L、KH 2PO 4 1g/L、K 2HPO 4 1g/L、MOPS 42g/L、MgSO 4 0.25g/L、FeSO 4·2H 2O 0.01g/L、MnSO 4·H 2O 0.01g/L、ZnSO 4·7H 2O 0.001g/L、CuSO 4 0.2mg/L、NiCl·6H 2O 0.02mg/L、CaCl 2 0.01g/L、原儿茶酸0.03g/L、生物素0.2mg/L、维生素B1 0.1mg/L，氯霉素终浓度为5μg/mL。检测结果见图1，数据显示添加0.6M不同盐都可以诱导NCgl1418启动子的转率和报告基因的表达(4.1-7.7倍)，因此确定该启动子受高盐诱导。
实施例4.不同渗透压强度对NCgl1418启动子的诱导作用
利用实施例3同样的方法，分别检测不同浓度硫酸钠对NCgl1418启动子的诱导作用。结果见图2，数据显示NCgl1418启动子的强度随硫酸钠浓度的升高而增强，在一定的范围内表现出明显的梯度诱导活性。
实施例5.NCgl1418启动子对高糖的响应
利用实施例3同样的方法，检测高浓度蔗糖(150g/L)对NCgl1418启动子的诱导作用。结果见图3，数据显示额外添加高浓度蔗糖对NCgl1418启动子的诱导活性没有明显增强作用，说明该启动子响应的不是所有的高渗压力，而针对性响应高浓度盐离子。
实施例6.NCgl1418启动子和tuf启动子强度比较
目前，已知通用的谷氨酸棒杆菌的内源强启动子是P tuf，因此，我们进一步将NCgl1418启动子与该启动子进行了对比，以验证NCgl1418启动子的强度。根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，设计引物tuf-F(SEQ ID NO:10)和tuf-R(SEQ ID NO:11)。以ATCC 13032基因组为模板通过PCR扩增得到带有tuf基因的启动子序列(SEQ ID NO:5)，PCR扩增参数为：95℃ 5min，95℃ 30s，55℃ 30s，72℃ 1min，循环30次，72℃延伸10min。同时，以pXM-P NCgl1418-gfp为模板，利用引物tuf-pGFP-F(SEQ ID NO:12)和tuf-pGFP-R(SEQ ID NO:13)，通过PCR扩增获得包含P NCgl1418的RBS的载体片段，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min。上述片段回收后利用Vazyme Clon Express Multies重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布氯霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pXM-P tuf-gfp。将该重组载体转化谷氨酸棒杆菌ATCC 13032，获得重组菌株。利用实施例3类似的方法，比较高盐或正常培养基条件下NCgl1418启动子与P tuf的强度。结果见图4，数据显示在正常渗透压条件下NCgl1418启动子强度低于P tuf，但在高渗透压(添加0.6M硫酸钠)条件下，其转录活性与P tuf基本一致，表明在高盐渗透压条件该启动子强度较高，可以用于目标基因的高效诱导表达。
实施例7.不同长度NCgl1418启动子的活性
以上述构建的pXM-P NCgl1418-gfp为模板，分别利用引物对1418-203-F/R(SEQ ID NO:14、15)、1418-145-F/R(SEQ ID NO:16、17)和1418-94-F/R(SEQ ID NO:18、19)，通过PCR扩增获得具有203bp(SEQ ID NO:2)、145bp(SEQ ID NO:3)和94bp(SEQ ID NO:4)的NCgl1418启动子片段，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min。上述三个片段回收后，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得新载体，将其分别命名为pXM-P 203-gfp、pXM-P 145-gfp和pXM-P 94-gfp。
将上述重组载体pXM-P 203-gfp、pXM-P 145-gfp和pXM-P 94-gfp分别转化谷氨酸棒杆菌ATCC 13032，获得重组菌株。利用实施例3类似的方法，分别比较高盐(添加0.6M硫酸钠)和正常培养基条件下不同长度NCgl1418启动子的强度。结果见图5，数据显示94bp长度的NCgl1418启动子尽管包含了核心序列(-35区和-10区)，但却基本丧失了启动子的正常功能；145bp长度的启动子在高盐条件下虽然诱导强度有所下降，但仍可以达到243bp启动子活性的74％以上；203bp长度的启动子在高盐渗透压条件下基本保持了243bp启动子的活性，为243bp启动子活性的94％；以上结果表明NCgl1418启动子的启动子活性以及在高盐渗透压条件下的活性至少需要包括SEQ ID NO:3所示的145bp长度的DNA序列。
实施例8.NCgl1418启动子调控dCpf1表达在赖氨酸合成中的应用
根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，设计引物1418-D-F(SEQ ID NO:20)和1418-D-R(SEQ ID NO:21)。以ATCC 13032基因组为模板通过PCR扩增得到NCgl1418基因的启动子序列，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 1min，循环10次，95℃ 30s，55℃ 30s，72℃ 1min，循环25次，72℃延伸10min。同时，以文献报道的pXM-07为模板 [11]，先利用引物pXM07-F1(SEQ ID NO:22)和pXM07-R1(SEQ ID NO:23)，通过PCR扩增获得带有dCpf1的载体片段一，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min；然后利用引物pXM07-F2(SEQ ID NO:24)和pXM07-R2(SEQ ID NO:25)，通过PCR扩增获得带有复制起点的载体片段二，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min。以文献报道的pEC-26为模板 [11]，利用引物pEC26-F(SEQ ID NO:26)和pEC26-R(SEQ ID NO:27)，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 1min，循环10次，95℃ 30s，55℃ 30s，72℃ 1min，循环25次，72℃延伸10min。将上述三个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布氯霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pXM-P NCgl1418-dCpf1。同时，以pXM-07为模板，利用引物pXM07-F3(SEQ ID NO:28)和pXM07-R3(SEQ ID NO:29)，通过PCR扩增获得载体片段三，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环30次，72℃延伸10min；将上述片段回收后与得到的NCgl1418基因的启动子序列片段通过Vazyme Clon Express Multies一步重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布氯霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得对照载体pXM-dCpf1-con。
根据文献中公开的赖氨酸菌株构建方法 [12]，利用基于pK18mobsacB的同源重组技术将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突 变为异亮氨酸，构建获得一株具有一定赖氨酸合成能力的菌株SCgL30。将上述重组载体pXM-P NCgl1418-dCpf1和pXM-dCpf1-con分别转化SCgL30菌株，获得重组菌株和对照菌株。将上述菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养，按照初始OD 0.3分别转接添加或不添加0.6M硫酸钠(模拟发酵后期高浓度产物积累造成的高盐高渗环境)的赖氨酸发酵培养基，培养体系为24孔板装液1mL，30℃，800r/min培养24h后终止发酵，检测剩余葡萄糖含量、OD 600和赖氨酸产量。其中赖氨酸发酵培养基配方为：葡萄糖80g/L、酵母粉8g/L、尿素9g/L、K 2HPO 4 1.5g/L、MOPS 42g/L、FeSO 4 0.01g/L、MnSO 4 0.01g/L、MgSO 4 0.6g/L，氯霉素终浓度为5μg/mL。检测结果见表2，数据显示不添加硫酸钠时，靶基因弱化菌株赖氨酸产量和葡萄糖转化率分别比对照菌株提高了23％和25％，表明NCgl1418启动子可以通过调控目标基因dCpf1的表达，实现该系统对靶基因的弱化功能。而在添加0.6M硫酸钠的高盐条件中，靶基因弱化菌株赖氨酸产量和葡萄糖转化率分别比对照菌株提高了49％和40％，表明高盐环境条件下dCpf1的表达强度更高，提高了靶基因的弱化程度，进一步促进了赖氨酸的合成和底物利用。
表2 NCgl1418启动子调控dCpf1表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000004
实施例9.NCgl1418启动子调控赖氨酸转运蛋白LysE表达在赖氨酸合成中的应用
根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，分别设计引物1418-L-F(SEQ ID NO:30)和1418-L-R(SEQ ID NO:31)，lysE-F(SEQ ID NO:32)和lysE-R(SEQ ID NO:33)。以ATCC 13032基因组为模板通过PCR扩增分别得到NCgl1418基因的启动子序列和lysE基因的DNA序列，PCR扩增参数为：95℃ 5min，95℃ 30s，65-55℃ 30s，72℃ 1min，循环10次，95℃ 30s，55℃ 30s，72℃ 1min，循环25次，72℃延伸10min。同时，以文献报道的pEC-XK99E为模板 [13]，利用引物pEC-F(SEQ ID NO:34)和pEC-R(SEQ ID NO:35)，通过PCR扩增获得载体片段，PCR扩增参数为：95℃ 10min，95℃ 30s，65-55℃ 30s，72℃ 3min，循环10次，95℃ 30s，55℃ 30s，72℃ 3min，循环25次，72℃延伸10min。将上述三个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布卡那霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pEC-P NCgl1418-lysE。
将上述重组载体pEC-P NCgl1418-lysE和pEC-XK99E分别转化谷氨酸棒杆菌ScgL30，获得重组菌株和对照菌株。利用如实施例8的方法(抗生素替换为终浓度为25μg/mL的卡那霉素)验证NCgl1418启动子调控的LysE表达菌株在赖氨酸合成中的应用效果，结果见表3。数据显示不添加硫酸钠时，NCgl1418启动子调控的LysE表达菌株赖氨酸产量和葡萄糖转化率为2.35g/L和0.039g/g，分别比对照菌株提高了38％和40％；而在添加0.6M硫酸钠的高盐条件中，LysE表达菌株赖氨酸产量和葡萄糖转化率达到了3.05g/L和0.063g/g，分别比对照菌株提高了49％和59％，表明高盐环境条件下LysE的表达强度更高，促进了赖氨酸的外排和胞外积累。
表3 NCgl1418启动子调控LysE表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000005
Figure PCTCN2022070499-appb-000006
进一步将上述两种策略组合，获得了NCgl1418启动子同时调控LysE和dCpf1表达的重组菌株。利用如实施例8的方法(抗生素替换为终浓度为5μg/mL的氯霉素和25μg/mL的卡那霉素)验证上述重组菌株在赖氨酸合成中的应用效果，结果见表4。数据显示不添加硫酸钠时，NCgl1418启动子调控的目标基因共表达菌株赖氨酸产量从1.75g/L提高到了3.15g/L(提高了80％)，葡萄糖转化率从0.03g/g提高到了0.053g/g(提高了78％)；而在添加0.6M硫酸钠的高盐条件中，目标基因共表达菌株赖氨酸产量和葡萄糖转化率达到了4.20g/L和0.1g/g，分别比对照菌株提高了115％和127％，效果非常显著。
表4 NCgl1418启动子同时调控dCpf1和LysE表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000007
实施例10.NCgl1418基因启动子突变文库构建
鉴于启动子-35区前后序列对启动子的强度可能具有重要的调控作用，本实施例分别对NCgl1418基因启动子核心区(SEQ ID NO:36)的-35区前后序列进行随机突变。NCgl1418基因启动子核心区序列为：
TATTAAAGATCACACCGAGTG GTGGAATTTCCTCAAGTGATTTAC CCACAAT GGACTTTG，下划线处分别为启动子-35区和-10区主要序列；
具体突变序列为：
TATTAAAGATCNNNNNNNNNN GTGGAATTTCCTCAAGTGATTTAC CCACAAT GGACTTTG。
分别利用1418mutant-F(SEQ ID NO:49)和1418mutant-R(SEQ ID NO:50)引物对实施例2中获得的pXM-P NCgl1418-gfp反向PCR扩增，通过对得到的线性化质粒片段进行磷酸化和连接，转化大肠杆菌T1感受态细胞，获得抗性克隆。对获得的所有克隆菌进行细胞收集并提取质粒，获得NCgl1418基因启动子的两种突变体文库。
实施例11.NCgl1418基因启动子突变文库筛选和突变启动子表征
将上述启动子突变体文库转化谷氨酸棒杆菌ATCC13032，使用实施例3中的菌株ATCC13032(pXM-Con)、ATCC13032(pXM-P NCgl1418-gfp)作为空载体和野生型对照，对突变库进行正反三次筛选。首先，接种上述菌株于含有5μg/mL氯霉素的TSB培养基，30℃，220r/min培养8～10h后，按照初始OD 1分别转接添加0.6M Na 2SO 4的CGXIIY培养基，培养体系为24孔板装液1mL，30℃，800r/min培养6h后，将所得菌液用PBS缓冲液稀释50倍，然后超声处理6min后，通过流式细胞仪进行荧光分选(正向筛选，top 0.01％)。将分选得到的混合菌株接种于含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养，用于下一次筛选。然后将混合菌株、空载体和野生型对照菌株接种于含有5μg/mL氯霉素的TSB培养基，30℃，220r/min培养8～10h后，按照初始OD 0.5分别转接CGXIIY培养基，培养体系为24孔板装液1mL，30℃，800r/min培养6h后，将所得菌液用PBS缓冲液稀释50倍，然后超声处理6min后，通过流式细胞仪进行荧光分选(反向筛选，bottom 1％)。最后，重复第一次正向筛选的过程。
将上述三次筛选获得的菌株及野生型NCgl1418启动子和无启动子对照菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养。其中，TSB液体培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，5g/L；大豆蛋白胨，9g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。
按照初始OD 0.5分别转接添加或不添加0.6M Na 2SO 4的CGXIIY培养基，培养体系为24孔板装液1mL，30℃，800r/min培养24h后检测不同菌株的GFP荧光强度及OD 600，利用单位菌体的荧光强度(扣除相同条件下对照菌株的单位菌体荧光强度)表征不同条件下突变启动子的相对强度。其中CGXIIY培养基配方为：葡萄糖50g/L、NH 4Cl 16.5g/L、尿素5g/L、KH 2PO 4 1g/L、K 2HPO 4 1g/L、MOPS 42g/L、MgSO 4 0.25g/L、FeSO 4·2H 2O 0.01g/L、MnSO 4·H 2O 0.01g/L、ZnSO 4·7H 2O 0.001g/L、CuSO 4 0.2mg/L、NiCl·6H 2O 0.02mg/L、CaCl 2 0.01g/L、原儿茶酸0.03g/L、生物素0.2mg/L、维生素B1 0.1mg/L，氯霉素终浓度为5μg/mL。根据检测获得的荧光强度筛选获得12个强诱导型突变启动子，高盐条件下启动子强度是野生型启动子的1.85-7.62倍，诱导活性均保持在1.64倍以上。其中35P7启动子强度是野生型启动子的3.9倍，而诱导活性达到了8.46，均显著高于野生型启动子(表5)。
表5
Figure PCTCN2022070499-appb-000008
Figure PCTCN2022070499-appb-000009
a高盐条件下(添加Na 2SO 4)各启动子荧光强度/野生型启动子荧光强度
b添加Na 2SO 4的荧光强度/不添加Na 2SO 4的荧光强度
实施例12.利用突变启动子调控dCpf1表达用于促进赖氨酸
根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，设计引物1418-D-F(SEQ ID NO:21)和1418-D-R(SEQ ID NO:22)，以ATCC 13032基因组为模板通过PCR扩增得到NCgl1418基因的启动子序列。同时，以文献报道的pXM-07为模板 [2]，先利用引物pXM07-F1(SEQ ID NO:23)和pXM07-R1(SEQ ID NO:24)，通过PCR扩增获得带有dCpf1的载体片段一；然后利用引物pXM07-F2(SEQ ID NO:25)和pGFP-R(SEQ ID NO:20)，通过PCR扩增获得带有复制起点的载体片段二。以文献报道的pEC-26为模板 [2]，利用引物pEC26-F(SEQ ID NO:26)和pEC26-R(SEQ ID NO:27)，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段。将上述三个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，获得重组载体pXM-P NCgl1418-dCpf1。同时，以pXM-07为模板，利用引物pXM07-F1(SEQ ID NO:23)和pGFP-R(SEQ ID NO:20)，通过PCR扩增获得载体片段三；将上述片段回收后与得到的NCgl1418基因的启动子序列片段通过Vazyme Clon Express Multies一步重组试剂盒进行重组连接，获得对照载体pXM-dCpf1-con。
以pXM-P NCgl1418-dCpf1为模板，分别利用引物35P7-F(SEQ ID NO:51)和pXM-R(SEQ ID NO:55)，pXM-F(SEQ ID NO:54)和35P7-R1(SEQ ID NO:52)，通过PCR扩增分别获得两个载体片段(包含35P7启动子)，利用Vazyme Clon Express Multies一 步重组试剂盒进行重组连接，获得重组载体pXM-P 35P7-dCpf1。类似地，以pXM-dCpf1-con为模板，利用上述相同引物和方法，构建获得相应的对照载体pXM-dCpf1-P 35P7con。
根据文献中公开的赖氨酸菌株构建方法 [3]，利用基于pK18mobsacB的同源重组技术将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸，构建获得一株具有一定赖氨酸合成能力的菌株SCgL30。将上述重组载体pXM-P 35P7-dCpf1、pXM-P NCgl1418-dCpf1、pXM-dCpf1-P 35P7con和pXM-dCpf1-con分别转化SCgL30菌株，获得重组菌株和对照菌株。将上述菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养，按照初始OD 0.3分别转接添加或不添加0.6M硫酸钠(模拟发酵后期高浓度产物积累造成的高盐高渗环境)的赖氨酸发酵培养基，培养体系为24孔板装液1mL，30℃，800r/min培养36h后终止发酵，检测剩余葡萄糖含量、OD 600和赖氨酸产量。其中赖氨酸发酵培养基配方为：葡萄糖80g/L、酵母粉8g/L、尿素9g/L、K 2HPO 4 1.5g/L、MOPS 42g/L、FeSO 4 0.01g/L、MnSO 4 0.01g/L、MgSO 4 0.6g/L，氯霉素终浓度为5μg/mL。检测结果见表6和表7，数据显示在不添加硫酸钠时，靶基因弱化菌株赖氨酸产量和葡萄糖转化率分别比对照菌株提高了28％和31％，均高于野生型NCgl1418启动子提升幅度为(23％和25％)。而在添加0.6M硫酸钠的高盐条件中，靶基因弱化菌株赖氨酸产量和葡萄糖转化率分别比对照菌株提高了52％和55％，明显高于野生型NCgl1418启动子提升幅度为(49％和40％)。上述结果说明高盐环境条件下dCpf1的表达强度更高，提高了靶基因的弱化程度，进一步促进了赖氨酸的合成和底物利用。
表6 35P7启动子调控dCpf1表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000010
表7 NCgl1418野生型启动子调控dCpf1表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000011
实施例13.利用突变启动子调控LysE表达在赖氨酸合成中的应用
根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列NC_003450.3)，分别设计引物1418-L-F(SEQ ID NO:28)和1418-L-R(SEQ ID NO:29)，lysE-F(SEQ ID NO:30)和lysE-R(SEQ ID NO:31)。以ATCC 13032基因组为模板通过PCR扩增分别得到NCgl1418基因的启动子序列和lysE基因的DNA序列。同时，以文献报道的pEC-XK99E为模板 [4]，利用引物pEC-F(SEQ ID NO:32)和pEC-R(SEQ ID NO:33)，通过PCR扩增获得载体片段。将上述三个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布卡那霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pEC-P NCgl1418-lysE。以pEC-P NCgl1418-lysE为模板，利用引物 35P7-F(SEQ ID NO:51)和35P7-R2(SEQ ID NO:53)，通过PCR扩增获得带有突变启动子35P7且包含lysE基因的载体片段。然后利用T4PNK将载体片段磷酸化，并通过自身环化构建获得pEC-P 35P7-lysE。
将上述重组载体pEC-P NCgl1418-lysE、pEC-P 35P7-lysE和pEC-XK99E分别转化谷氨酸棒杆菌ScgL30，获得重组菌株和对照菌株。利用如实施例12的方法(抗生素替换为终浓度为25μg/mL的卡那霉素)验证NCgl1418启动子调控的LysE表达菌株在赖氨酸合成中的应用效果，结果见表8和表9。数据显示，在不添加硫酸钠时，35P7启动子调控的LysE表达菌株赖氨酸产量和葡萄糖转化率为3.00g/L和0.050g/g，分别比对照菌株提高了45％和52％，且高于野生型NCgl1418启动子38％和40％的提升幅度；而在添加0.6M硫酸钠的高盐条件下，LysE表达菌株赖氨酸产量和葡萄糖转化率达到了5.53g/L和0.128g/g，分别比对照菌株提高了124％和176％，且高于野生型NCgl1418启动子49％和59％的提升幅度，显示出良好的应用效果。
表8 35P7启动子调控LysE表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000012
表9 野生型NCgl1418启动子调控LysE表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000013
此外，本公开图5中证明了145bp长度的启动子在高盐渗透压条件下可以达到243bp启动子活性的74％以上；203bp长度的启动子在高盐渗透压条件下基本保持了243bp启动子的活性，为243bp启动子活性的94％；说明了由于SEQ ID NO：2和SEQ ID NO：3的启动子片段含有NCgl1418基因的启动子的核心区，SEQ ID NO：2和SEQ ID NO：3的启动子片段在盐浓度、渗透压升高的环境下也同样能够表现出明显增强的启动子活性。因此，采用上述实施例中对启动子核心区进行改造的方法，即对SEQ ID NO：2所示序列的第130-139位中的一个或多个位置进行突变，或者对SEQ ID NO：3所示序列的第72-81位中的一个或多个位置进行突变，会获得同样诱导活性增强的启动子突变体。
实施例14.包含内源proP基因启动子序列的质粒构建
上述获得的Ncgl1418启动子突变体，是对野生型Ncgl1418启动子核心区(TATTAAAGATCACACCGA GTGGTGGAATTTCCTCAAGTGATTTAC CCACAATGGACTTTG，下划线处分别为启动子-35区和-10区主要序列)的部分序列进行突变获得的，与野生型启动子相比在盐浓度、渗透压升高的环境下表现出明显增强的启动子活性，也就是说明Ncgl1418的诱导活性可以通过改造进一步提升。因此，本公开拟进一步对该启动子的整个核心区序列、5’-UTR序列、其他区域序列进行整段序列的替换，以进一步提高启动子的诱导活性。
与Ncgl1418启动子在盐浓度、渗透压升高的环境下表现出增强的启动子活性近似，已有文献报道高渗条件下ProP的表达会明显上调，表明该基因的启动子可能也是一种高渗诱导型的启动子(Franzel,B.,et al.,Adaptation of Corynebacterium glutamicum to salt-stress conditions,Proteomics,2010,10(3):445–457.)。ProP是脯氨酸摄入蛋白，在高渗条件下诱导表达，通过提高相容性物质脯氨酸的摄入，增强菌株对高渗环境的耐受性。根据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC 13032的基因组序列(NC_003450.3)，设计引物proP-F(SEQ ID NO:59)和proP-R(SEQ ID NO:60)。以ATCC 13032基因组为模板通过PCR扩增得到带有proP基因的启动子(P proP)序列(SEQ ID NO:56)。同时，以文献报道的pXM-gfp为模板(Sun DH et al.,Journal of Industrial Microbiology&Biotechnology,2019,46(2):203-208.)，利用引物pGFP-F(SEQ ID NO:8)和pGFP-R(SEQ ID NO:9)，通过PCR扩增获得去除lacI基因和tac启动子的载体片段。上述片段回收后利用Vazyme Clon Express Multies重组试剂盒进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布氯霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pXM-P proP-gfp。同时，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得对照载体pXM-con。将上述重组载体转化谷氨酸棒杆菌ATCC 13032，获得重组菌株。
实施例15.高盐对proP基因启动子的诱导作用
将上述带有不同启动子重组载体的菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养。其中，TSB液体培养基成份为(g/L)：葡萄糖，5g/L；酵母粉，5g/L；大豆蛋白胨，9g/L；尿素，3g/L；丁二酸，0.5g/L；K 2HPO 4·3H 2O，1g/L；MgSO 4·7H 2O，0.1g/L；生物素，0.01mg/L；维生素B1，0.1mg/L；MOPS，20g/L。按照初始OD 0.5分别转接添加或不添加0.6M Na 2SO 4的CGXIIY培养基，培养体系为24孔板装液1mL，30℃，800r/min培养18h后检测不同菌株的GFP荧光强度及OD 600，利用单位菌体的荧光强度(扣除相同条件下对照菌株的单位菌体荧光强度)表征不同条件下不同启动子的相对强度。其中CGXIIY培养基配方为：葡萄糖50g/L、酵母粉2g/L、NH 4Cl 16.5g/L、尿素5g/L、KH 2PO 4 1g/L、K 2HPO 4 1g/L、MOPS 42g/L、MgSO 4 0.25g/L、FeSO 4·2H 2O 0.01g/L、MnSO 4·H 2O 0.01g/L、ZnSO 4·7H 2O 0.001g/L、CuSO 4 0.2mg/L、NiCl·6H 2O 0.02mg/L、CaCl 2 0.01g/L、原儿茶酸0.03g/L、生物素0.2mg/L、维生素B1 0.1mg/L，氯霉素终浓度为5μg/mL。检测结果见图6，数据显示proP基因启动子受高盐诱导，诱导活性达到了8.9倍。与实施例7中测定的NCgl1418启动子活性比较可以发现，proP基因启动子强度却明显低于NCgl1418启动子(图7)。
实施例16.Ncgl1418及proP启动子的杂合改造及表征质粒构建
鉴于proP启动子具有高诱导活性以及NCgl1418启动子具有高表达强度的特性，为了获得高诱导活性以及高表达强度的启动子，本发明进一步验证了Ncgl1418及proP启动子的杂合改造的效果。首先保留proP启动子可能的调控区，以保持高诱导活性，然后将proP启动子的5’-UTR替换为Ncgl1418启动子的5’-UTR(SEQ ID NO:57)，或者将proP启动子的-35区和-10区核心区以及5’-UTR同时替换为Ncgl1418启动子的-35区和-10区核心区以及5’-UTR(SEQ ID NO:58)，以保持启动子的高表达强度。通过上述设计得到P proP-1(SEQ ID NO:57)和P proP-2(SEQ ID NO:58)两种突变启动子。
以pXM-P proP-gfp为模板，利用引物proP-1-F(SEQ ID NO:61)和proP-1-R(SEQ ID NO:62)，通过PCR扩增替换proP启动子后的5’-UTR区，将上述PCR片段回收后，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得pXM-P proP-1-gfp。同时，以pXM-P proP-gfp为模板，利用引物proP-2-F(SEQ ID NO:63)和proP-2-R(SEQ ID NO:64)，通过PCR扩增替换proP启动子后的5’-UTR区以及-35区和-10区核心区，将上述片段回 收后，利用T4PNK将载体片段磷酸化，并通过自身环化构建获得pXM-P proP-2-gfp。将上述重组载体转化谷氨酸棒杆菌ATCC 13032，获得重组菌株。
实施例17.高盐对杂合启动子的诱导作用
利用实施例15类似的方法，比较高盐或正常培养基条件下突变启动子的强度和诱导活性。结果见图7，数据显示改造后的两种启动子在正常培养基及高盐条件下的强度均高于野生型proP启动子，P proP-1诱导活性基本保持了野生型proP启动子的性能(8.7倍)，但P proP-2由于本底表达水平较高，诱导活性降为3.2倍。
实施例18.利用突变启动子调控dCpf1表达用于促进赖氨酸
以pXM-P proP-gfp为模板，设计引物proP-D-F(SEQ ID NO:65)和proP-D-R(SEQ ID NO:66)，通过PCR扩增得到P proP启动子片段。同时，以文献报道的pXM-07为模板 [5]，先利用引物pXM07-F1(SEQ ID NO:22)和pXM07-R1(SEQ ID NO:23)，通过PCR扩增获得带有dCpf1的载体片段一；然后利用引物pXM07-F2(SEQ ID NO:24)和pXM07-R2(SEQ ID NO:25)，通过PCR扩增获得带有复制起点的载体片段二。以文献报道的pEC-26为模板(Li MY et al.,Frontiers in Bioengineering and Biotechnology,2020,8:357.)，利用引物pEC26-F(SEQ ID NO:26)和pEC26-R(SEQ ID NO:27)，通过PCR扩增获得靶向gltA、pgi、hom和pck基因的crRNA array片段。将上述四个片段回收后，利用Vazyme Clon Express Multies一步重组试剂盒进行重组连接，获得重组载体pXM-P proP-dCpf1。类似地，以pXM-P proP-1-gfp为模板，设计引物proP-D-F(SEQ ID NO:65)和proP1-D-R(SEQ ID NO:67)，通过PCR扩增得到P proP-1启动子片段。将上述启动子片段与载体片段一、载体片段二以及crRNA array片段重组，获得重组载体pXM-P proP-1-dCpf1。同时，以pXM-P proP-dCpf1为模板，利用引物pXM07-F1(SEQ ID NO:22)和pXM07-R2(SEQ ID NO:25)，通过PCR扩增获得载体片段三，将上述片段回收后分别与P proP和P proP-1启动子序列片段通过Vazyme Clon Express Multies一步重组试剂盒进行重组连接，获得相应的对照载体pXM-P proP-con和pXM-P proP-1-con。
根据文献中公开的赖氨酸菌株构建方法(Becker,J.,et al.,Metab.Eng.,2011,13,159-168.)，利用基于pK18mobsacB的同源重组技术将谷氨酸棒杆菌ATCC13032基因组上天冬氨酸激酶(lysC基因编码)第311位的苏氨酸突变为异亮氨酸，构建获得一株具有一定赖氨酸合成能力的菌株SCgL30。将上述重组载体pXM-P proP-dCpf1和pXM-P proP-1-dCpf1、以及各自的对照质粒pXM-P proP-con和pXM-P proP-1-con分别转化SCgL30菌株，获得重组菌株和对照菌株。将上述菌株分别接种含有5μg/mL氯霉素的TSB培养基，30℃，220r/min过夜培养，按照初始OD 0.3分别转接添加或不添加0.6M硫酸钠(模拟发酵后期高浓度产物积累造成的高盐高渗环境)的赖氨酸发酵培养基，培养体系为24孔板装液1mL，30℃，800r/min培养24h后终止发酵，检测剩余葡萄糖含量、OD600和赖氨酸产量。其中赖氨酸发酵培养基配方为：葡萄糖80g/L、酵母粉8g/L、尿素9g/L、K 2HPO 4 1.5g/L、MOPS 42g/L、FeSO 4 0.01g/L、MnSO 4 0.01g/L、MgSO 4 0.6g/L，氯霉素终浓度为5μg/mL。检测结果见表10，结果显示由于启动子强度较弱，野生型proP启动子调控的dCpf1弱化系统对赖氨酸产量及转化率效果非常有限，而改造后的突变启动子在正常培养条件下同样没有明显效果。但在高渗诱导条件下，突变启动子调控的dCpf1系统表现出良好的应用效果，赖氨酸产量及转化率均显著提高。
表10 杂合启动子调控dCpf1表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000014
Figure PCTCN2022070499-appb-000015
实施例19.利用突变启动子调控LysE表达在赖氨酸合成中的应用
以pXM-P proP-gfp为模板，利用引物proP-lysE-F(SEQ ID NO:68)和proP-lysE-R(SEQ ID NO:69)通过PCR扩增得到P proP-1启动子序列片段；以pXM-P proP-1-gfp为模板，利用引物proP-lysE-F(SEQ ID NO:68)和proP1-lysE-R(SEQ ID NO:70)，通过PCR扩增得到P proP启动子且序列片段。以ATCC 13032基因组为模板，利用引物lysE-F(SEQ ID NO:32)和lysE-R(SEQ ID NO:33)通过PCR扩增得到lysE基因片段。同时，以pXM-XK99E为模板，利用引物pEC-F(SEQ ID NO:34)和pEC-R(SEQ ID NO:35)，通过PCR扩增获得载体片段。将上述启动子片段分别与lysE片段和载体片段进行重组连接，并将连接产物转化到Trans T1感受态细胞，涂布卡那霉素抗性平板过夜培养，挑阳性克隆进行菌落PCR验证，并将正确转化子进行测序确认，获得的重组载体命名为pEC-P proP-lysE和pEC-P proP-1-lysE。
将上述重组载体pEC-P proP-lysE、pEC-P proP-1-lysE和pEC-XK99E分别转化谷氨酸棒杆菌ScgL30，获得重组菌株和对照菌株。利用如实施例17的方法(抗生素替换为终浓度为25μg/mL的卡那霉素)验证不同启动子调控的LysE表达菌株在赖氨酸合成中的应用效果，结果见表11。数据显示，野生型proP启动子调控的LysE过表达在高渗条件下可以少量提升赖氨酸产量及转化率，而改造后的杂合启动子在高渗诱导条件下赖氨酸产量及转化率明显提高，显示出良好的应用效果。
表11 不同启动子调控LysE表达在赖氨酸合成中的应用效果
Figure PCTCN2022070499-appb-000016
引用文献：
[10]Sun DH et al.,Journal of Industrial Microbiology&Biotechnology 2019,46(2):203-208.
[11]Li MY et al.,Frontiers in Bioengineering and Biotechnology,2020,8:357.
[12]Becker,J.,et al.,Metab.Eng.,2011,13,159-168.
[13]O Kirchner,et al.Journal of Biotechnology,2003,104:287-299.
本说明书公开的所有技术特征都可以任何组合方式进行组合。本说明所公开的每个特征也可以被其它具有相同、相等或相似作用的特征所替换。因此，除非特殊说明，所公开的每一特征仅仅是一系列相等或相似特征的实例。
此外，从上述描述中，本领域技术人员可从本公开中很容易清楚本公开的关键特征，在不脱离本公开的精神及范围的情况下，可对发明进行很多修改以适应各种不同的使用目的及条件，因此这类修改也旨在落入所附权利要求书的范围内。

Claims (15)

  1. 一种具有启动子活性的多核苷酸，其中，所述多核苷酸选自如下(i)-(viii)中组成的组中的任一项：
    (i)包含如SEQ ID NO：1-3任一序列所示的核苷酸序列；
    (ii)包含如SEQ ID NO：1所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：1所示序列的第170-179位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高于包含如SEQ ID NO：1所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：1所示序列的第170-179位中的核苷酸序列不为ACACCGAGTG；
    (iii)包含如SEQ ID NO：2所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：2所示序列的第130-139位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高于包含如SEQ ID NO：2所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：2所示序列的第130-139位中的核苷酸序列不为ACACCGAGTG；
    (iv)包含如SEQ ID NO：3所示序列的多核苷酸的突变体，所述突变体在SEQ ID NO：3所示序列的第72-81位中的一个或多个位置处具有突变的核苷酸；所述突变体的活性高于包含如SEQ ID NO：3所示序列的多核苷酸的启动子活性，且所述突变体在SEQ ID NO：3所示序列的第72-81位中的核苷酸序列不为ACACCGAGTG；
    (v)包含如SEQ ID NO：57-58任一序列所示的核苷酸序列；
    (vi)包含与(i)至(v)中任一项所示的核苷酸序列的反向互补序列的多核苷酸；
    (vii)包含在高严格性杂交条件或非常高严格性杂交条件下，能够与(i)至(v)中任一项所示的核苷酸序列杂交的序列的反向互补序列的多核苷酸；
    (viii)包含与(i)至(v)中任一项所示的核苷酸序列具有至少90％，可选至少95％，优选至少97％，更优选至少98％，最优选至少99％序列同一性的序列的多核苷酸。
  2. 根据权利要求1所述的具有启动子活性的多核苷酸，其中，所述多核苷酸在盐浓度或渗透压升高的环境中具有提高的启动子活性。
  3. 根据权利要求1或2所述具有启动子活性的多核苷酸，其中，所述突变体对应SEQ ID NO：1所示序列的第170-179位，或对应SEQ ID NO：2所示序列的第130-139位，或对应SEQ ID NO：3所示序列的第72-81位的核苷酸序列选自如下(p 1)-(p 12)组成的组中的任一项：
    (p 1)TACTTGCAGA，
    (p 2)AGTGCTGAAA，
    (p 3)GCACGAAAGG，
    (p 4)TATCTAGAGG，
    (p 5)AGGCTTGTCG，
    (p 6)CGCTTCTTTC，
    (p 7)TAACTCTTGG，
    (p 8)CCAAGTTCCA，
    (p 9)CGGTGCCACA，
    (p 10)AGCAGTTAGG，
    (p 11)AGATAAATAA，
    (p 12)ATCGATCTAG。
  4. 根据权利要求1-3任一项所述的具有启动子活性的多核苷酸，其中，所述突变体的核苷酸序列选自如SEQ ID NO：37-48任一项所示的序列。
  5. 一种转录表达盒，其中，所述转录表达盒包含根据权利要求1-4任一项所述的具有启动子活性的多核苷酸；可选地，所述转录表达盒还含有蛋白编码基因，所述蛋白编码基因与所述具有启动子活性的多核苷酸可操作地连接。
  6. 一种重组表达载体，其中，所述重组表达载体包含根据权利要求1-4任一项所述的具有启动子活性的多核苷酸，或根据权利要求5所述的转录表达盒。
  7. 一种重组宿主细胞，其中，所述重组宿主细胞包含根据权利要求5所述的转录表达盒，或根据权利要求6所述的重组表达载体。
  8. 根据权利要求7所述的重组宿主细胞，其中，所述宿主细胞来源于棒状杆菌属、短杆菌属、节杆菌属、微杆菌属或埃希氏菌属；优选地，所述宿主细胞为谷氨酸棒杆菌或大肠杆菌；更优选地，所述宿主细胞为谷氨酸棒杆菌ATCC 13032、谷氨酸棒杆菌ATCC 13869或谷氨酸棒杆菌ATCC 14067及其衍生菌株。
  9. 一种根据权利要求1-4任一项所述的多核苷酸，根据权利要求5所述的转录表达盒，根据权利要求6所述的重组表达载体，根据权利要求7或8所述的重组宿主细胞在如下至少一种中的用途：
    (a)调控基因的转录水平，或制备用于调控基因的转录水平的试剂或试剂盒；
    (b)制备蛋白，或制备用于制备蛋白的试剂或试剂盒；
    (c)生产目标化合物，或制备用于生产目标化合物的试剂或试剂盒。
  10. 根据权利要求9所述的用途，其中，所述蛋白选自基因表达调控蛋白或与目标化合物合成相关的蛋白、基因表达调控蛋白或与膜转运相关的蛋白。
  11. 根据权利要求9或10所述的用途，其中，所述目标化合物包括氨基酸和有机酸中的至少一种；可选地，所述氨基酸是如下的一种或两种以上的组合：脯氨酸、羟脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸或上述任一种的氨基酸的衍生物；可选地，所述有机酸是如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述任一种的有机酸的衍生物。
  12. 一种调控目标基因转录的方法，其中，所述方法包括将权利要求1-4任一项所述的具有启动子活性的多核苷酸与目标基因可操作地连接的步骤；或者，所述方法包括将权利要求1-4任一项所述的具有启动子活性的多核苷酸与目标RNA可操作地连接的步骤；
    可选地，所述目标基因包括与目标化合物合成相关的蛋白的编码基因、基因表达调控蛋白的编码基因、与膜转运相关的蛋白的编码基因中的至少一种；
    可选地，所述目标RNA是tRNA、sRNA中的至少一种。
  13. 一种制备蛋白的方法，其特征在于，包括利用根据权利要求5所述的转录表达盒，根据权利要求6所述的重组表达载体，或根据权利要求7-8任一项所述的重组宿主细胞表达所述蛋白的步骤；可选地，所述蛋白为与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白；
    任选地，所述方法还包括分离或纯化所述蛋白的步骤。
  14. 一种生产目标化合物的方法，其中，包括利用权利要求5所述的转录表达盒，权利要求6所述的重组表达载体，或权利要求7-8任一项所述的重组宿主细胞表达与目标化合物合成相关的蛋白、与膜转运相关的蛋白或基因表达调控蛋白，在所述与目标化合物合成相关的蛋白、与膜转运相关的蛋白或所述基因表达调控蛋白存在的环境下生产目标化合物的步骤；
    可选地，所述目标化合物包括氨基酸和有机酸中的至少一种；可选地，所述氨基酸是如下的一种或两种以上的组合：脯氨酸、羟脯氨酸、赖氨酸、谷氨酸、苏氨酸、甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、半胱氨酸、谷氨酰胺、甲硫氨酸、天冬氨酸、天冬酰胺、精氨酸、组氨酸、苯丙氨酸、酪氨酸、色氨酸、5-氨基乙酰丙酸 或上述任一种的氨基酸的衍生物；可选地，所述有机酸是如下的一种或两种以上的组合：柠檬酸、琥珀酸、乳酸、醋酸、丁酸、棕榈酸、草酸、草酰乙酸、酒石酸、丙酸、己烯酸、癸酸、辛酸、戊酸、苹果酸或上述任一种的有机酸的衍生物；
    任选地，所述方法还包括分离或纯化所述目标化合物的步骤。
  15. 根据权利要求14所述的方法，其中，所述与目标化合物合成相关的蛋白是丙酮酸羧化酶、磷酸烯醇式丙酮酸羧化酶、γ-谷氨酰激酶、谷氨酸半醛脱氢酶、吡咯啉-5-羧酸还原酶、氨基酸运输蛋白、ptsG系统、丙酮酸脱氢酶、高丝氨酸脱氢酶、草酰乙酸脱羧酶、葡萄糖酸阻遏蛋白、葡萄糖脱氢酶、天冬氨酸激酶、天冬氨酸半醛脱氢酶、天冬氨酸氨裂合酶、二氢吡啶二羧酸合成酶、二氢吡啶二羧酸还原酶、二氢吡啶甲酸还原酶、琥珀酰二氨基庚二酸氨基转移酶、四氢吡啶二羧酸酯琥珀酰酶、琥珀酰二氨基庚二酸脱酰基酶、二氨基庚二酸差向异构酶、二氨基庚二酸脱酰基酶、甘油醛-3-磷酸脱氢酶、转酮酶、二氨基庚二酸脱氢酶中的一种或两种以上的组合；
    可选地，所述与目标化合物合成相关的蛋白为与L-氨基酸合成相关的蛋白；
    可选地，所述与目标化合物合成相关的蛋白为与赖氨酸合成相关的蛋白。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024192935A1 (zh) * 2023-03-23 2024-09-26 赛诺根生物科学有限公司 一种催化性能提高的nadh焦磷酸酶突变体及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101605890A (zh) * 2007-01-15 2009-12-16 Cj第一制糖株式会社 源自谷氨酸棒杆菌的新型启动子核酸分子、包括所述启动子的重组载体、包括所述重组载体的宿主细胞和利用所述宿主细胞表达基因的方法
CN108350464A (zh) * 2015-12-07 2018-07-31 齐默尔根公司 来自谷氨酸棒杆菌的启动子
CN110709517A (zh) * 2018-03-09 2020-01-17 Cj第一制糖株式会社 新型启动子和利用其生产l-氨基酸的方法
CN113201538A (zh) * 2021-01-12 2021-08-03 中国科学院天津工业生物技术研究所 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
CN113278620A (zh) * 2021-07-26 2021-08-20 中国科学院天津工业生物技术研究所 一种突变的高渗诱导型启动子PproP及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101605890A (zh) * 2007-01-15 2009-12-16 Cj第一制糖株式会社 源自谷氨酸棒杆菌的新型启动子核酸分子、包括所述启动子的重组载体、包括所述重组载体的宿主细胞和利用所述宿主细胞表达基因的方法
CN108350464A (zh) * 2015-12-07 2018-07-31 齐默尔根公司 来自谷氨酸棒杆菌的启动子
CN110709517A (zh) * 2018-03-09 2020-01-17 Cj第一制糖株式会社 新型启动子和利用其生产l-氨基酸的方法
CN113201538A (zh) * 2021-01-12 2021-08-03 中国科学院天津工业生物技术研究所 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
CN113278620A (zh) * 2021-07-26 2021-08-20 中国科学院天津工业生物技术研究所 一种突变的高渗诱导型启动子PproP及其应用

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BECKER, J. ET AL., METAB. ENG., vol. 13, 2011, pages 159 - 168
FRANZEL, B. ET AL.: "Adaptation of Corynebacterium glutamicum to salt-stress conditions", PROTEOMICS, vol. 10, no. 3, 2010, pages 445 - 457
HU, T.H. ET AL., CHINA BREWING, vol. 37, no. 10, 2018, pages 51 - 56
KIM, M. J ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 100, 2016, pages 4473 - 4483
LI MY ET AL., FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, vol. 8, 2020, pages 357
MA, Y. C. ET AL., MICROB. CELL FACT., 2018, pages 17
O KIRCHNER ET AL., JOURNAL OF BIOTECHNOLOGY, vol. 104, 2003, pages 287 - 299
RYTTER, J. V ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 98, 2014, pages 2617 - 2623
SAMBROOK ET AL.: "Microbial Metabolic Engineering: Methods and Protocols", 1989, COLD SPRING HARBOR LABORATORY PRESS
SUN DH ET AL., JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, vol. 46, no. 2, 2019, pages 203 - 208
VARELA C ET AL., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 60, no. 5, 2003, pages 547 - 555
XU, J. Z. ET AL., MICROB CELL FACT, vol. 19, 2020, pages 39
ZHANG SHUANGHONG; LIU DINGYU; MAO ZHITAO; MAO YUFENG; MA HONGWU; CHEN TAO; ZHAO XUEMING; WANG ZHIWEN: "Model-based reconstruction of synthetic promoter library inCorynebacterium glutamicum", BIOTECHNOLOGY LETTERS, vol. 40, no. 5, 31 March 2018 (2018-03-31), Dordrecht , pages 819 - 827, XP036481708, ISSN: 0141-5492, DOI: 10.1007/s10529-018-2539-y *
ZHANG XIAN, ZHANG WEIYI;LI CHUANMIN;NIU SHUQI;ZANG WEIPING;XU DAQING: "Identification of Strong Promoter of the Cgl0864 Gene in Corynebacterium Glutamicum", JOURNAL OF HEBEI AGRICULTURAL UNIVERSITY, vol. 43, no. 2, 15 March 2020 (2020-03-15), CN , pages 69 - 75, XP055951967, ISSN: 1000-1573, DOI: 10.13320/j.cnki.jauh.2020.0032 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024192935A1 (zh) * 2023-03-23 2024-09-26 赛诺根生物科学有限公司 一种催化性能提高的nadh焦磷酸酶突变体及其应用

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