WO2023236634A1 - 一种ep6启动子与其相关生物材料及应用 - Google Patents

一种ep6启动子与其相关生物材料及应用 Download PDF

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WO2023236634A1
WO2023236634A1 PCT/CN2023/084969 CN2023084969W WO2023236634A1 WO 2023236634 A1 WO2023236634 A1 WO 2023236634A1 CN 2023084969 W CN2023084969 W CN 2023084969W WO 2023236634 A1 WO2023236634 A1 WO 2023236634A1
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seq
promoter
recombinant
strain
vector
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French (fr)
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魏爱英
孟刚
贾慧萍
米杰
杨立鹏
苏厚波
田斌
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宁夏伊品生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P13/14—Glutamic acid; Glutamine

Definitions

  • the present invention relates to an EP6 promoter and its related biological materials and applications in the field of biotechnology.
  • the purpose of the present invention is to provide an EP6 promoter, which can be used to produce amino acids.
  • the sequence of the EP6 promoter provided by the invention is SEQ ID No. 3 in the sequence list.
  • the present invention also provides biological materials related to the EP6 promoter, and the biological materials are any one of the following B1) to B7):
  • the expression cassette containing the EP6 promoter described in B1) refers to the DNA that can drive the expression of the target gene in the host cell with the EP6 promoter.
  • the DNA can not only include the target gene, but also include the termination of the target gene. Transcription terminator.
  • the expression cassette may also include an enhancer sequence.
  • the vector can be a plasmid, cosmid, phage or viral vector.
  • the microorganism may be yeast, bacteria, algae or fungi.
  • the bacterium may be Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniaphagogenes, Corynebacterium crenatum or Pantoea ).
  • the present invention also provides the use of EP6 promoter as a promoter.
  • the present invention also provides the use of EP6 promoter in the production of amino acids.
  • the present invention also provides the use of the biological material in the production of amino acids.
  • the amino acid may be lysine, glutamic acid or valine.
  • the EP6 promoter of the present invention can be used to produce a variety of products, including but not limited to lysine, glutamic acid and valine in the embodiments.
  • the products produced can also be glycine, alanine and leucine. , unusually bright Acid, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, sperm Amino acid, histidine, shikimic acid, protocatechuic acid, succinic acid, a-ketoglutaric acid, citric acid, ornithine, citrulline, etc.
  • the production of the target product can be achieved by placing the EP6 promoter of the present invention upstream of the gene in the synthesis pathway of the target product and allowing the EP6 promoter to drive the synthesis of the gene in the synthesis pathway of the target product.
  • the present invention also provides a method for producing amino acids.
  • the method includes: introducing an EP6 promoter into a biological cell capable of synthesizing a target amino acid, so that the EP6 promoter drives the expression of genes in the target amino acid synthesis pathway in the biological cell, Obtain recombinant biological cells; culture the recombinant biological cells to obtain the target amino acid.
  • the biological cells may be yeast, bacteria, algae, fungi, plant cells or animal cells that can synthesize the amino acid of interest.
  • the biological cell is any biological cell that can synthesize the amino acid of interest.
  • the bacterium may be Corynebacterium glutamicum, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniaphagogenes, Corynebacterium crenatum or Pantoea ).
  • the target amino acid is lysine
  • the bacterium is Corynebacterium glutamicum CGMCC No. 12856.
  • Bacteria used to produce lysine using the promoter of the present invention include, but are not limited to, Corynebacterium glutamicum CGMCC No. 12856.
  • the present invention can place the EP6 promoter of the present invention upstream of the genes in the lysine synthesis pathway of these bacteria, so that the EP6 promoter of the present invention can synthesize lysine by driving the expression of genes in the lysine synthesis pathway of these bacteria. acid.
  • the target amino acid is glutamic acid
  • the bacterium is Corynebacterium glutamicum CGMCC No. 21220.
  • Bacteria used to produce glutamic acid using the promoter of the present invention include, but are not limited to, Corynebacterium glutamicum CGMCC No. 21220.
  • the present invention can place the EP6 promoter of the present invention upstream of the genes in the glutamate synthesis pathway of these bacteria, so that the EP6 promoter of the present invention can synthesize glutamate by driving the expression of genes in the glutamate synthesis pathway of these bacteria. .
  • the target amino acid is valine
  • the bacterium is Corynebacterium glutamicum CGMCC No. 21260.
  • Bacteria used to produce valine using the promoter of the present invention include but are not limited to Corynebacterium glutamicum CGMCC No. 21260.
  • the present invention can place the EP6 promoter of the present invention upstream of the genes in the valine synthesis pathway of these bacteria, so that the EP6 promoter of the present invention can synthesize valine by driving the expression of genes in the valine synthesis pathway of these bacteria. acid.
  • the target amino acid may be lysine, glutamic acid or valine.
  • the EP6 promoter of the present invention can be used to produce a variety of products, including but not limited to lysine, glutamic acid and valine in the embodiments.
  • the products produced can also be glycine, alanine and leucine. , isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, Asparagine, glutamine, threonine, aspartic acid, arginine, histidine, shikimic acid, protocatechuic acid, succinic acid, alpha-ketoglutarate, citric acid, ornithine, Citrulline etc.
  • the production of the target product can be achieved by placing the EP6 promoter of the present invention upstream of the gene in the synthesis pathway of the target product and allowing the EP6 promoter to drive the synthesis of the gene in the synthesis pathway of the target product.
  • the target amino acid is lysine
  • the bacterium is Corynebacterium glutamicum CGMCC No. 12856
  • the gene in the lysine synthesis pathway is the lysA gene (SEQ ID No. .4 bits 149-1486).
  • the recombinant biological cell is achieved by replacing the EP6 promoter with the original promoter of the lysA gene in Corynebacterium glutamicum CGMCC No. 12856.
  • the target amino acid is glutamic acid
  • the bacterium is Corynebacterium glutamicum CGMCC No. 21220
  • the gene in the glutamic acid synthesis pathway is the BBD29_14295 gene (SEQ ID No. .11 bits 37-1161).
  • the recombinant biological cell is realized by replacing the EP6 promoter with the original promoter of the BBD29_14295 gene in Corynebacterium glutamicum CGMCC No. 21220.
  • the target amino acid is valine
  • the bacterium is Corynebacterium glutamicum CGMCC No. 21260
  • the gene in the valine synthesis pathway is the ilvC gene (SEQ ID No. .18 No. 180-1196).
  • the recombinant biological cell is realized by replacing the original promoter of the ilvC gene in Corynebacterium glutamicum CGMCC No. 21260 with the EP6 promoter.
  • the present invention also provides a product for producing amino acids, and the active ingredient of the product is the EP6 promoter or the biological material.
  • CGMCC Abbreviation of depository institution
  • Collection center registration number CGMCC No.21220.
  • CGMCC Abbreviation of depository institution
  • CGMCC Abbreviation of depository institution
  • Collection center registration number CGMCC No.12856.
  • Figure 1 shows the detection results of the promoter activities of EP6 and yfjB.
  • PEP6 is the EP6 promoter.
  • pEC-H10-mCherry vector It is recorded in the Chinese patent application with application number 202110256579.8 (publication number CN 112980867A). Its preparation method is as follows:
  • the strongest promoter H10 (GCTCAACCCTTACCGGTCGGCTCTAAGCCG) in Corynebacterium glutamicum reported by Wei et al. GCGGCGTATGGTAAGCTCTGTTATGTATAGTCCGAGCACGGCGAAAGGATACTC( SEQ ID No. 25)) as a template, as well as the gene sequence of mCherry protein and the sequence of the expression vector pEC-XK99E in Corynebacterium glutamicum, design and synthesize primers for constructing the vector of pEC-H10-mCherry.
  • the primers are designed as follows (synthesized by Guangzhou Jinweizhi Company):
  • Primer 1 5′-GGATCTAGAGTCGACCTGCAG-3′ (SEQ ID No. 26);
  • Primer 2 5′-TTAACTAGTATTGCGTTGCGCTCAC-3′ (SEQ ID No. 27);
  • Primer 3 5′-CAATACTAGTTAATGTGAGTTAGCGCG-3′ (SEQ ID No. 28);
  • Primer 4 5′-AGAGCTTACCATACGCCGCCGGCTTAGAGCCGACCGGTAAGGGTTGAGCCTAGAGGATCCCCGG GTAC-3′ (SEQ ID No. 29);
  • Primer 5 5′-GCGTATGGTAAGCTCTGTTATGTTATGTATAGTCCGAGCACGGCGAAAGGATACTCATGCGTAAAGGAGAAGAAG-3′ (SEQ ID No. 30);
  • Primer 6 5′-CGACTCTAGATCCGCCAAAACAGCC-3′ (SEQ ID No. 31).
  • Construction method Use pEC-XK99E as the template, use primer 1 and primer 2 to amplify the backbone region (6743bp) of the pEC plasmid; use promoter H10 as the template, use primer 3 and primer 4 to amplify the fragment containing the upstream region of the H10 promoter (387bp); using the plasmid pBblactam containing the mCherry gene (Zhang et al., Development of a transcription factor based lactam biosensor. ACS synthetic biology. 2017, 6, 439-445.) as a template, primers 5 and 6 were used to amplify the gene containing H10 The downstream region of the promoter and the fragment of mCherry gene (810bp).
  • bands with lengths of 387bp, 810bp and 6743bp were obtained respectively.
  • the above three bands were separated and recovered by agarose gel electrophoresis. After recovery, the DNA fragments of the 387bp and 810bp bands were used as templates. Fusion PCR amplification was performed using primer 3 and primer 6.
  • the obtained PCR amplified fragments were gel cut and recovered.
  • the recovered fragments i.e., the fragments containing H10 and mCherry
  • the pEC backbone fragment were subjected to enzyme digestion (SpeI, XbaI) respectively. After the enzyme digestion was completed, the fragments were purified through a column.
  • the vector is the pEC-H10-mCherry vector.
  • pk18 vector Addgene Company, product number is MLCC1103.
  • Corynebacterium glutamicum CGMCC No. 21220 in the following examples the strain number is YPGLU001, has been deposited in the General Microorganism Center of the China Microbial Culture Collection Committee on November 23, 2020, and the deposit number is CGMCC No.21220.
  • Corynebacterium glutamicum CGMCC No. 21260 in the following examples the strain number is YPFV1, has been deposited in the General Microorganism Center of the China Microbial Culture Collection Committee on November 30, 2020, and the deposit number is CGMCC No.21260.
  • NEBuilder recombination technology was used to construct the E. coli W3110yfjB promoter (SEQ ID No.1: gaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcggaa aaa)
  • pEC-PyfjB-mCherry vector based on the expression of the reporter protein mCherry, detects PyfjB initiation intensity and served as a control for subsequent mutated promoter sequences.
  • the primer sequence is as follows (synthesized by Shanghai Invitrogen Company):
  • pEC01 5′-ATGCGTAAAGGAGAAGAAGATAAC-3′ (SEQ ID No. 32);
  • pEC02 5′-CTAGAGGATCCCCGGGTAC-3′(SEQ ID No.33);
  • P2 5′- AATGATAGCCATGTTATCTTCTTCCTCTTACGCAT TTTTCCGAGGTCCTTGTTGC-3′ (SEQ ID No. 35).
  • Construction method Use the pEC-H10-mCherry vector as a template, use primers pEC01 and pEC02 to amplify the backbone region of the pEC-H10-mCherry vector 7772bp; use the DNA fragment shown in SEQ ID No. 1 as a template, use primers P1 and P2 amplified a 194 bp fragment of the yfjB promoter (referred to as PyfjB) containing the homology arm of the pEC-H10-mCherry vector.
  • PyfjB yfjB promoter
  • the above two DNA fragments (the backbone region of the pEC-H10-mCherry vector and the PyfjB fragment containing the homology arm of the pEC-H10-mCherry vector) were separated and purified by agarose gel electrophoresis, and then used with NEBuilder enzyme (NEB Company) at 50°C. After ligation for 30 minutes, the single clone grown after the ligation product was transformed into E. coli DH5a was modified with primers pECF/R (pECF: 5′-GTACCCGGGGATCCTCTAG-3′ (SEQ ID No. 36), pECR: 5′-GTTATCTTCTTCCTCTTACGCAT-3′ (SEQ ID No. 37)) were identified, and the plasmid was extracted to obtain a positive recombinant vector with the correct sequence, which is pEC-PyfjB-mCherry.
  • the sequence of the recombinant vector pEC-PyfjB-mCherry is SEQ ID No. 2 in the sequence listing.
  • the yfjB promoter activates the expression of the reporter gene mCherry.
  • the fluorescence value of mCherry is detected to obtain the strength of the yfjB promoter.
  • NEBuilder recombination technology was used for vector construction, and the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • Construction method Use pEC-PyfjB-mCherry vector as template, primers pEC01 and pEC02 to amplify 7772bp of the backbone region of pEC-H10-mCherry vector; use the DNA fragment shown in SEQ ID No. 3 as template, use primers P5 and P6 to amplify The 196bp EP6 promoter fragment containing the homology arm of pEC-H10-mCherry vector was added.
  • the above two DNA fragments (the backbone region of the pEC-H10-mCherry vector and the EP6 promoter fragment containing the homology arm of the pEC-H10-mCherry vector) were separated and purified by agarose gel electrophoresis, and then used NEBuilder enzyme (NEB Company) Ligate at 50°C for 30 minutes.
  • NEB Company NEBuilder enzyme Ligate at 50°C for 30 minutes.
  • the single clone grown after the ligation product is transformed into E. coli DH5a is modified with primers pECF/R (pECF: 5′-GTACCCGGGGATCCTCTAG-3′ (SEQ ID No.
  • pECR 5′-GTTATCTTCTTCCTTTTACGCAT-3′( SEQ ID No.37) was identified, and the plasmid was extracted to obtain a positive recombinant vector with the correct sequence, which was recorded as pEC-EP6-mCherry.
  • pEC-EP6-mCherry is a recombinant vector obtained by replacing PyfjB (SEQ ID No. 1) of pEC-PyfjB-mCherry with EP6 (SEQ ID No. 3).
  • the pEC-EP6-mCherry vector in step 2 and the pEC-PyfjB-mCherry vector in step 1 were respectively introduced into wild-type Corynebacterium glutamicum ATCC13032 to obtain recombinant bacteria ATCC13032/pEC-EP6-mCherry and ATCC13032/pEC-PyfjB.
  • LBHIS medium 5g Tryptone/L, 5g NaCl/L, 2.5g yeast extract/L, 18.5g BHI (Brain Heart Infusion)/L, 91g sorbitol (sorbose) alcohol)/L, pH 7.2.
  • Example 2 Replacement of the lysA gene promoter of the lysine metabolism pathway in Corynebacterium glutamicum and its effect on lysine production.
  • the pk18 vector was used to replace the original promoter of lysA, a key gene of the lysine metabolism pathway in the high-lysine-producing Corynebacterium glutamicum CGMCC No. 12856, by homologous recombination. (positions 1-148 of SEQ ID No. 4), thereby using an exogenous promoter to initiate the expression of lysA in the lysine metabolism pathway, and obtaining lysine-producing bacteria with an exogenous promoter-gene combination. Fermentation tests showed that the strain that starts lysA with EP6 can increase the production of lysine at a higher level, and the strain was named YPL-4-042. SEQ ID No.4 No. Positions 149-1486 are the sequence of lysA.
  • the vector was constructed using NEBuilder recombination technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • P8 5′-AAAAAAACGCGGAGAAATTCATGCCCCGTTCGACAATAAA-3′ (SEQ ID No. 41);
  • P9 5′-TTTATTGTCGAACGGGGCATGAATTTCTCCGGCGTTTTTTT-3′ (SEQ ID No. 42);
  • P10 5′-GCGAGATCAGCTGGTGTCATTTTTCCGAGGTCCTTGTTGC-3′ (SEQ ID No. 43);
  • P12 5′- CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCC TCGAACATCATCTCCACGCC-3′ (SEQ ID No. 45);
  • P8E 5′-AAAAAAACGCGAAGAAATTCATGCCCCGTTCGACAATAAA-3′ (SEQ ID No. 46);
  • P9E 5′-TTTATTGTCGAACGGGGCATGAATTTCTTCGCGTTTTTTT-3′ (SEQ ID No. 47);
  • P10E 5′-GCGAGATCAGCTGGTGTCATGATTTGGAGGTCCCTGCGTT-3′ (SEQ ID No. 48);
  • P11E 5′-AACGCAGGGACCTCCAAATCATGACACCAGCTGATCTCGC-3′ (SEQ ID No. 49).
  • PCR amplification was performed with primers P7/P8 (the PCR product sequence is SEQ ID No. 5 1-801), and the upstream homology arm fragment 801bp was obtained; with the vector pEC-PyfjB- mCherry was used as the template, and primers P9/P10 were used for PCR amplification to obtain PyfjB 162bp (the PCR product sequence is SEQ ID No. 5 No. 762-923); Corynebacterium glutamicum ATCC13032 was used as the template, and primers P11/P12 ( The PCR product sequence is SEQ ID No. 5 No. 884-1648) was PCR amplified to obtain a downstream homology arm fragment of 765 bp.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the ligation product was transformed into E. coli DH5a was used with the M13 primer (M13F: 5′-TGTAAAACGAGCGGCCAGT-3′ (SEQ ID No. 50), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No.
  • pK18-PyfjB-lysAOE contains the DNA fragment shown in SEQ ID No.5.
  • positions 1-801 are the sequences of the upstream homology arm fragment
  • positions 884-1648 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 762-923 are the sequence of PyfjB.
  • Corynebacterium glutamicum ATCC13032 was used as the template and primer P7/P8E (SEQ ID No. 6 No. 1-801) was used for PCR amplification to obtain the upstream homology arm fragment 801bp; using pEC-EP6-mCherry as the template, P9E/P10E was PCR amplified to obtain EP6 164bp (SEQ ID No. 6 No. 762-925); Corynebacterium glutamicum ATCC13032 was used as the template and primer P11E/P12 (SEQ ID No. 6 No. 886-1650) ) performed PCR amplification to obtain a 765bp downstream homology arm fragment.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the ligation product was transformed into E.
  • coli DH5a used M13 primers (M13F: 5′-TGTAAAACGGACGGCCAGT-3′ (SEQ ID No.50), M13R: 5′-CAGGAAACAGCTATGACC-3′ (SEQ ID No.51))
  • the positive integration vector was obtained after PCR identification.
  • the recombinant vector with correct sequence was extracted and recorded as pK18-EP6-lysAOE.
  • This recombinant vector contains a kanamycin resistance marker and can be screened with kanamycin to obtain the vector integrated into Recombinants on the genome.
  • pK18-EP6-lysAOE contains the DNA fragment shown in SEQ ID No.6.
  • positions 1-801 are the sequences of the upstream homology arm fragment
  • positions 886-1650 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 762-925 are the sequence of EP6.
  • the correctly sequenced recombinant vectors pK18-PyfjB-lysAOE and pK18-EP6-lysAOE were electrotransformed into the high-lysine-producing Corynebacterium glutamicum CGMCC No. 12856, and cultured in the recovery medium according to the recovery culture conditions.
  • the single colony produced by culture was identified by PCR with P13/P14 primers.
  • the recombinant strain introduced into pK18-PyfjB-lysAOE was PCR-amplified to produce an 885bp (SEQ ID No. 7) fragment as a positive strain, which was recorded as CGMCC No.
  • the positive strain was cultured on a medium containing 15% sucrose, and the single colony produced by the culture was further identified by PCR using P15/P16 primers, and a strain of 841bp (SEQ ID No. 9) was amplified to replace the high-yielding strain with PyfjB.
  • Corynebacterium glutamicum CGMCC No. 12856 a positive strain of the lysA original promoter, amplified a strain of 843 bp (SEQ ID No. 10) to replace EP6 with high lysine-producing Corynebacterium glutamicum CGMCC No. 12856 lysA original promoter-positive strains, those that cannot amplify the fragment are original bacteria.
  • YPL-4-041 (PyfjB-lysA integrated strain) is a high-lysine-producing Corynebacterium glutamicum CGMCC No. 12856 lysA original promoter (positions 1-148 of SEQ ID No.
  • yfjB promoter a recombinant strain obtained by keeping other nucleotides in its genome unchanged, which can increase the production of lysine
  • YPL-4-042 EP6-lysA integrated strain
  • the original promoter of CGMCC No.12856 lysA was replaced with the EP6 promoter, and the other nucleotides in the genome were kept unchanged.
  • the EP6 promoter was used as the mutated yfjB
  • the promoter, which activates the expression of lysA, can also increase the production of lysine.
  • the PCR identification primers are as follows:
  • P13 5′-GGCGACGATCTTGTTTGACC-3′ (inside the cg1332 gene) (SEQ ID No. 52);
  • P14 5′-ACTGATCCCCATAATAAGCG-3′ (internal to PyfjB) (SEQ ID No. 53);
  • P15 5′-CGCTTATTATGGGGATCAGT-3′ (internal to PyfjB) (SEQ ID No. 54);
  • P16 5′-GTAGTAGACATCGAAATCGG-3′ (inside the argS gene) (SEQ ID No. 55).
  • Recovery medium The solvent is water, and the solutes and their contents in the recovery medium are glucose 5g/L, urea 3g/L, yeast powder 10g/L, biotin 10 ⁇ g/L, soybean meal powder 15g/L, and diethylene glycol. Acid 0.5g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 2.5g/L, sorbitol 91g/L, brain heart infusion 18.5g/L, pH 7.0.
  • sucrose culture medium The solvent is water, and the solutes and their contents in the 15% sucrose culture medium are 15g/L sucrose, 3g/L urea, 10g/L yeast powder, 10 ⁇ g/L biotin, and soybean meal powder. 15g/L, succinic acid 0.5g/L, potassium dihydrogen phosphate 1g/L, sodium chloride 2.5g/L, sorbitol 91g/L, brain heart infusion 18.5g/L, agar powder 15g/L, pH7 .0.
  • Recovery culture conditions Resuspend the cells in recovery medium at 46°C, transfer to EP tubes, incubate on a 37°C shaker for 45-60 minutes at 220 rpm, then transfer to a 30°C shaker for 45-60 minutes, with the rotation speed unchanged.
  • Fermentation medium 1 The solvent is water, and the solutes and their contents in fermentation medium 1 are starch hydrolyzed sugar 30g/L, ammonium sulfate 12g/L, magnesium sulfate 0.87g/L, molasses 20g/L, and acidified corn steep liquor.
  • Acidified corn steep liquor is a product of Ningxia Yipin Biotechnology Co., Ltd.
  • Full process control Full process control 1. When the dissolved oxygen is ⁇ 30%, increase the speed in sequence to 750rpm ⁇ 800rpm ⁇ air volume 4L/min ⁇ 850rpm ⁇ 950rpm; 2.
  • the canning pressure is 0.01Mpa for 6 hours of fermentation; the canning pressure is 0.02Mpa ⁇ 0.03Mpa for 12h fermentation ⁇ 0.04Mpa ⁇ 0.05Mpa;
  • Residual sugar control 0.1-0.2% before F12h; after F12h combined with DO requirements, residual sugar control is 0.1-0.05%;
  • Ammonia nitrogen control 0.1-0.15 before F12h; 0.15-0.25 from F12-F32h; 0.1-0.15 after F32h;
  • Feeding materials 25% ammonia, 70% concentrated sugar, 50% ammonium sulfate, 10% ammonium sulfate;
  • the pk18 vector was used to replace the EP6 and yfjB promoters respectively with the original promoter of the key gene BBD29_14295 ( 1-36 of SEQ ID No. 11), thereby using an exogenous promoter to initiate the expression of BBD29_14295 in the glutamate metabolism pathway, obtaining exogenous promoter-gene combination glutamate-producing bacteria, and fermenting these production bacteria After testing, it was found that the strain activating BBD29_14295 with EP6 could increase the production of glutamic acid at a higher level.
  • the strain was named YPG-092. Positions 37-1161 of SEQ ID No. 11 are the sequence of BBD29_14295.
  • the vector was constructed using NEBuilder recombination technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • P20 5′-TCGTCGATTAGCTCGAACATTTTTCCGAGGTCCTTGTTGC-3′ (SEQ ID No. 59);
  • P21 5′-GCAACAAGGACCTCGGAAAAATGTTCGAGCTAATCGACGA-3′ (SEQ ID No. 60);
  • P18E 5′-AAAAAAACGCGAAGAAATTCTTAGAGGAATTTAACGCCTT-3′ (SEQ ID No. 62);
  • P19E 5′-AAGGCGTTAAATTCCTCTAAGAATTTCTTCGCGTTTTTTT-3′ (SEQ ID No. 63);
  • P20E 5′-TCGTCGATTAGCTCGAACATGATTTGGAGGTCCCTGCGTT-3′ (SEQ ID No. 64);
  • P21E 5′-AACGCAGGGACCTCCAAATCATGTTCGAGCTAATCGACGA-3′ (SEQ ID No. 65).
  • Corynebacterium glutamicum ATCC13869 was used as the template, PCR amplification was performed with primers P17/P18 (SEQ ID No. 12 1-792) to obtain the upstream homology arm fragment 792bp; with the vector pEC-PyfjB- mCherry was used as the template, and primers P19/P20 were used for PCR amplification to obtain PyfjB 162bp (SEQ ID No. 12 positions 753-914); Corynebacterium glutamicum ATCC13869 was used as the template, and primers P21/P22 (SEQ ID No. 12 No. 875-1699) was PCR amplified to obtain a downstream homology arm fragment of 825 bp.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the product was transformed into E. coli DH5a was treated with M13 primers (M13F: 5'-TGTAAAACGGACGGCCAGT-3' (SEQ ID No. 50), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No. 51)).
  • the positive integration vector (recombinant vector) was obtained by PCR identification, and the recombinant vector with the correct sequence was recorded as pK18-PyfjB-BBD29_14295OE.
  • the recombinant vector contains a kanamycin resistance marker, and the vector integration can be obtained through kanamycin screening. recombinants on the genome.
  • pK18-PyfjB-BBD29_14295OE contains the DNA fragment shown in SEQ ID No. 12.
  • positions 1-792 are the sequences of the upstream homology arm fragment
  • positions 875-1699 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 753-914 are the sequence of PyfjB.
  • PCR amplification was performed with primers P17/P18E (SEQ ID No. 13 No. 1-792) to obtain the upstream homology arm fragment. 792bp; using the vector pEC-EP6-mCherry as the template and using the primers P19E/P20E for PCR amplification, EP6 164bp (SEQ ID No. 13 positions 753-916) was obtained; using Corynebacterium glutamicum ATCC13869 as the template and using the primers P21E/P22 (positions 877-1701 of SEQ ID No. 13) was amplified by PCR, and a downstream homology arm fragment of 825 bp was obtained.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the product was transformed into E. coli DH5a was treated with M13 primers (M13F: 5'-TGTAAAACGGACGGCCAGT-3' (SEQ ID No. 50), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No. 51)).
  • the positive integration vector (recombinant vector) was obtained by PCR identification, and the recombinant vector with the correct sequence was recorded as pK18-EP6-BBD29_14295OE.
  • the recombinant vector contains a kanamycin resistance marker, and the vector integration can be obtained through kanamycin screening. recombinants on the genome.
  • pK18-EP6-BBD29_14295OE contains the DNA fragment shown in SEQ ID No. 13.
  • positions 1-792 are the sequences of the upstream homology arm fragment
  • positions 877-1701 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 753-916 are the sequence of EP6.
  • the correctly sequenced recombinant vectors (pK18-PyfjB-BBD29_14295OE, pK18-EP6-BBD29_14295OE) were respectively electrotransformed into the high-glutamic acid-producing Corynebacterium glutamicum CGMCC No. 21220, and in the recovery medium (same as Example 2) Cultivation was carried out according to the recovery culture conditions (same as Example 2), and the single colonies produced by the culture were identified by PCR with P23/P24 primers.
  • the PyfjB-BBD29_14295 integrated bacterium PCR amplified the 849bp (SEQ ID No. 14) fragment as a positive strain.
  • EP6-BBD29_14290 integrated bacteria that PCR amplify the 849bp (SEQ ID No. 15) fragment are positive strains, and those that cannot amplify the fragment are original bacteria.
  • the positive strain was cultured on a medium containing 15% sucrose (the same as the medium containing 15% sucrose in Example 2).
  • the single colony produced by the culture was further identified by PCR using P25/P26 primers, and the amplified size was
  • the strain of 889bp (SEQ ID No.16) is a strain in which PyfjB replaces the original promoter of BBD29_14295 on the genome of the high-glutamic acid-producing Corynebacterium glutamicum CGMCC No.21220; the strain of 891bp (SEQ ID No.17) was amplified
  • the strain is a strain in which EP6 replaces the original promoter of BBD29_14295 on the genome of the high-glutamic acid-producing Corynebacterium glutamicum CGMCC No. 21220. If the fragment cannot be amplified, it is the original strain.
  • YPG-091 The recombinant strain in which the original promoter of BBD29_14295 was replaced by PyfjB was named YPG-091 (PyfjB-BBD29_14295 integrated strain), and the recombinant strain in which the original promoter of BBD29_14295 was replaced by EP6 was named YPG-092 (EP6-BBD29_14295 integrated strain).
  • YPG-091 (PyfjB-BBD29_14295 integrated strain) is a high-glutamic acid-producing Corynebacterium glutamicum CGMCC No. 21220 BBD29_14295 original promoter (positions 1-36 of SEQ ID No. 11) replaced by PyfjB, maintaining its genome. A recombinant strain obtained by keeping other nucleotides unchanged, which can increase glutamic acid production; YPG-092 (EP6-BBD29_14295 integrated strain) is a high-glutamic acid-producing Corynebacterium glutamicum CGMCC No. 21220 BBD29_14295 original Promoter (positions 1-36 of SEQ ID No.
  • the EP6 promoter serves as a mutated yfjB promoter, which promotes the expression of BBD29_14295 and can increase glutamic acid production.
  • the PCR identification primers are as follows:
  • P24 5′-ACTGATCCCC ATAATAAGCG-3′ (internal to PyfjB) (SEQ ID No. 67);
  • P25 5′-CGCTTATTAT GGGGATCAGT-3′ (internal to PyfjB) (SEQ ID No. 68);
  • strains YPG-091, YPG-092 and the original strain CGMCC No. 21220 were fermented in a BLBIO-5GC-4-H fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) using fermentation medium 2 and culture conditions 2
  • L-glutamic acid production and OD value were detected at the end of fermentation.
  • Each strain was replicated three times.
  • Glutamic acid production was detected by a biosensor.
  • Fermentation medium 2 The solvent is water, the solute and its concentration in fermentation medium 2 are glucose 5.0g/L, phosphoric acid 0.38g/L, magnesium sulfate 1.85g/L, potassium chloride 1.6g/L, biotin 550 ⁇ g/L, vitamin B1 300 ⁇ g/L, ferrous sulfate 10mg/L, manganese sulfate 10g/dl, KH 2 PO 4 2.8g/L, vitamin C 0.75mg/L, vitamin B12 2.5 ⁇ g/L, para-aminobenzoic acid 0.75mg/L, defoaming agent 0.0015mL/dL, betaine 1.5g/L, sugar cane molasses 7mL/L, corn steep liquor 77mL/L, aspartic acid 1.7g/L.
  • Inoculation volume 13%; 0h: rotation speed 400rpm, air volume 3L/min, pressure 0.05MPA, culture temperature 32.5°C;
  • Bacterial liquid concentration OD 1.0: rotation speed 600rpm, air volume 5L/min, pressure 0.08MPA, culture temperature 37°C;
  • Flow sugar control The concentration of flow sugar in the fermentation tank is 50-55%, and the residual sugar in the fermentation tank is controlled at 0.5-1.0%.
  • Example 4 Increase in the ilvC gene promoter of the valine metabolism pathway in Corynebacterium glutamicum and its impact on valine production.
  • the pk18 vector was used to replace the original promoter of the key gene ilvC of the valine metabolism pathway in the high-valine-producing Corynebacterium glutamicum CGMCC No. 21260 with the EP6 and yfjB promoters using homologous recombination. (No. 1-179 of SEQ ID No. 18), thereby using an exogenous promoter to initiate the expression of the ilvC gene in the glutamate metabolism pathway, and obtaining a valine-producing bacterium with an exogenous promoter-gene combination.
  • Bacterial fermentation test it was found that the strain initiating ilvC with EP6 can increase the production of valine at a higher level, and the strain was named YPV-098. Positions 180-1196 of SEQ ID No. 18 are the sequence of ilvC.
  • the vector was constructed using NEBuilder recombination technology, and the primers were designed as follows (synthesized by Shanghai Invitrogen Company):
  • P28 5′-AAAAAAACGCGGAGAAATTCTTAGATCTTGGCCGGAGCCA-3′ (SEQ ID No. 71);
  • P29 5′-TGGCTCCGGCCAAGATCTAAGAATTTCTCCGGCGTTTTTTT-3′ (SEQ ID No. 72);
  • P31 5′-GCAACAAGGACCTCGGAAAAATGGCTATTGAACTGCTTTA-3′ (SEQ ID No. 74);
  • P28E 5′-AAAAAAACGCGAAGAAATTCTTAGATCTTGGCCGGAGCCA-3′(SEQ ID No.76);
  • P29E 5′-TGGCTCCGGCCAAGATCTAAGAATTTCTTCGCGTTTTTTT-3′ (SEQ ID No. 77);
  • P30E 5′-TAAAGCAGTTCAATAGCCATGATTTGGAGGTCCCTGCGTT-3′ (SEQ ID No. 78);
  • P31E 5′-AACGCAGGGACCTCCAAATCATGGCTATTGAACTGCTTTA-3′ (SEQ ID No. 79).
  • Corynebacterium glutamicum ATCC14067 was used as the template, and primers P27/P28 (SEQ ID No. 19 No. 1-805 position) to perform PCR amplification to obtain the upstream homology arm fragment 805bp; using the vector pEC-PyfjB-mCherry as the template and primers P29/P30 to perform PCR amplification to obtain PyfjB 162bp (SEQ ID No. 19 No. 766-927) Using Corynebacterium glutamicum ATCC14067 as a template, PCR amplification was performed with primers P31/P32 (SEQ ID No. 19 positions 888-1822), and a 935 bp downstream homology arm fragment was obtained.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the product was transformed into E. coli DH5a was treated with M13 primers (M13F: 5'-TGTAAAACGGACGGCCAGT-3' (SEQ ID No. 50), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No. 51)).
  • the positive integration vector (recombinant vector) was obtained by PCR identification, and the recombinant vector with correct sequence was recorded as pK18-PyfjB-ilvCOE.
  • This recombinant vector contains a kanamycin resistance marker, and the vector integration can be obtained through kanamycin screening. recombinants on the genome.
  • pK18-PyfjB-ilvCOE contains the DNA fragment shown in SEQ ID No. 19.
  • positions 1-805 are the sequences of the upstream homology arm fragment
  • positions 888-1822 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 766-927 are the sequence of PyfjB.
  • Corynebacterium glutamicum ATCC14067 was used as the template, and primers P27/P28E (SEQ ID No. 20 1-805) were used for PCR amplification to obtain an upstream homology arm fragment of 805 bp; using the vector pEC-EP6-mCherry as the template, PCR amplification was performed with primers P29E/P30E to obtain EP6 164bp (SEQ ID No. 20 No. 766-929); Corynebacterium glutamicum ATCC14067 was used as the template, and primers P31E/P32 (SEQ ID No. 20 No. 890- 1824) was PCR amplified to obtain a downstream homology arm fragment of 935 bp.
  • the three PCR products obtained were recovered by electrophoresis using column DNA gel recovery kits.
  • the three recovered PCR products were ligated with the purified pK18mobsacB vector (Addgene Company, which contains kanamycin resistance as a selection marker) digested by XbalI and BamHI, using NEBuilder enzyme (NEB Company) at 50°C for 30 minutes.
  • the single clone grown after the product was transformed into E. coli DH5a was treated with M13 primers (M13F: 5'-TGTAAAACGGACGGCCAGT-3' (SEQ ID No. 50), M13R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID No. 51)).
  • the positive integration vector (recombinant vector) was obtained by PCR identification, and the recombinant vector with correct sequence was recorded as pK18-EP6-ilvCOE.
  • This recombinant vector contains a kanamycin resistance marker, and the vector integration can be obtained through kanamycin screening. recombinants on the genome.
  • pK18-EP6-ilvCOE contains the DNA fragment shown in SEQ ID No. 20.
  • positions 1-805 are the sequences of the upstream homology arm fragment
  • positions 890-1824 are the sequences of the downstream homology arm fragment.
  • Sequence, positions 766-929 are the sequence of EP6.
  • the correctly sequenced integration vectors (pK18-PyfjB-ilvCOE, pK18-EP6-ilvCOE) were electrotransformed into the high-valine-producing Corynebacterium glutamicum CGMCC No. 21260, and in the recovery medium (same as Example 2) Cultivate according to the recovery culture conditions (same as Example 2), and perform PCR identification on the single colony produced by the culture using P33/P34 primers.
  • the PyfjB-ilvC integrated bacterium was PCR amplified to 901bp (SEQ ID No. 21). The fragment is a positive strain, the EP6-ilvC integrated strain has a 901bp (SEQ ID No. 22) fragment amplified by PCR, it is a positive strain, and the fragment cannot be amplified is the original strain.
  • the positive strain was cultured on a medium containing 15% sucrose (the same as the 15% sucrose medium in Example 2).
  • the single colony produced by the culture was further identified by PCR using P35/P36 primers, and the amplified size was 1023 bp.
  • the strain (SEQ ID No.23) is a positive strain in which PyfjB replaces the ilvC original promoter on the genome of the high-valine-producing Corynebacterium glutamicum CGMCC No.21260; the amplified strain is 1025bp (SEQ ID No.24)
  • the strain is a strain in which EP6 replaces the original ilvC promoter on the genome of the high-valine-producing Corynebacterium glutamicum CGMCC No. 21260.
  • the strain that cannot amplify the fragment is the original strain.
  • YPV-097 The recombinant strain in which the original promoter of ilvC was replaced by PyfjB was named YPV-097 (PyfjB-ilvC integrated strain), and the recombinant strain in which the original promoter of ilvC was replaced by EP6 was named YPV-098 (EP6-ilvC integrated strain).
  • YPV-097 (PyfjB-ilvC integrated strain) is a high-valine-producing Corynebacterium glutamicum CGMCC No. 21260 ilvC original promoter (positions 1-179 of SEQ ID No. 18) replaced by PyfjB, maintaining its genome A recombinant strain obtained by keeping other nucleotides unchanged, which can increase the production of valine;
  • YPV-098 (EP6-ilvC integrated strain) is the original promoter of Corynebacterium glutamicum CGMCC No. 21260 ilvC with high valine production (positions 1-179 of SEQ ID No. 18) was replaced with EP6 and other nucleotides in the genome were kept unchanged.
  • the EP6 promoter as a mutated yfjB promoter, is more capable of stimulating the expression of ilvC. Increase valine production.
  • the PCR identification primers are as follows:
  • P33 5′-CCCGACTTTG TTAACCCTTTC-3′ (inside CEY17_RS06885 gene) (SEQ ID No.80);
  • P34 5′-ACTGATCCCC ATAATAAGCG-3′ (internal to PyfjB) (SEQ ID No. 81);
  • P35 5′-CGCTTATTAT GGGGATCAGT-3′ (internal to PyfjB) (SEQ ID No.82);
  • P36 5′-GGGTGGTTGTTGTAGGAAGC-3′ (inside the i lvC gene) (SEQ ID No. 83).
  • the above strains YPV-097, YPV-098 and the original strain Corynebacterium glutamicum CGMCC No. 21260 were cultured in a BLBIO-5GC-4-H fermentation tank (Shanghai Bailun Biotechnology Co., Ltd.) with fermentation medium 3 and The fermentation experiment was carried out under culture condition 3. After the fermentation, the L-valine production and OD value were detected. Each strain was replicated three times.
  • the detection method of L-valine production is high performance liquid chromatography.
  • Fermentation medium 3 The solvent is water, the solutes and their concentrations are ammonium sulfate 14g/L, potassium dihydrogen phosphate 1g/L, dipotassium hydrogen phosphate 1g/L, magnesium sulfate 0.5g/L, yeast powder 2g/L, Ferrous sulfate 18mg/L, manganese sulfate 4.2mg/L, biotin 0.02mg/L, vitamin B1 2mg/L, antifoam (CB-442) defoaming agent 0.5mL/L, 70% glucose (base sugar) 40g/ L.
  • ammonium sulfate 14g/L ammonium sulfate 14g/L
  • potassium dihydrogen phosphate 1g/L dipotassium hydrogen phosphate 1g/L
  • magnesium sulfate 0.5g/L
  • yeast powder 2g/L yeast powder 2g/L
  • Ferrous sulfate 18mg/L manganese s
  • Residual sugar control 0.1-0.2% before F12h; after F12h combined with DO requirements, the residual sugar should be controlled to ⁇ 0.02%;
  • Feeding materials ammonia, 70% concentrated sugar, 5% soaked sugar;
  • the present invention uses the EP6 promoter to activate genes in the corresponding amino acid synthesis pathway to obtain the corresponding expression cassette, and then integrates the expression cassette as an exogenous strong promoter into bacteria with high amino acid production to replace the original key genes for amino acid synthesis. promoter, achieving an increase in the production of corresponding amino acids. It shows that the EP6 promoter of the present invention not only has high starting activity, but can also be used to produce amino acids, and has great application prospects.

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Abstract

提供了一种EP6启动子与其相关生物材料及应用。该EP6启动子的序列为序列表中SEQ ID No.3所示。将该EP6启动子作为外源强启动子整合到高产氨基酸的细菌中替换氨基酸合成关键基因的原始启动子，实现了相应氨基酸产量的提高。

Description

一种EP6启动子与其相关生物材料及应用 技术领域
本发明涉及生物技术领域中，一种EP6启动子与其相关生物材料及应用。
背景技术
目前工业上产氨基酸的水平已有较大提高，但如何在较高水平的基础上进一步提升氨基酸的产量，则需要全面考虑产氨基酸的菌生长所需的碳源，以及在发酵后期碳源如何最大限度的进入氨基酸的合成途径。为了解决该问题，对于产某种氨基酸的细菌而言，不同强度的启动子需要应用到相应氨基酸代谢通路的不同基因上，以达到氨基酸代谢通路与细菌生长的平衡。
发明公开
本发明的目的是提供一种EP6启动子，利用EP6启动子可以生产氨基酸。
本发明提供的EP6启动子，其序列为序列表中SEQ ID No.3。
本发明还提供了与EP6启动子相关的生物材料，所述生物材料为下述B1)至B7)中的任一种：
B1)含有EP6启动子的表达盒；
B2)含有EP6启动子的重组载体；
B3)含有B1)所述表达盒的重组载体；
B4)含有EP6启动子的重组微生物；
B5)含有B1)所述表达盒的重组微生物；
B6)含有B2)所述重组载体的重组微生物；
B7)含有B3)所述重组载体的重组微生物。
上述生物材料中，B1)所述的含有EP6启动子的表达盒，是指能够在宿主细胞中EP6启动子可驱动目的基因表达的DNA，该DNA不但可包括目的基因，还可包括终止目的基因转录的终止子。进一步，所述表达盒还可包括增强子序列。
可用现有的表达载体构建含有EP6启动子的重组载体。
上述生物材料中，所述载体可为质粒、黏粒、噬菌体或病毒载体。
上述生物材料中，所述微生物可为酵母、细菌、藻或真菌。其中，细菌可为谷氨酸棒杆菌(Corynebacterium glutamicum)、乳酸发酵短杆菌、黄色短杆菌(brevibacterium flavum)、北京棒杆菌(Corynebacterium pekinense)、噬氨短杆菌、钝齿棒状杆菌或泛菌(Pantoea)。
本发明还提供了EP6启动子在作为启动子中的应用。
本发明还提供了EP6启动子在生产氨基酸中的应用。
本发明还提供了所述生物材料在生产氨基酸中的应用。
上述应用中，所述氨基酸可为赖氨酸、谷氨酸或缬氨酸。
本发明的EP6启动子可以用于生产多种产物，包括但不限于实施例中的赖氨酸、谷氨酸与缬氨酸，所生产的产物还可为甘氨酸、丙氨酸、亮氨酸、异亮 氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、精氨酸、组氨酸、莽草酸、原儿茶酸、丁二酸、a酮戊二酸、柠檬酸、鸟氨酸，瓜氨酸等。各种产物的生产时，将本发明的EP6启动子置于目的产物合成途径中基因上游并使EP6启动子驱动目的产物合成途径中基因的合成即可实现目的产物的生产。
本发明还提供了一种生产氨基酸的方法，所述方法包括：将EP6启动子导入能合成目的氨基酸的生物细胞中，使EP6启动子驱动所述生物细胞中目的氨基酸合成途径中基因的表达，得到重组生物细胞；培养所述重组生物细胞，得到目的氨基酸。
上述方法中，所述生物细胞可为能合成目的氨基酸的酵母、细菌、藻、真菌、植物细胞或动物细胞。
所述生物细胞为任何可以合成目的氨基酸的生物细胞。
所述细菌可为谷氨酸棒杆菌(Corynebacterium glutamicum)、乳酸发酵短杆菌、黄色短杆菌(brevibacterium flavum)、北京棒杆菌(Corynebacterium pekinense)、噬氨短杆菌、钝齿棒状杆菌或泛菌(Pantoea)。
在本发明的一个实施例中，所述目的氨基酸为赖氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856。
利用本发明启动子生产赖氨酸所用细菌包括但不限于谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856。本发明可将本发明的EP6启动子置于这些细菌的赖氨酸合成途径中基因上游，使本发明的EP6启动子可以通过驱动这些细菌中赖氨酸合成途径中基因的表达进而合成赖氨酸。
在本发明的一个实施例中，所述目的氨基酸为谷氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220。
利用本发明启动子生产谷氨酸所用细菌包括但不限于谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220。本发明可将本发明的EP6启动子置于这些细菌的谷氨酸合成途径中基因上游，使本发明的EP6启动子可以通过驱动这些细菌中谷氨酸合成途径中基因的表达进而合成谷氨酸。
在本发明的一个实施例中，所述目的氨基酸为缬氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260。
利用本发明启动子生产缬氨酸所用细菌包括但不限于谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260。本发明可将本发明的EP6启动子置于这些细菌的缬氨酸合成途径中基因上游，使本发明的EP6启动子可以通过驱动这些细菌中缬氨酸合成途径中基因的表达进而合成缬氨酸。
上述方法中，所述目的氨基酸可为赖氨酸、谷氨酸或缬氨酸。
本发明的EP6启动子可以用于生产多种产物，包括但不限于实施例中的赖氨酸、谷氨酸与缬氨酸，所生产的产物还可为甘氨酸、丙氨酸、亮氨酸、异亮氨酸、甲硫氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、 天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、精氨酸、组氨酸、莽草酸、原儿茶酸、丁二酸、a酮戊二酸、柠檬酸、鸟氨酸，瓜氨酸等。各种目的产物的生产时，将本发明的EP6启动子置于目的产物合成途径中基因上游并使EP6启动子驱动目的产物合成途径中基因的合成即可实现目的产物的生产。
在本发明的一个实施例中，所述目的氨基酸为赖氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856，赖氨酸合成途径中基因为lysA基因(SEQ ID No.4的第149-1486位)。
所述重组生物细胞通过将EP6启动子替换谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856中lysA基因原始启动子而实现。
在本发明的一个实施例中，所述目的氨基酸为谷氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220，谷氨酸合成途径中基因为BBD29_14295基因(SEQ ID No.11的第37-1161位)。
所述重组生物细胞通过将EP6启动子替换谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220中BBD29_14295基因原始启动子而实现。
在本发明的一个实施例中，所述目的氨基酸为缬氨酸，所述细菌为谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260，缬氨酸合成途径中基因为ilvC基因(SEQ ID No.18第180-1196位)。
所述重组生物细胞通过将EP6启动子替换谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260中ilvC基因原始启动子而实现。
本发明还提供了一种生产氨基酸的产品，所述产品的活性成分为EP6启动子或所述生物材料。
生物材料保藏说明。
分类命名：谷氨酸棒杆菌(Corynebacterium glutamicum)。
菌株编号：YPGLU001。
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心。
保藏单位简称：CGMCC。
保藏单位地址：北京市朝阳区北辰西路1号院3号，邮政编码：100101。
保藏日期：2020年11月23日。
保藏中心登记入册编号：CGMCC No.21220。
生物材料保藏说明。
分类命名：谷氨酸棒杆菌(Corynebacterium glutamicum)。
菌株编号：YPFV1。
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心。
保藏单位简称：CGMCC。
保藏单位地址：北京市朝阳区北辰西路1号院3号，邮政编码：100101。
保藏日期：2020年11月30日。
保藏中心登记入册编号：CGMCC No.21260。
生物材料保藏说明。
分类命名：谷氨酸棒杆菌(Corynebacterium glutamicum)。
菌株编号：YP097158。
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心。
保藏单位简称：CGMCC。
保藏单位地址：北京市朝阳区北辰西路1号院3号，邮政编码：100101。
保藏日期：2016年8月16日。
保藏中心登记入册编号：CGMCC No.12856。
附图说明
图1为EP6与yfjB的启动子活性的检测结果。PEP6即为EP6启动子。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA/RNA的5′末端核苷酸，末位均为相应DNA/RNA的3′末端核苷酸。
pEC-H10-mCherry载体：记载在申请号为202110256579.8的中国专利申请(公布号为CN 112980867A)中，其制备方法如下：
以Wei等人(Promoter library-based module combination(PLMC)technology foroptimizationofthreoninebiosynthes isinCorynebacteriumglutamicum.A pplied Microbiology and Biotechnology.2018(102)4117-4130.)报道的在谷氨酸棒杆菌中最强启动子H10(GCTCAACCCTTACCGGTCGGCTCTAAGCCGGCGGCGTATGGTAAGCTCTGTTATGTATAGTCCGAGCACGGCGAAAGGATACTC(SEQ ID No.25))为模板，以及mCherry蛋白的基因序列和谷氨酸棒杆菌中表达载体pEC-XK99E的序列，设计并合成引物用于构建pEC-H10-mCherry的载体。引物设计如下(广州金唯智公司合成)：
引物1：5′-GGATCTAGAGTCGACCTGCAG-3′(SEQ ID No.26)；
引物2：5′-TTAACTAGTATTGCGTTGCGCTCAC-3′(SEQ ID No.27)；
引物3：5′-CAATACTAGTTAATGTGAGTTAGCGCG-3′(SEQ ID No.28)；
引物4：5′-AGAGCTTACCATACGCCGCCGGCTTAGAGCCGACCGGTAAGGGTTGAGCCTAGAGGATCCCCGG GTAC-3′(SEQ ID No.29)；
引物5：5′-GCGTATGGTAAGCTCTGTTATGTTATGTATAGTCCGAGCACGGCGAAAGGATACTCATGCGTAAAGGAGAAGAAG-3′(SEQ ID No.30)；
引物6：5′-CGACTCTAGATCCGCCAAAACAGCC-3′(SEQ ID No.31)。
构建方法：以pEC-XK99E为模板，以引物1和引物2扩增pEC质粒的骨架区域(6743bp)；以启动子H10为模板，以引物3和引物4扩增含有H10启动子上游区域的片段(387bp)；以含有mCherry基因的质粒pBblactam(Zhang et al.,Development of a transcription factor based lactam biosensor.ACS synthetic biology.2017,6,439-445.)为模板，以引物5和引物6扩增含有H10启动子下游区域以及mCherry基因的片段(810bp)。
PCR扩增结束分别获得长度为387bp，810bp以及6743bp的条带，将上述三个条带分别进行琼脂糖凝胶电泳切胶回收，回收完后，以387bp和810bp条带的DNA片段为模板，利用引物3和引物6进行融合PCR扩增。对得到的PCR扩增片段进行切胶回收。然后将回收后的片段(即含有H10和mCherry的片段)与pEC骨架片段分别进行酶切(SpeI,XbaI)，酶切完成后过柱纯化片段。以1：2的摩尔比将pEC骨架片段与含有H10和mCherry的片段混合，并加入T4DNA连接酶反应液，16℃连接1h后，将片段转入大肠杆菌DH5α菌株中，获得的序列正确的重组载体即为pEC-H10-mCherry载体。
pk18载体：Addgene公司，货号为MLCC1103。
下述实施例中的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856，菌株号为YP097158，已于2016年8月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.12856。
下述实施例中的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220，菌株号为YPGLU001，已于2020年11月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.21220。
下述实施例中的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260，菌株号为YPFV1，已于2020年11月30日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.21260。
实施例1、EP6具有启动子活性。
一、构建大肠杆菌中强启动子PyfjB启动mCherry表达的pEC-PyfjB-mCherry载体。
以pEC-H10-mCherry载体为骨架，采用NEBuilder重组技术构建含大肠杆菌W3110yfjB启动子(SEQ ID No.1：gaatttctccgcgtttttttcgcattcatctcgctaacttcgcttattatggggatcagtttcagggtttcaagggaagcactcacattgtcatcaatcttcgcaacaaggacctcggaaaa)的pEC-PyfjB-mCherry载体，依据报告蛋白mCherry的表达情况，检测PyfjB启动 强度，并作为后续突变启动子序列的对照处理。引物序列如下(上海invitrogen公司合成)：
pEC01：5′-ATGCGTAAAGGAGAAGAAGATAAC-3′(SEQ ID No.32)；
pEC02：5′-CTAGAGGATCCCCGGGTAC-3′(SEQ ID No.33)；
P1：5′-CATGGAATTCGAGCTCGGTACCCGGGGATCCTCTAGGAATTTCTCCGCGTTTTTTT-3′(SEQ ID No.34)；
P2：5′-AATGATAGCCATGTTATCTTCTTCTCCTTTACGCATTTTTCCGAGGTCCTTGTTGC-3′(SEQ ID No.35)。
构建方法：以pEC-H10-mCherry载体为模板，利用引物pEC01和pEC02扩增得到pEC-H10-mCherry载体的骨架区域7772bp；以SEQ ID No.1所示的DNA片段为模板，利用引物P1和P2扩增得到含有pEC-H10-mCherry载体同源臂的yfjB启动子(简称为PyfjB)片段194bp。
将上述两条DNA片段(pEC-H10-mCherry载体的骨架区域和含有pEC-H10-mCherry载体同源臂的PyfjB片段)经琼脂糖凝胶电泳分离纯化后，用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆经引物pECF/R(pECF：5′-GTACCCGGGGATCCTCTAG-3′(SEQ ID No.36)，pECR：5′-GTTATCTTCTTCTCCTTTACGCAT-3′(SEQ ID No.37))进行鉴定，提取质粒获得序列正确的阳性重组载体，即为pEC-PyfjB-mCherry。
重组载体pEC-PyfjB-mCherry的序列为序列表中SEQ ID No.2，其中yfjB启动子启动报告基因mCherry的表达，检测mCherry的荧光数值而获得yfjB启动子的强度。
二、构建突变的yfjB启动子表达载体pEC-EP6-mCherry。
对SEQ ID No.1所示的PyfjB突变，得到EP6启动子，大小124bp，EP6启动子的序列为SEQ ID No.3。
采用NEBuilder重组技术进行载体构建，引物设计如下(上海invitrogen公司合成)：
P5：5′-CATGGAATTCGAGCTCGGTACCCGGGGATCCTCTAGGAATTTCTTCGCGTTTTTTT-3′(SEQ ID No.38)；
P6：5′-AATGATAGCCATGTTATCTTCTTCTCCTTTACGCATGATTTGGAGGTCCCTGCGTT-3′(SEQ ID No.39)。
构建方法：以pEC-PyfjB-mCherry载体为模板，引物pEC01和pEC02扩增pEC-H10-mCherry载体的骨架区域7772bp；以SEQ ID No.3所示的DNA片段为模板，以引物P5和P6扩增含有pEC-H10-mCherry载体同源臂的EP6启动子片段196bp。
将上述两条DNA片段(pEC-H10-mCherry载体的骨架区域和含有pEC-H10-mCherry载体同源臂的EP6启动子片段)经琼脂糖凝胶电泳分离纯化后，用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆经引物pECF/R(pECF：5′-GTACCCGGGGATCCTCTAG-3′(SEQ ID No.36)，pECR：5′-GTTATCTTCTTCTCCTTTACGCAT-3′(SEQ ID No.37))进行鉴定，提取质粒获得序列正确的阳性重组载体，记为pEC-EP6-mCherry。
pEC-EP6-mCherry为将pEC-PyfjB-mCherry的PyfjB(SEQ ID No.1)替换为EP6(SEQ ID No.3)得到的重组载体。
三、启动子活性的测定。
将步骤二的pEC-EP6-mCherry和步骤一pEC-PyfjB-mCherry载体分别导入野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032中，得到重组菌ATCC13032/pEC-EP6-mCherry与ATCC13032/pEC-PyfjB-mCherry；将步骤二的pEC-EP6-mCherry和步骤一pEC-PyfjB-mCherry载体分别导入野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13869中，得到重组菌ATCC13869/pEC-EP6-mCherry与ATCC13869/pEC-PyfjB-mCherry；将步骤二的pEC-EP6-mCherry和步骤一pEC-PyfjB-mCherry载体分别导入野生型谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC14067中，得到重组菌ATCC14067/pEC-EP6-mCherry与ATCC14067/pEC-PyfjB-mCherry。比较这两种启动子在谷氨酸棒杆菌中的启动强度。
挑选上述6个重组菌的单克隆分别等量接种至含有900μL LBHIS培养基的96深孔板中，其中每个单克隆均含有3个重复，在30℃，800rpm条件下培养24h后用酶标仪检测mCherry的荧光数值，并根据荧光强度比较yfjB和EP6启动子活性。
LBHIS培养基：5g Tryptone(胰蛋白胨)/L,5g NaCl/L,2.5g yeast extract(酵母提取物)/L,18.5g BHI(Brain Heart Infusion脑心浸液)/L,91g sorbitol(山梨糖醇)/L,pH 7.2.
结果显示，EP6启动子具有启动子活性，且其启动活性明显高于yfjB启动子的启动活性(图1)。
实施例2、谷氨酸棒杆菌中赖氨酸代谢通路lysA基因启动子的替换及其对赖氨酸产量的影响。
一、重组菌的制备。
采用pk18载体，以同源重组的方法将EP6和yfjB启动子分别替换高产赖氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.12856中赖氨酸代谢通路的关键基因lysA的原始启动子(SEQ ID No.4的第1-148位)，从而利用外源启动子启动赖氨酸代谢通路中lysA的表达，获得外源启动子-基因组合的赖氨酸生产菌，对这些生产菌发酵测试，发现以EP6启动lysA的菌株，能更高的增加赖氨酸的产量，将该菌株命名为YPL-4-042。SEQ ID No.4的第 149-1486位为lysA的序列。
采用NEBuilder重组技术构建载体，引物设计如下(上海invitrogen公司合成)：
P7：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCCCAGGTGAAGAAGTCGTTG-3′(SEQ ID No.40)；
P8：5′-AAAAAAACGCGGAGAAATTCATGCCCCGTTCGACAATAAA-3′(SEQ ID No.41)；
P9：5′-TTTATTGTCGAACGGGGCATGAATTTCTCCGCGTTTTTTT-3′(SEQ ID No.42)；
P10：5′-GCGAGATCAGCTGGTGTCATTTTTCCGAGGTCCTTGTTGC-3′(SEQ ID No.43)；
P11：5′-GCAACAAGGACCTCGGAAAAATGACACCAGCTGATCTCGC-3′(SEQ ID No.44)；
P12：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTCGAACATCATCTCCACGCC-3′(SEQ ID No.45)；
P8E：5′-AAAAAAACGCGAAGAAATTCATGCCCCGTTCGACAATAAA-3′(SEQ ID No.46)；
P9E：5′-TTTATTGTCGAACGGGGCATGAATTTCTTCGCGTTTTTTT-3′(SEQ ID No.47)；
P10E：5′-GCGAGATCAGCTGGTGTCATGATTTGGAGGTCCCTGCGTT-3′(SEQ ID No.48)；
P11E：5′-AACGCAGGGACCTCCAAATCATGACACCAGCTGATCTCGC-3′(SEQ ID No.49)。
同源重组的具体方法如下：
以谷氨酸棒杆菌ATCC13032为模板，以引物P7/P8(PCR产物序列为SEQ ID No.5第1-801位)进行PCR扩增，获得上游同源臂片段801bp；以载体pEC-PyfjB-mCherry为模板，以引物P9/P10进行PCR扩增，获得PyfjB 162bp(PCR产物序列为SEQ ID No.5第762-923位)；以谷氨酸棒杆菌ATCC13032为模板，以引物P11/P12(PCR产物序列为SEQ ID No.5第884-1648位)进行PCR扩增，获得下游同源臂片段765bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。将回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID  No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，提取序列正确的重组载体记为pK18-PyfjB-lysAOE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-PyfjB-lysAOE含有SEQ ID No.5所示的DNA片段，SEQ ID No.5中，第1-801位为上游同源臂片段的序列，第884-1648位为下游同源臂片段的序列，第762-923位为PyfjB的序列。
以谷氨酸棒杆菌ATCC13032为模板，以引物P7/P8E(SEQ ID No.6第1-801位)进行PCR扩增，获得上游同源臂片段801bp；以pEC-EP6-mCherry为模板，以P9E/P10E进行PCR扩增，获得EP6 164bp(SEQ ID No.6第762-925位)；以谷氨酸棒杆菌ATCC13032为模板，以引物P11E/P12(SEQ ID No.6第886-1650位)进行PCR扩增，获得下游同源臂片段765bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。将回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，提取序列正确的重组载体记为pK18-EP6-lysAOE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-EP6-lysAOE含有SEQ ID No.6所示的DNA片段，SEQ ID No.6中，第1-801位为上游同源臂片段的序列，第886-1650位为下游同源臂片段的序列，第762-925位为EP6的序列。
将测序正确的重组载体pK18-PyfjB-lysAOE、pK18-EP6-lysAOE分别电转化入高产赖氨酸的谷氨酸棒杆菌CGMCC No.12856中，在恢复培养基中按照复苏培养条件进行培养，对培养产生的单菌落通过P13/P14引物进行PCR鉴定，导入pK18-PyfjB-lysAOE的重组菌PCR扩增出885bp(SEQ ID No.7)片段的为阳性菌株，记为CGMCC No.12856/pK18-PyfjB-lysAOE；导入pK18-EP6-lysAOE的重组菌PCR扩增出 885bp(SEQ ID No.8)片段的为阳性菌株，记为CGMCC No.12856/pK18-EP6-lysAOE，扩增不到片段的为原菌。
将阳性菌株在含15％蔗糖的培养基上培养，对培养产生的单菌落进一步采用P15/P16引物进行PCR鉴定，扩增出大小为841bp(SEQ ID No.9)的菌株为PyfjB替换高产赖氨酸的谷氨酸棒杆菌CGMCC No.12856lysA原始启动子的阳性菌株，扩增出大小为843bp(SEQ ID No.10)的菌株为EP6替换高产赖氨酸的谷氨酸棒杆菌CGMCC No.12856 lysA原始启动子的阳性菌株，扩增不到片段的为原菌。
将由导入pK18-PyfjB-lysAOE和pK18-EP6-lysAOE的重组菌得到的阳性菌 株分别命名为YPL-4-041(PyfjB-lysA整合菌株)和YPL-4-042(EP6-lysA整合菌株)。
YPL-4-041(PyfjB-lysA整合菌株)是将高产赖氨酸的谷氨酸棒杆菌CGMCC No.12856 lysA原始启动子(SEQ ID No.4的第1-148位)替换为yfjB启动子，保持其基因组中的其它核苷酸不变得到的重组菌，其可以提高赖氨酸的产量；YPL-4-042(EP6-lysA整合菌株)是将高产赖氨酸的谷氨酸棒杆菌CGMCC No.12856 lysA原始启动子(SEQ ID No.4的第1-148位)替换为EP6启动子，保持其基因组中的其它核苷酸不变得到的重组菌，EP6启动子作为突变的yfjB启动子，其启动lysA的表达，更能提高赖氨酸的产量。
PCR鉴定引物如下所示：
P13：5′-GGCGACGATCTTGTTTGACC-3′(cg1332基因内部)(SEQ ID No.52)；
P14：5′-ACTGATCCCCATAATAAGCG-3′(PyfjB内部)(SEQ ID No.53)；
P15：5′-CGCTTATTATGGGGATCAGT-3′(PyfjB内部)(SEQ ID No.54)；
P16：5′-GTAGTAGACATCGAAATCGG-3′(argS基因内部)(SEQ ID No.55)。
恢复培养基：溶剂为水，溶质及其在恢复培养基中的含量分别为葡萄糖5g/L、尿素3g/L、酵母粉10g/L、生物素10μg/L、豆粕粉15g/L、丁二酸0.5g/L、磷酸二氢钾1g/L、氯化钠2.5g/L、山梨醇91g/L、脑心浸液18.5g/L、pH7.0.
15％蔗糖的培养基：溶剂为水，溶质及其在15％蔗糖的培养基中的含量分别为蔗糖15g/L、尿素3g/L、酵母粉10g/L、生物素10μg/L、豆粕粉15g/L、丁二酸0.5g/L、磷酸二氢钾1g/L、氯化钠2.5g/L、山梨醇91g/L、脑心浸液18.5g/L、琼脂粉15g/L、pH7.0。
复苏培养条件：46℃恢复培养基重悬细胞，转移至EP管，220rpm，37℃摇床培养45-60min，之后转移至30℃摇床培养45-60min，转速不变。
二、L-赖氨酸发酵实验。
将上述菌株YPL-4-041、YPL-4-042和原始菌株谷氨酸棒杆菌CGMCC No.12856在BLBIO-5GC-4-H型号的发酵罐(上海百仑生物科技有限公司)中以发酵培养基1和培养条件1进行发酵实验，发酵结束检测L-赖氨酸产量，每个菌株重复三次。
以高效液相色谱法检测L-赖氨酸的产量。
发酵培养基1：溶剂为水，溶质及其在发酵培养基1中的含量分别为淀粉水解糖 30g/L、硫酸铵 12g/L、硫酸镁 0.87g/L、糖蜜 20g/L、酸化玉米浆 3mL/L、磷酸 0.4mL/L、氯化钾 0.53g/L、消泡剂(2％泡敌)4mL/L、硫酸亚铁 120mg/L、硫酸锰 120mg/L、烟酰胺 42mg/L、泛酸钙 6.3mg/L、维生素B1 6.3mg/L、硫酸铜 0.6g/L、硫酸锌 0.6g/L、生物素 0.88mg/L。酸化玉米浆为宁夏伊品生物科技股份有限公司产品。
培养条件1：
校正DO 100％，温度37℃、风量4L/min、转速1000rpm、罐压0Mpa，5min 后标定；
接种量：10％；
培养温度：37℃；
pH：6.9±0.05；
溶氧DO：10-30％；
初始条件：温度37℃、pH6.9、罐压0Mpa、风量3L/min、转速550rpm；
全程控制：全程控制 1、溶氧＜30％时，依次提转速750rpm→800rpm→风量4L/min→850rpm→950rpm；2、发酵6h提罐压0.01Mpa；12h提罐压0.02Mpa→0.03Mpa→0.04Mpa→0.05Mpa；
残糖控制：F12h前0.1-0.2％；F12h后结合DO要求控制残糖0.1-0.05％；
氨氮控制：F12h前0.1-0.15；F12-F32h0.15-0.25；F32h后0.1-0.15；
流加物料：25％氨水、70％浓糖、50％硫铵、10％泡敌；
发酵周期：48h左右。
结果显示，菌株CGMCC No.12856的L-赖氨酸产量为18.9g/100mL；YPL-4-041的L-赖氨酸产量为19.2g/100mL，显著高于菌株CGMCC No.12856(P<0.05)；YPL-4-042的L-赖氨酸产量为19.7g/100mL，显著高于菌株CGMCC No.12856(P<0.05)。由此表明，在赖氨酸生产菌CGMCC No.12856中以yfjB或者EP6启动子替换lysA原始启动子可以增加赖氨酸的产量，并且作为突变过的EP6启动lysA的表达，赖氨酸产量更高。
实施例3、谷氨酸棒杆菌中谷氨酸代谢通路BBD29_14295基因启动子的增加及其对谷氨酸产量的影响。
一、重组菌的制备。
采用pk18载体，以同源重组的方法将EP6和yfjB启动子分别替换高产谷氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21220中谷氨酸代谢通路的关键基因BBD29_14295的原始启动子(SEQ ID No.11的第1-36位)，从而利用外源启动子启动谷氨酸代谢通路中BBD29_14295的表达，获得外源启动子-基因组合的谷氨酸生产菌，对这些生产菌发酵测试，发现以EP6启动BBD29_14295的菌株，能更高的增加谷氨酸的产量，将该菌株命名为YPG-092。SEQ ID No.11的第37-1161位为BBD29_14295的序列。
采用NEBuilder重组技术构建载体，引物设计如下(上海invitrogen公司合成)：
P17：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGGAAACAACGCAGCCATAG-3′(SEQ ID No.56)；
P18：5′-AAAAAAACGCGGAGAAATTCTTAGAGGAATTTAACGCCTT-3′(SEQ ID No.57)；
P19：5′-AAGGCGTTAAATTCCTCTAAGAATTTCTCCGCGTTTTTTT-3′(SEQ ID  No.58)；
P20：5′-TCGTCGATTAGCTCGAACATTTTTCCGAGGTCCTTGTTGC-3′(SEQ ID No.59)；
P21：5′-GCAACAAGGACCTCGGAAAAATGTTCGAGCTAATCGACGA-3′(SEQ ID No.60)；
P22：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCATGACGTCCTGGAGCGCTGC-3′(SEQ ID No.61)；
P18E：5′-AAAAAAACGCGAAGAAATTCTTAGAGGAATTTAACGCCTT-3′(SEQ ID No.62)；
P19E：5′-AAGGCGTTAAATTCCTCTAAGAATTTCTTCGCGTTTTTTT-3′(SEQ ID No.63)；
P20E：5′-TCGTCGATTAGCTCGAACATGATTTGGAGGTCCCTGCGTT-3′(SEQ ID No.64)；
P21E：5′-AACGCAGGGACCTCCAAATCATGTTCGAGCTAATCGACGA-3′(SEQ ID No.65)。
同源重组的具体方法如下：
以谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13869为模板，以引物P17/P18(SEQ ID No.12第1-792位)进行PCR扩增，获得上游同源臂片段792bp；以载体pEC-PyfjB-mCherry为模板，以引物P19/P20进行PCR扩增，获得PyfjB 162bp(SEQ ID No.12第753-914位)；以谷氨酸棒杆菌ATCC13869为模板，以引物P21/P22(SEQ ID No.12第875-1699位)进行PCR扩增，获得下游同源臂片段825bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，将得到的序列正确的重组载体记为pK18-PyfjB-BBD29_14295OE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-PyfjB-BBD29_14295OE含有SEQ ID No.12所示的DNA片段，SEQ ID No.12中，第1-792位为上游同源臂片段的序列，第875-1699位为下游同源臂片段的序列，第753-914位为PyfjB的序列。
以谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13869为模板，以引物P17/P18E(SEQ ID No.13第1-792位)进行PCR扩增，获得上游同源臂片段 792bp；以载体pEC-EP6-mCherry为模板，以引物P19E/P20E进行PCR扩增，获得EP6 164bp(SEQ ID No.13第753-916位)；以谷氨酸棒杆菌ATCC13869为模板，以引物P21E/P22(SEQ ID No.13第877-1701位)进行PCR扩增，获得下游同源臂片段825bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，将得到的序列正确的重组载体记为pK18-EP6-BBD29_14295OE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-EP6-BBD29_14295OE含有SEQ ID No.13所示的DNA片段，SEQ ID No.13中，第1-792位为上游同源臂片段的序列，第877-1701位为下游同源臂片段的序列，第753-916位为EP6的序列。
将测序正确的重组载体(pK18-PyfjB-BBD29_14295OE、pK18-EP6-BBD29_14295OE)分别电转化入高产谷氨酸的谷氨酸棒杆菌CGMCC No.21220中，在恢复培养基(同实施例2)中按照复苏培养条件(同实施例2)进行培养，对培养产生的单菌落通过P23/P24引物进行PCR鉴定，PyfjB-BBD29_14295整合菌PCR扩增出849bp(SEQ ID No.14)片段的为阳性菌株，EP6-BBD29_14290整合菌PCR扩增出849bp(SEQ ID No.15)片段的为阳性菌株，扩增不到片段的为原菌。
将阳性菌株在含15％蔗糖的培养基(同实施例2中的含15％蔗糖培养基。)上培养，对培养产生的单菌落进一步采用P25/P26引物进行PCR鉴定，扩增出大小为889bp(SEQ ID No.16)的菌株为PyfjB替换高产谷氨酸的谷氨酸棒杆菌CGMCC No.21220基因组上BBD29_14295原始启动子的菌株；扩增出大小为891bp(SEQ ID No.17)的菌株为EP6替换高产谷氨酸的谷氨酸棒杆菌CGMCC No.21220基因组上BBD29_14295原始启动子的菌株，扩增不到片段的为原菌。
将由PyfjB替换BBD29_14295原始启动子的重组菌株命名为YPG-091(PyfjB-BBD29_14295整合菌株)，由EP6替换BBD29_14295原始启动子的重组菌株命名为YPG-092(EP6-BBD29_14295整合菌株)。
YPG-091(PyfjB-BBD29_14295整合菌株)是将高产谷氨酸的谷氨酸棒杆菌CGMCC No.21220 BBD29_14295原始启动子(SEQ ID No.11的第1-36位)替换为PyfjB，保持其基因组中的其它核苷酸不变得到的重组菌，其可以提高谷氨酸的产量；YPG-092(EP6-BBD29_14295整合菌株)是将高产谷氨酸的谷氨酸棒杆菌CGMCC No.21220 BBD29_14295原始启动子(SEQ ID No.11的第1-36位)替 换为EP6，保持其基因组中的其它核苷酸不变得到的重组菌，EP6启动子作为突变的yfjB启动子，其启动BBD29_14295的表达更能提高谷氨酸的产量。
PCR鉴定引物如下所示：
P23：5′-GAAGTAGGTC CTCGTGTTGC-3′(BBD29_14290基因内部)(SEQ ID No.66)；
P24：5′-ACTGATCCCC ATAATAAGCG-3′(PyfjB内部)(SEQ ID No.67)；
P25：5′-CGCTTATTAT GGGGATCAGT-3′(PyfjB内部)(SEQ ID No.68)；
P26：5′-GACGAGTTGT GCTTGTAGTC-3′(BBD29_14295基因内部)(SEQ ID No.69)。
二、L-谷氨酸发酵实验。
将上述菌株YPG-091、YPG-092和原始菌株CGMCC No.21220在BLBIO-5GC-4-H型号的发酵罐(上海百仑生物科技有限公司)中以发酵培养基2和培养条件2进行发酵实验，发酵结束检测L-谷氨酸产量与OD值。每个菌株重复三次。
谷氨酸产量由生物传感仪检测。
发酵培养基2：溶剂是水，溶质及其在发酵培养基2中的浓度为葡萄糖 5.0g/L、磷酸 0.38g/L、硫酸镁 1.85g/L、氯化钾 1.6g/L、生物素 550μg/L、维生素B1 300μg/L、硫酸亚铁 10mg/L、硫酸锰 10g/dl、KH2PO4 2.8g/L、维生素C 0.75mg/L、维生素B12 2.5μg/L、对氨基苯甲酸 0.75mg/L、消泡剂 0.0015mL/dL、甜菜碱 1.5g/L、甘蔗糖蜜 7mL/L、玉米浆 77mL/L、天冬氨酸 1.7g/L。
培养条件2：
校正DO 100％，温度32.5℃、风量7L/min、转速700rpm、罐压0Mpa，5min后标定；
接种量：13％；0h：转速400rpm，风量3L/min，压力0.05MPA，培养温度32.5℃；
菌液浓度OD＝1.0：转速600rpm，风量5L/min，压力0.08MPA，培养温度37℃；
菌液浓度OD＝1.0-菌液浓度OD＝1.4：转速700rpm，风量7L/min，压力0.11MPA，培养温度38℃；
培养32h-34h发酵结束，控制过程以溶氧50-20％做提降风量标准；
pH：0h控制7.0，14h控制6.8；
流加糖控制：发酵罐流加糖浓度50-55％，控制发酵罐残糖0.5-1.0％。
结果显示，CGMCC No.21220的L-谷氨酸产量为102.1g/L，OD(562nm)为46.1；YPG-091的L-谷氨酸产量为103.3g/L，显著高于菌株CGMCC No.21220(P<0.05)，OD(562nm)为46.6；YPG-092的L-谷氨酸产量为105.1g/L，显著高于菌株CGMCC No.21220(P<0.05)，OD(562nm)为47.1。由此表明，在谷氨酸生产菌CGMCC No.21220中以yfjB或者EP6启动子替换BBD29_14295原始启动子可以增加谷氨酸 的产量，并且作为突变过的EP6启动BBD29_14295的表达，谷氨酸产量更高。
实施例4、谷氨酸棒杆菌中缬氨酸代谢通路ilvC基因启动子的增加及其对缬氨酸产量的影响。
一、重组菌的制备。
采用pk18载体，以同源重组的方法将EP6和yfjB启动子分别替换高产缬氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260中缬氨酸代谢通路的关键基因ilvC的原始启动子(SEQ ID No.18的第1-179位)，从而利用外源启动子启动谷氨酸代谢通路中ilvC基因的表达，获得外源启动子-基因组合的缬氨酸生产菌，对这些生产菌发酵测试，发现以EP6启动ilvC的菌株，能更高的增加缬氨酸的产量，将该菌株命名为YPV-098。SEQ ID No.18的第180-1196位为ilvC的序列。
采用NEBuilder重组技术构建载体，引物设计如下(上海invitrogen公司合成)：
P27：5′-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGAGATCAACGACCGCCCAG-3′(SEQ ID No.70)；
P28：5′-AAAAAAACGCGGAGAAATTCTTAGATCTTGGCCGGAGCCA-3′(SEQ ID No.71)；
P29：5′-TGGCTCCGGCCAAGATCTAAGAATTTCTCCGCGTTTTTTT-3′(SEQ ID No.72)；
P30：5′-TAAAGCAGTTCAATAGCCATTTTTCCGAGGTCCTTGTTGC-3′(SEQ ID No.73)；
P31：5′-GCAACAAGGACCTCGGAAAAATGGCTATTGAACTGCTTTA-3′(SEQ ID No.74)；
P32：5′-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGGCGCTTGGTGAAGGTG-3′(SEQ ID No.75)；
P28E：5′-AAAAAAACGCGAAGAAATTC TTAGATCTTGGCCGGAGCCA-3′(SEQ ID No.76)；
P29E：5′-TGGCTCCGGCCAAGATCTAAGAATTTCTTCGCGTTTTTTT-3′(SEQ ID No.77)；
P30E：5′-TAAAGCAGTTCAATAGCCATGATTTGGAGGTCCCTGCGTT-3′(SEQ ID No.78)；
P31E：5′-AACGCAGGGACCTCCAAATCATGGCTATTGAACTGCTTTA-3′(SEQ ID No.79)。
同源重组的具体方法如下：
以谷氨酸棒杆菌ATCC14067为模板，以引物P27/P28(SEQ ID No.19第1-805 位)进行PCR扩增，获得上游同源臂片段805bp；以载体pEC-PyfjB-mCherry为模板，以引物P29/P30进行PCR扩增，获得PyfjB 162bp(SEQ ID No.19第766-927位)以谷氨酸棒杆菌ATCC14067为模板，以引物P31/P32(SEQ ID No.19第888-1822位)进行PCR扩增，获得下游同源臂片段935bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，将得到的序列正确的重组载体记为pK18-PyfjB-ilvCOE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-PyfjB-ilvCOE含有SEQ ID No.19所示的DNA片段，SEQ ID No.19中，第1-805位为上游同源臂片段的序列，第888-1822位为下游同源臂片段的序列，第766-927位为PyfjB的序列。
以谷氨酸棒杆菌ATCC14067为模板，以引物P27/P28E(SEQ ID No.20第1-805位)进行PCR扩增，获得上游同源臂片段805bp；以载体pEC-EP6-mCherry为模板，以引物P29E/P30E进行PCR扩增，获得EP6 164bp(SEQ ID No.20第766-929位)；以谷氨酸棒杆菌ATCC14067为模板，以引物P31E/P32(SEQ ID No.20第890-1824位)进行PCR扩增，获得下游同源臂片段935bp。
PCR反应结束后，对得到的3种PCR产物采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3种PCR产物与经过XbalI和BamHI酶切后纯化的pK18mobsacB载体(Addgene公司，该载体上含有卡那霉素抗性作为筛选标记)用NEBuilder酶(NEB公司)50℃连接30min，连接产物转化大肠杆菌DH5a后长出的单克隆用M13引物(M13F：5′-TGTAAAACGACGGCCAGT-3′(SEQ ID No.50)，M13R：5′-CAGGAAACAGCTATGACC-3′(SEQ ID No.51))经PCR鉴定获得阳性整合载体(重组载体)，将得到的序列正确的重组载体记为pK18-EP6-ilvCOE，该重组载体上含有卡那霉素抗性标记，可以通过卡那霉素筛选获得载体整合到基因组上的重组子。
pK18-EP6-ilvCOE含有SEQ ID No.20所示的DNA片段，SEQ ID No.20中，第1-805位为上游同源臂片段的序列，第890-1824位为下游同源臂片段的序列，第766-929位为EP6的序列。
将测序正确的整合载体(pK18-PyfjB-ilvCOE、pK18-EP6-ilvCOE)分别电转化入高产缬氨酸的谷氨酸棒杆菌CGMCC No.21260中，在恢复培养基(同实施例2)中按照复苏培养条件(同实施例2)进行培养，对培养产生的单菌落通过P33/P34引物进行PCR鉴定，PyfjB-ilvC整合菌PCR扩增出901bp(SEQ ID No.21) 片段的为阳性菌株，EP6-ilvC整合菌株PCR扩增出901bp(SEQ ID No.22)片段的为阳性菌株，扩增不到片段的为原菌。
将阳性菌株在含15％蔗糖的培养基(同实施例2中的15％蔗糖培养基。)上培养，对培养产生的单菌落进一步采用P35/P36引物进行PCR鉴定，扩增出大小为1023bp(SEQ ID No.23)的菌株为PyfjB替换高产缬氨酸的谷氨酸棒杆菌CGMCC No.21260基因组上ilvC原始启动子的阳性菌株；扩增出大小为1025bp(SEQ ID No.24)的菌株为EP6替换高产缬氨酸的谷氨酸棒杆菌CGMCC No.21260基因组上ilvC原始启动子的菌株，扩增不到片段的为原菌。
将由PyfjB替换ilvC原始启动子的重组菌株命名为YPV-097(PyfjB-ilvC整合菌株)，由EP6替换ilvC原始启动子的重组菌株命名为YPV-098(EP6-ilvC整合菌株)。
YPV-097(PyfjB-ilvC整合菌株)是将高产缬氨酸的谷氨酸棒杆菌CGMCC No.21260ilvC原始启动子(SEQ ID No.18的第1-179位)替换为PyfjB，保持其基因组中的其它核苷酸不变得到的重组菌，其可以提高缬氨酸的产量；YPV-098(EP6-ilvC整合菌株)是将高产缬氨酸的谷氨酸棒杆菌CGMCC No.21260ilvC原始启动子(SEQ ID No.18的第1-179位)替换为EP6，保持其基因组中的其它核苷酸不变得到的重组菌，EP6启动子作为突变的yfjB启动子，其启动ilvC的表达更能提高缬氨酸的产量。
PCR鉴定引物如下所示：
P33：5′-CCCGACTTTG TTACCCTTTC-3′(CEY17_RS06885基因内部)(SEQ ID No.80)；
P34：5′-ACTGATCCCC ATAATAAGCG-3′(PyfjB内部)(SEQ ID No.81)；
P35：5′-CGCTTATTAT GGGGATCAGT-3′(PyfjB内部)(SEQ ID No.82)；
P36：5′-GGGTGGTTGT TGTAGGAAGC-3′(i lvC基因内部)(SEQ ID No.83)。
二、缬氨酸发酵实验。
将上述菌株YPV-097、YPV-098和原始菌株谷氨酸棒杆菌CGMCC No.21260在BLBIO-5GC-4-H型号的发酵罐(上海百仑生物科技有限公司)中以发酵培养基3和培养条件3进行发酵实验，发酵结束检测L-缬氨酸产量与OD值。每个菌株重复三次。
L-缬氨酸产量的检测方法为高效液相色谱法。
发酵培养基3：溶剂为水，溶质及其浓度分别为硫酸铵 14g/L、磷酸二氢钾 1g/L、磷酸氢二钾 1g/L、硫酸镁 0.5g/L、酵母粉 2g/L、硫酸亚铁 18mg/L、硫酸锰 4.2mg/L、生物素 0.02mg/L、维生素B1 2mg/L、antifoam(CB-442)消泡剂 0.5mL/L、70％葡萄糖(底糖)40g/L。
培养条件3：
校正DO 100％，温度33℃、风量1L/min、转速400rpm、罐压0.01Mpa，5min后标定；
接种量3.5％；培养温度33℃；
pH7.0±0.05；溶氧DO10-20％；
初始条件：温度33℃、pH 7.0、罐压0Mpa、风量0.1L/min、转速400rpm；
全程控制：温度33℃、pH 7.0、罐压0Mpa、风量0.2L/min、转速400rpm；
残糖控制：F12h前0.1-0.2％；F12h后结合DO要求控制残糖≤0.02％；
培养成熟标准：OD610 30-35；OD610＞30后停止通风静止2小时(根据批次实验目的，进行菌体分离或连续催化)；
流加物料：氨水、70％浓糖、5％泡敌；
发酵周期：18-20h左右。
结果显示，谷氨酸棒杆菌CGMCC No.21260的L-缬氨酸产量为84.1g/L，OD610为98.2；YPV-097的L-缬氨酸产量为85.7g/L，显著高于菌株CGMCC No.21260(P<0.05)，OD610为99.2；YPV-098的L-缬氨酸产量为87.6g/L，显著高于菌株CGMCC No.21260(P<0.05)，OD610为100.3。由此表明，在缬氨酸生产菌CGMCC No.21260中以yfjB或者EP6启动子替换ilvC原始启动子可以增加缬氨酸的产量，并且作为突变过的EP6启动ilvC的表达，缬氨酸产量更高。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
上述实施例涉及的序列1-24如下：
序列1：
序列2：


序列3：
序列4：
序列5：
序列6：

序列7：
序列8：
序列9：
序列10：
序列11：
序列12：

序列13：
序列14：

序列15：
序列16：
序列17：

序列18：
序列19：
序列20：

序列21：
序列22：

序列23：
序列24：
工业应用
对产氨基酸的细菌，本发明将EP6启动子启动相应氨基酸合成途径中基因得到相应的表达盒，然后将该表达盒作为外源强启动子整合到高产氨基酸的细菌中替换氨基酸合成关键基因的原始启动子，实现了相应氨基酸产量的提高。说明，本发明的EP6启动子不仅具有高启动活性，还可以用于生产氨基酸，具有很要的应用前景。

Claims (10)

  1. DNA分子，其序列为序列表中SEQ ID No.3。
  2. 与权利要求1所述DNA分子相关的生物材料，为下述B1)至B7)中的任一种：
    B1)含有权利要求1所述DNA分子的表达盒；
    B2)含有权利要求1所述DNA分子的重组载体；
    B3)含有B1)所述表达盒的重组载体；
    B4)含有权利要求1所述DNA分子的重组微生物；
    B5)含有B1)所述表达盒的重组微生物；
    B6)含有B2)所述重组载体的重组微生物；
    B7)含有B3)所述重组载体的重组微生物。
  3. 权利要求1所述DNA分子在作为启动子中的应用。
  4. 权利要求1所述DNA分子或权利要求2所述生物材料在生产氨基酸中的应用。
  5. 根据权利要求4所述的应用，其特征在于：所述氨基酸为赖氨酸、谷氨酸或缬氨酸。
  6. 一种生产氨基酸的方法，包括：将权利要求1所述DNA分子导入能合成目的氨基酸的生物细胞中，使权利要求1所述DNA分子驱动所述生物细胞中目的氨基酸合成途径中基因的表达，得到重组生物细胞；培养所述重组生物细胞，得到目的氨基酸。
  7. 根据权利要求6所述的方法，其特征在于：所述生物细胞为能合成目的氨基酸的酵母、细菌、藻、真菌、植物细胞或动物细胞。
  8. 根据权利要求7所述的方法，其特征在于：所述细菌为谷氨酸棒杆菌。
  9. 根据权利要求6-8中任一所述的方法，其特征在于：所述目的氨基酸为赖氨酸、谷氨酸或缬氨酸。
  10. 一种生产氨基酸的产品，其活性成分为权利要求1所述DNA分子或权利要求2所述生物材料。
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