WO2015120775A1 - 一种产l-氨基酸的重组菌、其构建方法及l-氨基酸生产方法 - Google Patents

一种产l-氨基酸的重组菌、其构建方法及l-氨基酸生产方法 Download PDF

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WO2015120775A1
WO2015120775A1 PCT/CN2015/072220 CN2015072220W WO2015120775A1 WO 2015120775 A1 WO2015120775 A1 WO 2015120775A1 CN 2015072220 W CN2015072220 W CN 2015072220W WO 2015120775 A1 WO2015120775 A1 WO 2015120775A1
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gene
promoter
homology
bacterium
strain
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温廷益
商秀玲
张芸
刘树文
梁勇
张宇
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中国科学院微生物研究所
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Priority to JP2016515651A priority Critical patent/JP6188930B2/ja
Priority to US14/891,622 priority patent/US9796991B2/en
Priority to CN201580000936.7A priority patent/CN106459886B/zh
Priority to EP15748508.7A priority patent/EP3009505B1/en
Priority to KR1020157032480A priority patent/KR101937682B1/ko
Publication of WO2015120775A1 publication Critical patent/WO2015120775A1/zh

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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/24—Proline; Hydroxyproline; Histidine
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    • 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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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1235—Diphosphotransferases (2.7.6)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01043—Phosphogluconate 2-dehydrogenase (1.1.1.43)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01049—Glucose-6-phosphate dehydrogenase (1.1.1.49)
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    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01009—Glucose-6-phosphate isomerase (5.3.1.9)

Definitions

  • the present invention relates to the field of microbial fermentation, and in particular, to a method for producing L-amino acid by microbial fermentation and a dedicated recombinant strain thereof.
  • L-amino acid by microbial fermentation is currently the most widely used method for the production of amino acids.
  • the fermentation performance of amino acid producing bacteria is a key factor affecting the feasibility of large-scale industrial application of fermentation.
  • the acid production level and sugar acid conversion rate still need to be further improved.
  • L-histidine is the ninth essential amino acid in humans and animals. It is an important physiological process involved in the growth and development of the body, antioxidants and immune regulation. It is an important medicinal amino acid and can be used.
  • L-histidine production mainly uses protein hydrolysis extraction method using pig (bovine) blood meal as raw material.
  • protein hydrolysis extraction method has the disadvantages of high raw material cost, low utilization rate, complicated extraction process and large environmental pollution.
  • L-histidine is expensive to produce and expensive.
  • the production of L-histidine by microbial fermentation has not yet been applied to large-scale industrial applications.
  • the biosynthesis of L-histidine has the characteristics of competing with nucleotide synthesis, complex metabolic regulation mechanism and high energy demand in the synthesis process, resulting in relatively low acid production and conversion rate of the engineered bacteria.
  • L-histidine producing strains mainly adopts multiple rounds of conventional mutagenesis screening and genetic engineering on the basis of mutagenized strains.
  • the strains obtained by mutagenesis screening will accumulate a large number of negative effect mutations, resulting in problems such as slow growth of the strain, decreased environmental tolerance, and increased nutritional requirements. These defects limit the industrial application of strains.
  • the wild type Escherichia coli MG1655 was used as a starting strain to reduce the feedback inhibition of histidine by introducing the E271K mutation into the hisG gene; knockdown of the histidine synthesis operon transcriptional weakening factor hisL, up-regulation of histidine
  • the expression of the synthetic operon was simultaneously knocked out, and the purR gene was knocked out to increase the synthesis of the histidine synthesis precursor PRPP, and an L-histidine-producing engineering strain was constructed.
  • only the L-histidine terminal synthesis pathway was modified, and the yield of L-histidine was only 4.9 g/L, which was far from the industrial application.
  • the main carrier-passing pathway for L-histidine biosynthesis is the pentose phosphate pathway.
  • L-histidine synthesis precursor phosphoribosyl pyrophosphate (PRPP) is produced via the pentose phosphate pathway.
  • PRPP phosphoribosyl pyrophosphate
  • the pentose phosphate pathway is also a cofactor NADPH required for the synthesis of various amino acids (such as L-lysine, L-valine, L-threonine, L-valine, and L-hydroxyproline).
  • NADPH a cofactor required for the synthesis of various amino acids
  • the main production pathway in which one molecule of L-lysine is synthesized, it takes 4 molecules of NADPH, and the synthesis of one molecule of L-threonine, L-valine and L-hydroxyproline requires 3 molecules of NADPH to synthesize 1 molecule.
  • L-valine requires consumption of 2 molecules of NADPH.
  • the 6-phosphate glucose isomerase which inactivates the glycolytic pathway, can direct the carbon metabolic flux to the pentose phosphate pathway, but it leads to the growth of the strain and the weakening of glucose metabolism, which is not conducive to the application of the strain in fermentation production ( Marx, A., Hans, S., Mockel, B., Bathe, B., de Graaf, AA, McCormack, AC, Stapleton, C., Burke, K., O'Donohue, M., Dunican, LK, 2003. Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum. J Biotechnol. 104, 185-197.).
  • the preliminary results of the present inventors confirmed that knocking out the 6-phosphate glucose isomerase-encoding gene pgi caused a serious decrease in the growth and glucose metabolism ability of the strain, and the L-histidine production also decreased. Further, the present inventors have also found that the increase in the expression level of glucose-6-phosphate dehydrogenase alone has a poor effect on the increase in L-histidine production.
  • L-amino acids which are capable of increasing L-amino acids, in particular by providing a precursor substance or a cofactor NADPH, such as L-histidine, L-lysine, L-oxime.
  • a precursor substance or a cofactor NADPH such as L-histidine, L-lysine, L-oxime.
  • Recombinant bacteria for the production of lysine, L-threonine, L-valine, and L-hydroxyproline and methods for constructing the same, and methods for producing L-amino acids using the recombinant bacteria.
  • an aspect of the present invention provides a recombinant bacterium producing L-amino acid having reduced expression of a glucose-phosphate isomerase Pgi compared to a starting bacterium, and an increased glucose-phosphate of 6-phosphate Expression of the hydrogenase Zwf-OpcA, wherein the starting strain is a strain capable of accumulating an amino acid of interest.
  • the starting bacteria are obtained by mutagenesis or genetic engineering of the chromosomes of the original bacteria.
  • the starting strain may be an existing strain capable of accumulating the amino acid of interest, or may be a genetically engineered suitable original bacteria to obtain a strain capable of accumulating the amino acid of interest.
  • those having a higher yield for the amino acid of interest are preferred as starting bacteria.
  • the amino acid of interest referred to in the present invention refers to an L-amino acid synthesized by providing a precursor substance or a cofactor NADPH by a pentose phosphate pathway.
  • the amino acid of interest is L-histidine, L-lysine, L-valine, L-threonine, L-valine or L-hydroxyproline.
  • the recombinant strain can attenuate expression of the pgi gene compared to the starting strain while enhancing expression of the zwf-opcA gene.
  • the pgi gene on the chromosome of the recombinant strain has been inactivated, preferably has been knocked out, or the regulatory element of the pgi gene has been replaced with a regulatory element of low transcription or low expression activity, and the recombinant strain has The promoter of two or more copies of the zwf-opcA gene, or the tkt-tal-zwf-opcA-devB operon, is replaced with a strong promoter, for example, the P eftu promoter replaced by the original strain.
  • the starting bacteria can enhance the expression of the L-histidine synthesis operon hisEG gene and the hisDCB gene relative to the original bacteria.
  • a strong promoter can be substituted for the promoter of the gene.
  • the promoter of hisEG and hisDCB on the chromosomes of the original bacteria are replaced by the P glyA promoter on the original bacterial chromosome.
  • the starting bacteria are capable of enhancing expression of the PRPP synthase PrsA compared to the original bacteria. More preferably, the original strain having two or more copies of the prsA gene, or a promoter with a strong promoter instead of the prsA gene, for example, available in the original strain P sod promoter instead of prsA gene promoter.
  • the starting strain can enhance the expression of dapA gene (dihydropyridine II) compared with the original bacteria.
  • the gene encoding the carboxylic acid synthase) or the lysC gene (the gene encoding the aspartokinase) (Cremer, J., Eggeling, L., Sahm, H., 1991. Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed By overexpression of the individual corresponding genes. Appl Environ Microbiol. 57, 1746-1752).
  • the starting bacteria may have two or more copies of the dapA gene or the lysC gene, or a strong promoter to replace the promoter of the dapA gene or the lysC gene.
  • the starting strain can enhance the expression of the proline synthesis gene ilvBNCE (Blombach, B., Schreiner, ME, Holátko, J., Bartek, T., Oldiges) compared to the original bacteria. M., Eikmanns, BJ, 2007. L-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum. Appl Environ Microbiol. 73, 2079-2084).
  • the starting bacteria may have two or more copies of the ilvBNCE gene, or a strong promoter in place of the promoter of the ilvBNCE gene.
  • the starting bacteria can enhance the expression of the threonine synthesis pathway genes hom and thrB compared to the original bacteria (Reinscheid, DJ, Kronemeyer, W., Eggeling, L., Eikmanns, BJ, Sahm, H., 1994. Stable expression of hom-1-thrB in Corynebacterium glutamicum and its effect on the carbon flux to threonine and related amino acids. Appl Environ Microbiol. 60, 126-132).
  • the starting bacteria may have two or more copies of the hom and thrB genes, or a strong promoter to replace the promoters of the hom gene and the thrB gene, respectively.
  • the starting bacteria can enhance the expression of the ocd gene (ornithine cyclase deaminase-encoding gene) compared to the original bacteria (Jensen, JVK, Wendisch, V., 2013. Ornithine cyclodeaminase -based proline production by Corynebacterium glutamicum. Microb Cell Fact. 12, 63).
  • the starting bacterium may have two or more copies of the ocd gene, or a promoter with a strong promoter instead of the ocd gene.
  • the starting strain can enhance the expression of the p4hD gene (proline hydroxylase-encoding gene) compared to the original bacteria (Yi, Y., Sheng, H., Li, Z. , Ye, Q., 2014. Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli. BMC Biotechnol. 14, 44.).
  • the starting strain may have two or more copies of the p4hD gene, or a promoter with a strong promoter instead of the p4hD gene.
  • the recombinant bacterium can express the AICAR methyltransferase/IMP cyclase PurH more than the starting bacterium.
  • the recombinant strain has two or more copies of the purH gene, or a promoter that replaces the purH gene with a strong promoter, for example, a promoter that replaces the purH gene with the P eftu promoter of the original bacterium .
  • the recombinant bacterium has a weakened expression of the phosphoribosyltransferase PurF compared to the starting bacterium.
  • a weak promoter can be used in place of the promoter of the purF gene.
  • the promoter of the purF gene is replaced by a P hom promoter in the original bacterium on the chromosome of the recombinant bacterium.
  • strong promoter in various embodiments, there is no particular limitation on the strong promoter in the present invention as long as it can enhance the expression of the activated gene.
  • the strong promoter which can be used in the present invention may be, for example, P eftu , P sod , P glyA , P pck , P pgk promoter or the like of the original bacteria, but is not limited thereto.
  • the original bacteria is preferably a bacterium selected from the group consisting of Corynebacterium, Bacillus, and Brevibacterium.
  • the bacterium of the genus Corynebacterium is preferably selected from the group consisting of Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium ammoniagenes
  • the bacterium of the genus Bacillus is preferably selected from a strain of bacteria belonging to Microbacterium ammoniaphilum.
  • the bacterium of the genus Brevibacterium is preferably selected from the group consisting of Brevibacteriaceae flvum, Brevibacteriaceae lactofermentum, and Brevibacteriaceae ammoniagenes.
  • the original strain is wild-type C. glutamicum ATCC13032.
  • the P glyA promoter represented by the nucleotide sequence of the 5' end of SEQ ID NO: 863-1038 in the sequence of the starting strain is used for the respective Instead of the promoter of the L-histidine synthesis operon hisEG and hisDCB on the C. glutamicum ATCC13032 chromosome, and the starting bacterium capable of expressing the mutated ATP-phosphoribosyltransferase.
  • the mutated ATP-phosphoribosyltransferase is the 215th asparagine mutated to lysine, the 231th leucine to phenylalanine and the 235th of the ATP-phosphoribosyltransferase shown in SEQ ID NO: 6.
  • An enzyme that mutates threonine to alanine is used in place of the hisG gene on the chromosome of Corynebacterium glutamicum ATCC13032.
  • the starting strain has two or more copies of the prsA gene and the hisG fbr gene.
  • the prsA gene may be selected from the gene encoding PrsA shown in SEQ ID NO: 5; and encodes at least 60% homology, preferably at least 70% homology, more preferably at least 80% homology to the PrsA. Further, one of genes having at least 95% homology, even more preferably having at least 98%, or even 99% homology, and having PrsA activity is further preferred. Specifically, it may be the nucleotide sequence of position 15-99 shown by SEQ ID NO: 4 in the Sequence Listing.
  • the pgi gene may be selected from the group consisting of the gene encoding Pgi shown in SEQ ID NO: 14 in the sequence listing; and encoding at least 60% homology, preferably at least 70% homology to the Pgi. More preferably, having at least 80% homology, more preferably having at least 95% homology, even more preferably having at least 98%, or even 99% homology, and having said 6-phosphate glucose isomerase Pgi activity
  • the genes may be the nucleotide sequence shown in SEQ ID NO: 13.
  • the zwf-opcA gene may be selected from the group consisting of the gene encoding Zwf-OpcA shown in SEQ ID NO: 3 in the sequence listing; and encoding having at least 60% homology, preferably at least 70% homology to the Zwf-OpcA. And more preferably in a gene having at least 80% homology, more preferably at least 95% homology, even more preferably at least 98%, or even 99% homology, and having said Zwf-OpcA activity One. Specifically, it may be the nucleotide sequence shown in SEQ ID NO: 2.
  • the P eftu promoter may be the nucleotide sequence at positions 635-834 of the 5' end of the sequence 12 .
  • the purH gene may be selected from the group consisting of the gene encoding PurH shown in SEQ ID NO: 16 in the sequence listing; and encoding having at least 60% homology, preferably at least 70% homology, more preferably at least 80 compared to the PurH. % homology, more preferably one having at least 95% homology, even more preferably having at least 98%, or even 99% homology, and having the PurH activity, preferably, the purH gene It may be the nucleotide sequence shown in SEQ ID NO: 15 in the Sequence Listing.
  • the promoter may be P hom 'nucleotide sequence of position 736-865 18 5-terminal sequence.
  • a recombinant plasmid containing a gene can be introduced to increase the copy number of the gene, and a gene can be directly inserted into a suitable site on the chromosome of the strain.
  • the vector used to construct the recombinant plasmid is not limited and may be any suitable plasmid such as pXMJ19.
  • a method of constructing a recombinant strain producing L-amino acid comprises the steps of: reducing the expression of the 6-phosphate glucose isomerase Pgi in the starting bacteria, and increasing the expression of the 6-phosphate glucose dehydrogenase Zwf-OpcA in the starting bacteria, wherein the recombinant bacteria is obtained, wherein The starting bacteria are strains capable of accumulating amino acids of interest.
  • the starting bacteria can be obtained by a method such as mutagenesis or genetic engineering according to a known method, and an existing strain capable of producing an amino acid of interest can also be used as a starting strain. Those high-yield strains are preferred.
  • the amino acid of interest referred to in the present invention is preferably L-histidine, L-lysine, L-valine, L-threonine, L-valine or L-hydroxyproline.
  • reducing the expression of Pgi in the starting bacteria is achieved by the following A) or B):
  • obtaining the starting bacteria may comprise replacing the promoters of the L-histidine synthesis operons hisEG and hisDCB on the chromosome of the original bacteria with strong promoters, respectively, for example The step of the P glyA promoter on the original bacterial chromosome. Further preferably, obtaining the starting bacteria may further comprise the step of increasing the expression of the PRPP synthase PrsA in the starting bacteria. More preferably, said increasing the expression of PrsA in said starting bacteria is achieved by the following E) or F):
  • L-lysine For L-lysine, according to one embodiment, it is possible to accumulate L- by enhancing expression of the dapA gene (a gene encoding a dihydrodipicolinate synthase) or a lysC gene (a gene encoding an aspartokinase).
  • the starting bacteria of lysine Specifically, the copy number of the dapA gene or the lysC gene in the starting strain is increased, or the promoter of the dapA gene or the lysC gene is replaced with a strong promoter.
  • the starting strain can be obtained by enhancing expression of the proline synthesis gene ilvBNCE. Specifically, the copy number of the ilvBNCE gene in the starting strain can be increased, or the promoter of the ilvBNCE gene can be replaced with a strong promoter.
  • obtaining the starting bacteria may comprise the step of enhancing expression of the threonine synthesis pathway genes hom and thrB. Specifically, the copy number of the hom gene and the thrB gene in the starting strain may be increased, or the promoter of the hom and thrB genes may be replaced by a strong promoter, respectively.
  • obtaining the starting strain may comprise the step of enhancing expression of an ocd gene (ornithine cyclase deaminase encoding gene). Specifically, the copy number of the ocd gene in the starting strain may be increased, or the promoter of the ocd gene may be replaced by a strong promoter.
  • obtaining the starting strain may comprise the step of enhancing expression of the p4hD gene (proline hydroxylase encoding gene). Specifically, the copy number of the p4hD gene in the starting strain may be increased, or the promoter of the p4hD gene may be replaced with a strong promoter.
  • the method may further comprise the step of increasing the expression of the AICAR methyltransferase/IMP cyclase PurH in the recombinant strain.
  • said increasing the expression of PurH in said recombinant strain can be achieved by the following G) or H):
  • the method may further comprise the step of attenuating expression of the phosphoribosyltransferase PurF in the recombinant strain.
  • a weak promoter can be used in place of the promoter of the purF gene.
  • the weakened expression of the recombinant bacteria by replacing PurF purF gene promoter on the chromosome in strain departure P hom promoter on the chromosome of the original bacteria.
  • the strong promoter as long as it can enhance the expression of the activated gene.
  • the P eftu , P sod , P glyA , P pck or P pgk promoter of the original strain may be mentioned, but is not limited thereto.
  • the strain which can be used as the original bacteria can be selected from a strain of bacteria belonging to the genus Corynebacterium, Bacillus, and Brevibacterium.
  • the bacterium of the genus Corynebacterium is preferably selected from the group consisting of Corynebacterium glutamicum, Corynebacterium pekinense, Corynebacterium efficiens, Corynebacterium crenatum, Corynebacterium thermoaminogenes, Corynebacterium ammoniagenes
  • the bacterium of the genus Bacillus is preferably selected from a strain of bacteria belonging to Microbacterium ammoniaphilum.
  • the bacterium of the genus Brevibacterium is preferably selected from the group consisting of Brevibacteriaceae flvum, Brevibacteriaceae lactofermentum, and Brevibacteriaceae ammoniagenes. Most preferred is Corynebacterium glutamicum or Brevibacteriaceae flvum.
  • the original strain is wild-type C. glutamicum ATCC13032.
  • the starting bacteria can pass the following bacteria on the original bacteria. Restructuring and obtaining:
  • the promoters of the L-histidine synthesis operons hisEG and hisDCB on the C. glutamicum ATCC13032 chromosome were replaced with the nucleotide sequence 863-1038 of the 5' end of the sequence 7 (or 5 in the sequence 8).
  • mutant gene is a gene shown hisG -br 41007-1852 sequence of the nucleotide sequence.
  • the chromosome of Corynebacterium glutamicum ATCC13032 is further modified, and the promoter of the prsA gene on the chromosome is replaced with the sequence 11 P sod shown in the 5 'end of the promoter sequence of nucleotides 656-847.
  • L-histidine accumulation can be obtained by increasing the copy number of the prsA gene in the C. glutamicum ATCC13032 and increasing the copy number of the hisG fbr gene in the C. glutamicum ATCC13032. A better starting bacteria.
  • the prsA gene may be selected from the gene encoding PrsA shown in SEQ ID NO: 5; and encodes at least 60% homology, preferably at least 70% homology, more preferably at least 80% homology to the PrsA. Further, one of genes having at least 95% homology, even more preferably having at least 98%, or even 99% homology, and having PrsA activity is further preferred. Specifically, it may be the nucleotide sequence of position 15-99 shown in SEQ ID NO: 4 in the Sequence Listing.
  • the pgi gene may be selected from the group consisting of the gene encoding the Pgi shown in SEQ ID NO: 14 in the sequence listing; and encoding at least 60% homology, preferably at least 70% homology, more preferably at least 80% compared to the Pgi. Homology, more preferably one of genes having at least 95% homology, even more preferably 98%, or even 99% homology, and having said 6-phosphate glucose isomerase activity. Specifically, it may be the nucleotide sequence shown in SEQ ID NO: 13,
  • the zwf-opcA gene may be selected from the group consisting of the gene encoding Zwf-OpcA shown in SEQ ID NO: 3 in the sequence listing; and encoding having at least 60% homology, preferably at least 70% homology to the Zwf-OpcA. More preferably, one of the genes having at least 80% homology, more preferably at least 95% homology, even more preferably 98%, or even 99% homology, and having said Zwf-OpcA activity. Specifically, it may be the nucleotide sequence shown in SEQ ID NO: 2.
  • the P eftu promoter is the nucleotide sequence of positions 635-834 at the 5' end of the sequence 12 (or positions 634-833 at the 5' end of the sequence 20).
  • the purH gene is selected from the group consisting of the gene encoding PurH shown in SEQ ID NO: 16 in the sequence listing; and encoding at least 60% homology, preferably at least 70% homology, more preferably at least 80% compared to the PurH Homology, more preferably one of genes having at least 95% homology, even more preferably 98%, or even 99% homology, and having the PurH activity. Specifically, it may be a nucleotide sequence shown by SEQ ID NO: 15 in the Sequence Listing.
  • the promoter is a P hom 'nucleotide sequence of position 736-865 18 5-terminal sequence.
  • increasing the copy number of a gene can be achieved by constructing a recombinant plasmid containing the gene and introducing the recombinant plasmid into the starting/primitive bacteria.
  • the vector used to construct the recombinant plasmid is not limited and may be any suitable plasmid such as pXMJ19.
  • the recombinant strain of the present invention may be a recombinant strain obtained by the above construction method.
  • a method for producing an L-amino acid comprising the step of fermenting the above recombinant bacteria is provided.
  • the L-amino acid is preferably L-histidine, L-lysine, L-valine, L-threonine, L-valine or L-hydroxyproline.
  • the method for constructing a recombinant strain comprises the steps of: reducing the expression of glucose-6 isomerase in the starting bacteria, and increasing the expression of glucose-6-phosphate dehydrogenase and PRPP synthase in the starting bacteria, thereby obtaining Recombinant bacteria.
  • the reducing the expression of glucose-6 isomerase in the starting bacteria is achieved by the following A) or B):
  • the method for constructing the recombinant bacteria is as follows: I or II:
  • the method shown by I is to knock out the pgi gene of the starting bacterium chromosome, and increase the copy number of the zwf-opcA gene and the prsA gene in the starting bacterium to obtain a recombinant bacterium;
  • the method shown in II is to knock out the pgi gene of the starting bacterium chromosome, and replace the promoter of the tkt-tal-zwf-opcA-devB operon on the chromosome of the starting bacterium with the P eftu promoter, and the The promoter of the prsA gene on the chromosome of the starting strain was replaced with the P sod promoter.
  • the knock-out is to introduce a fragment containing the upstream and downstream homology arms of the gene to be knocked out pgi into the starting bacteria for homologous recombination;
  • the recombinant vector is a recombinant vector obtained by inserting the zwf-opcA gene and the prsA-hisG fbr fragment into an expression vector;
  • the expression vector may be an IPTG inducible expression vector pXMJ19;
  • the recombinant vector is pXMJ19-zwf-opcA-prsA-hisG fbr , which inserts the zwf-opcA gene (SEQ ID NO: 2) between the Hind III and Xba I sites of pXMJ19, and prsA-hisG
  • the fbr fragment (SEQ ID NO: 4) was inserted between the Xba I and Sma I sites.
  • the promoter of the original strain tkt-tal-zwf-opcA- devB operon on the chromosome is replaced with the P eftu promoter is a promoter fragment containing the promoter P eftu introduced in the original strain homologous recombination;
  • the promoter of the original strain prsA gene on the chromosome is replaced with P sod promoter is a fragment containing promoter P sod introduced into the starting bacteria homologous recombination.
  • the nucleotide sequence of the fragment containing the upstream and downstream homology arms of the gene to be knocked out is the sequence 1 in the sequence listing, wherein the nucleotide 1 to position 834 from the 5' end is the gene to be knocked out pgi
  • the upstream homology arm, sequence 1 from the 5' end of the nucleotides 835-1672 is the downstream homology arm of the gene to be knocked out pgi; the nucleotide sequence of the gene pgi is the sequence 13;
  • nucleotide sequence of the zwf-opcA gene is sequence 2 in the sequence listing;
  • the nucleotide sequence of the prsA-hisG fbr fragment is sequence 4 in the sequence listing ;
  • the recombinant vector is a vector obtained by inserting the zwf-opcA gene and the prsA-hisG fbr fragment into an expression vector;
  • the nucleotide sequence of the P eftu promoter is the nucleotide sequence 635-834 from the 5' end of the sequence 12 in the sequence listing;
  • the nucleotide sequence of the fragment containing the P eftu promoter is the sequence 12 in the sequence listing ;
  • the nucleotide sequence of the fragment containing the Psod promoter is the sequence 11 in the sequence listing.
  • the starting bacteria are prepared according to the method comprising the steps of: replacing the promoter of the L-histidine synthesis operon on the bacterial chromosome with the P glyA promoter, and performing the hisG gene on the bacterial chromosome Point mutation to obtain the starting bacteria;
  • the L-histidine synthesis operon is hisEG and hisDCB;
  • the nucleotide sequence of the P glyA promoter is nucleotides 863-1038 of SEQ ID NO: 7 in the sequence listing or nucleotides 752-927 of SEQ ID NO: 8 in the sequence listing;
  • the point mutation is such that the asparagine at position 215 of the protein encoded by the hisG gene of the bacterial chromosome is changed to lysine, the 231th leucine is changed to phenylalanine, and the 235th threonine is changed. Alanine.
  • the promoter of the L-histidine synthesis operon on the bacterial chromosome is replaced with a P glyA promoter which is a fragment of a P glyA promoter containing hisEG and a fragment of a P glyA promoter containing hisDCB.
  • bacterial homologous recombination wherein the nucleotide sequence of the fragment containing hisEG P glyA promoter sequence is sequence Listing 7; hisDCB nucleotide sequence fragment containing the P glyA promoter sequence table Sequence 8.
  • the hisG gene on the bacterial chromosome is point-mutated to introduce the nucleotide sequence shown in SEQ ID NO:9 into the bacterium for homologous recombination, and the nucleotide sequence shown in SEQ ID NO:10
  • the homologous recombination was carried out into the intermediate bacteria.
  • the bacterium is a bacterium belonging to the genus Corynebacterium, and the bacterium belonging to the genus Corynebacterium is specifically Corynebacterium glutamicum.
  • the recombinant bacteria prepared by the above methods are also within the scope of protection of the present invention.
  • the present invention also provides a method for preparing L-histidine, comprising the steps of: fermenting the above-mentioned recombinant bacteria to obtain L-histidine.
  • the "inactivation" of the pgi gene of the inactivated bacteria of the present invention refers to a change in the corresponding engineered subject, thereby achieving certain effects including, but not limited to, site-directed mutagenesis, insertional inactivation, and/or knockout.
  • the methods for chromosomal gene knockout, insertion inactivation, gene knock-in, promoter substitution, and site-directed mutagenesis used in the present invention are
  • the suicide vector pK18mobsacB carries the homologous recombination of the homologous arm of the engineered target gene to achieve homologous recombination.
  • the L-histidine engineering bacteria of the present invention has an L-histidine production intensity of 0.01 to 1 g/L/h for 24 hours of fermentation, and an L-histidine production of 1 to 60 g/L at the end of fermentation.
  • the fermentation yield can reach 2 g/L or more.
  • the recombinant strain provided by the present invention by using a knock-in pgi gene to block the upstream glycolytic pathway while overexpressing the zwf-opcA gene to enhance the metabolic ability of the pentose phosphate pathway, the growth of the engineered bacteria and The glucose consumption capacity was not significantly reduced compared with the wild-type strain, and the L-amino acid yield was significantly increased.
  • the recombinant bacteria provided by the present invention grow well in the basic medium (used in the shake flask fermentation experiment), and have no nutrient-deficient phenotype, which is convenient for industrial control.
  • the fermentation period provided by the present invention has a short fermentation period, and the maximum accumulation amount can be reached in about 45-72 hours in the fermentation tank enlargement experiment (the highest yield of the L-histidine engineered bacteria reported at the present time is up to a long time) 120 hours) (Mizukami, T., Hamu, A., Ikeda, M., Oka, T., Katsumata, R., 1994. Cloning of the ATP phosphoribosyl transferase gene of Corynebacterium glutamicum and application of the gene to L-histidine Production. Biosci. Biotechnol. Biochem. 58, 635-638.), easy process and cost control.
  • the present invention first proposed a combination modification strategy for enhancing the expression of glucose dehydrogenase 6-phosphate on the basis of the deletion of the pgi gene, thereby eliminating the restriction of growth and glucose metabolism caused by the deletion of the pgi gene, and maximizing the degree.
  • the central carbon metabolic flux is directed to the pentose phosphate pathway, while maintaining high growth metabolism and ATP levels of the bacteria, significantly increasing the yield of amino acids, and thus can be practically used for industrial production of bacterial fermentation.
  • the present invention also proposes for the first time a strategy of coupling the histidine synthesis pathway with a nucleotide synthesis pathway, and synthesizing the precursor ATP of histidine by using the histochemical synthesis by-product AICAR, significantly increasing the L-group.
  • the yield of the acid is thus practically used for the industrial production of L-histidine by bacterial fermentation.
  • the beneficial effects of the present invention are that a new method for improving the fermentation yield of L-amino acid is developed and practiced, and the corresponding engineering bacteria are constructed, and the effect of superimposing the yield can be observed, so that it can be practically used.
  • L-amino acid is produced by bacterial fermentation, which is convenient for popularization and application.
  • Figure 1 is a schematic representation of the recombinant plasmid pXMJ19-prsA-hisG fbr .
  • Fig. 2 is an electrophoresis pattern of PCR identification of genomic DNA of CG161 strain (pgi gene knocked out).
  • Figure 3 is a schematic representation of the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr .
  • Figure 4 is a SDS-PAGE diagram of the protein expressed by L-histidine engineered bacteria CG171.
  • Fig. 5 is a graph showing the measurement of glucose-dehydrogenase activity of 6-phosphate in L-histidine engineered bacteria CG171.
  • Figure 6 is a schematic representation of the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr -purH.
  • Figure 7 is an electrophoresis pattern of PCR identification of plasmid DNA carrying CG328 strain.
  • Fig. 8 is an electrophoresis map of PCR identification of genomic DNA of CG353 strain (purF gene weakened).
  • the phosphoglucose isomerase encoded by the pgi gene is a key enzyme of the glycolytic pathway.
  • the precursor PRPP for L-histidine synthesis is synthesized via the pentose phosphate pathway, so it is envisaged that knocking out the pgi gene will attenuate the metabolic flux of the glycolytic pathway and direct the central carbon metabolic pathway to the pentose phosphate pathway to enhance L- Histidine synthesis pathway metabolic flux.
  • the inventors have found that knocking out the pgi gene leads to excessive accumulation of intermediate metabolites in sugar metabolism, causing stress in glucose metabolism, which in turn leads to slowing of glucose metabolism and growth of the cells.
  • the present inventors also found that the L-histidine production of the L-histidine-producing engineering bacteria did not increase, but decreased significantly, after knocking out the pgi gene. The main reason is that histidine provides a precursor for the synthesis of its molecular skeleton through the pentose phosphate pathway, while lysine and proline provide the cofactor NADPH of its synthetase via the pentose phosphate pathway.
  • the histidine synthesis process consumes a large amount of energy carrier ATP, in order to use the weakened pgi gene expression and enhance the metabolic flux of the pentose phosphate pathway to increase histidine production, it needs to be maintained.
  • the balance of the metabolic flux of the pentose phosphate pathway and the glycolysis pathway ensures its synthesis precursor and energy supply.
  • the present inventors have found through experiments that over-expressing the zwf-opcA gene (which encodes a glucose-dehydrogenase 6-phosphate, a key rate-limiting enzyme in the pentose phosphate pathway) can enhance the ability of the cell's glucose metabolism and alleviate
  • the glucose metabolism pressure restores the glucose metabolism and growth ability of the strain, and balances the metabolic flux of the pentose phosphate pathway and the glycolysis pathway, balances the supply of the histidine synthesis precursors PRPP and ATP, and further increases the L-histidine production.
  • a strain obtained by recombinantly modifying a strain having enhanced prsA gene and L-histidine synthesis operon gene expression by weakening (e.g., knocking out) the pgi gene and simultaneously overexpressing the zwf-opcA gene transformation strategy L-histidine production has been significantly improved.
  • the present invention further proposes a strategy of coupling the L-histidine synthesis pathway to a nucleotide synthesis pathway.
  • the imidazole glycerol phosphate and the 5-phosphate ribose-4-carboxamido-5-aminoimidazole (AICAR) are catalyzed by the histH and hisF genes, and the imidazole glycerol phosphate L-histidine is finally synthesized along the histidine synthesis pathway, while AICAR can enter the purine synthesis pathway to form purine nucleotides (AMP, ATP, etc.).
  • ATP is one of the precursors of histidine synthesis and also provides energy for histidine synthesis.
  • the purH gene encodes a bifunctional enzyme, AICAR methyltransferase/IMP cyclase, which catalyzes a two-step reaction to generate IMP from AICAR.
  • AICAR methyltransferase/IMP cyclase which catalyzes a two-step reaction to generate IMP from AICAR.
  • the present inventors have found that enhancing the expression of the purH gene in C. glutamicum has a significant promoting effect on the accumulation of L-histidine, and the combination with the above-described transformation strategy can further improve the effect.
  • the L-histidine synthesis pathway is coupled to the purine nucleotide synthesis pathway at the metabolite AICAR, while the two pathways share the precursor substance PRPP.
  • the present inventors have found that weakening the catalytic ruthenium nucleotide synthesis first-step reaction enzyme (phosphory riboyltransferase) encoding gene purF, can be used for metabolic coupling of nucleotide synthesis and histidine synthesis pathway, using histidine synthesis
  • the by-product AICAR synthesizes nucleotides, increases the supply of histidine synthesis precursor PRPP, and simultaneously stimulates the metabolic flux of histidine synthesis pathway and promotes L-histidine accumulation. This genetic modification can also further increase the production of L-histidine.
  • the present invention combines multiple targets in the histidine synthesis-related pathway of microorganisms, and effectively realizes the accumulation of L-histidine.
  • the histidine synthesis pathway is coupled with the nucleotide synthesis pathway, and the coupling node AICAR of histidine synthesis and nucleotide synthesis is effectively utilized to generate the purine nucleotide.
  • the pathway saves the synthetic precursor PRPP, thereby providing more precursor substances PRPP and ATP for histidine synthesis, further increasing the accumulation of L-histidine.
  • starting bacteria refers to the initial strain used in the genetic engineering strategy of the present invention.
  • the strain may be a naturally occurring strain or may be mutagenized Or strains selected by genetic engineering or the like.
  • the starting bacterium is preferably a strain which can accumulate the L-amino acid (e.g., L-histidine).
  • original bacteria refers to a strain that has not been genetically engineered, and may be a strain that exists in nature or a strain that has been artificially mutagenized.
  • homology refers to the degree of similarity between the nucleotide sequence of a DNA or the amino acid sequence of a protein, and the DNA having a (some degree) homology as described herein is encoded.
  • the protein has the same or better activity at least for the function of the present invention, and the same protein having (a certain degree of homology) has the same or better activity at least for the function of the present invention.
  • the hisG gene has a high degree of similarity to the hisG fbr gene, which encodes ATP-phosphoribosyltransferase, which encodes ATP-phosphate, which abolishes histidine feedback inhibition regulation.
  • Ribotransferase which functions differently in function and activity as a whole, but the functions of the "catalytic enzyme for the first step reaction of histidine synthesis" used in the present invention are the same, therefore,
  • the hisG gene and the hisG fbr gene, as well as the enzymes encoded by the two, belong to homologous DNA and proteins in the sense of the present invention, respectively. They are all within the scope of the invention.
  • the invention is further illustrated by the following examples.
  • the experimental methods used in the following examples are conventional methods unless otherwise specified.
  • the materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
  • the technical means used in the examples are conventional means well known to those skilled in the art, and can be found in "Molecular Cloning Experiment Guide (3rd Edition)” (Science Press), “Microbiology Experiment (4th Edition) ) (Higher Education Press) and the manufacturer's instructions for the corresponding instruments and reagents.
  • the apparatus and reagents used in the examples are commercially available instruments and reagents. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • this example was carried out to modify the histidine synthesis of wild type C. glutamicum ATCC13032 to obtain the above-mentioned multi-target modified chassis bacteria of the present invention.
  • the promoters of hisEG and hisDCB two histidine synthase operons were replaced with the endogenous strong promoter of G.
  • glutamicum P glyA (nucleotide sequence 863-1038 at the 5' end of SEQ ID NO:7) Shown, or shown in nucleotide sequence 752-927 at the 5' end of Sequence 8) (Zhang, Y., Shang, X., Lai, S., Zhang, G., Liang, Y., Wen, T., 2012. Development and application of an arabinose-inducible expression aystem by facilitating inducer uptake in Corynebacterium glutamicum. Appl Environ Microbiol.
  • the coding gene hesG a key rate-limiting enzyme of histidine synthesis pathway, ATP-phosphoribosyltransferase (HisG, sequence 6), was replaced with a hisG fbr gene containing three amino acid site mutations (5 in sequence 4).
  • HisG a key rate-limiting enzyme of histidine synthesis pathway, ATP-phosphoribosyltransferase
  • HisG sequence 6
  • HisG fbr gene containing three amino acid site mutations 5 in sequence 4
  • the nucleotide sequence of the 1007-1852 nucleotides' is shown to relieve the feedback inhibition of histidine to enhance the catalytic activity of the enzyme (Zhang, Y., Shang, X., Deng, A., Chai) , X., Lai, S., Zhang, G., Wen, T., 2012. Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutant
  • Primers were designed according to the hisEG operon of C. glutamicum ATCC13032 in Genbank and its upstream and downstream sequences and the P glyA promoter sequence, respectively.
  • the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and P1 and P2 were used as primers to amplify the homologous arm upstream of the hisEG operon promoter. P3 and P4 were used as primers to amplify the promoter P glyA . P5 and P6 were used as primers. Primers amplify homologous arms downstream of the hisEG promoter.
  • the purified PCR product was used as a template, and P1 and P6 were used as primers, and amplified by overlap extension PCR (SOE) to obtain a 1920 bp PCR product containing the replacement promoter P glyA and the replaced promoter P hisEG . Fragment of the downstream homology arm (sequence 7).
  • sequence 7 from the 5' end of position 1 - 862 nucleotide is the upstream promoter of the replaced promoter P hisEG
  • sequence 7 from the 5' end of the 863-1038 nucleotides as the promoter P glyA sequence 7 from the 5' end of the 863-1038 nucleotides as the promoter P glyA
  • sequence The nucleotide from position 1053-1920 at the 5' end is the downstream homology arm of the replaced promoter P hisEG .
  • the above 1920 bp PCR product was digested with Xba I and BamH I, and ligated with the same double-digested homologous recombinant vector pK18mobsacB (purchased from American Type Culture Collection ATCC, Cat. No. 87097).
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • a positive transformant of 2132 bp was obtained, and the plasmid was extracted from the correct transformant, and the plasmid was subjected to double digestion with Xba I and BamH I to obtain 1920 bp.
  • the positive plasmid was sent for sequencing, and the plasmid was designated as pK18mobsacB-P glyA ::P hisEG by inserting the nucleotide shown in SEQ ID NO: 7 into the vector pK18mobsacB .
  • the homologous recombination plasmid pK18mobsacB-P glyA ::P hisDCB was constructed by the same method as follows: the homologous arm upstream of the hisDCB operon promoter was amplified with P7 and P8 as primers; the promoter was amplified with P9 and P10 as primers. glyA ; a homologous arm downstream of the promoter of hisDCB was amplified with P11 and P12 as primers. P7 and P12 were used as primers and amplified by overlap extension PCR (SOE).
  • SOE overlap extension PCR
  • the 1694 bp PCR product was obtained as a long fragment (sequence 8) containing the replacement promoter P glyA and the homologous arm upstream and downstream of the replaced promoter P hisDCB , wherein the sequence 8 was from the 1st to the 751th nucleotide at the 5' end.
  • the upstream homologous arm of the promoter P hisDCB was replaced, the sequence 8 from the 5' end of the nucleotides 752-927 is the promoter P glyA , and the sequence 8 is replaced by the 5' end of the nucleotides 942-1694.
  • the downstream homology arm of the child P hisDCB was obtained as a long fragment (sequence 8) containing the replacement promoter P glyA and the homologous arm upstream and downstream of the replaced promoter P hisDCB , wherein the sequence 8 was from the 1st to the 751th nucleotide at the 5' end.
  • the above 1694 bp PCR product was digested with Hind III and BamH I and ligated with the homologous recombinant vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • the 1906 bp positive transformant was obtained, and the correct transformant was used to extract the plasmid, and the plasmid was subjected to restriction enzyme digestion with Hind III and BamH I to obtain 1694 bp.
  • the positive plasmid was sent for sequencing, and the plasmid was designated as pK18mobsacB-P glyA ::P hisDCB by inserting the nucleotide shown in SEQ ID NO: 5 into the vector pK18mobsacB.
  • P15 TTTTATATATGGGTATCGGCGGTCTATGCT (SEQ ID NO: 35).
  • the sequence homologous recombinant plasmid pK18mobsacB-P glyA ::P hisEG was electrotransformed into Corynebacterium glutamicum wild type ATCC13032. Colonies that integrate the recombinant plasmid into the chromosome are positively screened by kanamycin resistance. Positive colonies in which two homologous recombinations occurred were obtained by reverse sucrose screening. The positive colonies were identified by PCR amplification with P15 and P6 as primers, and 948 bp was obtained as a recombinant strain, which was named Corynebacterium glutamicum WT-P glyA ::P hisEG .
  • sequence homologous recombinant plasmid pK18mobsacB-P glyA ::P hisDCB was electrotransformed into Corynebacterium glutamicum WT-P glyA ::P hisEG . Colonies that integrate the recombinant plasmid into the chromosome are positively screened by kanamycin resistance. Positive colonies in which two homologous recombinations occurred were obtained by reverse sucrose screening.
  • the positive colonies were identified by PCR amplification with P15 and P12 as primers, and 833 bp was obtained as a recombinant strain, which was named Corynebacterium glutamicum CG158 (WT-P glyA ::P hisEG -P glyA ::P hisDCB ).
  • the genomic DNA was extracted by the recombinant strain for sequencing, and it was confirmed that the promoters of hisEG and hisDCB in C. glutamicum wild type ATCC13032 were successfully replaced with the endogenous strong promoter P glyA of C. glutamicum, and hisE was Replace the RBS of the hisD gene with the conserved RBS sequence of the highly expressed gene of C. glutamicum (AAAGGAGGA), and replace the start codon GTG of the hisE gene with ATG with high expression intensity, Corynebacterium glutamicum CG158 (WT-P) glyA ::P hisEG -P glyA ::P hisDCB ) was successfully built.
  • the site-directed mutagenesis of the chromosome hisG gene adopts a two-step substitution method in order to simultaneously achieve three site-directed mutagenesis of the gene.
  • the chloramphenicol resistance gene Cm r and the hisG gene mutation fragment shown in SEQ ID NO: 9 in the sequence listing are upstream and downstream.
  • the long fragment of the source arm was homologously recombined with CG158 to obtain the recombinant WT-P glyA ::P hisEG -Cm r ::hisG-P glyA ::P hisDCB ; and then the three points shown in the sequence 10 in the sequence listing
  • the 264 bp fragment of the mutated hisG gene and the long fragment of the upstream and downstream homology arms were homologously recombined with the recombinant WT-P glyA ::P hisEG -Cm r ::hisG-P glyA ::P hisDCB to obtain CG160.
  • the purified PCR product was used as a template, and P16 and P21 were used as primers, and amplified by overlap extension PCR (SOE) to obtain 1689 bp chloramphenicol resistance gene Cm r and hisG gene mutation fragment upstream and downstream homology arms. Long fragment (sequence 9).
  • SOE overlap extension PCR
  • sequence 9 from the 5' end of the 1-420 nucleotides is the homologous arm upstream of the hisG gene mutation fragment
  • sequence 9 from the 5' end of the 421-42-181 nucleotide is the chloramphenicol resistance gene Cm r
  • Sequence 9 from the 5' end of nucleotides 1282-1689 is the homologous arm downstream of the hisG gene mutant fragment.
  • P31 was used as a primer and amplified by overlap extension PCR (SOE) to obtain a 846 bp hisG gene containing three point mutations (SEQ ID NO: 4 from nucleotides 1007-1852 at the 5' end).
  • SOE overlap extension PCR
  • the upstream homologous arm of the hisG gene site-directed mutagenesis was amplified by PCR with P16 and P22 as primers, and the downstream homologous arm of the hisG gene site-directed mutagenesis was amplified with P25 and P21 as primers; P23 and P24 were used as primers, and the above three were obtained.
  • the point-mutated hisG gene was used as a template to amplify a 264 bp fragment of the hisG gene containing three point mutations.
  • the purified PCR product was used as a template, and P16 and P21 were used as primers, and amplified by overlap extension PCR (SOE) to obtain a 226 bp fragment of the 1092 bp hisG gene containing three point mutations and the length of the upstream and downstream homology arms. Fragment (sequence 10).
  • sequence 10 is the upstream homologous arm from nucleotides 1 to 420 of the 5' end, and 264 bp fragment of the hisG gene of sequence 10 from the nucleotides 421-48 of the 5' end, sequence 10 From the 5' end, nucleotides 685-1092 are downstream homology arms.
  • the two PCR products recovered by purification were digested with BamH I and EcoR I, respectively, and ligated with the knock-out vector pK18mobsacB which was treated with the same double digestion.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • the 1901 bp and 1304 bp positive transformants carrying the two recombinant plasmids were obtained, and the correct transformant was used to extract the plasmid, and the plasmid was subjected to double digestion with BamH I and EcoR I to obtain 1689 bp and 1092 bp respectively.
  • Recombinant plasmid The recombinant plasmids pK18mobsacB-Cm r ::hisG and pK18mobsacB-hisG fbr ::Cm r were successfully constructed by further sequencing.
  • pK18mobsacB-Cm r :hisG is a recombinant vector obtained by inserting a long fragment (SEQ ID NO: 9) containing the chloramphenicol resistance gene Cm r and the homologous arm of the hisG gene mutation fragment into the vector pK18mobsacB.
  • pK18mobsacB-hisG fbr ::Cm r is a recombinant vector obtained by inserting a 264 bp fragment of the hisG gene containing a three point mutation and a long fragment (SEQ ID NO: 10) of the upstream and downstream homology arms into the vector pK18mobsacB.
  • P31 CTAGATGCGGGCGATGCGG (SEQ ID NO: 51).
  • the correct homologous recombination plasmid pK18mobsacB-Cm r ::hisG was electrotransformed into Corynebacterium glutamicum CG158, and the recombinant plasmid was integrated into the chromosome by positive screening of kanamycin resistance. To the screening, positive bacteria in which homologous recombination occurred twice were obtained.
  • the positive bacteria were identified by PCR amplification, and the 1872 bp recombinant strain WT-P glyA ::P hisEG -Cm r ::hisG-P glyA ::P hisDCB was obtained .
  • the sequence homologous recombinant plasmid pK18mobsacB-hisG fbr ::Cm r was electrotransformed into the above constructed recombinant WT-P glyA ::P hisEG -Cm r ::hisG-P glyA ::P hisDCB , by card
  • the natamycin resistance was positively screened to obtain a colony in which the recombinant plasmid was integrated into the chromosome, and the positive colony in which two homologous recombinations occurred was obtained by reverse screening of sucrose.
  • the genomic DNA was extracted by the recombinant strain for sequencing, and the result was a successful N215K/L231F/T235A point mutation of the chromosome hisG gene of Corynebacterium glutamicum CG158, Helicobacter glutamicum CG160 (WT-P glyA ::P hisEG - hisG fbr -P glyA ::P hisDCB ) was successfully built.
  • the N215K/L231F/T235A point mutation of hisG gene is changed to lysine at position 215 of ATP-phosphoribosyltransferase (HisG) encoded by hisG gene, and leucine to phenylalanine at position 231 And the 235th threonine becomes alanine.
  • HisG ATP-phosphoribosyltransferase
  • the prsA gene was further overexpressed and the hisG fbr gene (SEQ ID NO: 1007-1852 nucleotide sequence) was simultaneously overexpressed, and then knocked out.
  • the pgi gene (SEQ ID NO: 13) and the overexpression of the zwf-opcA gene (SEQ ID NO: 2) were combined to obtain a high-yield engineering strain CG171.
  • the prsA gene encodes a PRPP synthetase (PrsA, sequence 5, PRPP is a precursor for histidine synthesis), enhances the expression of the prsA gene, increases the synthesis of the histidine-producing precursor PRPP, and provides a synthesis for histidine synthesis. More precursor substances.
  • the prsA gene (shown as the nucleotide sequence 15-99 of the 5' end of the sequence 4) was overexpressed and the hisG fbr gene was overexpressed (5' in the sequence 4).
  • the nucleotide sequence of nucleotides 1007-1852 is shown) to obtain a primary engineered strain CG176 having a better histidine yield, in order to obtain a better effect after carrying out the strategy of the present invention.
  • the modification strategy of the present invention is not limited to the recombinant engineering of the primary engineering bacteria obtained in the present embodiment, and can also be used in other histological engineering bacteria.
  • the prsA gene (992 bp) and hisG fbr (860 bp) were amplified by PCR using the genomic DNA of strain CG160 as a template with P32/P33 and P34/P35 as primers.
  • the two genes were ligated by overlap extension PCR, and the amplified hisG fbr and prsA genes were used as templates, and P32 and P35 were used as primers for PCR amplification, and the 1852 bp PCR product was obtained as a prsA-hisG fbr fragment (sequence 4).
  • the sequence 4 is prsA from the 15th to the 992th nucleotide of the 5' end, and the sequence 4 is the hisG fbr (the hisG gene containing three point mutations) from the nucleotides 1007-1852 at the 5' end.
  • the above PCR product was digested with Xba I and Sma I, and ligated with the same double-digested C. glutamicum-E. coli shuttle expression plasmid pXMJ19.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ by chemical transformation, and the transformants were selected on LB plates containing chloramphenicol (20 ⁇ g/mL).
  • the transformants were subcultured for three generations, and P36 and P37 were used as primers to identify transformants by colony PCR.
  • a positive transformant of 2054 bp was obtained, and the plasmid was extracted from the correct transformant, and the plasmid was subjected to double digestion with Xba I and Sma I to obtain 1852 bp.
  • the plasmid pXMJ19-prsA-hisG fbr was transformed into the above constructed constructed substrate CG160, and P36 and P37 were used as primers. The transformants were identified by colony PCR to obtain 2054 bp positive transformants, and the correct transformant extraction plasmid was identified. It was further confirmed that the overexpression plasmid was successfully transformed into the engineered bacteria, and L-histidine engineered bacteria CG176 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB /pXMJ19-prsA-hisG fbr ) was successfully constructed.
  • the pgi gene encodes phosphoglucose isomerase (Pgi, shown in SEQ ID NO: 14).
  • the pgi gene (sequence 13) was knocked out to obtain the primary engineered strain CG161; on the basis of CG161, the prsA gene was overexpressed and the hisG fbr gene was overexpressed, and the pgi gene was knocked out.
  • the primary engineered strain CG161 was obtained by knocking out the pgi gene (sequence 13) in the L-histidine chassis engineering strain CG160, as follows:
  • primers were designed based on the pgi gene of C. glutamicum ATCC13032 in Genbank and its upstream and downstream sequences, respectively.
  • the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the homologous arm of pgi gene was amplified by PCR with P38 and P39 as primers.
  • the homologous arm of pgi gene was amplified by P40 and P41.
  • the purified PCR product was used as a template, and P38 and P41 were used as primers, and amplified by overlap extension PCR (SOE) to obtain a 1672 bp fragment containing the upstream and downstream homology arms of the pgi gene to be knocked out (sequence 1).
  • SOE overlap extension PCR
  • sequence 1 from position 1 to position 834 of the 5' end is the upstream homologous arm of the gene to be knocked out pgi
  • sequence 1 is from the 5' end of the 5' end of the nucleotides 835-1672 to the downstream of the gene to be knocked out pgi Homologous arm.
  • the purified PCR product was digested with BamH I and EcoR I and ligated with the homologous recombination vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL).
  • the transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • a 1884 bp positive transformant was obtained, and the correct transformant was used to extract the plasmid, and the plasmid was subjected to double digestion with BamH I and EcoR I to obtain a positive 1672 bp.
  • sequence analysis confirmed that the recombinant plasmid pK18mobsacB- ⁇ pgi was successfully constructed by inserting the fragment of the upstream and downstream homologous arm containing the pgi gene pgi (sequence 1) into the BamH I and EcoR I restriction sites of the vector pK18mobsacB. Carrier.
  • the primer sequences used are as follows:
  • the homologous recombinant plasmid pK18mobsacB- ⁇ pgi was correctly transformed into Corynebacterium glutamicum CG160, and the colonies integrated into the chromosome were obtained by positive screening of kanamycin resistance, and the reverse staining of sucrose was carried out. A colony of the second homologous recombination occurred. Using P42 and P43 as primers, the colonies were subjected to genomic DNA extraction and PCR amplification to obtain 1759 bp positive (see Figure 2), named CG161 (WT-P glyA ::P hisEG -hisG fbr -P glyA :: P hisDCB - ⁇ pgi).
  • CG161 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi) was analyzed by further sequence analysis, and the result was that the chromosome pgi gene knockout of L-histidine chassis engineering strain CG160 was successful, and CG161 was constructed. success.
  • the engineered strain CG172 is a recombinant strain obtained by introducing the plasmid pXMJ19-prsA-hisG fbr into the engineering strain CG161 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi/pXMJ19-prsA-hisG fbr ).
  • the specific operation method is a conventional method, and thus is omitted here.
  • the zwf-opcA gene encodes a 6-phosphate glucose dehydrogenase (Zwf-OpcA, shown in SEQ ID NO: 3, wherein the Zwf subunit consists of amino acids 1-514 from the 5' end, and the amino acid residues 515-833 form the OpcA subunit. ).
  • Zwf-OpcA 6-phosphate glucose dehydrogenase
  • SEQ ID NO: 3 6-phosphate glucose dehydrogenase
  • Primers were designed based on the zwf-opcA gene sequence of Corynebacterium glutamicum ATCC13032 in Genbank.
  • the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the 2519 bp zwf-opcA fragment was amplified by PCR using primers P44 and P45 (from the zwf gene).
  • the start codon is replaced by GTG to ATG to enhance its expression) (sequence 2).
  • the recombinant plasmid pXMJ19-zwf-opcA, pXMJ19-zwf-opcA was ligated with the same double-digested expression plasmid pXMJ19, and then digested with XbaI and SmaI.
  • plasmid pXMJ19-prsA-hisG fbr prepared above connected via prsA-hisG fbr fragment Sma I and Xba I double digestion of the obtained 1852bp.
  • the zwf-opcA fragment wherein the sequence 2 is the zwf gene from the 1st to the 545th nucleotide of the 5' end, and the sequence 2 is the opcA gene from the 5th to the 15th to 25th nucleotides of the 5' end.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ by chemical transformation, and the transformants were selected on LB plates containing chloramphenicol (20 ⁇ g/mL). The transformants were subcultured for three generations, and P36 and P37 were used as primers to identify transformants by colony PCR. , 4587 bp was obtained as a positive transformant.
  • the plasmid was extracted from the correct transformants, and the plasmids were identified by Xba I/Sma I and Hind III/Xba I double digestion, and 1852 bp and 2533 bp were positive, respectively.
  • the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr was successfully constructed and sequenced as recombinant plasmid pWYE 1229 (shown in Figure 3) for inserting the zwf-opcA gene (SEQ ID NO: 2) into Hind III and Xba of pXMJ19. Between the I sites, the prsA-hisG fbr fragment (SEQ ID NO: 4) was inserted between the Xba I and Sma I sites.
  • the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr was electrotransformed into the engineered CG160 not deleted by pgi and the engineered CG161 deleted by pgi, respectively.
  • the transformants were identified by colony PCR to obtain a positive transformant of 4587 bp, and the correct transformant was used to extract the plasmid.
  • the L-histidine engineered strain CG173 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB /pXMJ19-zwf-opcA-prsA-hisG fbr ) contains the plasmid recombinant plasmid pXMJ19-zwf -opcA-prsA-hisG fbr is a bacterium obtained by transferring the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr into the engineered CG160.
  • CG171 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi/pXMJ19-zwf-opcA-prsA-hisG fbr ) contains the plasmid recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr , The recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr was transferred into the bacterium CG161.
  • the expression of the overexpression plasmid carrying gene in the engineered bacteria was further verified.
  • the cell lysate of CG171 was prepared and subjected to SDS-PAGE. The results are shown in Fig. 4.
  • Lanes 1 and 2 are cell lysates of CG171, and lane 3 is cell lysate of ATCC13032/pXMJ19 (the pXMJ19 plasmid was introduced into ATCC13032).
  • As a control it was shown that the zwf (57.5 kDa), opcA (34.8 kDa) prsA (35.6 kDa) and hisG fbr (30.2 kDa) genes carried by the overexpression plasmid were successfully expressed in the engineered bacteria.
  • the specific enzyme activity of 6-phosphate glucose dehydrogenase (Zwf-opcA) in the engineered strain CG171 was further determined.
  • the reaction system was determined as follows (0.5 mL): 100 mmol/L Tris-HCl (pH 7.8), 200 mmol/L KCl, 1 mmol/L NADP, 10 mmol/L MgCl 2 , 5 mmol/L 6-phosphate glucose (G6P), an appropriate amount of cells. Lysate. The reaction was carried out for 5 min at 30 °C. The amount of product NADPH produced was reflected by detecting the change in absorbance at 340 nm.
  • the enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 nmol of reduced nicotinamide adenine dinucleotide phosphate (NADPH) per minute.
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • the histidine synthesis pathway will be enhanced.
  • the increased by-product AICAR is more directed to the purine nucleotide synthesis pathway, and the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr -purH is constructed and introduced into the primary strain CG161 to obtain the high-yield engineering strain CG319.
  • Primers were designed based on the purH gene sequence of Corynebacterium glutamicum ATCC13032 in Genbank, and the purH gene (1563 bp) was amplified by PCR using the ATCC13032 genomic DNA as a template and P46 and P47 as primers (SEQ ID NO: 15).
  • the above PCR product was digested with Sma I and EcoR I, and ligated with the same double-digested Corynebacterium glutamicum-E. coli shuttle expression plasmid pXMJ19.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and Transformants were screened on LB plates of chloramphenicol (20 ⁇ g/mL). After subculture of the transformants for three generations, P52 and P53 were used as primers, and the transformants were identified by colony PCR to obtain 1779 bp positive transformants, and the correct transformation was identified.
  • the plasmid was extracted and the plasmid was digested with Xba I and Sma I to obtain a positive of 1577 bp, which was designated as recombinant plasmid pXMJ19-purH.
  • the recombinant plasmid pXMJ19-zwf-opcA-prsA-hisG fbr was used as a template to amplify zwf-opcA (2519 bp) and prsA-hisG fbr fragment (1852 bp) with P48/P49 and P50/P51 as primers, respectively.
  • the two fragments were ligated using overlap extension PCR to obtain a 4385 bp zwf-opcA-prsA-hisG fbr fragment (SEQ ID NO: 17).
  • sequence 17 is zwf-opcA from nucleotides 15 to 2533 at the 5' end, and prsA-hisG fbr is from nucleotides 2534 to 4385 at the 5' end.
  • the above PCR product was digested with Xba I and Sma I, and ligated with the same double-digested recombinant plasmid pXMJ19-purH.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ by chemical transformation, and the transformants were selected on LB plates containing chloramphenicol (20 ⁇ g/mL). The transformants were subcultured for three generations, and P52 and P53 were used as primers to identify transformants by colony PCR.
  • a positive transformant of 6164 bp was obtained, and the correct transformant was used to extract the plasmid, and the plasmid was subjected to double digestion with Xba I and Sma I to obtain a positive result of 4385 bp, which was named recombinant plasmid pWYE1507 (pXMJ19-zwf-opcA- prsA-hisG fbr -purH) (shown in Figure 6).
  • pXMJ19-zwf-opcA-prsA-hisG fbr- purH was further sequenced and analyzed by inserting the zwf-opcA-prsA-hisG fbr fragment (SEQ ID NO: 17) into the Xba I and Sma I restriction sites of plasmid pXMJ19.
  • purH was inserted into a vector obtained between the Sma I and EcoR I restriction sites of the plasmid pXMJ19.
  • the plasmid pXMJ19-zwf-opcA-prsA-hisG fbr- purH was transformed into the engineered strain CG161, and the transformants were identified by colony PCR using P52 and P53 as primers to obtain a positive transformant of 6164 bp, and the correct transformant was extracted.
  • Plasmid identification further confirmed that the overexpression plasmid was successfully transformed into engineered bacteria, L-histidine engineered strain CG319 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi/pXMJ19-zwf-opcA- prsA-hisG fbr -purH) was successfully constructed.
  • the promoter of the purF gene of the primary engineered strain CG161 was replaced with P hom , and the engineered strain CG327 was obtained as follows:
  • primers were designed based on the purF gene of C. glutamicum ATCC13032 in Genbank and its upstream and downstream sequences, respectively.
  • the genomic DNA of Corynebacterium glutamicum ATCC13032 was used as a template, and the homologous arms of the purF gene were amplified by PCR using P54 and P55 as primers.
  • the P hom promoter was amplified by using P56 and P57 as primers.
  • the homologous arms downstream of the purF gene were amplified using P58 and P59 as primers.
  • the purified PCR product was used as a template, and P54 and P59 were used as primers, and amplified by overlap extension PCR (SOE) to obtain a 1654 bp fragment containing the P hom promoter and the homologous arm of the upstream and downstream promoters of the purF gene promoter. 18).
  • SOE overlap extension PCR
  • sequence 18 from the 5' end of the 1st to 735th nucleotide is the upstream homologous arm of the purF gene promoter
  • sequence 18 from the 5' end of the 736th to the 865th nucleotide is the P hom promoter
  • sequence 18 is The nucleotide at positions 86-1654 at the 5' end is the downstream homology arm of the purF gene promoter.
  • the purified PCR product was digested with BamH I and EcoR I and ligated with the homologous recombination vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL).
  • the transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • the 1866 bp positive transformant was obtained, and the correct transformant was used to extract the plasmid, and the plasmid was subjected to double digestion with BamH I and EcoR I to obtain 1654 bp.
  • sequence analysis confirmed that the recombinant plasmid pK18mobsacB-P hom ::P purF was successfully constructed, and inserted the fragment containing the promoter P hom and the upstream and downstream homology arms of the promoter (sequence 18) into the vector pK18mobsacB and BamH I and EcoR I cleaves the vector between the sites.
  • the correct homologous recombinant plasmid pK18mobsacB-P hom ::P purF was electrotransformed into Corynebacterium glutamicum CG161, and the recombinant plasmid was integrated into the chromosome by colony through kanamycin resistance. Reverse screening resulted in colonies in which a second homologous recombination occurred.
  • CG327 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi::P hom ::P purF ) was analyzed by further sequence analysis and the result was the chromosome of L-histidine primary engineering strain CG161 The purF gene promoter was replaced with P hom and CG327 was successfully constructed.
  • the engineered strain CG328 is a recombinant strain obtained by introducing the plasmid pXMJ19-zwf-opcA-prsA-hisG fbr -purH into the engineered CG327 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB - ⁇ pgi:: P hom ::P purF /pXMJ19-zwf-opcA-prsA-hisG fbr -purH).
  • the specific operation method is similar to the above-mentioned preparation of the engineering bacteria CG319, and is a conventional method, so a detailed description thereof is omitted herein.
  • the plasmid carried by the CG328 strain was identified by PCR, and P612 and P53 were used as primers to obtain a 6164 bp fragment (Fig. 7).
  • the DNA fragment was sequenced and the result was zwf-opcA-prsA-hisG fbr- purH fragment.
  • the CG328 strain was successfully constructed. .
  • Carrying the plasmid increases the metabolic burden of the engineered bacteria, and is not conducive to the industrial fermentation control of the engineered bacteria and the safety of the fermented product.
  • the plasmid carrying gene is enhanced on the chromosome, and a plasmid-free histidine engineered bacteria is constructed to reduce the metabolic burden of the engineered bacteria and achieve maximum conversion of the fermentation substrate to the product.
  • the homologous arm downstream of the promoter was used as a template, and P60 and P65 were used as primers, and amplified by overlap extension PCR (SOE) to obtain a 1455 bp PCR product, which was substituted with the promoter P sod and the replaced promoter P prsA . Fragment of the downstream homology arm (sequence 11).
  • sequence 11 from the 5' end of the 1-55th nucleotide is the upstream promoter of the replaced promoter P prsA
  • sequence 11 from the 5' end of the nucleotides 656-847 is the promoter P sod
  • sequence 11 nucleotides 848-1455 from the 5' end are the homologous arms of the downstream promoter P prsA .
  • the 1455 bp PCR product was digested with Hind III and BamH I and ligated with the homologous recombination vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • the 1667 bp positive transformant was obtained, and the correct transformant was used to extract the plasmid, and the plasmid was identified by Hind III and BamH I double digestion, and 1455 bp was positive.
  • the positive plasmid was sent for sequencing, and as a result, the plasmid was a recombinant plasmid obtained by inserting the nucleotide shown in SEQ ID NO: 11 into the vector pK18mobsacB, and named pK18mobsacB-P sod ::P prsA .
  • the homologous recombination plasmid pK18mobsacB-P eftu ::P tkt was constructed in the same manner, and the promoter of the tkt-tal-zwf-opcA-devB operon was replaced with the strong promoter P eftu .
  • P66 and P67 are used as primers to amplify the homologous arm upstream of the tkt-tal-zwf-opcA-devB operon promoter;
  • P68 and P69 are used as primers to amplify the promoter P eftu ;
  • P70 and P71 are used as primers to amplify The homologous arm downstream of the tkt-tal-zwf-opcA-devB operon promoter.
  • P66 and P71 were used as primers and amplified by overlap extension PCR (SOE).
  • the 1512 bp PCR product was obtained as a long fragment (sequence 12) containing the replacement promoter P etfu and the homologous arm upstream of the replaced promoter P tkt , wherein the sequence 12 was from nucleotides 1-346 at the 5' end.
  • the upstream homologous arm of the promoter P tkt was replaced, the sequence 12 from the 5' end of the 635-834 nucleotide is the promoter P eftu , and the sequence 12 from the 5' end of the 835-1512 nucleotide is replaced.
  • the downstream homology arm of the sub-P tkt was replaced.
  • the 1512 bp PCR product was digested with Hind III and BamH I and ligated with the homologous recombination vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • the 1724 bp positive transformant was obtained, and the plasmid was extracted from the correct transformant, and the plasmid was identified by Hind III and BamH I double digestion, and 1512 bp was positive.
  • the positive plasmid was sent for sequencing, and as a result, the plasmid was a recombinant plasmid obtained by inserting the nucleotide shown in SEQ ID NO: 12 in the sequence listing into the vector pK18mobsacB, and named pK18mobsacB-P eftu ::P tkt .
  • the correct homologous recombination plasmid pK18mobsacB-P sod ::P prsA was electrotransformed into L-histidine recombinant CG161, and the colony of the recombinant plasmid was integrated into the chromosome by forward screening of kanamycin resistance. Positive colonies in which two homologous recombinations occurred were obtained by reverse sucrose screening.
  • the correct homologous recombination plasmid pK18mobsacB-P eftu ::P tkt was electrotransformed into Corynebacterium glutamicum CG350, and the recombinant plasmid was integrated into the chromosome by colony through kanamycin resistance. Reverse screening resulted in positive colonies with two homologous recombinations.
  • the genomic DNA was extracted by the recombinant strain for sequencing, and the result was that the promoters of the tkt-tal-zwf-opcA-devB operon and the prsA gene in the L-histidine recombinant CG161 were successfully replaced with glutamate rods, respectively.
  • Bacillus endogenous strong promoters P eftu and P sod , plasmid-free L-histidine recombinant CG351 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB -P eftu ::P tkt -P sod :: P prsA - ⁇ pgi) was successfully constructed.
  • the promoter of the purH gene was replaced with the strong promoter P eftu to enhance the purH-encoded bifunctional enzyme AICAR methyltransferase/IMP cyclase (PurH, Expression of sequence 16) to further construct CG352; then replacing the purF gene promoter with the P hom promoter to attenuate the first enzyme in the nucleotide synthesis pathway, phosphoribosyltransferase (PurF, sequence 19), thereby constructing CG353.
  • the homologous recombinant plasmid pK18mobsacB-P eftu ::P purH was constructed by the same method as in Example 5 above, and the promoter of the purH gene was replaced with the strong promoter P eftu .
  • Primers were designed based on the upstream and downstream sequences of the purH gene of Corynebacterium glutamicum ATCC13032 in Genbank.
  • the P eftu promoter was amplified by using P70 and P75 as primers, and the upstream homologous arm was amplified by P76 and P77 as primers.
  • the downstream homologous arm was further amplified by the overlap extension PCR technique (SOE) using the purified PCR product as a template and P76 and P79 as primers to obtain a 1473 bp fragment containing the upstream and downstream homology arms and the promoter P eftu ( Sequence 20).
  • SOE overlap extension PCR technique
  • sequence 20 is the upstream homologous arm from nucleotides 1-336 at the 5' end, and sequence 634-833 from the 5' end is P eftu , and sequence 20 is from the 5' end of 834-1473.
  • the nucleotide is a downstream homology arm.
  • the above 1473 bp PCR product was digested with Xba I and Sma I, and ligated with the homologous recombination vector pK18mobsacB which was double-digested.
  • the ligation product was transformed into E. coli DH5 ⁇ by chemical transformation, and the transformants were screened on LB plates containing kanamycin (50 ⁇ g/mL). The transformants were subcultured for three generations, and P13 and P14 were used as primers to identify and transform by colony PCR.
  • a 1685 bp positive transformant was obtained, and the plasmid was extracted from the correct transformant, and the plasmid was subjected to double digestion with Xba I and Sma I to obtain 1473 bp.
  • the positive plasmid was sent for sequencing, and the plasmid was a recombinant plasmid obtained by inserting the nucleotide shown in SEQ ID NO: 20 into the vector pK18mobsacB, and named pK18mobsacB-P eftu ::P purH .
  • the correct homologous recombination plasmid pK18mobsacB-P eftu ::P purH was electrotransformed into L-histidine recombinant strain CG351, and the recombinant plasmid was positively screened by kanamycin resistance to obtain the recombinant plasmid integrated into the chromosome. Colonies were reverse screened by sucrose to obtain positive colonies with two homologous recombinations. The positive colonies were extracted into genomic DNA, and the extracted genomic DAN was used as a template. P74 and P79 were used as primers for PCR amplification, and 840 bp was positive clone.
  • L-histidine recombinant CG351 The sequence was verified to have successfully purH in L-histidine recombinant CG351.
  • the promoter of the gene was replaced by the endogenous strong promoter P eftu of C. glutamicum , and the recombinant L-histidine recombinant CG352 (WT-P glyA ::P hisEG -hisG fbr -P glyA ::P hisDCB -P Eftu ::P tkt -P sod ::P prsA - ⁇ pgi-P eftu ::P purH ) was successfully constructed.
  • Example 4 The sequence prepared in Example 4 was correctly transformed into the homologous recombinant plasmid pK18mobsacB-P hom ::P purF and transformed into Corynebacterium glutamicum CG352, and the recombinant plasmid was positively screened by kanamycin resistance to obtain a recombinant plasmid integrated into the chromosome. The colonies on the colon were reverse-screened by sucrose to obtain colonies in which the second homologous recombination occurred.
  • CG353 was further sequenced and analyzed, and the result was that the chromosome purF gene promoter of the engineered strain CG352 was replaced with P hom , and CG353 was successfully constructed.
  • the fermentation medium used in shake flask fermentation is as follows: glucose 40g/L, (NH 4 ) 2 SO 4 20g/L, KH 2 PO 4 0.5g/L, K 2 HPO 4 ⁇ 3H 2 O 0.5g/L, MgSO 4 ⁇ 7H 2 O 0.25g/L, FeSO 4 ⁇ 7H 2 O 0.01g/L, MnSO 4 ⁇ H 2 O 0.01g/L, ZnSO 4 ⁇ 7H 2 O 0.001g/L, CuSO 4 0.0002g/L, NiCl 2 ⁇ 6H 2 O 0.00002 g/L, biotin 0.0002 g/L, pH 7.0-7.2, CaCO 3 20 g/L.
  • Glucose was sterilized separately and autoclaved at 115 ° C for 15 min.
  • MgSO 4 ⁇ 7H 2 O and inorganic salt ions were separately sterilized and autoclaved at 121 ° C for 20 min.
  • the vitamins were sterilized by filtration using a 0.22 ⁇ m sterile filter. The remaining components were autoclaved at 121 ° C for 20 min.
  • the seed medium is specifically as follows: glucose 20 g / L, ammonium sulfate 5 g / L, K 2 HPO 4 ⁇ 3H 2 O 1 g / L, MgSO 4 ⁇ 7H 2 O 0.4 g / L, biotin 50 ⁇ g, vitamin B 1 1 mg, Ann Qi yeast powder (FM802) 10g / L, Angel Peptone (FP318) 10g / L.
  • the engineering bacteria CG176, CG172, CG173 and CG171 prepared in the above Example 2 were inoculated into the seed culture medium respectively, and the seed liquid culture conditions were a culture temperature of 32 ° C, a shaking speed of 220 r / min, and a culture time of 8 h to obtain a seed liquid.
  • the OD 600 is 20.
  • the seed solution was inoculated to a fermentation medium (with a final concentration of 10 ⁇ g/ml chloramphenicol) in a fermentation medium (500 mL of a baffled flask containing 30 mL) at a volume percentage of 3%, and cultured at 32 ° C, 220 r / min for 72 h.
  • the induced expression of the target gene was carried out by adding isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) at a final concentration of 1 mmol/L for 6 h.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the pH of the fermentation broth was controlled by intermittent addition of concentrated ammonia water between 7.0 and 7.2. According to the residual sugar condition, a glucose mother liquor with a concentration of 400 g/L was added to control the residual sugar of the fermentation broth at 5-10 g/L.
  • the fermentation product was collected at 12000 x g, centrifuged for 5 min, and the supernatant was collected.
  • High-performance liquid phase method the specific method is as follows (2,4-dinitrofluorobenzene pre-column derivatization high-performance liquid phase method): Take 50 ⁇ L of the above supernatant in a 2 mL centrifuge tube, and add 200 ⁇ L of NaHCO 3 aqueous solution (0.5 mol/L).
  • the column used was a C18 column (ZORBAX Eclipse XDB-C18, 4.6*150 mm, Agilent, USA); column temperature: 40 ° C; UV detection wavelength: 360 nm; mobile phase A was 0.04 mol/L KH 2 PO 4 aqueous solution (pH 7.2) ⁇ 0.05, pH was adjusted with 40 g/L KOH aqueous solution), mobile phase B was 55% acetonitrile aqueous solution (volume ratio), mobile phase flow rate was 1 mL/min, and the elution process is shown in Table 1 below:
  • the wild type strain C. glutamicum ATCC13032 was used as a control to measure glucose consumption, OD 600 and final L-histidine production during fermentation. The results are shown in Table 2.
  • Table 2 shows the glucose consumption, maximum OD 600 , specific growth rate and L-histidine production of L-histidine engineered bacteria CG160, CG176, CG172, CG173 and CG171 in shake flask fermentation experiments.
  • the accumulation of L-histidine was not detected in the wild-type strain C. glutamicum ATCC13032 for 72 h, and the L-histidine yield of the substrate CG160 was 0.03 g/L.
  • the L-histidine yield of the chassis engineered strain CG176 which was only transformed with the L-histidine terminal metabolic pathway, was 1.18 g/L.
  • the L-histidine yield of the engineered strain CG172 lacking the pgi gene alone was 0.77 g/L; the L-histidine yield of the engineered strain CG173 overexpressing zwf-opcA alone was 1.50 g/L.
  • the L-histidine yield of the engineered strain CG171 overexpressing the pgi gene and overexpressing zwf-opcA was 2.40 g/L, which was 2.1 times higher than that of the engineered strain CG172 lacking the pgi gene alone, and overexpressed the zwf-opcA gene alone.
  • the strain CG173 was increased by 60% compared to the strain CG176 which was only transformed with the L-histidine terminal metabolic pathway, and was increased by 102%.
  • the seed medium is specifically as follows: glucose 20 g / L, ammonium sulfate 5 g / L, K 2 HPO 4 ⁇ 3H 2 O 1 g / L, MgSO 4 ⁇ 7H 2 O 0.9 g / L, biotin 50 ⁇ g, vitamin B 1 1 mg, Qi yeast powder (FM802) 2g / L, Angel protein ⁇ (FP318) 2g / L.
  • the fermentation medium used for the fermentation is specifically as follows: glucose 20 g/L, ammonium sulfate 5 g/L, KH 2 PO 4 0.5 g/L, K 2 HPO 4 ⁇ 3H 2 O 0.5 g/L, MgSO 4 ⁇ 7H 2 O 0.25 g /L, FeSO 4 ⁇ 7H 2 O 10 mg/L, MnSO 4 ⁇ H 2 O 10 mg/L, vitamin B 1 0.5 mg/L, Angel Yeast powder (FM802) 5 g/L.
  • the engineering bacteria CG171, CG319 and CG328 were inoculated into the seed culture medium.
  • the seed culture conditions were culture temperature 32 ° C, shaking speed 220 r / min, culture time 8 h, and the seed liquid was obtained, and the OD 600 was 20.
  • the seed solution was inoculated to a fermentation medium containing a final concentration of 10 ⁇ g/ml chloramphenicol at a volume percentage of 10%.
  • the fermenter used is a 7.5L fermenter (BioFlo115, NBS): a built-in fixed-speed programmable pump that can achieve constant feed.
  • 600 g/L of glucose was added by a peristaltic pump, and the concentration of glucose sugar in the fermentation system was controlled to be 5 to 10 g/L, and 10 g/L of Angel Yeast powder (FM802) was added thereto.
  • the heating temperature is controlled to be maintained at 32 ° C by heating jacket and cooling water; dissolved oxygen is supplied through the air, and the dissolved oxygen is maintained at 30% by the cascade of the rotational speed and the dissolved oxygen signal; the pH is adjusted by adding concentrated ammonia water, and maintained at about 6.9.
  • the fermentation was continued for 52 h.
  • IPTG isopropyl thiogalactoside at a final concentration of 0.5 mmol/L
  • the fermentation product was collected and centrifuged at 12,000 x g for 5 min, and the supernatant was collected.
  • the L-histidine content in the supernatant was examined according to the method of 3) above.
  • the highest yield of L-histidine of the engineered strain CG171 was 10.87 g/L, and the production intensity was 0.21 g/ L/h
  • the highest yield of L-histidine of engineering bacteria CG319 is 14.15g / L
  • the production intensity is 0.30g / L / h
  • the highest yield of L-histidine of engineering bacteria CG328 is 15.96g / L
  • production intensity It is 0.32 g/L/h.
  • Table 3 The results are shown in Table 3 below.
  • the fermenter experiment showed that the CG171 strain achieved good results, and the histidine yield reached 10.87 g/L in 52 hours of fermentation.
  • the engineering strain CG328 which weakens purF relative to CG171 the histidine yield increased by about 30% and 50%, respectively, when the fermentation time was shorter. That is to say, on the basis of weakening pgi and over-expressing zwf-opcA, the histidine synthesis pathway is coupled with the nucleotide synthesis pathway, which drives the metabolic flux of the histidine synthesis pathway, further increasing the yield of histidine. .
  • the plasmid-free L-histidine engineering bacteria CG350, CG351, CG352 and CG353 shake flask fermentation to produce L-histidine
  • the preparation of the seed CG350, CG351, CG352 and CG353 seed liquids and the shake flask fermentation method are the same as described in the above, except that chloramphenicol and the inducer IPTG are not required to be added during the fermentation.
  • the L-histidine content assay is as described in the first item of this example.
  • the wild type strain C. glutamicum ATCC13032 was used as a control.
  • the accumulation of L-histidine was not detected in the wild-type strain C. glutamicum ATCC13032, and the L-histidine yield of the engineered strain CG350 lacking the pgi gene alone was 0.65 g/L.
  • the L-histidine yield of the engineered CG351 constructed by simultaneously increasing the expression level of zwf-opcA was 1.86 g/L, which was 186% higher than that of the engineered CG350 lacking the pgi gene alone.
  • the L-histidine yield of the strain CG352 which further improved the purH gene expression was 2.23 g/L
  • the L-histidine yield of the strain CG353 which further reduced the purF gene expression was 2.34 g/L.
  • Table 4 The results are shown in Table 4 below.

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Abstract

提供了一种产L-氨基酸的重组菌、其构建方法及L-氨基酸生产方法。该产L-氨基酸的重组菌相比于出发菌具有降低的6-磷酸葡萄糖异构酶Pgi的表达，和提高的6-磷酸葡萄糖脱氢酶Zwf-OpcA的表达，其中该出发菌是能够累积目的氨基酸的菌株。发酵培养该重组菌可显著提高L-氨基酸的产量。还提供了新的提高L-氨基酸的发酵产量的方法，可用于细菌发酵生产L-氨基酸。

Description

一种产L-氨基酸的重组菌、其构建方法及L-氨基酸生产方法 技术领域
本发明涉及微生物发酵领域，具体而言，本发明涉及微生物发酵生产L-氨基酸的方法及其专用重组菌。
背景技术
微生物发酵法生产L-氨基酸是目前应用最广泛的氨基酸生产方法，氨基酸生产菌的发酵生产性能是影响发酵法能否实现大规模工业化应用的关键因素。目前，仍有少数氨基酸品种由于缺少发酵性能优良的生产菌株而尚未实现发酵法生产。对于已实现发酵法生产的氨基酸生产菌株，为节约生产成本，其产酸水平和糖酸转化率仍有待进一步提高。以L-组氨酸为例，L-组氨酸是人和动物的第九种必需氨基酸，参与机体生长发育、抗氧化和免疫调节等重要的生理过程，是重要的药用氨基酸，可用于心脏病、贫血、胃肠溃疡治疗的输液制剂。目前，L-组氨酸生产主要采用以猪(牛)血粉为原料的蛋白质水解提取法，然而，蛋白质水解提取法存在原料成本高且利用率低、提取工艺复杂和环境污染大等缺点，使得L-组氨酸的生产成本高，价格昂贵。微生物发酵法生产L-组氨酸尚未得到大规模的工业化应用。L-组氨酸的生物合成具有与核苷酸合成竞争前体物质、复杂的代谢调控机制以及合成过程中高能量需求等特点，导致其工程菌的产酸水平和转化率相对较低。L-组氨酸生产菌株的选育主要采用多轮传统诱变筛选和在诱变菌株的基础上进行基因工程改造的方法。通过诱变筛选获得的菌株会积累大量的负效应突变，导致菌株生长缓慢、环境耐受性降低以及营养需求增高等问题。这些缺陷限制了菌株的工业化应用。目前通过系统代谢工程改造构建L-组氨酸工程菌的研究仅有一篇报道(Doroshenko,V.G.,Lobanov,A.O.,Fedorina,E.A.,2013.The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants.Appl Biochem Microbiol.49,130-135.)。该研究是以野生型大肠杆菌MG1655为出发菌，通过在hisG基因中引入E271K突变，减弱组氨酸对其反馈抑制调节；敲除组氨酸合成操纵子的转录弱化因子hisL，上调组氨酸合成操纵子的表达；同时敲除purR基因，增加组氨酸合成前体PRPP的合成，构建了一株产L-组氨酸的工程菌。该研究只进行了L-组氨酸终端合成途径的改造，其L-组氨酸的产量仅为4.9g/L，与实现工业化应用存在较大差距。
L-组氨酸生物合成的主要载流途径是戊糖磷酸途径，当以葡萄糖为碳源时，经由戊糖磷酸途径生成L-组氨酸合成前体磷酸核糖焦磷酸(PRPP)，PRPP同时进入核苷酸合成途径和L-组氨酸合成途径，经由核苷酸合成途径生成L-组氨酸合成的另一个前体ATP。
另外，戊糖磷酸途径也是多种氨基酸(如L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸和L-羟脯氨酸等)合成所需辅因子NADPH的主要生成途径，其中合成1分子L-赖氨酸需要消耗4分子NADPH，合成1分子L-苏氨酸、L-脯氨酸和L-羟脯氨酸需要消耗3分子NADPH，合成1分子L-缬氨酸需要消耗2分子NADPH。
通过失活糖酵解途径的6-磷酸葡萄糖异构酶，能够将碳代谢流导向戊糖磷酸途径，但会导致菌株的生长和葡萄糖代谢能力的减弱而不利于菌株在发酵生产中的应用(Marx,A.,Hans,S.,Mockel,B.,Bathe,B.,de Graaf,A.A.,McCormack,A.C.,Stapleton,C.,Burke,K.,O'Donohue,M.,Dunican,L.K.,2003.Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.J Biotechnol.104,185-197.)。本发明人的前期的研究结果证实，敲除6-磷酸葡萄糖异构酶编码基因pgi导致菌株的生长和葡萄糖代谢能力的严重下降，同时L-组氨酸产量也随之下降。另外，本发明人还发现单独提高6-磷酸葡萄糖脱氢酶的表达量，对于L-组氨酸产量提高的效果不佳。
发明内容
本发明的目的是提供能够提高L-氨基酸，特别是由戊糖磷酸途径提供前体物质或辅因子NADPH而合成的L-氨基酸，例如L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸和L-羟脯氨酸产量的重组菌及其构建方法，及利用所述重组菌生产L-氨基酸的方法。
为此，本发明一方面提供提供一种产L-氨基酸的重组菌，所述重组菌相比于出发菌具有降低的6-磷酸葡萄糖异构酶Pgi的表达，和提高的6-磷酸葡萄糖脱氢酶Zwf-OpcA的表达，其中所述出发菌是能够累积目的氨基酸的菌株。
根据一种实施方式，通过对原始菌的染色体进行诱变或遗传工程改造获得所述出发菌。为获得目的氨基酸，所述出发菌可以是现有的可以累积目的氨基酸的菌株，也可以是对合适的原始菌进行遗传工程改造而获得能够累积目的氨基酸的菌株。为获得高产工程菌，优选那些针对目的氨基酸有较高产量的菌株作为出发菌。
本发明所说的目的氨基酸是指由戊糖磷酸途径提供前体物质或辅因子NADPH而合成的L-氨基酸。优选地，所述目的氨基酸是L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸。
根据一种实施方式，所述重组菌相比于出发菌可弱化pgi基因的表达，同时增强表达zwf-opcA基因。具体地，所述重组菌的染色体上的pgi基因已失活，优选已被敲除，或者pgi基因的调控元件已被替换为低转录或低表达活性的调控元件，同时所述重组菌中具有两个或更多个拷贝的zwf-opcA基因，或tkt-tal-zwf-opcA-devB操纵子的启动子替换为强启动子，例如替换为原始菌的Peftu启动子。
针对产L-组氨酸的重组菌，其出发菌相对于原始菌可增强表达L-组氨酸合成操纵子hisEG基因和hisDCB基因。具体地，可以强启动子代替所述基因的启动子。例如以原始菌染色体上的PglyA启动子分别代替原始菌中染色体上hisEG和hisDCB的启动子。进一步优选地，所述出发菌相比于原始菌还能够增强表达PRPP合成酶PrsA。更优选地，所述出发菌中具有两个或更多个拷贝的prsA基因，或者以强启动子代替prsA基因的启动子，例如可用原始菌的Psod启动子代替prsA基因的启动子。
针对产L-赖氨酸的重组菌，其出发菌相比于原始菌可增强表达dapA基因(二氢吡啶二 羧酸合成酶的编码基因)或lysC基因(天冬氨酸激酶的编码基因)(Cremer,J.,Eggeling,L.,Sahm,H.,1991.Control of the lysine biosynthesis sequence in Corynebacterium glutamicum as analyzed by overexpression of the individual corresponding genes.Appl Environ Microbiol.57,1746-1752)。具体地，所述出发菌中可具有两个或更多个拷贝的dapA基因或lysC基因，或者以强启动子代替dapA基因或lysC基因的启动子。
针对产L-缬氨酸的重组菌，其出发菌相比于原始菌可增强表达缬氨酸合成基因ilvBNCE(Blombach,B.,Schreiner,M.E.,Holátko,J.,Bartek,T.,Oldiges,M.,Eikmanns,B.J.,2007.L-Valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum.Appl Environ Microbiol.73,2079-2084)。具体地，所述出发菌中可具有两个或更多个拷贝的ilvBNCE基因，或者以强启动子代替ilvBNCE基因的启动子。
针对产L-苏氨酸的重组菌，其出发菌相比于原始菌可增强表达苏氨酸合成途径基因hom和thrB(Reinscheid,D.J.,Kronemeyer,W.,Eggeling,L.,Eikmanns,B.J.,Sahm,H.,1994.Stable expression of hom-1-thrB in Corynebacterium glutamicum and its effect on the carbon flux to threonine and related amino acids.Appl Environ Microbiol.60,126-132)。具体地，所述出发菌中可具有两个或更多个拷贝的hom和thrB基因，或者以强启动子分别代替hom基因和thrB基因的启动子。
针对产L-脯氨酸的重组菌，其出发菌相比于原始菌可增强表达ocd基因(鸟氨酸环化脱氨酶编码基因)(Jensen,J.V.K.,Wendisch,V.,2013.Ornithine cyclodeaminase-based proline production by Corynebacterium glutamicum.Microb Cell Fact.12,63)。具体地，所述出发菌中可具有两个或更多个拷贝的ocd基因，或者以强启动子代替ocd基因的启动子。
针对产L-羟脯氨酸的重组菌，其出发菌相比于原始菌可增强表达p4hD基因(脯氨酸羟化酶编码基因)(Yi,Y.,Sheng,H.,Li,Z.,Ye,Q.,2014.Biosynthesis of trans-4-hydroxyproline by recombinant strains of Corynebacterium glutamicum and Escherichia coli.BMC Biotechnol.14,44.)。具体地，所述出发菌中可具有两个或更多个拷贝的p4hD基因，或者以强启动子代替p4hD基因的启动子。
针对产L-组氨酸的重组菌，根据一种优选的实施方式，所述重组菌相比于所述出发菌能更多地表达AICAR甲基转移酶/IMP环水化酶PurH。优选地，所述重组菌中具有两个或更多个拷贝的purH基因，或者以强启动子替换purH基因的启动子，例如，以所述原始菌的Peftu启动子替换purH基因的启动子。
根据一种更优选的实施方式，所述重组菌相比于所述出发菌具有弱化的磷酸核糖酰胺转移酶PurF的表达。具体地，可以弱启动子代替purF基因的启动子。优选地，所述重组菌的染色体上以所述原始菌中的Phom启动子代替purF基因的启动子。
虽然以上在不同实施方式中给出了强启动子的示例，但是对于强启动子，在本发明中均没有特别限制，只要能够起到增强所启动基因的表达即可。可以列举的可用于本发明的强启动子有原始菌的Peftu、Psod、PglyA、Ppck、Ppgk启动子等，但不限于此。
所述原始菌优选为选自棒杆菌属、小杆菌属、短杆菌属中的一株细菌。所述棒杆菌属的细菌优选选自谷氨酸棒杆菌Corynebacterium glutamicum、北京棒杆菌Corynebacterium pekinense、有效棒杆菌Corynebacterium efficiens、钝齿棒杆菌Corynebacterium crenatum、嗜热产氨棒杆菌Corynebacterium thermoaminogenes、产氨棒杆菌Corynebacterium aminogenes、百合棒杆菌Corynebacterium lilium、美棒杆菌Corynebacterium callunae和力士棒杆菌Corynebacterium herculis中的一株细菌。所述小杆菌属的细菌优选选自嗜氨小杆菌Microbacterium ammoniaphilum中的一株细菌。所述短杆菌属的细菌优选选自黄色短杆菌Brevibacteriaceae flvum、乳酸发酵短杆菌Brevibacteriaceae lactofermentum和产氨短杆菌Brevibacteriaceae ammoniagenes中的一株细菌。
根据一种具体实施方式，所述原始菌为野生型谷氨酸棒杆菌ATCC13032。
在这种情况下，针对产L-组氨酸的重组菌，所述出发菌的染色体上具有序列7中5’末端第863-1038位核苷酸序列所示的PglyA启动子用于分别代替所述谷氨酸棒杆菌ATCC13032染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子，和所述出发菌能够表达突变的ATP-磷酸核糖转移酶。
所述突变的ATP-磷酸核糖转移酶为序列6所示的ATP-磷酸核糖转移酶的第215位天冬酰胺突变为赖氨酸、第231位亮氨酸突变为苯丙氨酸和第235位苏氨酸突变为丙氨酸的酶。优选地，所述出发菌的染色体上具有序列4中第1007-1852位核苷酸序列所示的hisGfbr基因用于代替所述谷氨酸棒杆菌ATCC13032染色体上的hisG基因。
根据一种优选实施方式，所述出发菌的染色体上具有序列11中5’末端第656-847位核苷酸序列所示的Psod启动子用于代替所述谷氨酸棒杆菌ATCC13032染色体上的prsA基因的启动子。
根据另一种优选的实施方式，所述出发菌中具有两个或更多个拷贝的prsA基因和hisGfbr基因。所述prsA基因可选自编码序列5所示的PrsA的基因；和编码与所述PrsA相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、进一步优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有PrsA活性的基因中的一个。具体地可为序列表中序列4所示的第15-992位的核苷酸序列。
本发明的重组菌中，所述pgi基因可选自编码序列表中序列14所示Pgi的基因；和编码与所述Pgi相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述6-磷酸葡萄糖异构酶Pgi活性的基因中的一个。具体可为序列13所示的核苷酸序列。
所述zwf-opcA基因可选自编码序列表中序列3所示的Zwf-OpcA的基因；和编码与所述Zwf-OpcA相比具有有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述Zwf-OpcA活性的基因中的一个。具体可为序列2所示的核苷酸序列。
所述Peftu启动子可为序列12所示5’末端第635-834位的核苷酸序列。
所述purH基因可选自编码序列表中序列16所示的PurH的基因；和编码与所述PurH相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述PurH活性的基因中的一个，优选地，所述purH基因可为序列表中序列15所示的核苷酸序列。
所述Phom启动子可为序列18所示5’末端第736-865位的核苷酸序列。
在本发明的重组菌中，可导入包含某基因的重组质粒来增加该基因的拷贝数，也可直接将某基因插入菌株染色体上的合适位点。用于构建重组质粒的载体没有限制，可以是任何适宜的质粒，例如pXMJ19。
根据本发明的第二方面，提供一种构建产L-氨基酸的重组菌的方法。所述方法包括如下步骤：降低出发菌中6-磷酸葡萄糖异构酶Pgi的表达，且提高所述出发菌中6-磷酸葡萄糖脱氢酶Zwf-OpcA的表达，得到所述重组菌，其中，所述出发菌是能够积累目的氨基酸的菌株。
可根据已知的方法通过例如诱变或遗传工程改造等方法获得出发菌，也可以采用已有的可产目的氨基酸的菌株作为出发菌。优选那些高产菌株。
本发明所说的目的氨基酸优选为L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸等。
根据一种实施方式，降低出发菌中Pgi的表达通过如下A)或B)方式实现：
A)失活所述出发菌染色体的pgi基因；优选所述失活为敲除；
B)将所述出发菌中的pgi基因的调控元件替换为低转录或低表达活性的调控元件。
所述提高所述出发菌中Zwf-OpcA的表达通过如下C)或D)方式实现：
C)增加所述出发菌中zwf-opcA基因的拷贝数；
D)将所述出发菌染色体上的tkt-tal-zwf-opcA-devB操纵子的启动子替换为强启动子，例如所述原始菌染色体上的Peftu启动子。
针对L-组氨酸，根据一种实施方式，获得所述出发菌可包括将原始菌染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子分别替换为强启动子，例如所述原始菌染色体上的PglyA启动子的步骤。进一步优选地，获得所述出发菌可进一步包括提高所述出发菌中PRPP合成酶PrsA的表达的步骤。更优选地，所述提高所述出发菌中PrsA的表达通过如下E)或F)方式实现：
E)增加所述出发菌中prsA基因的拷贝数；
F)将所述出发菌染色体上的prsA基因的启动子替换为强启动子，例如所述原始菌染色体上的Psod启动子。
针对L-赖氨酸，根据一种实施方式，可通过增强表达dapA基因(二氢吡啶二羧酸合成酶的编码基因)或lysC基因(天冬氨酸激酶的编码基因)获得能够积累L-赖氨酸的出发菌。具体地，增加所述出发菌中dapA基因或lysC基因的拷贝数，或者以强启动子代替dapA基因或lysC基因的启动子。
针对L-缬氨酸，根据一种实施方式，可通过增强表达缬氨酸合成基因ilvBNCE获得所述出发菌。具体地，可增加所述出发菌中ilvBNCE基因的拷贝数，或者以强启动子代替ilvBNCE基因的启动子。
针对L-苏氨酸，根据一种实施方式，获得所述出发菌可包括增强表达苏氨酸合成途径基因hom和thrB的步骤。具体地，可增加所述出发菌中hom基因和thrB基因的拷贝数，或者以强启动子分别代替hom和thrB基因的启动子。
针对L-脯氨酸，根据一种实施方式，获得所述出发菌可包括增强表达ocd基因(鸟氨酸环化脱氨酶编码基因)的步骤。具体地，可增加所述出发菌中ocd基因的拷贝数，或者以强启动子代替ocd基因的启动子。
针对L-羟脯氨酸，根据一种实施方式，获得所述出发菌可包括增强表达p4hD基因(脯氨酸羟化酶编码基因)的步骤。具体地，可增加所述出发菌中p4hD基因的拷贝数，或者以强启动子代替p4hD基因的启动子。根据一种优选实施方式，针对L‐组氨酸，所述方法可进一步包括提高所述重组菌中AICAR甲基转移酶/IMP环水化酶PurH的表达的步骤。优选地，所述提高所述重组菌中PurH的表达可通过如下G)或H)方式实现：
G)增加所述出发菌中purH基因的拷贝数；
H)将所述出发菌染色体上的purH基因的启动子替换为强启动子，例如所述原始菌染色体上的Peftu启动子。
根据更优选实施方式，针对L-组氨酸，所述方法可进一步包括弱化所述重组菌中磷酸核糖酰胺转移酶PurF的表达的步骤。具体地，可以弱启动子代替purF基因的启动子。优选地，所述弱化所述重组菌中PurF的表达通过将所述出发菌中染色体上的purF基因的启动子替换为所述原始菌中染色体上的Phom启动子。
同样的，对于强启动子没有特别限制，只要能够起到增强所启动基因的表达即可。可以列举的有原始菌的Peftu、Psod、PglyA、Ppck或Ppgk启动子，但不限于此。
优选地，可以用作原始菌的菌株可选自棒杆菌属、小杆菌属、短杆菌属中的一株细菌。所述棒杆菌属的细菌优选选自谷氨酸棒杆菌Corynebacterium glutamicum、北京棒杆菌Corynebacterium pekinense、有效棒杆菌Corynebacterium efficiens、钝齿棒杆菌Corynebacterium crenatum、嗜热产氨棒杆菌Corynebacterium thermoaminogenes、产氨棒杆菌Corynebacterium aminogenes、百合棒杆菌Corynebacterium lilium、美棒杆菌Corynebacterium callunae和力士棒杆菌Corynebacterium herculis中的一株细菌。所述小杆菌属的细菌优选选自嗜氨小杆菌Microbacterium ammoniaphilum中的一株细菌。所述短杆菌属的细菌优选选自黄色短杆菌Brevibacteriaceae flvum、乳酸发酵短杆菌Brevibacteriaceae lactofermentum和产氨短杆菌Brevibacteriaceae ammoniagenes中的一株细菌。最优选谷氨酸棒杆菌Corynebacterium glutamicum或黄色短杆菌Brevibacteriaceae flvum。
根据一种具体实施方式，原始菌为野生型谷氨酸棒杆菌ATCC13032。
针对该实施方式，针对产L-组氨酸的重组菌，所述出发菌可通过对该原始菌进行如下的 重组改造获得：
将所述谷氨酸棒杆菌ATCC13032染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子分别替换为序列7中5’末端第863-1038位核苷酸序列(或者序列8中5’末端第752-927位核苷酸序列)所示的PglyA启动子，和
将所述谷氨酸棒杆菌ATCC13032表达的序列6所示的ATP-磷酸核糖转移酶的第215位天冬酰胺突变为赖氨酸、第231位亮氨酸突变为苯丙氨酸和第235位苏氨酸突变为丙氨酸。上述突变的基因为序列4中第1007-1852位核苷酸序列所示的hisG-br基因。
根据一种优选实施方式，为获得L-组氨酸累积效果更好的出发菌，进一步对所述谷氨酸棒杆菌ATCC13032染色体进行改造，将染色体上的prsA基因的启动子替换为序列11中5’末端第656-847位核苷酸序列所示的Psod启动子。
根据另一种优选实施方式，可通过增加所述谷氨酸棒杆菌ATCC13032中prsA基因的拷贝数和增加所述谷氨酸棒杆菌ATCC13032中hisGfbr基因的拷贝数来获得L-组氨酸累积效果更好的出发菌。
所述prsA基因可选自编码序列5所示的PrsA的基因；和编码与所述PrsA相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、进一步优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有PrsA活性的基因中的一个。具体可为序列表中序列4所示的第15-992位的核苷酸序列。
所述pgi基因可选自编码序列表中序列14所示Pgi的基因；和编码与所述Pgi相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选98％、甚至99％同源性，且具有所述6-磷酸葡萄糖异构酶活性的基因中的一个。具体可为序列13所示的核苷酸序列，
所述zwf-opcA基因可选自编码序列表中序列3所示的Zwf-OpcA的基因；和编码与所述Zwf-OpcA相比具有有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选98％、甚至99％同源性，且具有所述Zwf-OpcA活性的基因中的一个。具体可为序列2所示的核苷酸序列。
所述Peftu启动子是序列12所示5’末端第635-834位(或者是序列20所示5’末端第634-833位)的核苷酸序列。
所述purH基因选自编码序列表中序列16所示的PurH的基因；和编码与所述PurH相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选98％、甚至99％同源性，且具有所述PurH活性的基因中的一个。具体可为序列表中序列15所示的核苷酸序列。
所述Phom启动子是序列18所示5’末端第736-865位的核苷酸序列。
在本发明的方法中，增加某基因的拷贝数可通过构建包含该基因的重组质粒，再将重组质粒导入出发菌/原始菌中实现。这些方法都是本领域常用的，因此不再赘述。用于构建重组质粒的载体没有限制，可以是任何适宜的质粒，例如pXMJ19。
本发明的重组菌可为通过上述构建方法获得的重组菌。
根据本发明的第三方面，提供一种生产L-氨基酸的方法，包括发酵培养上述重组菌的步骤。所述L-氨基酸，优选地为L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸。
本发明提供的构建重组菌的方法，包括如下步骤：降低出发菌中6-磷酸葡萄糖异构酶的表达，且提高所述出发菌中6-磷酸葡萄糖脱氢酶和PRPP合成酶的表达，得到重组菌。
上述方法中，所述降低出发菌中6-磷酸葡萄糖异构酶的表达通过如下A)或B)方式实现：
A)失活所述出发菌染色体的pgi基因；所述失活具体为敲除；
B)将所述出发菌中的pgi基因的调控元件替换为低转录和低表达活性的调控元件实现；
所述提高所述出发菌中6-磷酸葡萄糖脱氢酶和PRPP合成酶的表达通过如下C)或D)方式实现：
C)增加所述出发菌中zwf-opcA基因和prsA基因的拷贝数；
D)将所述出发菌染色体上的tkt-tal-zwf-opcA-devB操纵子的启动子替换为Peftu启动子，且将所述出发菌染色体上的prsA基因的启动子替换为Psod启动子。
上述方法中，所述构建重组菌的方法为如下Ⅰ或Ⅱ：
Ⅰ所示的方法为敲除所述出发菌染色体的pgi基因，且增加所述出发菌中zwf-opcA基因和prsA基因的拷贝数，得到重组菌；
Ⅱ所示的方法为敲除所述出发菌染色体的pgi基因，将所述出发菌染色体上的tkt-tal-zwf-opcA-devB操纵子的启动子替换为Peftu启动子、且将所述出发菌染色体上的prsA基因的启动子替换为Psod启动子。
上述方法，
所述敲除为将含欲敲除基因pgi的上下游同源臂的片段导入所述出发菌中进行同源重组；
所述增加所述出发菌中zwf-opcA基因和prsA基因的拷贝数为将zwf-opcA基因和prsA-hisGfbr片段通过重组载体导入所述出发菌；
上述重组载体为将zwf-opcA基因和prsA-hisGfbr片段插入表达载体得到的重组载体；所述表达载体可以为IPTG诱导型表达载体pXMJ19；
在本发明的实施例2中，重组载体为pXMJ19-zwf-opcA-prsA-hisGfbr，为将zwf-opcA基因(序列2)插入pXMJ19的Hind III和Xba I位点间，且将prsA-hisGfbr片段(序列4)插入Xba I和Sma I位点间得到的载体。
。
所述将所述出发菌染色体上的tkt-tal-zwf-opcA-devB操纵子的启动子替换为Peftu启动子为将含有Peftu启动子的片段导入所述出发菌中进行同源重组；
所述将所述出发菌染色体上的prsA基因的启动子替换为Psod启动子为将含有Psod启动子的片段导入所述出发菌中进行同源重组。
上述方法中，
所述含欲敲除基因pgi的上下游同源臂的片段的核苷酸序列为序列表中的序列1，其中序列1自5’末端第1-834位核苷酸为欲敲除基因pgi的上游同源臂，序列1自5’末端第835-1672位核苷酸为欲敲除基因pgi的下游同源臂；基因pgi的核苷酸序列为序列13；
所述zwf-opcA基因的核苷酸序列为序列表中的序列2；
所述prsA-hisGfbr片段的核苷酸序列为序列表中的序列4；
所述重组载体为将所述zwf-opcA基因和所述prsA-hisGfbr片段插入表达载体得到的载体；
所述Peftu启动子的核苷酸序列为序列表中的序列12自5’末端第635-834位核苷酸；
所述含有Peftu启动子的片段的核苷酸序列为序列表中的序列12；
所述含有Psod启动子的片段的核苷酸序列为序列表中的序列11。
上述方法中，所述出发菌按照包括如下步骤的方法制备：将细菌染色体上的L-组氨酸合成操纵子的启动子替换为PglyA启动子，且将所述细菌染色体上的hisG基因进行点突变，得到出发菌；
所述L-组氨酸合成操纵子为hisEG和hisDCB；
所述PglyA启动子的核苷酸序列为序列表中序列7的第863-1038位核苷酸或序列表中序列8的第752-927位核苷酸；
所述点突变为将所述细菌染色体的hisG基因编码的蛋白的第215位天冬酰胺变为赖氨酸、第231位亮氨酸变为苯丙氨酸和第235位苏氨酸变为丙氨酸。
上述方法中，所述将细菌染色体上的L-组氨酸合成操纵子的启动子替换为PglyA启动子为将含有hisEG的PglyA启动子的片段和含有hisDCB的PglyA启动子的片段导入细菌中进行同源重组；其中，含有hisEG的PglyA启动子的片段的核苷酸序列为序列表中的序列7；含有hisDCB的PglyA启动子的片段的核苷酸序列为序列表中的序列8。
上述方法中，所述将所述细菌染色体上的hisG基因进行点突变为将序列9所示的核苷酸序列导入所述细菌中进行同源重组，再将序列10所示的核苷酸序列导入中间菌中进行同源重组。
上述方法中，所述细菌为棒杆菌属细菌，所述棒杆菌属细菌具体为谷氨酸棒杆菌。
由上述方法制备的重组菌也是本发明保护的范围。
上述的重组菌在制备L-组氨酸中的应用也是本发明保护的范围。
本发明还提供一种制备L-组氨酸的方法，包括如下步骤：发酵培养上述的重组菌，即得到L-组氨酸。
本发明所述的失活细菌的pgi基因，“失活”指的是相应被改造的对象发生变化，从而达到一定的效果，包括但是不限于，定点突变、插入失活和/或敲除。
本发明所用的染色体基因敲除、插入失活、基因敲入、启动子替换和定点突变的方法是 通过自杀性载体pK18mobsacB携带改造靶基因的同源臂发生同源重组实现的。
本发明所述的L-组氨酸工程菌，发酵24小时的L-组氨酸生产强度为0.01～1g/L/h，发酵结束时的L-组氨酸产量为1～60g/L，一般地，发酵产量可达2g/L以上。
本发明的实验证明，与现有的L-组氨酸工程菌和L-组氨酸发酵生产方法相比，本发明的优点在于：
(1)本发明所提供的重组菌，通过采用敲除pgi基因阻断上游糖酵解途径的同时过表达zwf-opcA基因增强戊糖磷酸途径的代谢能力的组合改造策略，工程菌的生长和葡萄糖消耗能力与野生型菌株相比未见明显减弱，同时L-氨基酸产量显著提高。
(2)本发明所提供的重组菌在基本培养基(摇瓶发酵实验所用)中生长良好，无营养物质缺陷表型，便于工业化控制。
(3)本发明所提供的重组菌的发酵周期短，在发酵罐放大实验中约45-72小时可达到最大积累量(而目前报道的最高产量的L-组氨酸工程菌发酵时间长达120小时)(Mizukami,T.,Hamu,A.,Ikeda,M.,Oka,T.,Katsumata,R.,1994.Cloning of the ATP phosphoribosyl transferase gene of Corynebacterium glutamicum and application of the gene to L-histidine production.Biosci.Biotechnol.Biochem.58,635-638.)，易于过程和成本控制。
(4)本发明首次提出了在pgi基因缺失的基础上，同时增强6-磷酸葡萄糖脱氢酶的表达的组合改造策略，解除了pgi基因缺失导致的菌株生长和葡萄糖代谢的限制，能够最大程度地将中心碳代谢流导向戊糖磷酸途径，同时维持细菌较高的生长代谢和ATP水平，显著提高氨基酸的产量，从而在实践上可用于细菌发酵工业化生产。
(5)本发明还首次提出了通过将组氨酸合成途径与核苷酸合成途径相偶联的策略，利用组氨酸合成副产物AICAR合成组氨酸的前体ATP，显著提高L-组氨酸产量，从而在实践上可用于细菌发酵工业化生产L-组氨酸。
综上所述，本发明的有益效果在于，开辟并且实践证明了新的提高L-氨基酸的发酵产量的方法并构建了相应的工程菌，观察到了可以叠加提高产量的效果，从而在实践上可用于细菌发酵生产L-氨基酸，便于推广应用。
为了便于理解，以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见的。
另外，本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
附图说明
参考以下附图，将有助于更好地理解本发明的方案及有益效果。
图1为重组质粒pXMJ19-prsA-hisGfbr的示意图。
图2为CG161菌株(pgi基因已敲除)基因组DNA的PCR鉴定电泳图。
图3为重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr的示意图。
图4为L-组氨酸工程菌CG171表达蛋白的SDS-PAGE图。
图5为L-组氨酸工程菌CG171中6-磷酸葡萄糖脱氢酶酶活性测定图。
图6为重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr-purH的示意图。
图7为CG328菌株携带质粒DNA的PCR鉴定电泳图。
图8为CG353菌株(purF基因被弱化)基因组DNA的PCR鉴定电泳图。
具体实施方式
以下结合附图和实施例，对本发明的具体实施方式进行更加详细地说明，以便能够更好地理解本发明的方案以及其各个方面的优点。然而，以下描述的具体实施方式和实施例仅是说明的目的，而不是对本发明的限制。具体来说，以下描述均以(野生型)谷氨酸棒杆菌为例对重组工程菌的构建以及L-组氨酸的生产进行说明和实验，然而本领域技术人员应理解，本发明对氨基酸代谢途径的改造策略可用于其他合适的菌株以构建用于提高L-组氨酸的产量的工程菌。不但如此，通过组合适当的插件，可用于提高其他具有类似代谢途径的氨基酸，特别是L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸和L-羟脯氨酸等)的产量。
如背景技术中提及的，pgi基因编码的磷酸葡萄糖异构酶，是糖酵解途径的关键酶。L-组氨酸合成的前体PRPP是经由戊糖磷酸途径合成的，因此设想敲除pgi基因将减弱糖酵解途径的代谢流量，将中心碳代谢流导向戊糖磷酸途径，以增强L-组氨酸合成途径代谢流量。
通过敲除pgi基因增强戊糖磷酸途径代谢流量的改造策略，在文献和专利中都有报道(用于生产L-赖氨酸、L-缬氨酸和核苷等产物Marx,A.,Hans,S.,Mockel,B.,Bathe,B.,de Graaf,A.A.,McCormack,A.C.,Stapleton,C.,Burke,K.,O'Donohue,M.,Dunican,L.K.,2003.Metabolic phenotype of phosphoglucose isomerase mutants of Corynebacterium glutamicum.J Biotechnol.104,185-197；Blombach,B.,Schreiner,M.E.,Bartek,T.,Oldiges,M.,Eikmanns,B.J.,2008.Corynebacterium glutamicum tailored for high-yield L-valine production.Appl Microbiol Biotechnol.79,471-479；Peifer,S.,Barduhn,T.,Zimmet,S.,Volmer,D.,Heinzle,E.,Schneider,K.,2012.Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine.Microb Cell Fact.11,138；US6586214B1；EP1087015A2)。
然而实际上，本发明人研究发现，敲除pgi基因会导致糖代谢中间代谢物的过度积累，造成糖代谢压力，进而导致菌体葡萄糖代谢和生长减慢。本发明人还发现敲除pgi基因后，产L-组氨酸的工程菌的L-组氨酸产量非但没有增加，反而明显下降。其主要原因在于：组氨酸通过戊糖磷酸途径提供合成其分子骨架的前体，而赖氨酸和缬氨酸通过戊糖磷酸途径提供其合成酶的辅因子NADPH。此外，因组氨酸合成过程消耗大量的能量载体ATP，要想利用弱化pgi基因的表达，增强戊糖磷酸途径代谢流量的策略来增加组氨酸的产量，则需要维持 戊糖磷酸途径和糖酵解途径代谢流量的平衡，以保证其合成前体和能量供应。
针对这样的问题，本发明通过实验发现，过表达zwf-opcA基因(该基因编码6-磷酸葡萄糖脱氢酶，是戊糖磷酸途径的关键限速酶)可以增强菌体的糖代谢能力，缓解糖代谢压力，恢复菌株的葡萄糖代谢和生长能力，同时平衡戊糖磷酸途径和糖酵解途径的代谢流量，平衡组氨酸合成前体PRPP和ATP的供应，进而提高L-组氨酸产量。
根据本发明，通过弱化(如敲除)pgi基因并同时过表达zwf-opcA基因的改造策略，对已增强prsA基因和L-组氨酸合成操纵子基因表达的菌株进行重组改造所获得菌株的L-组氨酸产量有了显著提高。
同样的，对于诸如L-苏氨酸、L-赖氨酸、L-缬氨酸、L-脯氨酸和L-羟脯氨酸等其他氨基酸来说，它们通过戊糖磷酸途径提供其合成酶的辅因子NADPH，因此，本发明的弱化pgi基因并同时过表达zwf-opcA基因的改造策略，在增加了NADPH的同时，也平衡戊糖磷酸途径和糖酵解途径的代谢流量，能消除因pgi基因弱化造成菌体葡萄糖代谢和生长减慢的问题，从而同样会进一步增加这些氨基酸的产量。
在此基础上，本发明进一步提出了将L-组氨酸合成途径与核苷酸合成途径相偶联的策略。L-组氨酸合成过程中，由hisH和hisF基因编码的咪唑甘油磷酸合酶催化生成咪唑甘油磷酸和5-磷酸核糖-4-甲酰胺基-5-氨基咪唑(AICAR)，其中咪唑甘油磷酸沿组氨酸合成途径最终合成L-组氨酸，而AICAR则可进入嘌呤合成途径，最终生成嘌呤核苷酸(AMP，ATP等)。而ATP是组氨酸合成的前体物质之一，同时也为组氨酸合成提供能量。purH基因编码的双功能酶，AICAR甲基转移酶/IMP环水化酶，催化从AICAR生成IMP的两步反应。本发明人发现增强谷氨酸棒杆菌中的purH基因的表达对L-组氨酸积累有明显的促进作用，与上述改造策略组合可以进一步提高效果。
此外，L-组氨酸合成途径与嘌呤核苷酸合成途径在代谢物AICAR处偶联，同时两个途径共用前体物质PRPP。本发明人发现弱化催化嘌呤核苷酸合成第一步反应酶(磷酸核糖酰胺转移酶)的编码基因purF，可以将核苷酸合成与组氨酸合成途径进行代谢偶联，利用组氨酸合成副产物AICAR合成核苷酸，增加组氨酸合成前体物质PRPP的供应，同时拉动组氨酸合成途径代谢流量，促进L-组氨酸积累。该基因改造同样可以更进一步地提高L-组氨酸的产量。
由上述可见，本发明对微生物的组氨酸合成相关途径中的多个靶点进行组合改造，有效实现了L-组氨酸的积累。此外，在改造组氨酸合成途径的同时，将组氨酸合成途径与核苷酸合成途径进行偶联，有效利用组氨酸合成与核苷酸合成的偶联节点AICAR生成嘌呤核苷酸的途径，节约合成前体PRPP，从而为组氨酸的合成提供更多的前体物质PRPP和ATP，进一步增加L-组氨酸的积累。
定义：
本文提及的“出发菌(starting bacteria)”(或在文中又称为底盘菌(base bacteria))是指用于本发明基因改造策略的初始菌株。该菌株可以是天然存在的菌株，也可以是通过诱变 或遗传工程改造等方式选育的菌株。为构建用于生产某种L-氨基酸(如L-组氨酸)的工程菌，所述出发菌优选为可积累该种L-氨基酸(如L-组氨酸)的菌株。
本文提及的“原始菌(original bacteria)”是指未经任何遗传工程改造的菌株，可以是自然界存在的菌株，也可以是经人工诱变培育的菌株。
本文提及的“同源性(homology)”是指DNA的核苷酸序列或蛋白质的氨基酸序列之间的相似程度，同时本文所说的具有(一定程度)同源性的DNA其所编码的蛋白至少在用于本发明的功能方面具有相同或更好的活性，同样的具有(一定程度)同源性的蛋白质至少在用于本发明的功能方面具有相同或更好的活性。举例来说，hisG基因与进行了三个位点突变后的hisGfbr基因具有高度的相似性，其中前者编码ATP-磷酸核糖转移酶，而后者编码解除了组氨酸反馈抑制调节的ATP-磷酸核糖转移酶，这两个酶整体上看功能和活性有所不同，但是对于用于本发明的“组氨酸合成第一步反应的催化酶”这一功能，二者是相同的，因此，hisG基因和hisGfbr基因，以及二者编码的酶分别属于本发明意义上的具有同源性的DNA和蛋白质。它们均在本发明的保护范围之内。
本文提及的方法中各步骤的执行顺序，除特别说明外，并不限于本文的文字所体现出来的顺序，也就是说，各个步骤的执行顺序是可以改变的，而且两个步骤之间根据需要可以插入其他步骤。
以下通过具体实施例进一步说明本发明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，可参见《分子克隆实验指南(第3版)》(科学出版社)、《微生物学实验(第4版)》(高等教育出版社)以及相应仪器和试剂的厂商说明书等。实施例中所用仪器设备和试剂为市售的常用仪器、试剂。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。
实施例1、L-组氨酸底盘工程菌CG160的获得
根据本发明人的前期研究，本实施例对野生型谷氨酸棒杆菌ATCC13032进行增强组氨酸合成的改造，以获得实施本发明上述多靶点改造的底盘菌。首先，将hisEG和hisDCB(两个组氨酸合成基因操纵子)的启动子替换为谷氨酸棒杆菌内源强启动子PglyA(序列7中5’末端第863-1038位核苷酸序列所示，或者序列8中5’末端第752-927位核苷酸序列所示)(Zhang,Y.,Shang,X.,Lai,S.,Zhang,G.,Liang,Y.,Wen,T.,2012.Development and application of an arabinose-inducible expression aystem by facilitating inducer uptake in Corynebacterium glutamicum.Appl Environ Microbiol.78,5831-5838.)，同时将hisE和hisD基因的核糖体结合位点(RBS)替换为谷氨酸棒杆菌高表达基因的保守RBS序列(AAAGGAGGA)(序列7中5’端末第1039-1047位核苷酸序列所示，或者序列8中5’末端第928-936位核苷酸序列所示)，从而解除这两个组氨酸合成基因操纵子的转录和翻译的弱化调节，将hisE基因的起始 密码子GTG替换为ATG(序列7中5’端第1053-1055位核苷酸序列所示)，以增强其表达。其次，将组氨酸合成途径的关键限速酶，ATP-磷酸核糖转移酶(HisG，序列6所示)的编码基因hisG替换为含三个氨基酸位点突变的hisGfbr基因(序列4中5’端第1007-1852位核苷酸序列所示)，以解除组氨酸对该酶的反馈抑制调节，增强该酶催化活性(Zhang,Y.,Shang,X.,Deng,A.,Chai,X.,Lai,S.,Zhang,G.,Wen,T.,2012.Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine.Biochimie.94,829-838.)。
一、谷氨酸棒杆菌野生型ATCC13032中L-组氨酸合成操纵子启动子替换为强启动子PglyA
根据Genbank中谷氨酸棒杆菌ATCC13032的hisEG操纵子及其上下游序列和PglyA启动子序列分别设计引物。
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P1和P2为引物，PCR扩增hisEG操纵子启动子上游同源臂；以P3和P4为引物扩增启动子PglyA；以P5和P6为引物扩增hisEG启动子下游同源臂。再以纯化的上述PCR产物为模板，以P1和P6为引物，采用重叠延伸PCR技术(SOE)扩增，得到1920bp的PCR产物，为含替换启动子PglyA及被替换启动子PhisEG的上下游同源臂的片段(序列7)。
其中，序列7自5’末端第1-862位核苷酸为被替换启动子PhisEG的上游同源臂，序列7自5’末端第863-1038位核苷酸为启动子PglyA，序列7自5’末端第1053-1920位核苷酸为被替换启动子PhisEG的下游同源臂。
将上述1920bp的PCR产物经Xba I和BamH I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB(购自美国典型微生物保藏中心ATCC，货号87097)连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到2132bp为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Xba I和BamH I双酶切鉴定，得到1920bp的为阳性。
将阳性质粒送去测序，结果该质粒为将序列表中序列7所示的核苷酸插入载体pK18mobsacB中得到的重组质粒，命名为pK18mobsacB-PglyA::PhisEG。
采用相同的方法构建同源重组质粒pK18mobsacB-PglyA::PhisDCB，具体如下：以P7和P8为引物扩增hisDCB操纵子启动子上游同源臂；以P9和P10为引物扩增启动子PglyA；以P11和P12为引物扩增hisDCB的启动子下游同源臂。以P7和P12为引物，采用重叠延伸PCR技术(SOE)扩增。得到1694bp的PCR产物，为含替换启动子PglyA及被替换启动子PhisDCB上下游同源臂的长片段(序列8)，其中，序列8自5’末端第1-751位核苷酸为被替换启动子PhisDCB的上游同源臂，序列8自5’末端第752-927位核苷酸为启动子PglyA，序列8自5’末端第942-1694位核苷酸为被替换启动子PhisDCB的下游同源臂。
将上述1694bp的PCR产物经Hind III和BamH I双酶切后，与经同样双酶切处理的同源 重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1906bp为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Hind III和BamH I双酶切鉴定，得到1694bp的为阳性。将阳性质粒送去测序，结果该质粒为将序列表中序列5所示的核苷酸插入载体pK18mobsacB中得到的重组质粒，命名为pK18mobsacB-PglyA::PhisDCB。
上述所用的引物序列如下：
P1:GCTCTAGAGTATCGGCGTGGAGTTGTC(Xba I)(序列21)
P2:TAGTGGAGTAGCTTTATTTTGCGACACCTGCC(序列22)
P3:GTCGCA AAATAAAGCTACTCCACTAGTGTGATCG(序列23)
P4:GGTTCCTCCTTTGCGTAAGACCTCACTCGC(序列24)
P5:GAGGTCTTACGCAAAGGAGGAACCGAATGAAGACATTTGA(序列25)
P6:CGCGGATCCCAGGATCTGCTGCTCTGG(BamH I)(序列26)
P7:CCCAAGCTTCGAGGAAACCGTTGAGGA(Hind III)(序列27)
P8:TAGTGGAGTAGCTATGGATTTCACCTCTGTGAATG(序列28)
P9:TCTCCACTTTAGGTAAGCTACTCCACTAGTGTGATCG(序列29)
P10:CGATCCTCCTTTGCGTAAGACCTCACTCGC(序列30)
P11:GAGGTCTTACGCAAAGGAGGATCGCCATGTTGAATGTC(序列31)
P12:CGCGGATCCGGCAGAGGCATCAGCAAG(BamH I)(序列32)
P13:ATGTGCTGCAAGGCGATTAA(序列33)
P14：TATGCTTCCGGCTCGTATGT(序列34)
P15:TTTTATATATGGGTATCGGCGGTCTATGCT(序列35)。
将序列测定正确的同源重组质粒pK18mobsacB-PglyA::PhisEG电转化至谷氨酸棒杆菌野生型ATCC13032中。通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落。通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。将阳性菌落以P15和P6为引物进行PCR扩增鉴定，得到948bp的为重组菌，命名为谷氨酸棒杆菌WT-PglyA::PhisEG。
将序列测定正确的同源重组质粒pK18mobsacB-PglyA::PhisDCB电转化至谷氨酸棒杆菌WT-PglyA::PhisEG中。通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落。通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。将阳性菌落以P15和P12为引物进行PCR扩增鉴定，得到833bp的为重组菌，命名为谷氨酸棒杆菌CG158(WT-PglyA::PhisEG-PglyA::PhisDCB)。
经该重组菌提取基因组DNA进行测序，结果为证实已成功将谷氨酸棒杆菌野生型ATCC13032中的hisEG和hisDCB的启动子均替换为谷氨酸棒杆菌内源强启动子PglyA，将hisE和hisD基因的RBS替换为谷氨酸棒杆菌高表达基因的保守RBS序列(AAAGGAGGA)，将hisE基因的起始密码子GTG替换为高表达强度的ATG，谷氨酸棒杆菌CG158 (WT-PglyA::PhisEG-PglyA::PhisDCB)构建成功。
二、染色体上基因hisG的定点突变获得L-组氨酸底盘工程菌CG160
染色体hisG基因定点突变采用两步替换的方法，以期同时实现该基因的三个定点突变，先将序列表中序列9所示的含氯霉素抗性基因Cmr及hisG基因突变片段上下游同源臂的长片段与CG158同源重组，得到重组菌WT-PglyA::PhisEG-Cmr::hisG-PglyA::PhisDCB；再将序列表中序列10所示的含三个点突变的hisG基因末端264bp片段及其上下游同源臂的长片段与重组菌WT-PglyA::PhisEG-Cmr::hisG-PglyA::PhisDCB同源重组，得到CG160。
具体如下：
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P16和P17为引物PCR扩增hisG基因突变片段的上游同源臂，以P18和P19为引物扩增hisG基因突变片段的下游同源臂；以P20和P21为引物，质粒pXMJ19(购自于Biovector Science Lab,Inc,货号SMD1168H)为模板，扩增氯霉素抗性基因Cmr。再以纯化的上述PCR产物为模板，以P16和P21为引物，采用重叠延伸PCR技术(SOE)扩增，得到1689bp含氯霉素抗性基因Cmr及hisG基因突变片段上下游同源臂的长片段(序列9)。
其中，序列9自5’末端第1-420位核苷酸为hisG基因突变片段上游同源臂，序列9自5’末端第421-1281位核苷酸为氯霉素抗性基因Cmr，序列9自5’末端第1282-1689位核苷酸为hisG基因突变片段下游同源臂。
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P28和P29为引物扩增含C645G(第215位天冬酰胺变为赖氨酸)突变位点的hisG基因片段，以P30和P31为引物扩增含A693C和A703G(第231位亮氨酸变为苯丙氨酸和第235位苏氨酸变为丙氨酸)突变位点的hisG片段，再以纯化的上述PCR产物为模板，以P28和P31为引物，采用重叠延伸PCR技术(SOE)扩增，得到846bp含三个点突变的hisG基因(序列4自5’末端第1007-1852位核苷酸)。以P16和P22为引物PCR扩增hisG基因定点突变的上游同源臂，以P25和P21为引物扩增hisG基因定点突变的下游同源臂；以P23和P24为引物，以上述获得的含三个点突变的hisG基因为模板，扩增含三个点突变的hisG基因末端264bp片段。再以纯化的上述PCR产物为模板，以P16和P21为引物，采用重叠延伸PCR技术(SOE)扩增，得到1092bp含三个点突变的hisG基因末端264bp片段及其上下游同源臂的长片段(序列10)。
其中，序列10自5’末端第1-420位核苷酸为上游同源臂，序列10自5’末端第421-684位核苷酸为三个点突变的hisG基因末端264bp片段，序列10自5’末端第685-1092位核苷酸为下游同源臂。
纯化回收的两个PCR产物分别经BamH I和EcoR I双酶切后，分别与经同样双酶切处理的敲除载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1901bp和1304bp的分别为携带两种重组质粒的阳性转化 子，对鉴定正确的转化子提取质粒，并对质粒进行BamH I和EcoR I双酶切鉴定，得到1689bp和1092bp的分别为两种重组质粒。经进一步序列测定验证，重组质粒pK18mobsacB-Cmr::hisG和pK18mobsacB-hisGfbr::Cmr构建成功。
pK18mobsacB-Cmr::hisG为将含氯霉素抗性基因Cmr及hisG基因突变片段上下游同源臂的长片段(序列9)插入到载体pK18mobsacB中得到的重组载体。
pK18mobsacB-hisGfbr::Cmr为将含三个点突变的hisG基因末端264bp片段及其上下游同源臂的长片段(序列10)插入到载体pK18mobsacB中得到的重组载体。
上述所用的引物序列如下：
P16:CGCGGATCCATCTACGTTGCTGGTGGC(BamH I)(序列36)
P17:ACGGGCAACAGCTGCTGCTCTGGGGTGAC(序列37)
P18:CAGAGCAGCAGCTGTTGCCCGTCTCACTGGT(序列38)
P19:GGTAGTTAAAATTACGCCCCGCCCTGCCACT(序列39)
P20:GCGGGGCGTAATTTTAACTACCCCCGAAAAT(序列40)
P21:CCGGAATTCCGAATGAAATCTGGGACG(EcoR I)(序列41)
P22:CGAAGCAGGATCTGCTGCTCTGGGGTGAC(序列42)
P23:CAGAGCAGCAGATCCTGCTTCGCCGCATCCA(序列43)
P24:GGTAGTTAAAACTAGATGCGGGCGATGCG(序列44)
P25:CCCGCATCTAGTTTTAACTACCCCCGAAAAT(序列45)
P26:TCCCAAACAAAGGCTCGC(序列46)
P27:CAGTCGGCGGTTTGCTAA(序列47)
P28:ATGTTGAAAATCGCTG(序列48)
P29:TTACTGCAGTGGCAGCGTCCAGGTTGTCGCGGTCGACCTTGTAATCCAGCAT(序列49)
P30:ACCTGGACGCTGCCACTGCAGTAACCCCAGGCTTCTCCGGCCCAGCGGTATC(序列50)
P31:CTAGATGCGGGCGATGCGG(序列51)。
将序列测定正确的同源重组质粒pK18mobsacB-Cmr::hisG电转化至谷氨酸棒杆菌CG158中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生两次同源重组的阳性菌。
以P26和P27为引物，对阳性菌进行PCR扩增鉴定，得到1872bp的为重组菌WT-PglyA::PhisEG-Cmr::hisG-PglyA::PhisDCB。
将序列测定正确的同源重组质粒pK18mobsacB-hisGfbr::Cmr电转化至上述构建的重组菌WT-PglyA::PhisEG-Cmr::hisG-PglyA::PhisDCB中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。
以P26和P27为引物，对阳性菌落进行PCR扩增鉴定，得到1275bp的为重组菌，命名 为谷氨酸棒杆菌CG160(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB)。
经该重组菌提取基因组DNA进行测序，结果为证实成功对谷氨酸棒杆菌CG158的染色体hisG基因的N215K/L231F/T235A点突变，谷氨酸棒杆菌CG160(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB)构建成功。
hisG基因的N215K/L231F/T235A点突变为将hisG基因编码的ATP-磷酸核糖转移酶(HisG)的第215位天冬酰胺变为赖氨酸、第231位亮氨酸变为苯丙氨酸和第235位苏氨酸变为丙氨酸。
实施例2、含有质粒的高产L-组氨酸的重组菌CG171的构建
在本实施例中，在实施例1中获得的初级工程菌的基础上，进一步过表达prsA基因并同时过表达hisGfbr基因(序列4第1007-1852位核苷酸序列)，然后将敲除pgi基因(序列13)和过表达zwf-opcA基因(序列2)的改造进行组合，获得高产工程菌CG171。
一、L-组氨酸初级工程菌CG176的构建
prsA基因编码PRPP合成酶(PrsA，序列5所示，PRPP为组氨酸合成的前体物质)，增强prsA基因的表达，以增加组氨酸合成前体PRPP的合成，为组氨酸合成提供更多的前体物质。
在实施例1获得的底盘工程菌CG160的基础上，过表达prsA基因(序列4中5’末端第15-992位核苷酸序列所示)并同时过表达hisGfbr基因(序列4中5’端第1007-1852位核苷酸序列所示)，以获得有较好组氨酸产量的初级工程菌CG176，以便实施本发明的策略后能够获得更好的效果。当然，本领域技术人员应理解，本发明的改造策略并不仅限于对本实施例获得的初级工程菌进行重组改造，也可用于其他组氨酸工程菌中。
以菌株CG160的基因组DNA为模板，分别以P32/P33和P34/P35为引物PCR扩增prsA基因(992bp)和hisGfbr(860bp)。采用重叠延伸PCR将两基因连接，以扩增的hisGfbr和prsA基因为模板，用P32和P35为引物进行PCR扩增，得到1852bp的PCR产物为prsA-hisGfbr片段(序列4)。
其中，序列4自5’末端第15-992位核苷酸为prsA，序列4自5’末端第1007-1852位核苷酸为hisGfbr(含三个点突变的hisG基因)。
将上述PCR产物经Xba I和Sma I双酶切后，与经同样双酶切处理的谷氨酸棒杆菌-大肠杆菌穿梭表达质粒pXMJ19连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含氯霉素(20μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P36和P37为引物，采用菌落PCR鉴定转化子，得到2054bp的为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Xba I和Sma I双酶切鉴定，得到1852bp的为阳性。
pXMJ19-prsA-hisGfbr经进一步序列测定分析，该质粒为将prsA-hisGfbr片段(序列4)插入质粒pXMJ19的Xba I和Sma I酶切位点间，得到的载体pXMJ19-prsA-hisGfbr命名为重组质粒pWYE 1230(图1所示)。
将质粒pXMJ19-prsA-hisGfbr转化至上述构建的底盘工程菌CG160中，以P36和P37为引物，采用菌落PCR鉴定转化子，得到2054bp的为阳性转化子，对鉴定正确的转化子提取质粒鉴定进一步确定过表达质粒成功转化至工程菌中，L-组氨酸工程菌CG176(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB/pXMJ19-prsA-hisGfbr)构建成功。
上述所用的引物序列如下：
P32：GCTCTAGAAAAGGAGGATCCTCATGACTGCTCACTGG(Xba I)(序列52)
P33：TTGTCCTCCTTTTTAGGCCTCGCCCTCGAA(序列53)
P34：GGCGAGGCCTAAAAAGGAGGACAATCATGTTGAAAATCGCTG(序列54)
P35：TCCCCCGGGCTAGATGCGGGCGATGCGG(Sma I)(序列55)
P36：CAATTAATCATCGGCTCGTA(序列56)
P37：ACCGCTTCTGCGTTCTGATT(序列57)。
二、L-组氨酸初级工程菌CG161和CG172的获得
pgi基因编码磷酸葡萄糖异构酶(Pgi，序列14所示)。在以上获得的底盘菌160的基础上，敲除pgi基因(序列13)，获得初级工程菌CG161；在CG161的基础上，过表达prsA基因并同时过表达hisGfbr基因，获得了敲除了pgi基因的工程菌CG172。
初级工程菌CG161为将L-组氨酸底盘工程菌CG160中pgi基因(序列13)敲除获得，具体如下：
首先，根据Genbank中谷氨酸棒杆菌ATCC13032的pgi基因及其上下游序列分别设计引物。
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P38和P39为引物PCR扩增pgi基因上游同源臂；以P40和P41为引物扩增pgi基因下游同源臂。再以纯化的上述PCR产物为模板，以P38和P41为引物，采用重叠延伸PCR技术(SOE)扩增，得到1672bp的含欲敲除基因pgi的上下游同源臂的片段(序列1)。
其中，序列1自5’末端第1-834位核苷酸为欲敲除基因pgi的上游同源臂，序列1自5’末端第835-1672位核苷酸为欲敲除基因pgi的下游同源臂。
纯化回收的PCR产物经BamH I和EcoR I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1884bp的为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行BamH I和EcoR I双酶切鉴定，得到1672bp的为阳性。经进一步序列测定验证，重组质粒pK18mobsacB-Δpgi构建成功，为将含欲敲除基因pgi的上下游同源臂的片段(序列1)插入载体pK18mobsacB的BamH I和EcoR I酶切位点间得到的载体。
所用的引物序列如下：
P38：CGCGGATCCGCTCTTTCGGAGTGACCT(BamH I)(序列58)
P39：TAAGCAAGCGAGAAAACTCCTTTATTGTCG(序列59)
P40：TAAAGGAGTTTTCTCGCTTGCTTATAGGGTC(序列60)
P41：CCGGAATTCTCGGGAAGCAGTTAGTGAAA(EcoR I)(序列61)
P42：TTGACGACGCAAGAGCCA(序列62)
P43：CACCATTACCGATGAGAAAC(序列63)。
将序列测定正确的同源重组质粒pK18mobsacB-Δpgi电转化至谷氨酸棒杆菌CG160中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生第二次同源重组的菌落。以P42和P43为引物，对菌落进行基因组DNA提取及PCR扩增鉴定，得到1759bp的为阳性(见图2)，命名为CG161(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi)。
CG161(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi)经进一步序列测定分析，结果为L-组氨酸底盘工程菌CG160的染色体pgi基因敲除成功，CG161构建成功。
工程菌CG172为质粒pXMJ19-prsA-hisGfbr导入工程菌CG161中得到的重组菌(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi/pXMJ19-prsA-hisGfbr)。具体操作方法为常规方法，故在此省略。
三、L-组氨酸高产工程菌CG171以及作为比较的工程菌CG173的构建
zwf-opcA基因编码6-磷酸葡萄糖脱氢酶(Zwf-OpcA，序列3所示，其中自5’末端第1-514位氨基酸构成Zwf亚单元，第515-833位氨基酸残基构成OpcA亚单元)。实施敲除pgi基因和过表达zwf-opcA基因(序列2)的组合改造，获得高产工程菌CG171。作为比较，获得未敲除pgi基因但过表达zwf-opcA基因的工程菌CG173。
根据Genbank中谷氨酸棒杆菌ATCC13032的zwf-opcA基因序列设计引物，以谷氨酸棒杆菌ATCC13032的基因组DNA为模板，采用引物P44和P45，PCR扩增2519bp的zwf-opcA片段(zwf基因的起始密码子由GTG替换为ATG，以增强其表达)(序列2)。经Hind III和Xba I双酶切后，先与经同样双酶切处理的表达质粒pXMJ19连接，获得的重组质粒pXMJ19-zwf-opcA，pXMJ19-zwf-opcA再经XbaI和SmaI双酶切，与以上制备的质粒pXMJ19-prsA-hisGfbr经Xba I和Sma I双酶切获得的1852bp的prsA-hisGfbr片段连接。
zwf-opcA片段，其中，序列2自5’末端第1-1545位核苷酸为zwf基因，序列2自5’末端第1560-2519位核苷酸为opcA基因。
连接产物采用化学转化法转化至大肠杆菌DH5α，在含氯霉素(20μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P36和P37为引物，采用菌落PCR鉴定转化子，得到4587bp的为阳性转化子。对鉴定正确的转化子提取质粒，并对质粒分别进行Xba I/Sma I和Hind III/Xba I双酶切鉴定，分别得到1852bp和2533bp的为阳性。
经序列测定验证，重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr构建成功，命名为重组质粒pWYE 1229(图3所示)，为将zwf-opcA基因(序列2)插入pXMJ19的Hind III和Xba I位 点间，且将prsA-hisGfbr片段(序列4)插入Xba I和Sma I位点间得到的载体。
P44：CCCAAGCTTAAAGGAGGACCATCATGAGCACAAACACGACCCCCT(Hind III)(序列64)
P45：GCTCTAGATTAGACGGTTTCCAGCTTG(Xba I)(序列65)
将重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr分别电转化至pgi未缺失的工程菌CG160和pgi缺失的工程菌CG161中。以P36和P37为引物，采用菌落PCR鉴定转化子，得到4587bp的为阳性转化子，对鉴定正确的转化子提取质粒。
送去测序，L-组氨酸工程菌CG173(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB/pXMJ19-zwf-opcA-prsA-hisGfbr)含有质粒重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr，为将重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr转入工程菌CG160得到的菌。
CG171(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi/pXMJ19-zwf-opcA-prsA-hisGfbr)含有质粒重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr，为将重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr转入工程菌CG161得到的菌。
进一步验证过表达质粒携带基因在工程菌中的表达情况。制备CG171的细胞裂解液，进行SDS-PAGE检测，结果如图4所示，泳道1和2为CG171的细胞裂解液，泳道3为ATCC13032/pXMJ19(将pXMJ19质粒导入ATCC13032中得到)的细胞裂解液，为对照，表明过表达质粒携带的zwf(57.5kDa)、opcA(34.8kDa)prsA(35.6kDa)和hisGfbr(30.2kDa)基因在工程菌中成功表达。
进一步测定工程菌CG171中6-磷酸葡萄糖脱氢酶(Zwf-opcA)的比酶活性。测定反应体系如下(0.5mL)：100mmol/L Tris-HCl(pH 7.8),200mmol/L KCl,1mmol/L NADP,10mmol/L MgCl2,5mmol/L 6-磷酸葡萄糖(G6P)，适量的细胞裂解液。30℃，反应5min。通过检测340nm处吸光值的变化反映产物NADPH的生成量。酶活单位(U)定义为每分钟生成1nmol还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)所需的酶量。结果如图5所示，与野生型菌株相比，通过质粒过表达zwf-opcA，6-磷酸葡萄糖脱氢酶的比活性提高了34倍。
实施例3、含有质粒的L-组氨酸高产工程菌CG319的构建
在以上获得的高产工程菌CG171的基础上，为了进一步过表达编码AICAR甲基转移酶/IMP环水化酶(PurH，序列16所示)的purH基因，从而将因组氨酸合成途径增强而增多的副产物AICAR更多地导向嘌呤核苷酸合成途径，构建重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr-purH，并导入初级菌CG161中，获得高产工程菌CG319。
根据Genbank中谷氨酸棒杆菌ATCC13032的purH基因序列设计引物，以ATCC13032基因组DNA为模板，以P46和P47为引物PCR扩增purH基因(1563bp)(序列15)。
将上述PCR产物经Sma I和EcoR I双酶切后，与经同样双酶切处理的谷氨酸棒杆菌-大肠杆菌穿梭表达质粒pXMJ19连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含 氯霉素(20μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P52和P53为引物，采用菌落PCR鉴定转化子，得到1779bp的为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Xba I和Sma I双酶切鉴定，得到1577bp的为阳性，命名为重组质粒pXMJ19-purH。
以重组质粒pXMJ19-zwf-opcA-prsA-hisGfbr为模板，分别以P48/P49和P50/P51为引物PCR扩增zwf-opcA(2519bp)和prsA-hisGfbr片段(1852bp)。采用重叠延伸PCR将两片段连接获得4385bp的zwf-opcA-prsA-hisGfbr片段(序列17)。
其中，序列17自5’末端第15-2533位核苷酸为zwf-opcA，序列17自5’末端第2534-4385位核苷酸为prsA-hisGfbr。
将上述PCR产物经Xba I和Sma I双酶切后，与经同样双酶切处理的重组质粒pXMJ19-purH连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含氯霉素(20μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P52和P53为引物，采用菌落PCR鉴定转化子，得到6164bp的为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Xba I和Sma I双酶切鉴定，得到4385bp的为阳性，命名为重组质粒pWYE1507(pXMJ19-zwf-opcA-prsA-hisGfbr-purH)(图6所示)。
pXMJ19-zwf-opcA-prsA-hisGfbr-purH经进一步序列测定分析，该质粒为将zwf-opcA-prsA-hisGfbr片段(序列17)插入质粒pXMJ19的Xba I和Sma I酶切位点间，同时将purH插入质粒pXMJ19的Sma I和EcoR I酶切位点间得到的载体。
将质粒pXMJ19-zwf-opcA-prsA-hisGfbr-purH转化至工程菌CG161中，以P52和P53为引物，采用菌落PCR鉴定转化子，得到6164bp的为阳性转化子，对鉴定正确的转化子提取质粒鉴定进一步确定过表达质粒成功转化至工程菌中，L-组氨酸工程菌CG319(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi/pXMJ19-zwf-opcA-prsA-hisGfbr-purH)构建成功。
上述所用的引物序列如下：
P46：TCCCCCGGGAAAGGAGGACCTTCATGAGCGATGATCGTAAG(Sma I)(序列66)
P47：CCGGAATTCTTAGTGAGCGAAGTGTCGCG(EcoR I)(序列67)
P48：GCTCTAGAAAAGGAGGACCATCATGAGCACAAACACGACCC(Xba I)(序列68)
P49：AGTCATGAGGATCCTCCTTTTTAGACGGTTTCCAGCTTG(序列69)
P50：TCAAGCTGGAAACCGTCTAAAAAGGAGGATCCTCATGACTGCTCACTG(序列70)
P51：TCCCCCGGGCTAGATGCGGGCGATGCGGATTTC(Sma I)(序列71)
P52：CAATTAATCATCGGCTCGTA(序列72)
P53：ACCGCTTCTGCGTTCTGATT(序列73)
实施例4、含有质粒的L-组氨酸高产工程菌CG328的构建
在以上获得CG319的基础上，为弱化编码磷酸核糖酰胺转移酶(PurF，序列19)的purF基因，增加组氨酸合成前体物质PRPP向组氨酸合成途径的分流，将初级工程菌CG161中purF 的启动子替换为Phom，获得CG327，再导入质粒pXMJ19-zwf-opcA-prsA-hisGfbr-purH，获得高产工程菌CG328。
将初级工程菌CG161的purF基因的启动子替换为Phom，得到工程菌CG327，具体如下：
首先，根据Genbank中谷氨酸棒杆菌ATCC13032的purF基因及其上下游序列分别设计引物。
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P54和P55为引物PCR扩增purF基因上游同源臂；以P56和P57为引物扩增Phom启动子。以P58和P59为引物扩增purF基因下游同源臂。再以纯化的上述PCR产物为模板，以P54和P59为引物，采用重叠延伸PCR技术(SOE)扩增，得到1654bp的含Phom启动子及purF基因启动子上下游同源臂的片段(序列18)。
其中，序列18自5’末端第1-735位核苷酸为purF基因启动子的上游同源臂，序列18自5’末端第736-865位核苷酸为Phom启动子，序列18自5’末端第866-1654位核苷酸为purF基因启动子的下游同源臂。
纯化回收的PCR产物经BamH I和EcoR I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1866bp的为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行BamH I和EcoR I双酶切鉴定，得到1654bp的为阳性。经进一步序列测定验证，重组质粒pK18mobsacB-Phom::PpurF构建成功，为将含启动子Phom及欲替换启动子的上下游同源臂的片段(序列18)插入载体pK18mobsacB的BamH I和EcoR I酶切位点间得到的载体。
上述所用的引物序列如下：
P54：CGCGGATCCTCCGCAGAAAGCACCTCA(BamH I)(序列74)
P55：TTTAGTTTTCAACGGCTAAAGTTTGACCACTGG(序列75)
P56：GTGGTCAAACTTTAGCCGTTGAAAACTAAAAAGC(序列76)
P57：TCCGGTCCTCCTTTTACTTTGTTTCGGCCACCC(序列77)
P58：GGCCGAAACAAAGTAAAAGGAGGACCGGAATGACCCAGGTAAACCAC(序列78)
P59：CCGGAATTCAACCTTTGCGGGTTGTCT(EcoR I)(序列79)
将序列测定正确的同源重组质粒pK18mobsacB-Phom::PpurF电转化至谷氨酸棒杆菌CG161中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生第二次同源重组的菌落。以P56和P59为引物，对菌落进行基因组DNA提取及PCR扩增鉴定，得到905bp的为阳性，命名为CG327(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi::Phom::PpurF)。
CG327(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi：：Phom::PpurF)经进一步序列测定分析，结果为L-组氨酸初级工程菌CG161的染色体purF基因启动子替换为Phom，CG327构建成功。
工程菌CG328为将质粒pXMJ19-zwf-opcA-prsA-hisGfbr-purH导入工程菌CG327中得到的 重组菌(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Δpgi::Phom::PpurF/pXMJ19-zwf-opcA-prsA-hisGfbr-purH)。具体操作方法与以上制备工程菌CG319类似，为常规方法，故在此省略其详细描述。对CG328菌株携带的质粒进行PCR鉴定，以P52和P53为引物，得到6164bp片段(图7)，此DNA片段进行序列测定，结果为zwf-opcA-prsA-hisGfbr-purH片段，CG328菌株构建成功。
实施例5、无质粒L-组氨酸高产重组菌CG351的构建
携带质粒会增加工程菌的代谢负担，同时不利于工程菌的工业化发酵控制和发酵产品的安全性。为此，本实施例中，将质粒携带基因在染色体上增强表达，构建无质粒的组氨酸工程菌，以减轻工程菌的代谢负担，实现发酵底物到产物的最大转化。
在本实施例中，在敲除pgi基因的初级菌CG161的基础上进行进一步改造：用Psod启动子替换prsA基因的启动子，以提高PRPP合成酶(PrsA)的表达，从而获得CG350；进一步地，以Peftu启动子替换tkt-tal-zwf-opcA-devB操纵子的启动子以提高6-磷酸葡萄糖脱氢酶(Zwf-OpcA)的表达，从而获得CG351。
根据Genbank中谷氨酸棒杆菌ATCC13032的prsA基因及其上下游序列和Psod启动子序列分别设计引物。
以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P60和P61为引物扩增prsA启动子上游同源臂；以P62和P63为引物扩增Psod启动子；以P64和P65为引物扩增prsA启动子下游同源臂。再以纯化的上述PCR产物为模板，以P60和P65为引物，采用重叠延伸PCR技术(SOE)扩增，得到1455bp的PCR产物，为含替换启动子Psod及被替换启动子PprsA的上下游同源臂的片段(序列11)。
其中，序列11自5’末端第1-655位核苷酸为被替换启动子PprsA的上游同源臂，序列11自5’末端第656-847位核苷酸为启动子Psod，序列11自5’末端第848-1455位核苷酸为被替换启动子PprsA的下游同源臂。
将上述1455bp的PCR产物经Hind III和BamH I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1667bp为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Hind III和BamH I双酶切鉴定，得到1455bp的为阳性。
将阳性质粒送去测序，结果该质粒为将序列表中序列11所示的核苷酸插入载体pK18mobsacB中得到的重组质粒，命名为pK18mobsacB-Psod::PprsA。
采用相同的方法构建同源重组质粒pK18mobsacB-Peftu::Ptkt，将tkt-tal-zwf-opcA-devB操纵子的启动子替换为强启动子Peftu。具体如下：以P66和P67为引物扩增tkt-tal-zwf-opcA-devB操纵子启动子上游同源臂；以P68和P69为引物扩增启动子Peftu；以P70和P71为引物扩增tkt-tal-zwf-opcA-devB操纵子启动子下游同源臂。以P66和P71为引物，采用重叠延伸PCR技术(SOE)扩增。得到1512bp的PCR产物，为含替换启动子Petfu及被替换启动子Ptkt上下游 同源臂的长片段(序列12)，其中，序列12自5’末端第1-634位核苷酸为被替换启动子Ptkt的上游同源臂，序列12自5’末端第635-834位核苷酸为启动子Peftu，序列12自5’末端第835-1512位核苷酸为被替换启动子Ptkt的下游同源臂。
将上述1512bp的PCR产物经Hind III和BamH I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1724bp为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Hind III和BamH I双酶切鉴定，得到1512bp的为阳性。将阳性质粒送去测序，结果该质粒为将序列表中序列12所示的核苷酸插入载体pK18mobsacB中得到的重组质粒，命名为pK18mobsacB-Peftu::Ptkt。
上述所用的引物序列如下：
P60:CCCAAGCTTTCCAGCAACCACCTGGAT(Hind III)(序列80)
P61:AATTGGCAGCTATTAGCCTTCCTGGTTGTG(序列81)
P62:CAGGAAGGCTAATAGCTGCCAATTATTCCG(序列82)
P63:TTGTCCTCCTTTGGGTAAAAAATCCTTTCG(序列83)
P64:GATTTTTTACCCAAAGGAGGACAACCATGACTGCTCACTGGAA(序列84)
P65:CGCGGATCCCGCCATTGGGGCATCGCC(BamH I)(序列85)
P66:CCCAAGCTTTCAACGATCACTGCCCAG(Hind III)(序列86)
P67:GGGTAACGGCCAGTGTGTCTTAGAAAATG(序列87)
P68:CTAAGACACACTGGCCGTTACCCTGCGAA(序列88)
P69:TTGTCCTCCTTTTGTATGTCCTCCTGGACT(序列89)
P70:GGAGGACATACAAAAGGAGGACAACCTTGACCACCTTGACGCTG(序列90)
P71:CGCGGATCCAAGCGATCTCAGTGTTGT(BamH I)(序列91)
P72:TGTGACCCGCTACCCGATAA(序列92)
P73:CGTTACCCTGCGAATGTC(序列93)
将序列测定正确的同源重组质粒pK18mobsacB-Psod::PprsA电转化至L-组氨酸重组菌CG161中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。将阳性菌落以P72和P65为引物进行PCR扩增鉴定，得到778bp的为重组菌WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Psod::PprsA-Δpgi，命名为CG350。
将序列测定正确的同源重组质粒pK18mobsacB-Peftu::Ptkt电转化至谷氨酸棒杆菌CG350中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。将阳性菌落以P73和P71为引物进行PCR扩增鉴定，得到874bp的为重组菌WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Peftu::Ptkt-Psod::PprsA-Δpgi，命名为谷氨酸棒杆菌CG351。
经该重组菌提取基因组DNA进行测序，结果为证实已成功将L-组氨酸重组菌CG161中的tkt-tal-zwf-opcA-devB操纵子和prsA基因的启动子分别替换为谷氨酸棒杆菌内源强启动子Peftu和Psod，无质粒L-组氨酸重组菌CG351(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Peftu::Ptkt-Psod::PprsA-Δpgi)构建成功。
实施例6、无质粒L-组氨酸高产重组菌CG352和CG353的构建
在本实施例中，在以上实施例5的基础上，用强启动子Peftu替换purH基因的启动子，以提高purH编码的双功能酶AICAR甲基转移酶/IMP环水化酶(PurH，序列16)的表达，从而进一步构建CG352；然后以Phom启动子替换purF基因启动子，以弱化核苷酸合成途径中的第一酶，磷酸核糖酰胺转移酶(PurF，序列19)，从而构建CG353。
采用如上实施例5中相同的方法构建同源重组质粒pK18mobsacB-Peftu::PpurH，将purH基因的启动子替换为强启动子Peftu。根据Genbank中谷氨酸棒杆菌ATCC13032的purH基因上下游序列设计引物。以谷氨酸棒杆菌ATCC13032基因组DNA为模板，以P74和P75为引物扩增得到Peftu启动子，以P76和P77为引物扩增得到上游同源臂，以P78和P79为引物，扩增得到下游同源臂，再以纯化的上述PCR产物为模板，以P76和P79为引物，采用重叠延伸PCR技术(SOE)扩增，得到1473bp的含上下游同源臂及启动子Peftu的片段(序列20)。
其中，序列20自5’末端第1-633位核苷酸为上游同源臂，序列20自5’末端第634-833位核苷酸为Peftu，序列20自5’末端第834-1473位核苷酸为下游同源臂。
将上述1473bp的PCR产物经Xba I和Sma I双酶切后，与经同样双酶切处理的同源重组载体pK18mobsacB连接。连接产物采用化学转化法转化至大肠杆菌DH5α，在含卡那霉素(50μg/mL)的LB平板上筛选转化子，转化子传代培养三代后，以P13和P14为引物，采用菌落PCR鉴定转化子，得到1685bp为阳性转化子，对鉴定正确的转化子提取质粒，并对质粒进行Xba I和Sma I双酶切鉴定，得到1473bp的为阳性。
将阳性质粒送去测序，结果该质粒为将序列表中序列20所示的核苷酸插入载体pK18mobsacB中得到的重组质粒，命名为pK18mobsacB-Peftu::PpurH。
P74:CTGGAGAGGCTAATGGCCGTTACCCTGCGAA(序列94)
P75:ATCATCGCTCATTGTATGTCCTCCTGGACT(序列95)
P76:GCTCTAGAATGATGGTTCCGAGGCCG(Xba I)(序列96)
P77：GGGTAACGGCCATTAGCCTCTCCAGTTGAG(序列97)
P78:GGAGGACATACAATGAGCGATGATCGTAAG(序列98)
P79：TCCCCCGGGTGGTGCCGATCCAACCTG(Sma I)(序列99)
将序列测定正确的同源重组质粒pK18mobsacB-Peftu::PpurH电转化至L-组氨酸重组菌工程菌CG351中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生两次同源重组的阳性菌落。将阳性菌落提取基因组DNA，以提取的基因组DAN为模板，以P74和P79为引物进行PCR扩增，得到840bp为阳性克隆，经测序验证， 已成功将L-组氨酸重组菌CG351中的purH基因的启动子替换为谷氨酸棒杆菌内源强启动子Peftu，无质粒L-组氨酸重组菌CG352(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Peftu::Ptkt-Psod::PprsA-Δpgi-Peftu::PpurH)构建成功。
将实施例4中制备的序列测定正确的同源重组质粒pK18mobsacB-Phom::PpurF电转化至谷氨酸棒杆菌CG352中，通过卡那霉素抗性正向筛选得到重组质粒整合至染色体上的菌落，通过蔗糖反向筛选，得到发生第二次同源重组的菌落。以P56和P59为引物，对菌落进行基因组DNA提取及PCR扩增鉴定，得到905bp的为阳性(见图8)，命名为CG353(WT-PglyA::PhisEG-hisGfbr-PglyA::PhisDCB-Peftu::Ptkt-Psod::PprsA-Δpgi-Peftu::PpurH-Phom::PpurF)。
CG353经进一步序列测定分析，结果为工程菌CG352的染色体purF基因启动子替换为Phom，CG353构建成功。
实施例7、L-组氨酸工程菌在生产L-组氨酸中的应用
一、含有质粒的高产L-组氨酸的重组菌发酵
1、含有质粒的高产L-组氨酸工程菌摇瓶发酵
摇瓶发酵采用的发酵培养基具体如下：葡萄糖40g/L，(NH4)2SO4 20g/L，KH2PO4 0.5g/L，K2HPO4·3H2O 0.5g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H2O 0.01g/L，MnSO4·H2O 0.01g/L，ZnSO4·7H2O 0.001g/L，CuSO4 0.0002g/L，NiCl2·6H2O 0.00002g/L，生物素0.0002g/L，pH 7.0-7.2，CaCO3 20g/L。葡萄糖单独灭菌，115℃高压灭菌15min。MgSO4·7H2O以及无机盐离子单独灭菌，121℃高压灭菌20min。维生素采用0.22μm无菌滤膜过滤除菌。其余组分在121℃高压灭菌20min。
种子培养基具体如下：葡萄糖20g/L，硫酸铵5g/L，K2HPO4·3H2O 1g/L，MgSO4·7H2O 0.4g/L，生物素50μg，维生素B1 1mg，安琪酵母粉(FM802)10g/L，安琪蛋白胨(FP318)10g/L。
1)、种子液的获得
将上述实施例2制备的工程菌CG176，CG172，CG173和CG171分别接种到种子培养基中，种子液培养条件为培养温度32℃，摇床转速为220r/min，培养时间8h，得到种子液，OD600为20。
2)、发酵
将种子液按照体积百分含量为3％接种到含有终浓度为10μg/ml氯霉素的发酵培养基(500mL挡板三角瓶装液量为30mL)中，32℃，220r/min，培养72h，并于发酵培养6h时加入终浓度为1mmol/L的异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行目的基因的诱导表达。间歇补加浓氨水控制发酵液的pH在7.0-7.2之间，根据残糖情况，补加浓度为400g/L的葡萄糖母液，控制发酵液残糖在5-10g/L。
收集发酵产物12000×g，离心5min，收集上清液。
3)、检测L-组氨酸含量
采用高效液相法，具体方法如下(2,4-二硝基氟苯柱前衍生高效液相法)：取50μL上述上清液于2mL离心管中，加入200μL NaHCO3水溶液(0.5mol/L，pH 9.0)和100μL 1％的2,4-二硝基氟苯-乙腈溶液(体积比)，于60℃水浴中暗处恒温加热60min，然后冷却至25℃，加入650μL KH2PO4水溶液(0.01mol/L，pH 7.2±0.05，用NaOH水溶液调整pH)，放置15min过滤后可进样，进样量为15μL。
所用色谱柱为C18柱(ZORBAX Eclipse XDB-C18，4.6*150mm，Agilent，USA)；柱温：40℃；紫外检测波长：360nm；流动相A为0.04mol/L KH2PO4水溶液(pH 7.2±0.05，用40g/L KOH水溶液调整pH)，流动相B为55％乙腈水溶液(体积比)，流动相流速为1mL/min，洗脱过程如下表1所示：
表1
Figure PCTCN2015072220-appb-000001
以野生型菌株C.glutamicum ATCC13032为对照，测定发酵过程中的葡萄糖消耗、OD600以及最终的L-组氨酸产量。结果如表2所示。
表2为摇瓶发酵实验中L-组氨酸工程菌CG160，CG176，CG172，CG173和CG171的葡萄糖消耗、最大OD600，比生长速率和L-组氨酸产量。
表2
Figure PCTCN2015072220-appb-000002
摇瓶发酵实验中，发酵72h，野生型菌株C.glutamicum ATCC13032未检测到L-组氨酸的积累，底盘菌CG160的L-组氨酸产量为0.03g/L。仅进行L-组氨酸终端代谢途径改造的底盘工程菌CG176的L-组氨酸产量为1.18g/L。在此基础上，单独缺失pgi基因的工程菌CG172的L-组氨酸产量为0.77g/L；单独过表达zwf-opcA的工程菌CG173的L-组氨酸产量为1.50g/L。 缺失pgi基因的同时过表达zwf-opcA的工程菌CG171的L-组氨酸产量为2.40g/L，较单独缺失pgi基因的工程菌CG172产量提高了2.1倍，与单独过表达zwf-opcA基因的菌株CG173相比提高了60％，与仅进行L-组氨酸终端代谢途径改造的菌株CG176相比提高了102％。
2、L-组氨酸工程菌CG171、CG319和CG328发酵罐发酵生产L-组氨酸
种子培养基具体如下：葡萄糖20g/L，硫酸铵5g/L，K2HPO4·3H2O 1g/L，MgSO4·7H2O 0.9g/L，生物素50μg，维生素B1 1mg，安琪酵母粉(FM802)2g/L，安琪蛋白胨(FP318)2g/L。
发酵采用的发酵培养基具体如下：葡萄糖20g/L，硫酸铵5g/L，KH2PO4 0.5g/L，K2HPO4·3H2O 0.5g/L，MgSO4·7H2O 0.25g/L，FeSO4·7H2O 10mg/L，MnSO4·H2O 10mg/L，维生素B1 0.5mg/L，安琪酵母粉(FM802)5g/L。
1)、种子液的获得
将工程菌CG171、CG319和CG328接种到种子培养基中，种子液培养条件为培养温度32℃，摇床转速为220r/min，培养时间8h，得到种子液，OD600为20。
2)、发酵
将种子液按照体积百分含量为10％接种到含有终浓度10μg/ml氯霉素的发酵培养基中。
采用的发酵罐为7.5L发酵罐(BioFlo115，NBS)：内置定速可编程控泵，可以实现恒速补料。发酵过程中通过蠕动泵补加600g/L的葡萄糖，控制发酵体系中葡糖糖的浓度为5～10g/L，同时流加10g/L的安琪酵母粉(FM802)。通过加热套和冷却水控制发酵温度维持在32℃；通入空气提供溶氧，转速与溶氧信号级联控制溶氧维持在30％；补加浓氨水调控pH，维持在6.9左右。发酵连续进行52h。当OD600＝4～5时，加入IPTG(异丙基硫代半乳糖苷，终浓度为0.5mmol/L)诱导重组质粒携带的基因表达。
收集发酵产物12000×g离心5min，收集上清液。
3)、检测L-组氨酸含量
按照上述1中3)的方法检上清液中的L-组氨酸含量，结果如下表所示，工程菌CG171的L-组氨酸最高产量为10.87g/L，生产强度为0.21g/L/h；工程菌CG319的L-组氨酸最高产量为14.15g/L，生产强度为0.30g/L/h；工程菌CG328的L-组氨酸最高产量为15.96g/L，生产强度为0.32g/L/h。结果见下表3。
表3
Figure PCTCN2015072220-appb-000003
由上表可以看出，发酵罐实验显示CG171菌株取得了很好的效果，组氨酸产量在发酵52小时达到了10.87g/L。而进一步过表达purH的工程菌CG319，以及进一步同时过表达purH 及弱化purF的工程菌CG328相对于CG171，在发酵时间更短的情况下，组氨酸产量分别提高了约30％和50％。也就说在弱化pgi和过表达zwf-opcA的基础上，将组氨酸合成途径与核苷酸合成途径偶联，拉动组氨酸合成途径的代谢流量，进一步使组氨酸的产量大大提高。
二、无质粒L-组氨酸工程菌CG350、CG351、CG352和CG353摇瓶发酵生产L-组氨酸
工程菌CG350、CG351、CG352和CG353种子液的获得和摇瓶发酵方法同上述一中所述，不同的是发酵过程中不需要加入氯霉素和诱导剂IPTG。L-组氨酸含量检测同本实施例第一项中所述。以野生型菌株C.glutamicum ATCC13032为对照。
摇瓶发酵实验中，发酵72h，野生型菌株C.glutamicum ATCC13032未检测到L-组氨酸的积累，单独缺失pgi基因构建的工程菌CG350的L-组氨酸产量为0.65g/L，在此基础上同时提高zwf-opcA的表达量构建的工程菌CG351的L-组氨酸产量为1.86g/L，相对于单独缺失pgi基因的工程菌CG350提高了186％。进一步提高purH基因表达的菌株CG352的L-组氨酸产量为2.23g/L，更进一步降低purF基因表达的菌株CG353的L-组氨酸产量为2.34g/L。结果见下表4。
表4
Figure PCTCN2015072220-appb-000004

Claims (25)

  1. 一种产L-氨基酸的重组菌，所述重组菌相比于出发菌具有降低的6-磷酸葡萄糖异构酶Pgi的表达，和提高的6-磷酸葡萄糖脱氢酶Zwf-OpcA的表达，其中所述出发菌是能够累积目的氨基酸的菌株，优选地，所述目的氨基酸是L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸。
  2. 根据权利要求1所述的重组菌，其中重组菌的染色体上的pgi基因已失活，优选已被敲除，或者pgi基因的调控元件已被替换为低转录或低表达活性的调控元件，同时所述重组菌中具有两个或更多个拷贝的zwf-opcA基因，或以强启动子代替所述出发菌染色体上tkt-tal-zwf-opcA-devB操纵子的启动子，优选地，所述强启动子为原始菌的Peftu启动子。
  3. 根据权利要求1所述的重组菌，其中所述出发菌相对于原始菌具有增强的L-组氨酸合成操纵子hisEG基因和hisDCB基因的表达，优选以强启动子代替所述hisEG基因和hisDCB基因的启动子，更优选以所述原始菌染色体上的PglyA启动子分别代替hisEG基因和hisDCB基因的启动子；
    进一步优选地，所述出发菌相比于原始菌具有增强的PRPP合成酶PrsA的表达，更优选地，所述出发菌中具有两个或更多个拷贝的prsA基因，或者以强启动子代替prsA基因的启动子，优选所述强启动子为所述原始菌的Psod启动子。
  4. 根据权利要求1所述的重组菌，其中，
    所述出发菌相对于原始菌具有增强的dapA基因或lysC基因的表达；或者
    所述出发菌相比于原始菌具有增强的缬氨酸合成基因ilvBNCE的表达；或者
    所述出发菌相比于原始菌具有增强的苏氨酸合成途径基因hom和thrB的表达；或者
    所述出发菌相比于原始菌具有增强的ocd基因的表达；或者
    所述出发菌相比于原始菌具有增强的p4hD基因的表达。
  5. 根据权利要求3所述的重组菌，其中所述重组菌相比于所述出发菌具有增强的AICAR甲基转移酶/IMP环水化酶PurH的表达；
    优选地，所述重组菌中具有两个或更多个拷贝的purH基因，或者以强启动子替换purH基因的启动子，优选所述强启动子为所述原始菌的Peftu启动子。
  6. 根据权利要求3或5所述的重组菌，其中所述重组菌相比于所述出发菌具有减弱的磷酸核糖酰胺转移酶PurF的表达；
    优选地，以弱启动子代替purF基因的启动子，更优选所述弱启动子为所述原始菌中的 Phom启动子。
  7. 根据权利要求1～6中任一项所述的重组菌，其中所述原始菌为选自棒杆菌属、小杆菌属、短杆菌属中的一株细菌，
    优选地，所述棒杆菌属的细菌选自谷氨酸棒杆菌Corynebacterium glutamicum、北京棒杆菌Corynebacterium pekinense、有效棒杆菌Corynebacterium efficiens、钝齿棒杆菌Corynebacterium crenatum、嗜热产氨棒杆菌Corynebacterium thermoaminogenes、产氨棒杆菌Corynebacterium aminogenes、百合棒杆菌Corynebacterium lilium、美棒杆菌Corynebacterium callunae和力士棒杆菌Corynebacterium herculis中的一株细菌；
    所述小杆菌属的细菌选自嗜氨小杆菌Microbacterium ammoniaphilum中的一株细菌；和
    所述短杆菌属的细菌选自黄色短杆菌Brevibacteriaceae flvum、乳酸发酵短杆菌Brevibacteriaceae lactofermentum和产氨短杆菌Brevibacteriaceae ammoniagenes中的一株细菌。
  8. 根据权利要求7所述的重组菌，其中所述原始菌为野生型谷氨酸棒杆菌ATCC13032。
  9. 根据权利要求8所述的重组菌，其中所述出发菌的染色体上具有序列7中5’末端第863-1038位核苷酸序列所示的PglyA启动子用于分别代替所述谷氨酸棒杆菌ATCC13032染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子，和
    所述出发菌能够表达突变的ATP-磷酸核糖转移酶，所述突变的ATP-磷酸核糖转移酶为序列6所示的ATP-磷酸核糖转移酶的第215位天冬酰胺突变为赖氨酸、第231位亮氨酸突变为苯丙氨酸和第235位苏氨酸突变为丙氨酸的酶，优选地，所述出发菌的染色体上具有序列4中第1007-1852位核苷酸序列所示的hisGfbr基因用于代替所述谷氨酸棒杆菌ATCC13032染色体上的hisG基因；
    优选地，所述出发菌的染色体上具有序列11中5’末端第656-847位核苷酸序列所示的Psod启动子用于代替所述谷氨酸棒杆菌ATCC13032染色体上的prsA基因的启动子；
    或者，所述出发菌中具有两个或更多个拷贝的prsA基因和hisGfbr基因，
    其中所述prsA基因选自编码序列5所示的PrsA的基因；和编码与所述PrsA相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、进一步优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有PrsA活性的基因中的一个，优选地，所述prsA基因为序列表中序列4所示的第15-992位的核苷酸序列。
  10. 根据权利要求9所述的重组菌，其中所述pgi基因选自编码序列表中序列14所示Pgi的基因；和编码与所述Pgi相比具有至少60％同源性、优选具有至少70％同源性、更优选具 有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述6-磷酸葡萄糖异构酶活性的基因中的一个，优选地，所述pgi基因为序列13所示的核苷酸序列，
    所述zwf-opcA基因选自编码序列表中序列3所示的Zwf-OpcA的基因；和编码与所述Zwf-OpcA相比具有有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述Zwf-OpcA活性的基因中的一个，优选地，所述zwf-opcA基因为序列2所示的核苷酸序列，和
    所述Peftu启动子是序列12所示5’末端第635-834位的核苷酸序列。
  11. 根据权利要求10所述的方法，其中所述purH基因选自编码序列表中序列16所示的PurH的基因；和编码与所述PurH相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述PurH活性的基因中的一个，优选地，所述purH基因为序列表中序列15所示的核苷酸序列。
  12. 根据权利要求11所述的方法，其中所述Phom启动子是序列18所示5’末端第736-865位的核苷酸序列。
  13. 一种构建产L-氨基酸的重组菌的方法，包括如下步骤：降低出发菌中6-磷酸葡萄糖异构酶Pgi的表达，且提高所述出发菌中6-磷酸葡萄糖脱氢酶Zwf-OpcA的表达，得到所述重组菌，
    其中，所述出发菌是能够积累目的氨基酸的菌株，更优选地，所述目的L-氨基酸是L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸。
  14. 根据权利要求13所述的方法，其中，
    所述降低出发菌中Pgi的表达通过如下A)或B)方式实现：
    A)失活所述出发菌染色体的pgi基因；优选所述失活为敲除；
    B)将所述出发菌中的pgi基因的调控元件替换为低转录和低表达活性的调控元件实现，和
    所述提高所述出发菌中Zwf-OpcA的表达通过如下C)或D)方式实现：
    C)增加所述出发菌中zwf-opcA基因的拷贝数；
    D)将所述出发菌染色体上的tkt-tal-zwf-opcA-devB操纵子的启动子替换为强启动子，优选所述强启动子为所述原始菌染色体上的Peftu启动子。
  15. 根据权利要求13所述的方法，其中获得所述出发菌包括将原始菌染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子分别替换为强启动子的步骤，优选所述强启动子为所述原始菌染色体上的PglyA启动子；
    优选地，获得所述出发菌进一步包括提高所述出发菌中PRPP合成酶PrsA的表达的步骤；
    更优选地，所述提高所述出发菌中PrsA的表达通过如下E)或F)方式实现：
    E)增加所述出发菌中prsA基因的拷贝数；
    F)将所述出发菌染色体上的prsA基因的启动子替换为强启动子，优选所述强启动子为所述原始菌染色体上的Psod启动子。
  16. 根据权利要求13所述的方法，其中获得所述出发菌包括：
    增强表达dapA基因或lysC基因的步骤；或者
    增强表达缬氨酸合成基因ilvBNCE的步骤；或者
    增强表达苏氨酸合成途径基因hom和thrB的步骤；或者
    增强表达ocd基因的步骤；或者
    增强表达p4hD基因的步骤。
  17. 根据权利要求15所述的方法，其中所述方法进一步包括提高所述重组菌中AICAR甲基转移酶/IMP环水化酶PurH的表达的步骤；
    优选地，所述提高所述重组菌中PurH的表达通过如下G)或H)方式实现：
    G)增加所述出发菌中purH基因的拷贝数；
    H)将所述出发菌染色体上的purH基因的启动子替换为强启动子，优选所述强启动子为所述原始菌染色体上的Peftu启动子。
  18. 根据权利要求15或17所述的方法，其中所述方法进一步包括弱化所述重组菌中磷酸核糖酰胺转移酶PurF的表达的步骤；
    优选地，所述弱化所述重组菌中PurF的表达通过以弱启动子代替purF基因的启动子的方式实现，更优选将所述出发菌中染色体上的purF基因的启动子替换为所述原始菌中染色体上的Phom启动子。
  19. 根据权利要求13～18中任一所述的方法，其中，用于获得所述出发菌的原始菌为选自棒杆菌属、小杆菌属、短杆菌属中的一株细菌，
    优选地，所述棒杆菌属的细菌选自谷氨酸棒杆菌Corynebacterium glutamicum、北京棒杆菌Corynebacterium pekinense、有效棒杆菌Corynebacterium efficiens、钝齿棒杆菌Corynebacterium crenatum、嗜热产氨棒杆菌Corynebacterium thermoaminogenes、产氨棒杆菌Corynebacterium aminogenes、百合棒杆菌Corynebacterium lilium、美棒杆菌Corynebacterium  callunae和力士棒杆菌Corynebacterium herculis中的一株细菌；
    所述小杆菌属的细菌选自嗜氨小杆菌Microbacterium ammoniaphilum中的一株细菌；和
    所述短杆菌属的细菌选自黄色短杆菌Brevibacteriaceae flvum、乳酸发酵短杆菌Brevibacteriaceae lactofermentum和产氨短杆菌Brevibacteriaceae ammoniagenes中的一株细菌。
  20. 根据权利要求19所述的方法，其中所述原始菌为野生型谷氨酸棒杆菌ATCC13032。
  21. 根据权利要求20所述的方法，其中，获得所述出发菌包括以下步骤：
    将所述谷氨酸棒杆菌ATCC13032染色体上的L-组氨酸合成操纵子hisEG和hisDCB的启动子分别替换为序列7中5’末端第863-1038位核苷酸序列所示的PglyA启动子，和
    将所述谷氨酸棒杆菌ATCC13032表达的序列6所示的ATP-磷酸核糖转移酶的第215位天冬酰胺突变为赖氨酸、第231位亮氨酸突变为苯丙氨酸和第235位苏氨酸突变为丙氨酸，优选地，用于进行上述突变的基因为序列4中第1007-1852位核苷酸序列所示的hisGfbr基因；
    优选地，获得所述出发菌进一步包括以下步骤：
    将所述谷氨酸棒杆菌ATCC13032染色体上的prsA基因的启动子替换为序列11中5’末端第656-847位核苷酸序列所示的Psod启动子；
    或者，进一步包括以下步骤：
    增加所述谷氨酸棒杆菌ATCC13032中prsA基因的拷贝数，和
    增加所述谷氨酸棒杆菌ATCC13032中hisGfbr基因的拷贝数，
    其中所述prsA基因选自编码序列5所示的PrsA的基因；和编码与所述PrsA相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、进一步优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有PrsA活性的基因中的一个，优选地，所述prsA基因为序列表中序列4所示的第15-992位的核苷酸序列。
  22. 根据权利要求21所述的方法，其中，所述pgi基因选自编码序列表中序列14所示Pgi的基因；和编码与所述Pgi相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性，且具有所述6-磷酸葡萄糖异构酶活性的基因中的一个，优选地，所述pgi基因为序列13所示的核苷酸序列，
    所述zwf-opcA基因选自编码序列表中序列3所示的Zwf-OpcA的基因；和编码与所述Zwf-OpcA相比具有有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述Zwf-OpcA活性的基因中的一个，优选地，所述zwf-opcA基因为序列2所示的核苷酸序列，和
    所述Peftu启动子是序列12所示5’末端第635-834位的核苷酸序列。
  23. 根据权利要求22所述的方法，其中所述purH基因选自编码序列表中序列16所示的PurH的基因；和编码与所述PurH相比具有至少60％同源性、优选具有至少70％同源性、更优选具有至少80％同源性、更为优选具有至少95％同源性、甚至更优选具有至少98％、甚至99％同源性，且具有所述PurH活性的基因中的一个，优选地，所述purH基因为序列表中序列15所示的核苷酸序列。
  24. 根据权利要求23所述的方法，其中所述Phom启动子是序列18所示5’末端第736-865位的核苷酸序列。
  25. 一种生产L-氨基酸的方法，包括发酵培养权利要求1～12中任一项所述的重组菌的步骤，所述L-氨基酸优选地为L-组氨酸、L-赖氨酸、L-缬氨酸、L-苏氨酸、L-脯氨酸或L-羟脯氨酸。
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