WO2021238184A1 - 生产l-缬氨酸的重组微生物、其构建方法及应用 - Google Patents

生产l-缬氨酸的重组微生物、其构建方法及应用 Download PDF

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WO2021238184A1
WO2021238184A1 PCT/CN2020/137779 CN2020137779W WO2021238184A1 WO 2021238184 A1 WO2021238184 A1 WO 2021238184A1 CN 2020137779 W CN2020137779 W CN 2020137779W WO 2021238184 A1 WO2021238184 A1 WO 2021238184A1
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gene
microorganism
valine
dna fragment
homologous recombination
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French (fr)
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张学礼
郭恒华
刘萍萍
张冬竹
唐金磊
韩成秀
唐思青
刘树蓬
马延和
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安徽华恒生物科技股份有限公司
中国科学院天津工业生物技术研究所
巴彦淖尔华恒生物科技有限公司
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Priority to EP20930653.9A priority Critical patent/EP3943595A4/en
Priority to US17/604,770 priority patent/US20230084158A1/en
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    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Definitions

  • the present invention relates to a method for constructing a recombinant microorganism for producing L-valine, the recombinant microorganism obtained by the constructing method, specifically recombinant Escherichia coli, and a method for producing L-valine through a fermentation method.
  • L-valine is an essential amino acid, which cannot be synthesized in humans and animals, and can only be obtained by supplementation from the outside world.
  • L-valine is widely used in the fields of food and medicine, mainly including food additives, nutritional supplements and flavoring agents; widely used in the preparation of cosmetics, as well as antibiotics or herbicide precursors; in addition, with With the increasing demand for feed quality and ratio, L-valine will play an increasingly important role in the feed additive industry in the future, the demand will increase, and the future market has great potential.
  • L-valine is mainly produced through fermentation in the world.
  • the production strains used for fermentation are mostly derived by mutagenesis, and the starting strains mainly include Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium flavum and so on. For example, Chen Ning et al.
  • Sang Yup Lee's research team started from Escherichia coli W3110 in 2007, combined with rational metabolic engineering, transcriptome analysis, genetic modification, and gene knockout to obtain an engineered strain capable of producing L-valine.
  • the strain is produced in batch culture under aerobic conditions, and continuous oxygen is required during the culture process, and L-isoleucine needs to be added during the culture process to ensure normal cell growth.
  • the research group used a similar strategy to reconstruct a new strain of L-valine engineered bacteria from Escherichia coli W, which is more resistant to L-valine. This strain also needs to be cultured under aerobic conditions.
  • the invention solves the problem of L-valine fermentation process by enhancing the amino acid dehydrogenase activity of L-valine fermentation strains and/or activating Entner-Doudoroff (ED) pathway
  • ED Entner-Doudoroff
  • the first aspect of the present invention provides a method for constructing a recombinant microorganism of L-valine.
  • the recombinant microorganism obtained by this method has a stable genetic background and a balanced L-valine reducing power, which is suitable for one step Method anaerobic fermentation.
  • the "enhancement" of enzyme activity refers to the increase of the intracellular activity of one or more enzymes in the microorganism that has the corresponding DNA encoding.
  • the enhancement of the activity can be achieved by any suitable method known in the art, for example, by Expression, including but not limited to increasing the copy number of the gene or allele, modifying the nucleotide sequence guiding or controlling gene expression, using a strong promoter to increase protein activity or concentration by 10%-500% compared to the initial microbial level .
  • activation refers to the production or enhancement of the metabolic pathway to be activated by means known in the art, such as the introduction of related genes on the metabolic pathway into the microorganism to generate the metabolic pathway; or The related genes of the metabolic pathway to be activated are placed under the control of appropriate regulatory elements, for example, placed under the enhancer sequence, so that the expression intensity of these genes is increased.
  • it includes introducing an amino acid dehydrogenase gene into the microorganism, so that the enzyme activity is enhanced.
  • the enzyme activity is enhanced by introducing an amino acid dehydrogenase gene into the microorganism.
  • a step of activating the ED pathway in the microorganism is included.
  • it includes the steps of introducing an amino acid dehydrogenase gene into the microorganism and activating the ED pathway in the microorganism.
  • the amino acid dehydrogenase gene introduced in the present invention is foreign to the introduced microorganism.
  • the amino acid dehydrogenase gene may be a corresponding gene from any microorganism such as Lactococcus, Bacillus and the like.
  • the amino acid dehydrogenase gene is NADH-dependent.
  • glucose metabolism is mainly through the glycolysis pathway (EMP pathway).
  • EMP pathway glycolysis pathway
  • 1 mol of glucose is metabolized to produce 2 mol of pyruvate
  • 2 mol of ATP and 2 mol of NADH are generated. Therefore, this leads to the problem of unbalanced supply of redox power in metabolically engineered Escherichia coli under anaerobic conditions, that is, the supply of NADH is excessive and the supply of NADPH is insufficient. Therefore, in order to efficiently produce L-valine under anaerobic conditions, the problem of cofactor imbalance must be solved.
  • the present invention selects NADH-dependent amino acid dehydrogenase gene to consume excess NADH under anaerobic conditions, and solves the problem of reducing power balance during anaerobic fermentation.
  • the amino acid dehydrogenase gene is a leucine dehydrogenase gene, preferably, the leucine dehydrogenase gene is leuDH.
  • the activation of the ED pathway includes increasing the 6-phosphate glucose dehydrogenase (zwf) gene, the lactonase encoding gene (pgl), the 6-phosphogluconate dehydratase gene (edd), and 2-ketone. -3-deoxy-6-phosphogluconate aldolase gene (eda) expression intensity step.
  • the expression intensity of the genes can be increased by increasing the copy number of the zwf, pgl, edd and eda genes, or by placing these genes under the control of appropriate regulatory elements.
  • the method further includes the step of introducing a gene encoding acetohydroxy acid reductoisomerase into the microorganism, so that the enzyme activity is enhanced; preferably, the gene encoding acetohydroxy acid reductoisomerase is selected from ilvC.
  • the "introduction" can be present in the microorganism in any suitable manner known in the art, for example, in the form of a plasmid or integrated into the genome.
  • the enzyme-encoding gene integrated into the genome is placed under the control of a suitable regulatory element.
  • the control element is selected from M1-93 artificial control element, MRS1 artificial control element, RBS artificial control element or M1-46 artificial control element;
  • the M1-93 artificial regulatory element regulates the leuDH gene, zwf gene and pgl gene;
  • the MRS1 artificial regulatory element regulates the edd gene.
  • the RBS artificial regulatory element regulates the gene eda.
  • the M1-46 artificial regulatory element regulates the ilvC gene.
  • the present invention also includes the step of knocking out the 6-phosphoglucokinase gene (pfkA) of the above-mentioned recombinant microorganism.
  • pfkA 6-phosphoglucokinase gene
  • the present invention also includes modifying one or more of the following enzyme genes of the above-mentioned recombinant microorganism as follows to reduce or inactivate the activity of these enzymes.
  • knockout refers to gene knockout using methods known in the art, so that the activity of the enzyme is reduced or inactivated.
  • the knock-out operation is aimed at the endogenous enzyme gene of the starting microorganism, so that the above-mentioned endogenous enzyme activity of the microorganism is reduced or inactivated.
  • the gene to replace these endogenous enzymes may be a gene to be expressed to be enhanced, such as the aforementioned ilvC gene or leuDH gene.
  • it further includes:
  • AHAS is selected from ilvBN, ilvGM or ilvIH, and the activity of at least one of them is enhanced.
  • the ilvBN and ilvGM genes can be placed under the control of appropriate regulatory elements to increase the expression of the genes.
  • the control element is preferably an M1-93 artificial control element.
  • the activity of ilvIH is enhanced by releasing the feedback inhibition of ilvIH by valine, for example, by mutating the ilvH gene to relieve the feedback inhibition of ilvIH by valine.
  • AHAS III isozyme III
  • AHASIII is encoded by the ilvIH operon, which is composed of ilvI encoding the large subunit (catalytic subunit) and ilvH encoding the small subunit (control subunit).
  • AHASIII is feedback inhibition by L-valine.
  • the ilvI gene can be mutated using reported methods, such as the amino acid substitution of ilvH 14Gly ⁇ Asp (Vyazmensky, M.
  • the activity of dihydroxy acid dehydratase (ilvD) in the recombinant microorganism of the present invention is enhanced, for example, by introducing the ilvD gene into the microorganism to enhance the activity of ilvD.
  • the operation is performed in conjunction with the modification of item (2).
  • the operation is performed in conjunction with the modification of item (6).
  • the operation is performed in conjunction with the modification of the items (2) and (5).
  • the operation is performed in conjunction with the modification of the items (2) and (6).
  • the operation is performed in conjunction with the modification of items (1) and (3)-(6).
  • the operation is performed in conjunction with the modification of items (1)-(6).
  • the endogenous mgsA gene of the microorganism is replaced with the ilvC gene to achieve the knockout of item (1).
  • the endogenous pflB gene of the microorganism is replaced with the ilvD gene, and/or the endogenous frd gene of the microorganism is replaced with the leuDH gene to achieve the knockout of item (6).
  • the replacement can integrate the coding sequence of the gene to be inserted into the replaced gene coding sequence site in the microbial chromosome in a manner known to those skilled in the art, so that the original site gene coding sequence is integrated into the inserted gene The coding sequence is replaced.
  • the replacement of ilvC, ilvD and leuDH occurs simultaneously.
  • the microorganism is Escherichia coli.
  • the microorganism is Escherichia coli ATCC 8739.
  • At least one regulatory element is used to regulate the genes of the aforementioned enzymes involved.
  • the control element is selected from M1-93 artificial control element, MRS1 artificial control element, RBS artificial control element or M1-46 artificial control element;
  • the M1-93 artificial regulatory element regulates the ilvD, leuDH, ilvBN, zwf, pgl, and ilvGM genes.
  • the MRS1 artificial regulatory element regulates the edd gene.
  • the RBS artificial regulatory element regulates the gene eda.
  • the M1-46 artificial regulatory element regulates the ilvC gene.
  • Regulatory elements can be inserted upstream of the gene by known genetic engineering methods.
  • the method includes, but is not limited to, inserting the sequence of the regulatory element into the upstream of the gene coding sequence of the target enzyme by means of gene recombination, for example, by means of homologous recombination, so as to enhance the intensity of expression of the target gene.
  • the gene encoding the enzyme and the regulatory element are integrated into the genome of the microorganism.
  • a plasmid containing the enzyme encoding gene and the regulatory element sequence is introduced into the microorganism.
  • the introduction, mutation or knockout of the target enzyme gene is accomplished by integrating into the genome of the microorganism.
  • the introduction, mutation or knockout of the enzyme gene is accomplished by the method of homologous recombination.
  • the introduction, mutation or knockout of the enzyme gene is accomplished by a two-step homologous recombination method.
  • Homologous recombination systems known in the art can be used, such as E. coli RecA recombination system, Red recombination system for homologous recombination to achieve the introduction, mutation or knockout of target genes.
  • the two-step homologous recombination method to introduce, mutate or knock out the target gene includes the following steps (take E. coli as an example):
  • DNA fragment I Using pXZ-CS plasmid (Tan, et al., Appl Environment Microbiol, 2013, 79: 4838-4844) DNA as a template, use amplification primer 1 to amplify DNA fragment I, and use In the first step, homologous recombination;
  • DNA fragment II was amplified with the amplification primer 2. DNA fragment II is used for the second homologous recombination.
  • the second step of homologous recombination transform the DNA fragment II into the colony obtained in the second step; use the detection primer 2 to verify the transformed bacteria and select the colony.
  • the second aspect of the present invention provides a recombinant microorganism for producing L-valine obtained by the above construction method, specifically a recombinant E. coli, which contains acetohydroxy acid isoreductase and/ Or the amino acid dehydrogenase gene,
  • E. coli ATCC 8739 is used as the starting strain, and the intracellular cofactor NADH supply and cell growth are coupled through genetic homologous recombination, so as to achieve cell growth and L-valine production under anaerobic conditions. Coupling ( Figure 1).
  • the recombinant Escherichia coli obtained by the above-mentioned construction method undergoes metabolic evolution, for example, 50 generations, 70 generations, 80 generations, 90 generations, 100 generations, and 120 generations to obtain recombinant L-valine-producing recombinants.
  • Escherichia coli In one embodiment, after 70 generations of metabolic evolution, a recombinant Escherichia coli strain producing L-valine was obtained, its preservation number is: CGMCC 19457, and its classification is named: Escherichia coli, biological materials
  • the deposit was submitted on March 6, 2020.
  • the depository unit General Microbiology Center of China Microbial Culture Collection Management Committee. The deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing.
  • the third aspect of the present invention is the application of the recombinant microorganism obtained by the above method in the production of L-valine.
  • the fourth aspect of the present invention is a method for fermentatively producing L-valine by using the recombinant microorganism obtained by the above construction, which includes: (1) fermentation and cultivation of the recombinant microorganism obtained by the construction; (2) separating and harvesting L-valine.
  • the fermentation culture is anaerobic fermentation culture.
  • the anaerobic fermentation includes the following steps:
  • Seed culture Pick the clones on the plate and inoculate them into the seed culture medium, and culture them with shaking at 37°C to obtain the seed culture solution;
  • Fermentation culture inoculate the seed culture liquid in the fermentation medium, ferment for 4 days at 37° C., 150 rpm, to obtain the fermentation liquid. Control the pH of the fermenter at 7.0. No gas is allowed to pass through the cultivation process.
  • the seed culture medium consists of the following components (the solvent is water): glucose 20g/L, corn steep liquor 10g/L, KH 2 PO4 8.8g/L, (NH 4 ) 2 SO 4 2.5g/L, MgSO 4 ⁇ 7H 2 O 2g/L.
  • Fermentation medium and seed medium have the same composition, the only difference is that the glucose concentration is 50g/L.
  • the present invention realizes the production of L-valine by one-step anaerobic fermentation, which reduces the production cost and improves the conversion rate.
  • the present invention preferably constructs a stable genetic L-valine production strain on the genome of the recombinant microorganism rather than in the form of a plasmid, without additional addition of antibiotics and inducers and other substances, and the production process is stable and easy to operate.
  • the recombinant Escherichia coli Sval049 constructed in the present invention is classified and named as Escherichia coli (Escherichia coli), and the biological material has been submitted for preservation on March 6, 2020.
  • the preservation unit the General Microbiology Center of China Microbial Culture Collection Management Committee, The preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355, and the preservation number is CGMCC No.19457.
  • Figure 1 L-valine synthesis pathway.
  • Figure 2 The standard for the determination of L-valine by high performance liquid chromatography.
  • FIG. 1 HPLC determination of Sval048 strain fermentation broth components.
  • FIG. 4 Metabolic evolution fermentation culture obtained Sval049 strain.
  • Figure 5 Standards for the determination of L-valine by high performance liquid chromatography.
  • FIG. 6 HPLC determination of Sval049 strain fermentation broth components.
  • strains and plasmids constructed in this study are shown in Table 1, and the primers used are shown in Table 2.
  • Example 1 Knockout of the mgsA gene encoding methylglyoxal synthase in ATCC 8739 strain
  • the primer mgsA-cs-up/mgsA-cs-down was used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system is: Phusion 5X buffer (New England Biolabs) 10 ⁇ l, dNTP (each dNTP 10 mM) 1 ⁇ l, DNA template 20ng, primer (10 ⁇ M) each 2 ⁇ l, Phusion High-Fidelity DNA polymerase (New England Biolabs) (2.5 U/ ⁇ l) 0.5 ⁇ l, distilled water 33.5 ⁇ l, the total volume is 50 ⁇ l.
  • the amplification conditions were 98°C pre-denaturation for 2 minutes (1 cycle); 98°C denaturation for 10 seconds, 56°C annealing for 10 seconds, 72°C extension for 2 minutes (30 cycles); 72°C extension for 10 minutes (1 cycle).
  • the electrotransformation conditions are as follows: first prepare electrotransformation competent cells of E. coli ATCC 8739 with pKD46 plasmid; place 50 ⁇ l of competent cells on ice, add 50ng DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2cm Bio -Rad shock cup. A MicroPulser (Bio-Rad company) electroporator was used, and the shock parameter was 2.5kv. After the electric shock, quickly transfer 1ml of LB medium to the electric shock cup, pipette 5 times and then transfer it to the test tube, incubate at 75 rpm and 30°C for 2 hours.
  • the genomic DNA of wild-type E. coli ATCC 8739 is used as a template, and the 566bp DNA fragment II is amplified with the primer XZ-mgsA-up/mgsA-del-down. DNA fragment II is used for the second homologous recombination.
  • the amplification conditions and system are the same as those described in the first step.
  • the DNA fragment II was electroporated into the strain Sval001.
  • Electrotransformation conditions are as follows: first prepare electrotransformation competent cells of Sval001 with pKD46 plasmid (Dower et al., 1988, Nucleic Acids Res 16:6127-6145); place 50 ⁇ l of competent cells on ice and add 50ng DNA fragments II. Place on ice for 2 minutes and transfer to a 0.2cm Bio-Rad shock cup. A MicroPulser (Bio-Rad company) electroporator was used, and the shock parameter was 2.5kv. After the electric shock, quickly transfer 1ml of LB medium to the electric shock cup, pipette 5 times and transfer to the test tube, incubate at 30°C for 4 hours at 75 revolutions.
  • Example 2 Knockout of the gene encoding ldhA for lactate dehydrogenase
  • the primer ldhA-cs-up/ldhA-cs-down was used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1. Electrotransfer DNA fragment I to Sval002.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval002 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval002 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-ldhA-up/XZ-ldhA-down was verified. The correct PCR product should be 3448bp. Pick a correct single colony and name it Sval003.
  • DNA fragment II was amplified with primers XZ-ldhA-up/ldhA-del-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electrotransformed to the strain Sval003.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-ldhA-up/XZ-ldhA-down, and the correct colony amplification product was a 829bp fragment.
  • a correct single colony was selected and named Sval004 (Table 1).
  • Example 3 Knockout of phosphoacetyltransferase encoding gene pta and acetate kinase encoding gene ackA
  • the pXZ-CS plasmid DNA is used as a template, and the primer ackA-cs-up/pta-cs-down is used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1. Electrotransfer DNA fragment I to Sval004.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval004 by electroporation, and then the DNA fragment I was electrotransformed into the E. coli Sval004 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-ackA-up/XZ-pta-down for verification. The correct PCR product should be 3351bp. Pick a correct single colony and name it Sval005.
  • DNA fragment II was amplified with primers XZ-ackA-up/ackA-del-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electroporated to the strain Sval005.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-ackA-up/XZ-pta-down, and the correct colony amplification product was a 732bp fragment.
  • a correct single colony was selected and named Sval006 (Table 1).
  • Example 4 Knockout of propionate kinase encoding gene tdcD and formate acetyltransferase encoding gene tdcE
  • the primer tdcDE-cs-up/tdcDE-cs-down was used to amplify a 2719bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1. Electrotransfer DNA fragment I to Sval006.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval006 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval006 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-tdcDE-up/XZ-tdcDE-down was verified. The correct PCR product should be 4380bp. Pick a correct single colony and name it Sval007.
  • primer XZ-tdcDE-up/tdcDE-del-down was used to amplify a 895bp DNA fragment II.
  • DNA fragment II is used for the second homologous recombination.
  • the DNA fragment II was electrotransformed to the strain Sval007.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-tdcDE-up/XZ-tdcDE-down, and the correct colony amplification product was a 1761bp fragment.
  • a correct single colony was selected and named Sval008 (Table 1).
  • the primers adhE-cs-up/adhE-cs-down are used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1.
  • DNA fragment I was used for the first homologous recombination: first, the plasmid pKD46 was transformed into E. coli Sval008 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval008 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-adhE-up/XZ-adhE-down was verified. The correct PCR product should be 3167bp. Pick a correct single colony and name it Sval009.
  • DNA fragment II was amplified with primers XZ-adhE-up/adhE-del-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electroporated into the strain Sval009.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-adhE-up/XZ-adhE-down, and the correct colony amplification product was a 548bp fragment.
  • a correct single colony was selected and named Sval010 (Table 1).
  • Example 6 Integration of acetohydroxy acid reductoisomerase encoding gene ilvC at the mgsA site of methylglyoxal synthase encoding gene
  • the acetohydroxy acid reductoisomerase encoding gene ilvC from E. coli was integrated into the mgsA site of the methylglyoxal synthase encoding gene through a two-step homologous recombination method.
  • the specific steps include:
  • the cat-sacB fragment was integrated into the mgsA site of the Sval010 strain.
  • the PCR, integration, and verification of the cat-sacB fragment were exactly the same as the first step of mgsA gene knockout in Example 1, and the obtained clone was named Sval011.
  • DNA fragment II was amplified with the primer mgsA-ilvC-up/mgsA-ilvC-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electroporated into the strain Sval011.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-mgsA-up/XZ-mgsA-down, and the correct colony amplification product was a 2503bp fragment.
  • a correct single colony was selected and named Sval012 (Table 1).
  • the primer mgsA-Pcs-up/mgsA-Pcs-down was used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1. Electrotransfer DNA fragment I to Sval012.
  • DNA fragment I for the first homologous recombination first, pKD46 plasmid (purchased from the CGSC Escherichia coli Collection of Yale University, CGSC#7739) was transformed into E. coli Sval012 by electroporation, and then DNA fragment I was electrotransformed to Escherichia coli Sval012 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-mgsA-up/ilvC-YZ347-down was verified. The correct PCR product should be 3482bp. Pick a correct single colony and name it Sval013.
  • the second step is to use the genomic DNA of M1-46 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) as a template, and use the primer mgsA-P46-up/ilvC-P46-down to amplify 188bp ⁇ DNA Fragment II.
  • DNA fragment II is used for the second homologous recombination.
  • the DNA fragment II was electrotransformed to the strain Sval013.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primer used was XZ-mgsA-up/ilvC-YZ347-down, and the correct colony amplification product was a 951bp fragment.
  • a correct single colony was selected and named Sval014 (Table 1).
  • the dihydroxy acid dehydratase encoding gene ilvD from E. coli was integrated into the pflB site of the pyruvate formate lyase encoding gene by two-step homologous recombination method and the pflB gene was replaced, that is, knocking out at the same time as ilvD was integrated.
  • the specific steps are as follows:
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval014 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval014 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-pflB-up600/XZ-pflB-down was verified. The correct PCR product should be 3675bp. Pick a correct single colony and name it Sval015.
  • Example 9 Expression regulation of ilvD encoding dihydroxy acid dehydratase gene
  • the primers pflB-Pcs-up/pflB-Pcs-down are used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1. Electrotransfer DNA fragment I to Sval016.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval016 by electroporation, and then the DNA fragment I was electrotransformed into the E. coli Sval016 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-pflB-up600/ilvD-YZ496-down was verified. The correct PCR product should be 3756bp. Pick a correct single colony and name it Sval017.
  • DNA fragment II is used for the second homologous recombination.
  • the DNA fragment II was electrotransformed to the strain Sval017.
  • the artificial regulatory element M1-93 is used to regulate the expression of the acetolactate synthase gene ilvBN through a two-step homologous recombination method. The specific steps are as follows:
  • the first step using pXZ-CS plasmid DNA as a template, use primers ilvB pro-catup/ilvB pro-catdown to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval018 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval018 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers ilvB pro-YZup/ilvB pro-YZdown for verification. The correct PCR product should be 2996bp. Pick a correct single colony and name it Sval019.
  • the second step is to use the genomic DNA of M1-93 as a template, and use primers ilvB pro-up/ilvB pro-down to amplify a 188bp DNA fragment II.
  • DNA fragment II is used for the second homologous recombination.
  • the DNA fragment II was electroporated into the strain Sval019.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were ilvB pro-YZup/ilvB pro-YZdown.
  • the correct colony amplification product was a 465bp fragment.
  • a correct single colony was selected and named Sval020.
  • the artificial regulatory element M1-93 is used to regulate the expression of the acetolactate synthase gene ilvGM through a two-step homologous recombination method. The specific steps are as follows:
  • the first step using pXZ-CS plasmid DNA as a template, use primers ilvG pro-catup/ilvG pro-catdown to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval020 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval020 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers ilvG pro-YZup/ilvG p-YZdown for verification. The correct PCR product should be 2993bp. Pick a correct single colony and name it Sval021.
  • a 188bp DNA fragment II was amplified with primers ilvG pro-up/ilvG pro-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electrotransformed to the strain Sval021.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were ilvG pro-YZup/ilvG p-YZdown, and the correct colony amplification product was a 462 bp fragment.
  • a correct single colony was selected and named Sval022.
  • a two-step homologous recombination method is used to introduce mutations into the ilvH gene to relieve the feedback inhibition of L-valine.
  • the specific steps are as follows:
  • primers ilvH*-cat-up/ilvH*-cat-down are used to amplify a 2719bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval022 by electrotransformation, and then the DNA fragment I was electrotransformed into the E. coli Sval022 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers ilvH*-mutYZ-up/ilvH*-mut-down for verification. The correct PCR product should be 3165bp. Pick a correct single colony and name it Sval023.
  • DNA fragment II was amplified with primers ilvH*-mut-up/ilvH*-mut-down. DNA fragment II is used for the second homologous recombination. The DNA fragment II was electroporated into the strain Sval023.
  • Example 13 Fermentation and production of L-valine using recombinant strain Sval024
  • the seed culture medium consists of the following components (the solvent is water):
  • the anaerobic fermentation of Sval024 includes the following steps:
  • Seed culture Inoculate fresh clones on the LB plate into a test tube containing 4ml of seed culture medium, and cultivate overnight at 37°C with shaking at 250 rpm. Then, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed culture medium according to the inoculum amount of 2% (V/V), and the seed culture was obtained by shaking culture at 37° C. and 250 rpm for 12 hours for fermentation medium inoculation.
  • Fermentation culture The volume of the fermentation medium in a 500ml anaerobic tank is 250ml.
  • the neutralizer is 5M ammonia water to control the pH of the fermenter at 7.0. Do not pass any gas during the cultivation process.
  • Analytical method use Agilent (Agilent-1260) high performance liquid chromatograph to determine the components in the fermentation broth after 4 days of fermentation.
  • concentration of glucose and organic acids in the fermentation broth is determined using Biorad's Aminex HPX-87H organic acid analytical column.
  • Amino acid determination uses Sielc amino acid analysis column primesep 100 250 ⁇ 4.6mm.
  • Sval024 strain can produce 1.3g/L of L-valine (the L-valine peak corresponding to the position of Figure 2) after fermentation for 4 days under anaerobic conditions, and the sugar-acid conversion rate is 0.31mol/mol .
  • the leuDH gene is a leuDH sequence derived from the Lysinibacillus sphaericus IFO 3525 strain according to literature reports (Ohshima, T.et.al, Properties of crystalline leucine dehydrogenase from Bacillus sphaericus. The Journal of biological chemistry 253, 5719-5725 (1978)) Codon optimization (optimized sequence shown in sequence number 90) is obtained through full gene synthesis. During synthesis, M1-93 artificial regulatory elements are added before the leuDH gene to initiate the expression of the leuDH gene, and inserted into the pUC57 vector to construct the plasmid pUC57. -M1-93-leuDH (Nanjing GenScript Biotechnology Co., Ltd.
  • the M1-93 artificial regulatory element and the leuDH gene were integrated into the frd site of the fumarate reductase encoding gene in the Sval024 strain through a two-step homologous recombination method, and the frd gene was replaced, that is, the frd gene was knocked out while leuDH was integrated .
  • the specific steps include:
  • the pXZ-CS plasmid DNA is used as a template, and the primer frd-cs-up/frd-cs-down is used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • the amplification system and amplification conditions are the same as those described in Example 1.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval024 by electrotransformation, and then the DNA fragment I was electrotransformed into the E. coli Sval024 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers XZ-frd-up/XZ-frd-down for verification, the correct PCR product should be 3493bp. Pick a correct single colony and name it Sval025.
  • primer frd-M93-up/frd-leuDH-down was used to amplify a 1283bp DNA fragment II.
  • DNA fragment II is used for the second homologous recombination.
  • the DNA fragment II was electrotransformed to the strain Sval025.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were XZ-frd-up/XZ-frd-down, and the correct colony amplification product was a 2057bp fragment.
  • a correct single colony was selected and named Sval026.
  • Example 15 Fermentation to produce L-valine using recombinant strain Sval026
  • composition and formulation of the seed medium and the fermentation medium are the same as those described in Example 13.
  • the fermentation was carried out in a 500 mL fermentor, and the fermentation process and analysis process were the same as the fermentation process and analysis process of Sval024 described in Example 13.
  • Example 16 Regulation of the gene encoding 6-phosphate glucose dehydrogenase zwf
  • a two-step homologous recombination method uses artificial regulatory elements to regulate the expression of the zwf gene to obtain recombinant E. coli Sval041, which specifically includes the following steps:
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval026 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval026 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers zwf-YZ442-up/zwf-YZ383-down, the correct colony amplification product is a 3339bp fragment. Pick a correct single colony and name it Sval041.
  • the genomic DNA of M1-93 was used as a template, and the primers zwf-RBS1-up/zwf-RBS1-down were used to amplify the 189bp DNA fragment II, which was used in the second step of homologous recombination.
  • the amplification conditions and system are the same as those described in (1).
  • the DNA fragment II was electroporated into the strain Sval041.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were zwf-YZ442-up/zwf-YZ383-down, and the correct colony amplification product was a 809bp fragment.
  • a correct single colony was selected and named Sval042 (Table 1).
  • a two-step homologous recombination method uses artificial regulatory elements to regulate the expression of pgl gene to obtain recombinant E. coli Sval042, which specifically includes the following steps:
  • the first step using pXZ-CS plasmid DNA as a template, using primers pgl-Pcat-up/pgl-PsacB-down to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval033 by electrotransformation, and then the DNA fragment I was electrotransformed into E. coli Sval042 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers For verification with pgl-YZ308-up/pgl-YZ341-down, the correct PCR product should be 3248 bp. Pick a correct single colony and name it Sval043.
  • the second step is to use the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) as a template, and use primers pgl-RBS2-up/pgl-RBS2-down to amplify to obtain 189bp DNA fragment II of, used in the second step of homologous recombination.
  • the amplification conditions and system are the same as those described in (1).
  • the DNA fragment II was electrotransformed to the strain Sval043.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were pgl-YZ308-up/pgl-YZ341-down, and the correct colony amplification product was a 718bp fragment.
  • a correct single colony was selected and named Sval044 (Table 1).
  • Example 18 Cloning and integration of edd and eda genes from Zymomonas mobilis mobilis ZM4 strain
  • the Zymomonas mobilis mobilis ZM4-derived edd and eda gene sequences are synthesized through the full gene synthesis of Zymomonas mobilis mobilis edd and eda genes derived from ZM4, where the edd gene is preceded by the artificial regulatory element MRS1, and the edd and eda genes are connected by the artificial RBS element (CAGGAAACAGCT).
  • the fully synthesized MRS1-edd-RBS-eda fragment was ligated into the pUC57 vector via EcoRV.
  • the first step is to use pXZ-CS plasmid (Tan, et al., Appl Environment Microbiol, 2013, 79: 4838-4844) DNA as a template, and use primers edd-cat-up/eda-sacB-down to amplify 2719bp DNA fragment I, used for the first step of homologous recombination.
  • the DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into E. coli Sval044 by electroporation, and then the DNA fragment I was electrotransformed into the E. coli Sval044 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers edd-YZ-up/edd-YZ-down for verification. The correct PCR product should be 3123bp. Pick a correct single colony and name it Sval045.
  • the plasmid DNA of pUC57-MRS1-edd-eda was used as a template, and the primers Edd-int-up/Eda-int-down were used to amplify DNA fragment II of 2651 bp, which was used in the second step of homologous recombination.
  • the amplification conditions and system are the same as those described in (1).
  • the DNA fragment II was electrotransformed to the strain Sval045.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were edd-YZ-up/edd-YZ-down, and the correct colony amplification product was a 3055bp fragment.
  • a correct single colony was selected and named Sval046 (Table 1).
  • the first step is to use pXZ-CS plasmid (Tan, et al., Appl Environment Microbiol, 2013, 79: 4838-4844) DNA as a template, and use primers pfkAdel-cat-up/pfkAdel-sacB-down to amplify 2719bp DNA fragment I, used for the first step of homologous recombination.
  • DNA fragment I for the first homologous recombination first use the pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the plasmid was purchased from the CGSC Escherichia coli Collection Center of Yale University, CGSC#7739) Transform into Escherichia coli Sval046 by electroporation, and then electrotransform DNA fragment I into Escherichia coli Sval046 with pKD46.
  • the electroporation conditions and steps are the same as the first step method for mgsA gene knockout described in Example 1. Take 200 ⁇ l of bacterial solution and apply it on the LB plate containing ampicillin (final concentration of 100 ⁇ g/ml) and chloramphenicol (final concentration of 34 ⁇ g/ml). After incubating at 30°C overnight, select a single colony for PCR verification, using primers pfkAdel-up/pfkAdel-YZ-down for verification. The correct PCR product should be 3145bp. Pick a correct single colony and name it Sval047.
  • the second step is to use wild-type ATCC 8739 genomic DNA as a template, and use primers pfkAdel-up/pfkAdel-down to amplify a 379 bp DNA fragment II, which is used in the second step of homologous recombination.
  • the amplification conditions and system are the same as those described in (1).
  • the DNA fragment II was electroporated into the strain Sval047.
  • the electroporation conditions and steps are the same as the second step method for mgsA gene knockout described in Example 1.
  • Colony PCR was used to verify the clone.
  • the primers used were pfkAdel-up/pfkAdel-YZ-down, and the correct colony amplification product was a 526 bp fragment.
  • a correct single colony was selected and named Sval048 (Table 1).
  • Example 20 Using recombinant strain Sval048 to produce L-valine
  • composition and formulation of the seed medium and the fermentation medium are the same as those described in Example 13.
  • the fermentation was carried out in a 500 mL fermentor, and the fermentation process and analysis process were the same as the fermentation process and analysis process of Sval024 described in Example 13.
  • the metabolic evolution process uses a 500ml fermentor, and the fermentation medium is 250ml. Use 5M ammonia water as the neutralizer to control the pH of the fermenter at 7.0.
  • the composition and formulation of the fermentation medium used for evolutionary metabolism are the same as those described in Example 18 for the fermentation medium. Every 24 hours, transfer the fermentation broth to a new fermentor so that the initial OD550 reaches 0.1. After 100 generations of evolution, the strain Sval049 was obtained ( Figure 4).
  • the preservation number of Sval049 strain is CGMCC 19457, and it is preserved in China Common Microorganism Collection Management Center (CGMCC).
  • Example 22 Fermentation of recombinant strain Sval049 to produce L-valine in a 500mL fermentor
  • composition and preparation of the seed culture medium are the same as those described in Example 13.
  • Fermentation is carried out in a 500mL fermentor, and the fermentation medium is 250ml.
  • the fermentation medium is basically the same as the seed medium. The difference is that the glucose concentration is 100g/L, and the neutralizer used is 5M ammonia, so that the pH of the fermenter is controlled at 7.0.
  • Example 23 The recombinant strain Sval049 was fermented to produce L-valine in a 5L fermentor
  • the composition, preparation and analysis method of the seed culture medium are the same as those described in Example 13.
  • the fermentation medium is basically the same as the seed medium, except that the glucose concentration is 140g/L.
  • the fermentation is carried out anaerobic in a 5L fermentor (Shanghai Baoxing, BIOTECH-5BG), including the following steps:
  • Seed culture 150ml seed culture medium in a 500ml Erlenmeyer flask, sterilized at 115°C for 15min. After cooling, the recombinant E. coli Sval041 was inoculated into the seed culture medium at an inoculum of 1% (V/V), and cultivated at 37° C. and 100 rpm for 12 hours to obtain the seed liquid, which was used to inoculate the fermentation medium.

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Abstract

生产L-缬氨酸的重组微生物、其构建方法及应用，通过增强L-缬氨酸发酵菌株的氨基酸脱氢酶活性，和/或激活恩特纳-杜德洛夫代谢(Entner-Doudoroff，ED)途径，解决了L-缬氨酸发酵过程中的还原力不平衡的问题，从而提高大肠杆菌生产L-缬氨酸的产量和转化率，并实现一步法厌氧发酵L-缬氨酸。

Description

生产L-缬氨酸的重组微生物、其构建方法及应用 技术领域
本发明涉及生产L-缬氨酸的重组微生物的构建方法，所述构建方法获得的重组微生物，具体是重组大肠杆菌，以及通过发酵法生产L-缬氨酸的方法。
背景技术
L-缬氨酸作为三大支链氨基酸(Branched-chain amino acid，BCAA)的一种，属于必需氨基酸，在人和动物中不能合成，只能通过从外界补充获得。目前，L-缬氨酸被广泛用于食品和医药领域，主要包括食品添加剂、营养增补液及风味剂等；广泛用于化妆品制备、以及用作抗生素或者除草剂前体等；另外，随着对饲料质量和配比需求的提高，在未来L-缬氨酸在饲料添加剂行业中的作用将越来越重要，需求量会越来越大，未来市场具有极大的潜力。
微生物细胞可以直接合成L-缬氨酸，但胞内大量反馈抑制等调控网络极大地限制了野生细胞的生产能力。要想获得能够高效生产L-缬氨酸的发酵菌株，必须有效解除微生物细胞这些自我调节机制。目前，世界范围内L-缬氨酸主要通过发酵法生产获得。用于发酵的生产菌株多通过诱变而来，出发菌株主要包括谷氨酸棒杆菌、黄色短杆菌、黄色短杆菌等。如陈宁等以黄色短杆菌为出发菌株，使用原生质体紫外诱变并结合DES化学诱变的策略，筛选获得了一株L-缬氨酸高产菌TV2564，L-缬氨酸产量高达29.39g/L。但是，传统诱变获得的菌株具有很强的随机性，并且遗传背景不清晰，副产物多，并且不容易进一步通过改造获得更加高产的菌株。
近年来，随着合成生物学和代谢工程的迅速发展，通过遗传改造获得能够高效生产L-缬氨酸、遗传背景清晰、且易于培养的重组工程菌株应运而生并取得了很好的效果。谢希贤等整合枯草芽孢杆菌乙酰乳酸合酶的编码基因alsS，解除了L-缬氨酸对合成通路的反馈抑制，同时整合大肠杆菌ppGpp3’-焦磷酸水解酶的突变基因spoTM，增强了丙酮酸供应，增强了出发菌株VHY03的摇瓶发酵生产L-缬氨酸的水平。Sang Yup Lee课题组2007年从大肠杆菌W3110出发，结合理性代谢工程、转录组分析和遗传改造、以及基因敲除等手段获得了一株能够生产L-缬氨酸的工程菌。该菌株在好氧条件下批式培养(batch culture)以生产，培养过程中需要持续通氧，且培养过程中需要添加L-异亮氨酸保证细胞正常生长。进一步的，课题组使用类似的策略从对L-缬氨酸具有更高耐受性的大肠杆菌W出发重新构建了1株新的L-缬氨酸工程菌。该菌株同样需要在好氧条件下培养。
上述报道虽然提高了L-缬氨酸的发酵产量，但是L-缬氨酸的生产过程多是好氧或者微好氧条件下完成的，无法实现一步法厌氧发酵。好氧工艺在生产过程中需要用空气，能耗很大。更关键的是，有相当一部分碳源进入三羧酸循环(TCA)从而被用于细胞生长，导致转化率比理论最大值要低很多。因此好氧工艺成本高，且对设备有要求，操作步骤繁杂。厌氧工艺和好氧相比，具有低能耗、高转化率的优点，生产过程中不需要通空气，大大节约能耗；产 品的转化率通常接近理论最大值。氨基酸生产的厌氧发酵最早是在丙氨酸生产过程中实现的，目前其他氨基酸的生产目前未有纯厌氧发酵工艺的报道。
另外，L-缬氨酸工程菌改造过程中关键基因的过表达都是通过外源质粒转化宿主菌实现，这就导致发酵过程中需要添加抗生素维持质粒存在，极大的增加了生产成本并存在在产业化生产中质粒丢失的风险。
因此，本领域仍需要提供高产、节能、简便、稳定的生产L-缬氨酸的重组微生物以及相应的L-缬氨酸的生产制备方法。
发明内容
本发明通过增强L-缬氨酸发酵菌株的氨基酸脱氢酶活性，和/或激活恩特纳-杜德洛夫代谢(Entner-Doudoroff，ED)途径，解决了L-缬氨酸发酵过程中的还原力不平衡的问题，从而提高大肠杆菌生产L-缬氨酸的产量和转化率，并实现一步法厌氧发酵L-缬氨酸。
本发明的第一个方面，是提供一种L-缬氨酸的重组微生物的构建方法，经该方法获得的重组微生物具有稳定遗传背景、且具备平衡的L-缬氨酸还原力，适宜一步法厌氧发酵。
在本申请中，酶的活性“增强”是指提高微生物中有相应的DNA编码的一种或多种酶的胞内活性，增强活性可以通过本领域已知的任何合适方法实现，例如通过过表达，包括但不限于提高所述基因或等位基因的拷贝数，修饰指导或控制基因表达的核苷酸序列，使用强启动子使蛋白质活性或浓度比起始微生物水平提高10％-500％。
在本申请中，“激活”是指通过本领域已知的方式使得所述的待激活的代谢途径产生或增强，如通过向微生物中导入代谢途径上的相关基因，使得代谢途径产生；或通过将待激活的代谢途径的相关基因置于合适的调控元件的控制下，例如置于增强子序列下，以使得这些基因的表达强度提高。
在一个实施方式中，包括向所述微生物中导入氨基酸脱氢酶基因，使得所述酶活性增强。
在一个优选的实施方式中，通过向所述微生物中导入氨基酸脱氢酶基因，使得所述酶活性增强。
在一个实施方式中，包括激活微生物中的ED途径的步骤。
在某些优选的实施方式中，包括向微生物中导入氨基酸脱氢酶基因，以及激活微生物中的ED途径的步骤。
在一个实施方式中，本发明导入的氨基酸脱氢酶基因对于被导入的微生物而言是外源的。所述氨基酸脱氢酶基因可以是来自任何微生物例如乳球菌、芽孢杆菌等的相应基因。
在一个实施方式中，所述氨基酸脱氢酶基因是NADH依赖型的。
在厌氧发酵时，葡萄糖的代谢主要是通过糖酵解途径(EMP途径)，1mol葡萄糖代谢生产 2mol丙酮酸的同时，会生成2molATP和2molNADH。因此，这就导致了厌氧条件下代谢工程改造的大肠杆菌中出现氧化还原力供给不平衡的问题，即NADH过剩，而NADPH供给不足。因此为实现在厌氧条件下高效生产L-缬氨酸，须解决辅因子不平衡问题。本发明选择NADH依赖型的氨基酸脱氢酶基因能够使得厌氧条件下过剩的NADH得到消耗，解决厌氧发酵时还原力平衡的问题。
在一个实施方式中，所述氨基酸脱氢酶基因是亮氨酸脱氢酶基因，优选地，所述的亮氨酸脱氢酶基因是leuDH。
在一个实施方式中，所述激活ED途径包括提高6-磷酸葡萄糖脱氢酶(zwf)基因、内酯酶编码基因(pgl)、6-磷酸葡糖酸脱水酶基因(edd)和2-酮-3-脱氧-6-磷酸葡糖酸醛缩酶基因(eda)的表达强度的步骤。
本领域技术人员能够理解，可通过增加zwf、pgl、edd和eda基因的拷贝数，或者通过将这些基因置于合适的调控元件的控制下，提高所述基因的表达强度。
在一个实施方式中，还包括向微生物中导入乙酰羟基酸还原异构酶编码基因的步骤，使得所述酶活性增强；优选地，所述乙酰羟基酸还原异构酶编码基因选自ilvC。
所述的“导入”可以以本领域已知的任何合适方式，例如以质粒形式，或者整合入基因组中的形式存在与所述微生物中。在一个实施方式中，所述整合入基因组中的酶编码基因置于合适的调控元件的控制下。
所述调控元件选自M1-93人工调控元件、MRS1人工调控元件、RBS人工调控元件或M1-46人工调控元件；
在一个实施方式中，M1-93人工调控元件调控leuDH基因、zwf基因和pgl基因；
在一个实施方式中，MRS1人工调控元件调控edd基因。
在一个实施方式中，RBS人工调控元件调控基因eda。
在一个实施方式中，M1-46人工调控元件调控ilvC基因。
在一个实施方式中，本发明还包括敲除上述重组微生物的6-磷酸葡萄糖激酶基因(pfkA)的步骤。
在一个实施方式中，本发明还包括对上述重组微生物的以下酶基因中的一种或几种进行如下改造，以使得这些酶的活性降低或失活。
(1)敲除甲基乙二醛合酶(mgsA)基因；
(2)敲除乳酸脱氢酶(ldhA)基因；
(3)敲除磷酸乙酰转移酶(pta)和/或乙酸激酶(ackA)基因；
(4)敲除丙酸激酶(tdcD)和/或甲酸乙酰转移酶(tdcE)基因；
(5)敲除醇脱氢酶(adhE)基因；
(6)敲除富马酸还原酶(frd)和/或丙酮酸甲酸裂解酶(pflB)基因。
本申请所述“敲除”，是指用本领域已知的方式进行基因敲除，使得所述酶的活性被降低或失活。所述的敲除操作针对的是出发微生物内源性的酶基因，使得微生物的上述内源性酶活性降低或失活。
还可以通过同源重组等基因工程的方式，以另一基因的编码序列取代上述(1)-(6)中所述酶基因的编码序列，从而使得微生物的上述内源性酶活性降低或失活。替代这些内源性酶的基因可以是待增强表达的基因，如上述的ilvC基因或leuDH基因。
在一个实施方式中，还包括：
(7)增强本发明的重组微生物中的乙酰乳酸合成酶(AHAS)和/或二羟酸脱水酶(ilvD)的活性。
在一个优选地实施方式中，AHAS选自ilvBN、ilvGM或ilvIH，它们中至少一种酶的活性被增强。
在一个优选地实施方式中，可以通过将ilvBN、ilvGM基因置于合适的调控元件的控制下，以使得所述基因的表达增强。所述调控原件优选M1-93人工调控元件。
通过在一个优选地实施方式中，ilvIH的活性通过解除缬氨酸对ilvIH的反馈抑制得到增强，例如通过突变ilvH基因解除缬氨酸对ilvIH的反馈抑制。
就涉及L-缬氨酸生物合成的乙酰羟酸合成酶而言，除了同功酶Ⅱ(这里也称为AHASⅡ)，还知道有同功酶Ⅲ(这里也称为AHASⅢ)。AHASⅢ由ilvIH操纵子编码，该操纵子由编码大亚基(催化亚基)的ilvI和编码小亚基(控制亚基)的ilvH组成。AHASⅢ受L-缬氨酸的反馈抑制。可采用已报道的方法突变ilvI基因，例如ilvH 14Gly→Asp的氨基酸取代(Vyazmensky,M.等，《生物化学》35:10339-10346(1996))和/或ilvH 17Ser→Phe(US6737255B2)；以及ilvH612(De Felice等，《细菌学杂志》120:1058-1067(1974))等。
在一个实施方式中，本发明的重组微生物中的二羟酸脱水酶(ilvD)的活性被增强，例如通过向微生物中导入ilvD基因增强ilvD的活性。
第(7)项操作，任选地结合上述(1)-(6)任一项或几项改造进行。
在一个实施方式中，结合第(2)项改造进行操作。
在一个实施方式中，结合第(6)项改造进行操作。
在一个实施方式中，结合第(2)项和第(5)项改造进行操作。
在一个实施方式中，结合第(2)项和第(6)项改造进行操作。
在一个实施方式中，结合第(1)项、第(3)-(6)项改造进行操作。
在一个实施方式中，结合第(1)-(6)项改造进行操作。
在一个实施方法中，任选地，以ilvC基因替换微生物内源性的mgsA基因来实现第(1)项敲除。
在一个实施方式中，以ilvD基因替换微生物内源性的pflB基因，和/或以leuDH基因替换微生物内源性的frd基因来实现第(6)项敲除。
所述替换可以本领域技术人员已知的方式，将待插入的基因的编码序列整合到所述微生物染色体中被替换的基因编码序列位点，使得原位点基因编码序列被整合插入的基因的编码序列所取代。
优选地，ilvC、ilvD和leuDH的替换同时发生。
在一个实施方式中，所述的微生物为大肠杆菌。
在一个实施方式中，所述的微生物为大肠杆菌ATCC 8739。
在一个实施方式中，使用至少一个调控元件调控上述涉及的酶的基因。
所述调控元件选自M1-93人工调控元件、MRS1人工调控元件、RBS人工调控元件或M1-46人工调控元件；
在一个实施方式中，M1-93人工调控元件调控ilvD、leuDH、ilvBN、zwf、pgl和ilvGM基因。
在一个实施方式中，MRS1人工调控元件调控edd基因。
在一个实施方式中，RBS人工调控元件调控基因eda。
在一个实施方式中，M1-46人工调控元件调控ilvC基因。
调控元件可通过已知的基因工程方法插入所述基因的上游。所述方法包括但不限于以基因重组的方式，例如以同源重组的方式调控元件的序列插入目标酶的基因编码序列上游，以增强目标基因表达的强度。
在一个实施方式中，其中所述酶编码基因和所述的调控元件整合入所述微生物的基因组中。
在一个实施方式中，将包含所述述酶编码基因和所述的调控元件序列的质粒导入所述微生物中。
在一个实施例中，以整合入所述微生物的基因组的方法完成目标酶基因的导入、突变或敲除。
在一个实施例中，以同源重组的方法完成所述酶基因的导入、突变或敲除。
在一个实施例中，以两步同源重组的方法完成所述酶基因的导入、突变或敲除。
可采用本领域已知的同源重组系统，如大肠杆菌RecA重组系统，Red重组系统进行同源重组以实现目标基因的导入、突变或敲除。
以两步同源重组的方法导入、突变或敲除目标基因包括如下步骤(以大肠杆菌为例)：
(1)DNA片段I的制备：以pXZ-CS质粒(Tan,et al.,Appl Environ Microbiol,2013,79:4838-4844)DNA为模板，使用扩增引物1扩增出DNA片段I，用于第一步同源重组；
(2)第一步同源重组：将pKD46质粒(Datsenko and Wanner 2000,Proc Natl Acad Sci USA 97:6640-6645)转化至大肠杆菌，然后将DNA片段I转至pKD46的大肠杆菌，使用检测引物1验证转化的菌并挑选菌落；
(3)DNA片段II的制备：以出发大肠杆菌为模板，用扩增引物2扩增出DNA片段II。DNA片段II用于第二次同源重组。
(4)第二步同源重组：将DNA片段II转化至第二步挑选获得的菌落；使用检测引物2验证转化的菌并挑选菌落。
本发明的第二个方面，是提供了一种利用上述构建方法得到的用于生产L-缬氨酸的重组微生物，具体是一种重组大肠杆菌，其包含乙酰羟酸异构还原酶和/或氨基酸脱氢酶基因，
在一个实施方式中，采用大肠杆菌ATCC 8739作为出发菌株，通过基因同源重组实现胞内辅因子NADH供给和细胞生长的偶联，从而实现厌氧条件下细胞生长和L-缬氨酸生产的偶联(图1)。
在一个实施方式中，利用上述构建方法得到的重组大肠杆菌，进一步经过代谢进化，经过例如50代、70代、80代、90代、100代、120代获得了高产L-缬氨酸的重组大肠杆菌。在一个实施方式中，经70代代谢进化获得了一株产L-缬氨酸的重组大肠杆菌，其保藏号为：CGMCC 19457，分类命名为：大肠埃希氏菌(Escherichia coli)，生物材料已于2020年3月6日提交保藏，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号。
本发明的第三方面，是上述方法获得的重组微生物在生产L-缬氨酸中的应用。
本发明的第四方面，是利用上述构建获得的重组微生物发酵生产L-缬氨酸的方法，包括：(1)发酵培养构建获得的重组微生物；(2)分离并收获L-缬氨酸。
在一个实施方式中，所述发酵培养为厌氧发酵培养。
在一个实施方式中，所述厌氧发酵包括如下步骤：
(1)种子培养：挑取平板上的克隆接种到种子培养基中，37℃，振荡培养，获得种子培养液；
(2)发酵培养：将种子培养液接种于发酵培养基，37℃，150rpm，发酵4天，得到发酵液。控制发酵罐的pH在7.0。培养过程不通任何气体。
其中种子培养基由以下成分组成(溶剂为水)：葡萄糖20g/L，玉米浆干粉10g/L，KH 2PO4 8.8g/L、(NH 4) 2SO 4 2.5g/L、MgSO 4·7H 2O 2g/L。
发酵培养基和种子培养基成分相同，区别仅在于葡萄糖浓度为50g/L。
本发明的有益效果：
(1)相对于之前的生产方法和菌株，本发明实现了一步法厌氧发酵生产L-缬氨酸，降低了生产成本、提高了转化率。
(2)本发明优选对重组微生物的基因组而非以质粒形式构建稳定遗传的L-缬氨酸生产菌株，不需要额外添加抗生素和诱导剂等物质，生产工艺稳定易操作。
(3)通过代谢进化方式，提高了重组微生物的L-缬氨酸的产量和转化率以及细胞耐受性。
生物材料保藏
本发明构建的重组大肠杆菌Sval049，分类命名为：大肠埃希氏菌(Escherichia coli)，生物材料已于2020年3月6日提交保藏，保藏单位：中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，电话：010-64807355，保藏编号为CGMCC No.19457。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1：L-缬氨酸合成途径。
图2：高效液相色谱测定L-缬氨酸的标准品。
图3：高效液相色谱测定Sval048菌株发酵液组分。
图4：代谢进化发酵培养获得Sval049菌株。
图5：高效液相色谱测定L-缬氨酸的标准品。
图6：高效液相色谱测定Sval049菌株发酵液组分。
具体实施方式
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改 和改变也包含在本发明的范围内。
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。
本研究中所构建的菌株和质粒详见表1，所用引物详见表2。
表1 本发明所用的菌株和质粒
Figure PCTCN2020137779-appb-000001
Figure PCTCN2020137779-appb-000002
表2 本发明所用的引物
Figure PCTCN2020137779-appb-000003
Figure PCTCN2020137779-appb-000004
Figure PCTCN2020137779-appb-000005
Figure PCTCN2020137779-appb-000006
Figure PCTCN2020137779-appb-000007
实施例1：ATCC 8739菌株中甲基乙二醛合酶编码基因mgsA的敲除
从大肠杆菌ATCC 8739出发，采用两步同源重组的方法敲除甲基乙二醛合酶编码基因mgsA，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物mgsA-cs-up/mgsA-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。
扩增体系为：Phusion 5X缓冲液(NewEngland Biolabs)10μl、dNTP(每种dNTP各10 mM)1μl、DNA模板20ng、引物(10μM)各2μl、Phusion High-Fidelity DNA聚合酶(NewEngland Biolabs)(2.5U/μl)0.5μl、蒸馏水33.5μl，总体积为50μl。
扩增条件为98℃预变性2分钟(1个循环)；98℃变性10秒、56℃退火10秒、72℃延伸2分钟(30个循环)；72℃延伸10分钟(1个循环)。
将上述DNA片段I用于第一次同源重组：首先将pKD46质粒(购买于美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌ATCC 8739，然后将DNA片段I电转至带有pKD46的大肠杆菌ATCC 8739。
电转条件为：首先准备带有pKD46质粒的大肠杆菌ATCC 8739的电转化感受态细胞；将50μl感受态细胞置于冰上，加入50ng DNA片段I，冰上放置2分钟，转移至0.2cm的Bio-Rad电击杯。使用MicroPulser(Bio-Rad公司)电穿孔仪，电击参数为电压2.5kv。电击后迅速将1ml LB培养基转移至电击杯中，吹打5次后转移至试管中，75rpm，30℃孵育2小时。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，所用引物XZ-mgsA-up/XZ-mgsA-down，正确的菌落扩增产物为3646bp的片段，挑选一个正确的单菌落，命名为Sval001。
第二步，野生型大肠杆菌ATCC 8739的基因组DNA为模板，用引物XZ-mgsA-up/mgsA-del-down扩增出566bp的DNA片段II。DNA片段II用于第二次同源重组。扩增条件和体系同第一步中所述。将DNA片段II电转至菌株Sval001。
电转条件为：首先准备带有pKD46质粒的Sval001的电转化感受态细胞(Dower et al.,1988,Nucleic Acids Res 16:6127-6145)；将50μl感受态细胞置于冰上，加入50ng DNA片段II，冰上放置2分钟，转移至0.2cm的Bio-Rad电击杯。使用MicroPulser(Bio-Rad公司)电穿孔仪，电击参数为电压2.5kv。电击后迅速将1ml LB培养基转移至电击杯中，吹打5次后转移至试管中，75转，30℃孵育4小时。将菌液转移至含有10％蔗糖的没有氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24小时后在含有6％蔗糖的没有氯化钠的LB固体培养基上划线培养。经过PCR验证，所用引物为XZ-mgsA-up/XZ-mgsA-down，正确的菌落扩增产物为1027bp的片段，挑选一个正确的单菌落，将其命名为Sval002(表1)。
实施例2：乳酸脱氢酶编码基因ldhA的敲除
从Sval002出发，通过两步同源重组的方法敲除乳酸脱氢酶编码基因ldhA，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ldhA-cs-up/ldhA-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Sval002。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval002，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval002。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液 涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ldhA-up/XZ-ldhA-down进行验证，正确的PCR产物应该3448bp，挑选一个正确的单菌落，命名为Sval003。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物XZ-ldhA-up/ldhA-del-down扩增出476bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval003。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-ldhA-up/XZ-ldhA-down，正确的菌落扩增产物为829bp的片段，挑选一个正确的单菌落，将其命名为Sval004(表1)。
实施例3：磷酸乙酰转移酶编码基因pta和乙酸激酶编码基因ackA的敲除
从Sval004出发，通过两步同源重组的方法敲除磷酸乙酰转移酶编码基因pta和乙酸激酶编码基因ackA，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ackA-cs-up/pta-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Sval004。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval004，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval004。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-ackA-up/XZ-pta-down进行验证，正确的PCR产物应该3351bp，挑选一个正确的单菌落，命名为Sval005。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物XZ-ackA-up/ackA-del-down扩增出371bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval005。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-ackA-up/XZ-pta-down，正确的菌落扩增产物为732bp的片段，挑选一个正确的单菌落，将其命名为Sval006(表1)。
实施例4：丙酸激酶编码基因tdcD和甲酸乙酰转移酶编码基因tdcE的敲除
从Sval006出发，通过两步同源重组的方法敲除丙酸激酶编码基因tdcD和甲酸乙酰转移酶编码基因tdcE，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物tdcDE-cs-up/tdcDE-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Sval006。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval006，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval006。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-tdcDE-up/XZ-tdcDE-down进行验证，正确的PCR产物应该4380bp，挑选一个正确的单菌落，命名为Sval007。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物XZ-tdcDE-up/tdcDE-del-down扩增出895bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval007。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-tdcDE-up/XZ-tdcDE-down，正确的菌落扩增产物为1761bp的片段，挑选一个正确的单菌落，将其命名为Sval008(表1)。
实施例5：醇脱氢酶基因adhE的敲除
从Sval008出发，通过两步同源重组的方法敲除醇脱氢酶基因adhE，具体步骤包括：
第一步，以pXZ-CS质粒DNA为模板，使用引物adhE-cs-up/adhE-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval008，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval008。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-adhE-up/XZ-adhE-down进行验证，正确的PCR产物应该3167bp，挑选一个正确的单菌落，命名为Sval009。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物XZ-adhE-up/adhE-del-down扩增出271bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval009。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-adhE-up/XZ-adhE-down，正确的菌落扩增产物为548bp的片段，挑选一个正确的单菌落，将其命名为Sval010(表1)。
实施例6：乙酰羟基酸还原异构酶编码基因ilvC在甲基乙二醛合酶编码基因mgsA位点的整合
从Sval010出发，通过两步同源重组的方法将来自大肠杆菌的乙酰羟基酸还原异构酶编码基因ilvC整合到甲基乙二醛合酶编码基因mgsA位点，具体步骤包括：
第一步，在Sval010菌株中mgsA位点整合cat-sacB片段，cat-sacB片段的PCR、整合、验证同实施例1中mgsA基因敲除的第一步完全一致，获得的克隆命名为Sval011。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物mgsA-ilvC-up/mgsA-ilvC-down扩增出1576bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval011。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-mgsA-up/XZ-mgsA-down，正确的菌落扩增产物为2503bp的片段，挑选一个正确的单菌落，将其命名为Sval012(表1)。
实施例7：乙酰羟基酸还原异构酶编码基因ilvC的调控
从Sval012出发，使用人工调控元件调控整合在甲基乙二醛合酶编码基因mgsA位点的乙酰羟基酸还原异构酶编码基因ilvC的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物mgsA-Pcs-up/mgsA-Pcs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Sval012。
将DNA片段I用于第一次同源重组：首先将pKD46质粒(购买于美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌Sval012，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval012。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-mgsA-up/ilvC-YZ347-down进行验证，正确的PCR产物应该3482bp，挑选一个正确的单菌落，命名为Sval013。
第二步，以M1-46(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物mgsA-P46-up/ilvC-P46-down扩增出188bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval013。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-mgsA-up/ilvC-YZ347-down，正确的菌落扩增产物为951bp的片段，挑选一个正确的单菌落，将其命名为Sval014(表1)。
实施例8：二羟酸脱水酶编码基因ilvD的整合
从Sval014出发，通过两步同源重组的方法将来自大肠杆菌的二羟酸脱水酶编码基因ilvD整合到丙酮酸甲酸裂解酶编码基因pflB位点并替换掉pflB基因，即在整合ilvD的同时敲除pflB基因，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物pflB-CS-up/pflB-CS-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA 片段I电转至Sval014。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval014，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval014。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-pflB-up600/XZ-pflB-down进行验证，正确的PCR产物应该3675bp，挑选一个正确的单菌落，命名为Sval015。
第二步，以大肠杆菌MG1655(来自ATCC，编号700926)的基因组DNA为模板，用引物pflB-ilvD-up/pflB-ilvD-down扩增出1951bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval015。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-pflB-up600/XZ-pflB-down，正确的菌落扩增产物为2907bp的片段，挑选一个正确的单菌落，将其命名为Sval016(表1)。
实施例9：二羟酸脱水酶编码基因ilvD的表达调控
从Sval016出发，使用人工调控元件调控整合在丙酮酸甲酸裂解酶编码基因pflB位点的二羟酸脱水酶编码基因ilvD的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物pflB-Pcs-up/pflB-Pcs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。将DNA片段I电转至Sval016。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval016，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval016。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-pflB-up600/ilvD-YZ496-down进行验证，正确的PCR产物应该3756bp，挑选一个正确的单菌落，命名为Sval017。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，用引物pflB-Pro-up/ilvD-Pro-down扩增出189bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval017。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-pflB-up600/ilvD-YZ496-down，正确的菌落扩增产物为1226bp的片段，挑选一个正确的单菌落，将其命名为Sval018(表1)。
实施例10：乙酰乳酸合成酶基因ilvBN的调控
使用人工调控元件M1-93通过两步同源重组的方法调控乙酰乳酸合成酶基因ilvBN的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvB pro-catup/ilvB pro-catdown扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval018，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval018。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvB pro-YZup/ilvB pro-YZdown进行验证，正确的PCR产物应该2996bp，挑选一个正确的单菌落，命名为Sval019。
第二步，以M1-93的基因组DNA为模板，用引物ilvB pro-up/ilvB pro-down扩增出188bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval019。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvB pro-YZup/ilvB pro-YZdown，正确的菌落扩增产物为465bp的片段，挑选一个正确的单菌落，将其命名为Sval020。
实施例11：乙酰乳酸合成酶基因ilvGM的调控
使用人工调控元件M1-93通过两步同源重组的方法调控乙酰乳酸合成酶基因ilvGM的表达，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvG pro-catup/ilvG pro-catdown扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval020，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval020。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvG pro-YZup/ilvG p-YZdown进行验证，正确的PCR产物应该2993bp，挑选一个正确的单菌落，命名为Sval021。
第二步，以M1-93的基因组DNA质粒DNA为模板，用引物ilvG pro-up/ilvG pro-down扩增出188bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval021。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvG pro-YZup/ilvG p-YZdown，正确的菌落扩增产物为462bp的片段，挑选一个正确的单菌落，将其命名为Sval022。
实施例12：乙酰乳酸合成酶基因ilvH的突变
通过两步同源重组的方法在ilvH基因中引入突变解除L-缬氨酸的反馈抑制，具体步骤如下：
第一步，以pXZ-CS质粒DNA为模板，使用引物ilvH*-cat-up/ilvH*-cat-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval022，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval022。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物ilvH*-mutYZ-up/ilvH*-mut-down进行验证，正确的PCR产物应该3165bp，挑选一个正确的单菌落，命名为Sval023。
第二步，以野生型大肠杆菌ATCC 8739的DNA为模板，用引物ilvH*-mut-up/ilvH*-mut-down扩增出467bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval023。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为ilvH*-mutYZ-up/ilvH*-mut-down，正确的菌落扩增产物为619bp的片段，挑选一个正确的单菌落，将其命名为Sval024。
实施例13：使用重组菌株Sval024发酵生产L-缬氨酸
种子培养基由以下成分组成(溶剂为水)：
葡萄糖20g/L，玉米浆干粉10g/L，KH 2PO 4 8.8g/L、(NH 4) 2SO 4 2.5g/L、MgSO 4·7H 2O 2g/L。
发酵培养基大部分和种子培养基相同，区别仅在于葡萄糖浓度为50g/L
Sval024的厌氧发酵包括以下步骤：
(1)种子培养：将LB平板上新鲜的克隆接种到含有4ml种子培养基的试管中，37℃，250rpm振荡培养过夜。然后，按照2％(V/V)的接种量将培养物转接到含有30ml种子培养基的250ml三角瓶中，在37℃，250rpm振荡培养12小时得到种子培养液用于发酵培养基接种。
(2)发酵培养：500ml厌氧罐中发酵培养基体积为250ml，将种子培养液按照终浓度OD550＝0.1的接种量接种于发酵培养基，37℃，150rpm，发酵4天，得到发酵液。中和剂为5M氨水，使发酵罐的pH控制在7.0。培养过程中不通任何气体。
分析方法：使用安捷伦(Agilent-1260)高效液相色谱仪对发酵4天的发酵液中的组分进行测定。发酵液中的葡萄糖和有机酸浓度测定采用伯乐(Biorad)公司的Aminex HPX–87H有机酸分析柱。氨基酸测定使用Sielc氨基酸分析柱primesep 100 250×4.6mm。
结果发现：Sval024菌株在厌氧条件下发酵4天，能够生产1.3g/L的L-缬氨酸(出现与图2对应位置的L-缬氨酸峰)，糖酸转化率0.31mol/mol。
实施例14：亮氨酸脱氢酶编码基因leuDH的克隆与整合
leuDH基因是根据文献报道(Ohshima,T.et.al,Properties of crystalline leucine dehydrogenase from Bacillus sphaericus.The Journal of biological chemistry 253,5719-5725(1978))的来自Lysinibacillus sphaericus IFO 3525菌株的leuDH序列并经过密码子优化(优化序列如序列号90所示)后通过全基因合成获得，合成时在leuDH基因前加上M1-93人工调控元件用于启动leuDH基因的表达，插入pUC57载体，构建获得质粒pUC57-M1-93-leuDH(南京金斯瑞生物科技有限公司完成基因合成和载体构建)。将M1-93人工调控元件和leuDH基因一起通过来两步同源重组的方法整合到Sval024菌株中富马酸还原酶编码基因frd位点并替换掉frd基因，即在整合leuDH的同时敲除frd基因。具体步骤包括：
第一步，以pXZ-CS质粒DNA为模板，使用引物frd-cs-up/frd-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。扩增体系和扩增条件与实施例1中所述一致。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval024，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval024。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物XZ-frd-up/XZ-frd-down进行验证，正确的PCR产物应该3493bp，挑选一个正确的单菌落，命名为Sval025。
第二步，以pUC57-M1-93-leuDH质粒DNA为模板，用引物frd-M93-up/frd-leuDH-down扩增出1283bp的DNA片段II。DNA片段II用于第二次同源重组。将DNA片段II电转至菌株Sval025。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为XZ-frd-up/XZ-frd-down，正确的菌落扩增产物为2057bp的片段，挑选一个正确的单菌落，将其命名为Sval026。
实施例15：使用重组菌株Sval026发酵生产L-缬氨酸
种子培养基和发酵培养基的组成和配制同实施例13中所述相同。
发酵在500mL的发酵罐中进行，发酵过程和分析过程同实施例13中所述Sval024的发酵过程和分析过程一致。
结果发现：Sval026菌株在厌氧条件下发酵4天，能够生产1.8g/L的L-缬氨酸(出现与图2对应位置的L-缬氨酸峰)，糖酸转化率0.56mol/mol。
实施例16：6-磷酸葡萄糖脱氢酶编码基因zwf的调控
从Sval026出发，通过两步同源重组的方法使用人工调控元件调控zwf基因的表达，获得重组大肠杆菌Sval041，具体包括以下步骤：
第一步，以pXZ-CS质粒DNA为模板，使用引物Zwf-Pcat-up/Zwf-PsacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval026，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval026。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物zwf-YZ442-up/zwf-YZ383-down，正确的菌落扩增产物为3339bp的片段，挑选一个正确的单菌落，命名为Sval041。
第二步，以M1-93的基因组DNA为模板，用引物zwf-RBS1-up/zwf-RBS1-down扩增获得189bp的DNA片段II，用于第二步同源重组。扩增条件和体系同(1)中所述。将DNA片段II电转至菌株Sval041。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为zwf-YZ442-up/zwf-YZ383-down，正确的菌落扩增产物为809bp的片段，挑选一个正确的单菌落，将其命名为Sval042(表1)。
实施例17：内酯酶编码基因pgl的调控
从Sval033出发，通过两步同源重组的方法使用人工调控元件调控pgl基因的表达，获得重组大肠杆菌Sval042，具体包括以下步骤：
第一步，以pXZ-CS质粒DNA为模板，使用引物pgl-Pcat-up/pgl-PsacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval033，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval042。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物pgl-YZ308-up/pgl-YZ341-down进行验证，正确的PCR产物应该3248bp，挑选一个正确的单菌落，命名为Sval043。
第二步，以M1-93(Lu,et al.,Appl Microbiol Biotechnol,2012,93:2455-2462)的基因组DNA为模板，使用引物pgl-RBS2-up/pgl-RBS2-down扩增获得189bp的DNA片段II，用于第二步同源重组。扩增条件和体系同(1)中所述。将DNA片段II电转至菌株Sval043。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为pgl-YZ308-up/pgl-YZ341-down，正确的菌落扩增产物为718bp的片段，挑选一个正确的单菌落，将其命名为Sval044(表1)。
实施例18：Zymomonas mobilis mobilis ZM4菌株来源edd和eda基因的克隆与整合
根据文献(The genome sequence of the ethanologenic bacterium Zymomonas mobilis ZM4,Nat.Biotechnol.,2005,23(1):63-68)报道的Zymomonas mobilis mobilis ZM4来源的edd和eda基因的序列，通过全基因合成Zymomonas mobilis mobilis ZM4来源的edd和eda基因，其中edd基因前加上人工调控元件MRS1，edd和eda基因之间通过人工RBS元件(CAGGAAACAGCT)连接。全基因合成的MRS1-edd-RBS-eda片段通过EcoRV连接到pUC57载体中。序列的全基因合成由南京金斯瑞生物科技有限公司完成，pUC57载体同样来自于南京金斯瑞生物科技有限公司。将MRS1-edd-RBS-eda通过两步同源重组的方法整合到大肠杆菌自身edd-eda位点并替换掉大肠杆菌自身edd-eda基因，具体步骤如下：
第一步，以pXZ-CS质粒(Tan,et al.,Appl Environ Microbiol,2013,79:4838-4844)DNA为模板，使用引物edd-cat-up/eda-sacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。
将DNA片段I用于第一次同源重组：首先将pKD46质粒通过电转化法转化至大肠杆菌Sval044，然后将DNA片段I电转至带有pKD46的大肠杆菌Sval044。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物edd-YZ-up/edd-YZ-down进行验证，正确的PCR产物应该3123bp，挑选一个正确的单菌落，命名为Sval045。
第二步，以pUC57-MRS1-edd-eda的质粒DNA为模板，使用引物Edd-int-up/Eda-int-down扩增获得2651bp的DNA片段II，用于第二步同源重组。扩增条件和体系同(1)中所述。将DNA片段II电转至菌株Sval045。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为edd-YZ-up/edd-YZ-down，正确的菌落扩增产物为3055bp的片段，挑选一个正确的单菌落，将其命名为Sval046(表1)。
实施例19：6-磷酸葡萄糖激酶编码基因pfkA的敲除
在Sval037菌株中通过敲除糖酵解途径中关键酶6-磷酸葡萄糖激酶基因pfkA实现重组菌株利用ED途径发酵生产L-缬氨酸。通过两步同源重组的方法敲除pfkA基因，具体步骤包括：
第一步，以pXZ-CS质粒(Tan,et al.,Appl Environ Microbiol,2013,79:4838-4844)DNA为模板，使用引物pfkAdel-cat-up/pfkAdel-sacB-down扩增出2719bp的DNA片段I，用于第一步同源重组。
将DNA片段I用于第一次同源重组：首先将pKD46质粒(Datsenko and Wanner 2000,Proc Natl Acad Sci USA 97:6640-6645；质粒购买于美国耶鲁大学CGSC大肠杆菌保藏中心，CGSC#7739)通过电转化法转化至大肠杆菌Sval046，然后将DNA片段I电转至带有pKD46 的大肠杆菌Sval046。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第一步方法一致。取200μl菌液涂在含有氨苄霉素(终浓度为100μg/ml)和氯霉素(终浓度为34μg/ml)的LB平板上，30℃过夜培养后，挑选单菌落进行PCR验证，使用引物pfkAdel-up/pfkAdel-YZ-down进行验证，正确的PCR产物应该3145bp，挑选一个正确的单菌落，命名为Sval047。
第二步，以野生型ATCC 8739基因组DNA为模板，使用引物pfkAdel-up/pfkAdel-down扩增获得379bp的DNA片段II，用于第二步同源重组。扩增条件和体系同(1)中所述。将DNA片段II电转至菌株Sval047。
电转条件和步骤同实施例1中所述用于mgsA基因敲除的第二步方法一致。菌落PCR对克隆进行验证，所用引物为pfkAdel-up/pfkAdel-YZ-down，正确的菌落扩增产物为526bp的片段，挑选一个正确的单菌落，将其命名为Sval048(表1)。
实施例20：使用重组菌株Sval048生产L-缬氨酸
种子培养基和发酵培养基的组成和配制同实施例13中所述相同。
发酵在500mL的发酵罐中进行，发酵过程和分析过程同实施例13中所述Sval024的发酵过程和分析过程一致。
结果发现：Sval048菌株在厌氧条件下发酵4天，能够生产1.9g/L的L-缬氨酸，糖酸转化率0.82mol/mol。图2为L-缬氨酸标准品的谱图，图3为Sval048发酵液的谱图。
实施例21：重组菌株Sval049构建
从Sval048开始，通过代谢进化同步提高细胞生长和L-缬氨酸的生产能力。
代谢进化过程使用500ml的发酵罐，发酵培养基为250ml。使用5M氨水为中和剂，使发酵罐的pH控制在7.0。进化代谢所用的发酵培养基组成和配制同实施例18中发酵培养基所述。每24小时，将发酵液转接到新的发酵罐中，使初始OD550达0.1。经过100代进化，获得菌株Sval049(图4)。Sval049菌株的保藏号为CGMCC 19457，保藏于中国普通微生物菌种保藏管理中心(CGMCC)。
实施例22：重组菌株Sval049在500mL发酵罐中发酵生产L-缬氨酸
种子培养基的组成和配制同实施例13中所述相同。
发酵在500mL的发酵罐中进行，发酵培养基为250ml。发酵培养基基本上和种子培养基相同，区别是葡萄糖浓度为100g/L，使用的中和剂为5M氨水，使发酵罐的pH控制在7.0。
结果发现：Sval049在500mL发酵罐发酵48小时后，L-缬氨酸产量达47g/L，产率达0.91mol/mol，无杂酸等杂质生成。
实施例23：重组菌株Sval049在5L发酵罐中发酵生产L-缬氨酸
种子培养基的组成和配制、分析方法同实施例13中所述相同。发酵培养基基本上和种子培养基相同，区别是葡萄糖浓度为140g/L。
发酵在5L发酵罐(上海保兴，BIOTECH-5BG)中厌氧进行，包括以下步骤：
(1)种子培养：500ml三角瓶中种子培养基为150ml，115℃灭菌15min。冷却后将重组大肠杆菌Sval041按照1％(V/V)的接种量接种于种子培养基，在37℃和100rpm的条件下培养12小时得到种子液，用于发酵培养基接种。
(2)发酵培养：5L中发酵培养基体积为3L，115℃灭菌25min。将种子液按照终浓度OD550＝0.2的接种量接种于发酵培养基，37℃厌氧培养48小时，搅拌转速200rpm，得到发酵液。发酵液为发酵罐内所有物质。培养过程没有通任何气体。
结果发现：Sval049在5L发酵罐中发酵48小时后，L-缬氨酸产量达87g/L，产率达0.92mol/mol，无杂酸等杂质生成。图5为L-缬氨酸标准品的谱图，图6为Sval049发酵液的谱图。

Claims (10)

  1. 一种生产L-缬氨酸的重组微生物的构建方法，其包括：向微生物中导入氨基酸脱氢酶基因、和/或激活微生物中恩特纳-杜德洛夫代谢途径的步骤；
    任选地，还包括敲除6-磷酸葡萄糖激酶基因pfkA的步骤；
    任选地，还包括向微生物中导入乙酰羟基酸还原异构酶编码基因的步骤；所述乙酰羟基酸还原异构酶编码基因优选ilvC；
    优选地，所述氨基酸脱氢酶基因是NADH依赖型的；
    优选地，所述氨基酸脱氢酶基因是亮氨酸脱氢酶基因；
    优选地，所述激活恩特纳-杜德洛夫代谢途径包括提高zwf基因、pgl基因、edd基因和eda基因的表达强度的步骤。
  2. 根据权利要求1所述的构建方法，其特征在于，还包括对权利要求1所述的重组微生物进行以下(1)-(7)中一种或几种的改造：
    (1)敲除基因mgsA；
    (2)敲除基因ldhA；
    (3)敲除基因pta和/或ackA；
    (4)敲除基因tdcD和/或tdcE；
    (5)敲除基因adhE；
    (6)敲除基因frd和/或pflB；
    (7)增强AHAS和/或ilvD的活性；
    优选地，AHAS为ilvBN或ilvGM或ilvIH；任选地，所述ilvIH的活性通过解除缬氨酸对所述ilvH的反馈抑制得到增强，优选的，通过突变ilvH基因得到增强；
    优选地，选择上述第(7)项进行改造；
    优选地，选择上述第(7)和第(2)项进行改造；
    优选地，选择上述第(7)和第(6)项进行改造；
    优选地，选择上述第(7)项、第(2)项和第(5)项进行改造；
    优选地，选择上述第(7)项、第(2)项和第(6)项进行改造；
    优选地，选择上述第(7)项、第(1)项、第(3)-(6)项进行改造；
    优选地，选择上述第(1)-(7)项进行改造；
    优选地，以ilvD基因替换微生物本身的pflB基因的方式实现第(6)项；
    优选地，以leuDH基因替换微生物本身的frd基因的方式实现第(6)项；
    优选地，以ilvC基因替换微生物本身的mgsA基因的方式实现第(1)项。
  3. 根据权利要求1或2所述的构建方法，其特征在于，所述微生物为大肠杆菌；更优选的，所述微生物为大肠杆菌ATCC 8739。
  4. 根据权利要求1-3中任一项所述的构建方法，其特征在于，使用至少一个调控元件激活或增强ilvD、leuDH、ilvBN、zwf、pgl、ilvGM、edd、eda或ilvC的编码基因的活性；
    优选地，所述调控元件选自M1-93人工调控元件、MRS1人工调控元件、RBS人工调控元件或M1-46人工调控元件；
    优选地，所述M1-93人工调控元件调控ilvD、leuDH、ilvBN、zwf、pgl和ilvGM基因；
    所述MRS1人工调控元件调控edd基因；
    所述RBS人工调控元件调控基因eda；
    所述M1-46人工调控元件调控ilvC基因。
  5. 根据权利要求1-4中任一项所述的构建方法，其特征在于，其中所述酶编码基因的一个或多个拷贝和所述的调控元件整合入所述微生物的基因组中，或者将包含所述酶编码基因的质粒导入所述微生物中；
    优选地，以整合入所述微生物的基因组的方法完成所述酶基因的导入、突变、敲除、激活或调控；
    优选地，以同源重组的方法完成所述酶基因的导入、突变、敲除、激活或调控；
    优选地，以两步同源重组的方法完成所述酶基因的导入、突变、敲除、激活或调控。
  6. 利用权利要求1-5任一所述构建方法得到的重组微生物。
  7. 一种获得高产L-缬氨酸的重组微生物的方法，其特征在于，在权利要求6所述的重组微生物的基础上，经过代谢进化获得。
  8. 一种重组微生物，其保藏号为CGMCC 19457。
  9. 权利要求6或权利要求8所述的重组微生物在生产L-缬氨酸中的应用。
  10. 一种生产L-缬氨酸的方法，包括：(1)发酵培养权利要求6或8所述的重组微生物；(2)分离并收获L-缬氨酸；优选地，所述发酵培养为厌氧发酵培养。
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