WO2023246071A1 - 一种mreC突变体及其在L-缬氨酸发酵生产中的应用 - Google Patents

一种mreC突变体及其在L-缬氨酸发酵生产中的应用 Download PDF

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WO2023246071A1
WO2023246071A1 PCT/CN2022/143630 CN2022143630W WO2023246071A1 WO 2023246071 A1 WO2023246071 A1 WO 2023246071A1 CN 2022143630 W CN2022143630 W CN 2022143630W WO 2023246071 A1 WO2023246071 A1 WO 2023246071A1
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valine
gene
mrec
strain
electroporation
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PCT/CN2022/143630
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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
刘树蓬
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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
合肥华恒生物工程有限公司
巴彦淖尔华恒生物科技有限公司
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Publication of WO2023246071A1 publication Critical patent/WO2023246071A1/zh

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Definitions

  • the invention belongs to the field of bioengineering technology, specifically relates to an mreC mutant and its application in the fermentation production of L-valine, and in particular to an mreC mutant and its nucleic acid molecules in the construction of L-valine engineering bacteria and Application in L-valine fermentation production.
  • L-Valine is one of the 20 amino acids that make up proteins, and is also one of the essential amino acids and glycogenic amino acids for mammals. L-Valine has important applications in the fields of feed, food and medicine, cosmetics, antibiotics and herbicides. Especially in the field of feed, in 2018, the T/CFIAS 001-2018 "Compound Feed for Piglets and Growing and Fattening Pigs" group standard drafted by the China Feed Industry Association specifically added a minimum limit for L-valine; during the period from 2017 to 2019 L-Valine shows a growth trend with an average annual compound growth rate of nearly 100%; therefore, the application and demand of L-Valine in the feed field will surely increase in the future, and the market potential is unlimited.
  • L-Valine can also be used as a food additive, nutritional supplement, flavoring agent, etc., so it is also widely used in the food and pharmaceutical fields.
  • L-valine With the rapid development of L-valine from traditional feed additives to high value-added industries such as food, medicine, and cosmetics, under the guidance of national policies, the market demand for L-valine is bound to lead to breakthrough growth. L-Valine will become the next limiting amino acid in pig and poultry diets. According to data from the China Biological Fermentation Industry Association, the global demand for valine in 2019 is approximately 32,500 tons, with a future growth rate of 24%, and the market potential is huge.
  • L-valine is mainly produced through fermentation.
  • L-valine fermentation production is mostly achieved through aerobic or two-step fermentation, which consumes a lot of energy and has a low conversion rate.
  • aerobic fermentation the anaerobic process does not require air ventilation during the production process, and the conversion rate of the product is usually close to the theoretical maximum. It has the advantages of low energy consumption and high conversion rate, and has become a research hotspot in recent years.
  • the wild-type strain synthesizes L-valine through anaerobic fermentation, and a large amount of intracellular feedback inhibition and other regulatory networks greatly limit its production capacity, resulting in extremely low L-valine production.
  • the existing technology involves strain modification for the anaerobic fermentation process of valine, including the introduction and knockout of relevant enzyme genes in the metabolic pathway to solve the problem of cofactor imbalance and thereby achieve the goal of valine anaerobic fermentation under anaerobic conditions.
  • the recombinant strain can efficiently produce L-valine, but the ability of the above-mentioned modified recombinant strain to fermentatively produce L-valine still needs to be improved without domestication.
  • the object of the present invention is to provide an mreC mutant and its application in the fermentation production of L-valine.
  • the present invention can significantly improve the production capacity and biomass of the valine engineering strain by introducing into the microbial genome a genome mutation that can significantly improve the growth and valine production of the engineered strain.
  • the present invention provides an mreC mutant, which is a protein consisting of the amino acid sequence shown in SEQ ID NO: 3.
  • the mreC mutant is a protein obtained by mutating proline at position 150 of the EcolC_0457 rod-shaped determinant protein amino acid sequence shown in SEQ ID NO: 2 to leucine.
  • the present invention provides nucleic acid molecules encoding the above mreC mutants.
  • nucleic acid molecule is the DNA molecule described in any one of (a1)-(a2):
  • the coding region is the DNA molecule shown in SEQ ID NO: 4;
  • nucleotide sequence is the DNA molecule shown in SEQ ID NO: 4.
  • the DNA molecule shown in SEQ ID NO: 4 is a point mutation introduced at position 449 of the nucleotide sequence of the coding region of the wild-type mreC gene shown in SEQ ID NO: 1, mutating C to T.
  • the present invention provides a biological material, which is any one of the following (b1)-(b3):
  • the recombinant vector may be a recombinant plasmid obtained by inserting the nucleic acid molecule into an expression vector or a cloning vector.
  • the recombinant microorganism can be a microorganism containing the above-mentioned recombinant vector; the microorganism can be yeast, bacteria, algae or fungi. Furthermore, the bacterium may be Escherichia coli.
  • the present invention also provides any application of the following (c1)-(c2):
  • the invention also provides a method for constructing L-valine engineering bacteria, which includes the following steps: introducing the above nucleic acid molecule into the microorganism.
  • the microorganism is any microorganism in the existing technology that has the ability to produce L-valine and the L-valine production pathway involves the rod-shaped determinant protein mreC; Escherichia coli has a clear genetic background, convenient genetic manipulation tools, and substrate utilization. It is widely used, easy to cultivate, and grows rapidly. It has become one of the most important model strains in metabolic engineering, and has realized the industrialization of using engineered strains to produce compounds such as L-alanine, D-lactic acid, and succinic acid.
  • Escherichia coli is preferred, and the Escherichia coli can be wild type, or any mutant type that does not affect the purpose of producing L-valine; Escherichia coli MG1655 is considered a biosafe microbial strain, so it is more preferred E. coli MG1655.
  • glucose metabolism is mainly through the glycolysis pathway.
  • 1 mol of glucose is metabolized to produce 2 mol of pyruvate, 2 mol of ATP and 2 mol of NADH will be generated, resulting in an imbalance in the supply of redox power in E. coli modified under anaerobic conditions.
  • the problem is that there is an excess of NADH and an insufficient supply of NADPH.
  • the present invention also performs any one of the following (d1)-(d2) transformations on the above-mentioned recombinant microorganisms:
  • the "introduction" may be present in the microorganism in any suitable manner known in the art, such as in the form of a plasmid, or in the form of integration into the genome.
  • the enzyme-encoding gene integrated into the genome is integrated into the genome of the microorganism under the control of appropriate regulatory elements.
  • a plasmid containing enzyme encoding genes and regulatory element sequences is introduced into the microorganism.
  • the "activation” can be done by any means known in the art, such as placing the enzyme encoding gene to be activated under the control of appropriate regulatory elements, so that the expression of the enzyme gene is enhanced.
  • Regulatory elements can be inserted upstream of the target gene through known genetic engineering methods.
  • the method includes, but is not limited to, inserting the sequence of the regulatory element upstream of the coding sequence of the target gene through genetic recombination, such as homologous recombination, to enhance the intensity of the expression of the target gene.
  • the control element may be selected from the group consisting of M1-93 artificial control element, M1-37 artificial control element, M1-46 artificial control element or RBS5 artificial control element.
  • the M1-93 artificial regulatory element regulates the acetolactate synthase gene ilvBN, the acetolactate synthase gene ilvGM, the leucine dehydrogenase encoding gene leuDH, the dihydroxy acid dehydratase encoding gene ilvD, and the transhydrogenase encoding gene.
  • Gene pntAB the acetolactate synthase gene ilvBN, the acetolactate synthase gene ilvGM, the leucine dehydrogenase encoding gene leuDH, the dihydroxy acid dehydratase encoding gene ilvD, and the transhydrogenase encoding gene.
  • the M1-37 artificial regulatory element regulates the NAD kinase encoding gene yfjB.
  • the M1-46 artificial regulatory element regulates the NADPH-dependent acetohydroxyacid reductoisomerase encoding gene ilvC.
  • the RBS5 artificial regulatory element NADH-dependent acetohydroxyacid reductoisomerase encodes the gene kari.
  • gene knockout can be carried out in a manner known in the art so that the activity of the enzyme is reduced or inactivated.
  • the knockout operation is aimed at activating the endogenous enzyme genes of microorganisms, so that the activity of the above-mentioned endogenous enzymes of microorganisms is reduced or inactivated.
  • the introduction, mutation or knockout of the target gene is accomplished by integrating into the genome of the microorganism.
  • the introduction, mutation or knockout of the enzyme gene is accomplished by homologous recombination.
  • the introduction, mutation or knockout of the enzyme gene is accomplished by a two-step homologous recombination method.
  • Introduction, mutation or knockout of target genes using a two-step homologous recombination method includes the following steps:
  • DNA fragment I Use pXZ-CS plasmid (Tan, et al., Appl Environ 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;
  • the "replacement” can be done in a manner known to those skilled in the art, such as integrating the coding sequence of the gene to be inserted into the replaced gene coding sequence site in the chromosome of the microorganism, so that the original site gene coding sequence is integrated and inserted.
  • the coding sequence of the gene is replaced.
  • the coding sequence of the enzyme gene described in (1)-(5) above can also be replaced by the coding sequence of another gene through genetic engineering such as homologous recombination, thereby improving the above-mentioned endogenous enzyme activity of the microorganism. reduced or inactivated.
  • Genes that replace these endogenous enzymes can be genes whose expression is to be enhanced, such as the above-mentioned ilvC gene or leuDH gene.
  • the knockout of item (4) is achieved by replacing the endogenous frd gene of the microorganism with the leuDH gene; in one embodiment, the pflB gene endogenous to the microorganism is replaced with the ilvD gene to achieve the knockout of item (4). ) item is deleted.
  • the knockout of item (5) is achieved by replacing the endogenous mgsA gene of the microorganism with the ilvC gene.
  • the acetolactate synthase AHAS includes ilvBN, ilvGM and ilvIH.
  • the activity of ilvIH is enhanced by releasing the feedback inhibition of ilvH by valine; more preferably, a mutation is introduced into the ilvH gene to relieve the feedback inhibition of L-valine.
  • isoenzyme III As for the acetohydroxyacid synthase involved in L-valine biosynthesis, in addition to isoenzyme II (also called AHAS II here), isoenzyme III (also called AHASIII here) is also known.
  • AHASIII is encoded by the ilvIH operon, which consists of ilvI encoding the large subunit (catalytic subunit) and ilvH encoding the small subunit (control subunit).
  • AHASIII is feedback inhibited by L-valine.
  • the ilvI gene can be mutated using reported methods, such as amino acid substitution of ilvH 14Gly ⁇ Asp (Vyazmensky, M.
  • the activity of the dihydroxyacid dehydratase ilvD is enhanced by introducing the ilvD gene into the microorganism.
  • the present invention also provides L-valine engineering bacteria obtained by using the above construction method.
  • the L-valine engineering bacterium introduces a mreC mutation point into the genome (a point mutation is introduced at position 449 of the wild-type mreC gene coding region, changing C to T, corresponding to the 150th amino acid Recombinant E. coli obtained by changing proline (P) into leucine (Leucine (L)).
  • the L-valine engineering bacterium introduces the above-mentioned mreC mutation point into the genome, and introduces the NADH-dependent acetohydroxyacid reductoisomerase gene KARI and the NADH-dependent leucine dehydrogenase gene. Recombinant E. coli derived from leuDH.
  • the L-valine engineering bacterium introduces the above-mentioned mreC mutation point into the genome, and introduces the NADPH-dependent acetohydroxyacid reductoisomerase gene ilvC and the NADH-dependent leucine dehydrogenase gene.
  • leuDH, recombinant E. coli obtained by activating the activities of transhydrogenase PntAB and NAD kinase YfjB.
  • the invention also provides the application of the above-mentioned L-valine engineering bacteria in the production of L-valine.
  • L-valine production is carried out by aerobic fermentation culture, microaerophilic fermentation culture or anaerobic fermentation culture, preferably anaerobic fermentation culture.
  • the invention also provides a method for producing L-valine, which includes the following steps:
  • the fermentation culture is an aerobic, microaerophilic or anaerobic fermentation culture, and anaerobic fermentation culture is preferred.
  • the anaerobic fermentation culture 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 liquid;
  • Fermentation culture Inoculate the seed culture liquid into the fermentation medium, and culture it with shaking at 37°C to obtain the fermentation liquid. Control the pH of the fermentor at 7.0. No gas is allowed to pass through the culture process.
  • the seed culture medium consists of the following ingredients (the solvent is water):
  • Macroelements glucose 20g/L, NH 4 H 2 PO 4 0.87g/L, (NH 4 ) 2 HPO 4 2.63g/L, MgSO 4 ⁇ 7H 2 O 0.18g/L, betaine-HCl 0.15g/L .
  • Trace elements FeCl 3 ⁇ 6H 2 O 1.5 ⁇ g/L, CoCl 2 ⁇ 6H 2 O 0.1 ⁇ g/L, CuCl 2 ⁇ 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L.
  • composition of the fermentation medium and the seed medium are the same, the only difference is that the glucose concentration is 50g/L.
  • the present invention has the following beneficial technical effects: by introducing point mutations of mreC into the microbial genome, the present invention effectively improves the fermentation capacity of the L-valine engineering bacteria and improves its fermentation production of L-valine. The production of amino acids and cell biomass were also significantly increased. In addition, through the knockout and introduction of relevant enzyme genes in the L-valine anaerobic fermentation pathway, the problem of reducing power imbalance in anaerobic metabolism was effectively solved, and appropriate carbon metabolism flow and flow during L-valine production were obtained.
  • Reducing power balance control through the regulation of multiple genes, coordinates and forms a metabolic pathway suitable for L-valine fermentation, realizing the one-step anaerobic high-level fermentation of L-valine in microorganisms, and reducing production costs. It has great application value.
  • Figure 1 is the synthesis pathway of L-valine.
  • Escherichia coli has become one of the most important model strains in metabolic engineering due to its clear genetic background, convenient genetic manipulation tools, extensive substrate utilization, easy culture, and rapid growth. It has also achieved the use of engineered strains to produce L-propanol. Industrialization of compounds such as amino acid, D-lactic acid and succinic acid. Escherichia coli MG1655 is considered a biosafe microbial strain, and the compounds produced by cell factories constructed using this strain will have great application potential in medicine, food and other fields.
  • the applicant of the present invention screened out a batch of recombinant strains that can efficiently produce L-valine through early strain domestication, and conducted genome sequencing on the starting strain and this batch of recombinant strains respectively, and found that the genome of some of the recombinant strains encoded EcolC_0457 rods. A point mutation occurred in the mreC gene that determines the protein. Therefore, the introduction of mreC gene point mutations into microbial genomes will be of great significance for the construction of L-valine production strains and efficient fermentation production of L-valine.
  • Escherichia coli MG1655 is used as the starting strain, and a genomic mutation (point mutation of mreC) that can significantly improve the growth and L-valine production of the engineering strain (producer strain obtained by transforming Escherichia coli MG1655) is provided.
  • the mreC gene encodes the EcolC_0457 rod-shaped determinant protein.
  • strains and plasmids used in this study are detailed in Table 1, and the primers used are shown in Table 2.
  • Example 1 Knockout of lactate dehydrogenase gene ldhA in MG1655 strain
  • the amplification system is: 10 ⁇ l Phusion 5X buffer (NewEngland Biolabs), 1 ⁇ l dNTP (10mM each dNTP), 20ng DNA template, 2 ⁇ l each primer (10 ⁇ M), Phusion High-Fidelity DNA polymerase (2.5U/ ⁇ l) 0.5 ⁇ l, distilled water 33.5 ⁇ l, the total volume is 50 ⁇ l.
  • Amplification conditions were: pre-denaturation at 98°C for 2 min (1 cycle); denaturation at 98°C for 10 s, annealing at 56°C for 10 s, and extension at 72°C for 2 min (30 cycles); extension at 72°C for 10 min (1 cycle).
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid (purchased from Yale University CGSC Escherichia coli Collection Center, CGSC#7739) was transformed into wild-type E. coli MG1655 (denoted as pKD46-MG1655) by electrotransformation. ), and then the DNA fragment I was electroporated into E. coli MG1655 with pKD46 (pKD46-MG1655) to obtain the recombinant strain SvalM001.
  • Electroporation conditions are: first prepare electrotransformation competent cells of E. coli MG1655 (denoted as pKD46-MG1655) carrying pKD46 plasmid (for the preparation method of competent cells, please refer to the literature: Dower et al., 1988, Nucleic Acids Res16: 6127- 6145); then place 50 ⁇ l of pKD46-MG1655 competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2cm Bio-Rad electroshock cup. Use a MicroPulser (Bio-Rad Company) electroporator.
  • the electroporation parameter is a voltage of 2.5kv.
  • step 1.1 Use the genomic DNA of wild-type E. coli MG1655 as a template, use the same amplification system and amplification conditions as in step 1.1, and use primers XZ-ldhA-up/ldhA-del-down to amplify 476 bp DNA fragment II, and use in the second homologous recombination.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into SvalM001 (denoted as pKD46-SvalM001) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM001 to obtain the SvalM002 strain.
  • Electroporation conditions are: first prepare electroporated competent cells of pKD46-SvalM001; then place 50 ⁇ l of pKD46-SvalM001 competent cells on ice, add 50ng of DNA fragment II, place on ice for 2 minutes, and transfer to a 0.2cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad Company) electroporator was used. The electroporation parameter was a voltage of 2.5kv. After electroporation, 1ml of LB liquid culture medium was quickly transferred to the electroporation cup. After pipetting 5 times, it was transferred to the test tube and incubated at 75 rpm at 30°C for 4 hours.
  • Example 2 Knockout of the phosphate acetyltransferase encoding gene pta and the acetate kinase encoding gene ackA
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers ackA-cs-up/pta-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM002 (denoted as pKD46-SvalM002) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM002.
  • step 2 The electroporation conditions and steps are the same as those in step 2) in step 1.1 of Example 1.
  • step 2 After electroporation, quickly transfer 1ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • ampicillin final concentration: 100 ⁇ g/ml
  • chloramphenicol final concentration: 34 ⁇ g/ml
  • the correct PCR product should be 3351bp. Select a correct single colony and name it SvalM003.
  • step 1.1 Use the genomic DNA of wild-type E. coli MG1655 as a template, use the same amplification system and amplification conditions as in step 1.1, and use primers XZ-ackA-up/ackA-del-down to amplify 371 bp DNA fragment II, and use in the second homologous recombination.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM003 (denoted as pKD46-SvalM003) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM003 to obtain SvalM004 strain.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use primers XZ-ackA-up/XZ-pta-down to perform colony PCR verification. The correct colony amplification product is a 732bp fragment. Select a correct single colony and name it SvalM004.
  • 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:
  • step 1.1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers ilvB pro-catup/ilvB pro-catdown to amplify 2719bp DNA fragment I for The first step is homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM004 (denoted as pKD46-SvalM004) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM004.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • primer ilvB After overnight culture at 30°C, single colonies are selected and primer ilvB is used.
  • Pro-YZup/ilvB pro-YZdown performs colony PCR verification.
  • the correct colony amplification product is a 2996bp fragment. Select a correct single colony and name it SvalM005.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM005 (denoted as pKD46-SvalM005) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM005 to obtain SvalM006 strain.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use the primers ilvB pro-YZup/ilvB pro-YZdown to perform colony PCR verification. The correct colony amplification product is a 465bp fragment. Select a correct single colony and name it SvalM006.
  • 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:
  • step 1.1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers ilvG pro-catup/ilvG pro-catdown to amplify 2719bp DNA fragment I for The first step is homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM006 (denoted as pKD46-SvalM006) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM006.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • primer ilvG After overnight culture at 30°C, single colonies are selected and primer ilvG is used. Pro-YZup/ilvG p-YZdown was used for colony PCR verification. The correct colony amplification product was a 2993bp fragment. A correct single colony was selected and named SvalM007.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM007 (denoted as pKD46-SvalM007) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM007 to obtain strain SvalM008.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use the primers ilvG pro-YZup/ilvG p-YZdown to perform colony PCR verification. The correct colony amplification product is a 462bp fragment. Select a correct single colony and name it SvalM008.
  • a mutation was introduced into the ilvH gene through a two-step homologous recombination method to relieve the feedback inhibition of L-valine.
  • the specific steps are as follows:
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM008 (denoted as pKD46-SvalM008) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM008.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • electroporation quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • After culturing overnight at 30°C select a single colony and use primer ilvH *-mutYZ-up/ilvH*-mut-down perform colony PCR verification.
  • the correct colony amplification product is a 3165bp fragment. Select a correct single colony and name it SvalM009.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM009 (denoted as pKD46-SvalM009) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM009 to obtain SvalM010 strain.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use the primers ilvH*-mutYZ-up/ilvH*-mut-down to perform colony PCR verification. The correct colony amplification product is a 619bp fragment. Select a correct single colony and name it SvalM010.
  • Example 6 Integration of the NADH-dependent acetohydroxyacid reductoisomerase encoding gene kari into the adhE site of the alcohol dehydrogenase gene
  • the acetohydroxy acid reductoisomerase encoding gene kari is based on literature reports (Brinkmann-Chen, S., Cahn, J.K.B. & Arnold, F.H. Uncovering rare NADH-preferring ketol-acid reductoisomerases. Metab Eng 26, 17-22, doi: 10.1016/ j.ymben.2014.08.003 (2014).)
  • the kari sequence from Thermacetogenium phaeum strain was obtained through full gene synthesis after codon optimization. During synthesis, an RBS5 artificial regulatory element was added in front of the kari gene to activate the kari gene.
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers adhE-cs-up/adhE-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM010 (denoted as pKD46-SvalM0010) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM010.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • After culturing overnight at 30°C select a single colony and use primer XZ -adhE-up/XZ-adhE-down perform colony PCR verification.
  • the correct colony amplification product is a 3167bp fragment. Select a correct single colony and name it SvalM011.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM011 (denoted as pKD46-SvalM011) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM011 to obtain SvalM012 strain.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use primers XZ-adhE-up/XZ-adhE-down to perform colony PCR verification. The correct colony amplification product is a 1636bp fragment. Select a correct single colony and name it SvalM012.
  • Example 7 Integration of the dihydroxyacid dehydratase encoding gene ilvD
  • the dihydroxy acid dehydratase encoding gene ilvD from Escherichia coli and the promoter RBSL4 that controls ilvD expression were integrated into the pflB site of the pyruvate formate lyase encoding gene through a two-step homologous recombination method and pflB was replaced. gene, that is, integrating ilvD and knocking out the pflB gene at the same time. Specific steps are as follows:
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM012 (denoted as pKD46-SvalM0012) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM012.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • electroporation quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • After culturing overnight at 30°C select a single colony and use primer XZ -pflB-up600/XZ-pflB-down performed colony PCR verification.
  • the correct colony amplification product was a 3675bp fragment. Select a correct single colony and name it SvalM013.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM013 (denoted as pKD46-SvalM013) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM013 to obtain SvalM014 strain.
  • step 2) of step 1.2 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.2 in Example 1.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 4 hours. Then transfer the bacterial solution to LB liquid culture medium containing 10% sucrose without sodium chloride (50ml culture medium in a 250ml flask). After culturing for 24 hours, incubate in LB solid culture medium containing 6% sucrose without sodium chloride. Line culture on culture medium. Use primers XZ-pflB-up600/XZ-pflB-down to perform colony PCR verification. The correct colony amplification product is a 2996bp fragment. Select a correct single colony and name it SvalM014.
  • Example 8 Cloning and integration of leucine dehydrogenase encoding gene leuDH
  • the leucine dehydrogenase gene leuDH is derived from 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 chemistry253, 5719-5725 (1978))
  • the leuDH sequence was obtained through full gene synthesis after codon optimization.
  • an M1-93 artificial regulatory element was added before the leuDH gene to start the expression of the leuDH gene. It was 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 SvalM014 strain through a two-step homologous recombination method and replaced the frd gene. That is, the frd gene was knocked out while integrating leuDH. Specific steps include:
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers frd-cs-up/frd-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM014 (denoted as pKD46-SvalM0014) by electroporation, and then DNA fragment I was electroporated into pKD46-SvalM014.
  • step 2) of step 1.1 in Example 1 The electroporation conditions and steps are the same as those in step 2) of step 1.1 in Example 1.
  • electroporation quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • After culturing overnight at 30°C select a single colony and use primer XZ -frd-up/XZ-frd-down perform colony PCR verification.
  • the correct colony amplification product is a 3493bp fragment. Select a correct single colony and name it SvalM015.
  • DNA fragment II was used for the second homologous recombination: first, the pKD46 plasmid was transformed into strain SvalM013 (denoted as pKD46-SvalM013) by electroporation, and then DNA fragment II was electroporated into pKD46-SvalM013 to obtain SvalM014 strain.
  • step 2) of step 1.2 in Example 1 Use primers XZ-pflB-up600/XZ-pflB-down to perform colony PCR verification.
  • the correct colony amplification product is a 2057bp fragment. Select a correct single colony and name it SvalM016.
  • Example 9 Knockout of the methylglyoxal synthase encoding gene mgsA and integration of the NADPH-dependent acetohydroxyacid reductoisomerase encoding gene ilvC into the methylglyoxal synthase encoding gene mgsA site
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use the primers mgsA-cs-up/mgsA-cs-down to amplify the 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into SvalM016 (denoted as pKD46-SvalM0016) by electroporation, and then DNA fragment I was electroporated into E. coli SvalM016 with pKD46 (pKD46-SvalM016 ).
  • step 2) After electroporation, quickly transfer 1ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • ampicillin final concentration: 100 ⁇ g/ml
  • chloramphenicol final concentration: 34 ⁇ g/ml
  • the correct colony amplification product is a 3646bp fragment. Select a correct single colony and name it SvalM017.
  • step 2) Electroporate DNA fragment II into the strain SvalM017 carrying pKD46 (denoted as pKD46-SvalM017) to obtain the SvalM018 strain.
  • the electroporation conditions and steps are the same as step 2) of step 1.2 in Example 1.
  • the correct colony amplification product is a 1027bp fragment. Select a correct one. A single colony was named SvalM018.
  • step 2) Electroporate DNA fragment II into strain SvalM017 carrying pKD46 to obtain strain SvalM019.
  • the electroporation conditions and steps are the same as step 2) of step 1.2 in Example 1.
  • the correct colony amplification product is a fragment of 259lbp. Select a correct A single colony was named SvalM019.
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers pntAB-cs-up/pntAB-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • step 2) After electroporation, quickly transfer 1ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • ampicillin final concentration: 100 ⁇ g/ml
  • chloramphenicol final concentration: 34 ⁇ g/ml
  • -YZ-up/pntAB-YZ-down perform colony PCR verification.
  • the correct colony amplification product is a 3459bp fragment. Select a correct single colony and name it SvalM020.
  • step 2) of step 1.2 in Example 1. Use the primers pntAB-YZ-up/pntAB-YZ-down to perform colony PCR verification.
  • the correct colony amplification product is a 928bp fragment. Select the correct one.
  • a single colony was named SvalM021.
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers yfjb-cs-up/yfjb-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • the correct colony amplification product is a 3542bp fragment. Select a correct single colony and name it SvalM022.
  • step 2) of step 1.2 in Example 1 Use the primers yfjb-YZ-up/yfjb-YZ-down to perform colony PCR verification.
  • the correct colony amplification product is a 1011bp fragment. Select a correct one.
  • a single colony was named SvalM023.
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use primers adhE-cs-up/adhE-cs-down to amplify 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml).
  • the correct colony amplification product is a 3167bp fragment. Select a correct single colony and name it SvalM024.
  • step 2) of step 1.2 in Example 1. Use primers XZ-adhE-up/XZ-adhE-down to perform colony PCR verification.
  • the correct colony amplification product is a 548bp fragment. Select a correct one.
  • a single colony was named SvalM025.
  • a point mutation was introduced at position 449 of the wild-type mreC gene coding region shown in SEQ ID NO: 1 through a two-step homologous recombination method, changing C into T (the nucleotide sequence after the mutation is as SEQ ID NO: 4), correspondingly, the 150th amino acid of the EcolC_0457 rod-shaped determinant protein encoded by SEQ ID NO: 2 is changed from proline (Proline, P) to leucine (Leucine, L) (the amino acid after mutation The sequence is shown in SEQ ID NO: 3), and strain SvalM027 was obtained. Specific steps are as follows:
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use the primers cat-mreC-up/SacB-mreC-down to amplify the 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • step 2) After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, pipet 5 times and then transfer to the test tube, incubate at 75 rpm and 30°C for 2 hours. Then take 200 ⁇ l of bacterial solution and apply it on an LB solid plate containing ampicillin (final concentration: 100 ⁇ g/ml) and chloramphenicol (final concentration: 34 ⁇ g/ml). After overnight culture at 30°C, single colonies are selected and primer mreC is used. -YZ490-up/mreC-YZ885-down performed colony PCR verification. The correct colony amplification product was a 3994bp fragment. Select a correct single colony and named it SvalM026.
  • step 2) of step 1.2 in Example 1 Use the primers mreC-YZ490-up/mreC-YZ885-down to perform colony PCR verification.
  • the correct colony amplification product is a 1375bp fragment. Select one The correct single colony was named SvalM027.
  • step 1.1 in Example 1 1) Using pXZ-CS plasmid DNA as a template, using the same amplification system and amplification conditions as step 1.1 in Example 1, use the primers cat-mreC-up/SacB-mreC-down to amplify the 2719bp DNA fragment I, Used for the first step of homologous recombination.
  • step 2) of step 1.1 in Example 1. Use the primers mreC-YZ490-up/mreC-YZ885-down to perform colony PCR verification.
  • the correct colony amplification product is a 3994bp fragment. Select a correct single colony and name it SvalM028.
  • step 2) of step 1.2 in Example 1 Use the primers mreC-YZ490-up/mreC-YZ885-down to perform colony PCR verification.
  • the correct colony amplification product is a 1375bp fragment. Select one The correct single colony was named SvalM029.
  • Example 14 Fermentation production of L-valine by recombinant strain
  • the seed culture medium consists of the following ingredients (the solvent is water):
  • Macroelements glucose 20g/L, NH 4 H 2 PO 4 0.87g/L, (NH 4 ) 2 HPO 4 2.63g/L, MgSO 4 ⁇ 7H 2 O 0.18g/L, betaine-HCl 0.15g/L .
  • Trace elements FeCl 3 ⁇ 6H 2 O 1.5 ⁇ g/L, CoCl 2 ⁇ 6H 2 O 0.1 ⁇ g/L, CuCl 2 ⁇ 2H 2 O 0.1 ⁇ g/L, ZnCl 2 0.1 ⁇ g/L, Na 2 MoO 4 ⁇ 2H 2 O 0.1 ⁇ g/L, MnCl 2 ⁇ 4H 2 O 0.2 ⁇ g/L, H 3 BO 3 0.05 ⁇ g/L.
  • the fermentation medium is mostly the same as the seed medium, the only difference is that the glucose concentration is 50g/L.
  • the production of L-valine by anaerobic fermentation by SvalM029 strain includes the following steps:
  • Seed culture Inoculate the fresh clones on the LB solid plate into a test tube containing 4 ml of seed culture medium, and culture overnight at 37°C and 250 rpm with shaking. The culture was then transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed medium according to an inoculation amount of 2% (V/V), and cultured with shaking at 37°C and 250 rpm for 12 hours to obtain a seed culture liquid, which was used for inoculation of the fermentation medium.
  • V/V inoculation amount
  • Fermentation culture The volume of the fermentation medium in the 500ml anaerobic tank is 250ml.
  • the neutralizing agent used is 7M ammonia water, so that the pH of the fermentation tank is controlled at 7.0, and no gas is allowed to flow during the cultivation process.
  • the introduction of point mutations in the genome can increase valine production by 291%.
  • the engineering strain that uses a combination of NADPH-dependent IlvC, NADH-dependent LeuDH, transhydrogenase PntAB and NAD kinase YfjB to produce valine
  • the introduction of point mutations in the genome can make valine Acid production increased by 325%.

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Abstract

本发明提供了一种mreC突变体及其在L-缬氨酸发酵生产中的应用，该mreC突变体为将野生型mreC氨基酸序列第150位的脯氨酸突变为亮氨酸后所得的蛋白质；通过在微生物基因组中引入mreC的点突变，即野生型mreC基因的编码区核苷酸序列的第449位位置上C突变为T，可以显著提高缬氨酸工程菌株的生产能力和生物量，并通过L-缬氨酸厌氧发酵途径中相关酶基因的敲除和导入，获得L-缬氨酸生产过程中适宜的碳代谢流和还原力平衡控制，通过多基因的调控，协调配合形成适宜L-缬氨酸发酵的代谢途径，实现了在微生物中L-缬氨酸的高水平合成。

Description

一种mreC突变体及其在L-缬氨酸发酵生产中的应用 技术领域
本发明属于生物工程技术领域，具体涉及一种mreC突变体及其在L-缬氨酸发酵生产中的应用，特别涉及一种mreC突变体及其核酸分子在构建L-缬氨酸工程菌和L-缬氨酸发酵生产中的应用。
背景技术
L-缬氨酸是组成蛋白质的20种氨基酸之一，也是哺乳动物的必需氨基酸和生糖氨基酸之一。L-缬氨酸在饲料、食品和医药、化妆品、抗生素和除草剂等领域有着重要的应用。尤其是在饲料领域，2018年，中国饲料工业协会起草的T/CFIAS 001-2018《仔猪、生长育肥猪配合饲料》团体标准中专门增设了L-缬氨酸的最低限量；2017-2019年期间L-缬氨酸呈现年均复合增长率接近100％的增长趋势；因此未来L-缬氨酸在饲料领域的应用和需求量必将越来越大，市场潜力无限。L-缬氨酸还可用作食品添加剂、营养增补液及风味剂等，因此在食品和医药领域也具有广泛的应用。随着L-缬氨酸由传统饲料添加剂向食品、医药、化妆品等高附加值行业的迅猛发展，在国家政策的引导下，L-缬氨酸的市场需求量势必会引来突破性增长，L-缬氨酸将成为猪禽日粮中下一个限制性氨基酸。据中国生物发酵产业协会数据显示，2019年全球缬氨酸需求量约3.25万吨，未来增长率达24％，市场潜力巨大。
随着合成生物学和代谢工程的快速发展，以可再生资源为原料，通过微生物发酵实现绿色、环保、高效生产大宗化学品、精细化工品和天然产物等化合物的技术越来越受到重视，并逐渐成为替代石油基化学品的重要力量，呈蓬勃发展之势。目前，L-缬氨酸主要通过发酵法生产获得。目前L-缬氨酸发酵生产多是通过好氧或者两步法发酵实现的，能耗大且转化率低。相较于好氧发酵，厌氧工艺在生产过程中不需要通空气，且产品的转化率通常接近理论最大值，具有低能耗、高转化率的优点，成为近年来的研究热点。野生型菌株厌氧发酵合成L-缬氨酸，胞内大量反馈抑制等调控网络极大地限制了其生产能力，导致L-缬氨酸产量极低。
针对上述技术弊端，现有技术中有针对缬氨酸厌氧发酵过程进行菌株改造，包括对代谢通路中相关酶基因的导入、敲除等以解决辅因子不平衡问题，进而实现厌氧条件下重组菌株高效生产L-缬氨酸，但上述改造的重组菌株在未经驯化的情况下，其发酵生产L-缬氨酸能力仍有待提高。
发明内容
本发明的目的在于提供一种mreC突变体及其在L-缬氨酸发酵生产中的应用。本发明通过在微生物基因组引入可显著改善工程菌株生长和缬氨酸产量的基因组突变，可以显著提高缬氨酸工程菌株的生产能力和生物量。
第一方面，本发明提供了一种mreC突变体，其为由SEQ ID NO：3所示氨基酸序列组成的蛋白质。
进一步地，所述mreC突变体为SEQ ID NO：2所示的EcolC_0457杆状决定蛋白氨基酸序列第150位的脯氨酸突变为亮氨酸后所得的蛋白质。
第二方面，本发明提供了编码上述mreC突变体的核酸分子。
进一步地，所述核酸分子为(a1)-(a2)中任一种所述的DNA分子：
(a1)编码区是SEQ ID NO：4所示的DNA分子；
(a2)核苷酸序列是SEQ ID NO：4所示的DNA分子。
其中，SEQ ID NO：4所示DNA分子为SEQ.IDNO：1所示野生型mreC基因的编码区核苷酸序列的第449位引入点突变，将C突变为T。
第三方面，本发明提供了一种生物材料，其为下述(b1)-(b3)中任一种：
(b1)含有上述核酸分子的基因表达盒；
(b2)含有上述核酸分子的重组载体；
(b3)含有上述核酸分子的重组微生物。
进一步地，所述重组载体可为向表达载体或克隆载体插入所述核酸分子得到的重组质粒。
进一步地，所述重组微生物可为含有上述重组载体的微生物；所述微生物可为酵母、细菌、藻类或真菌。更进一步地，所述细菌可为大肠杆菌。
本发明还提供了下述(c1)-(c2)中任一种应用：
(c1)上述mreC突变体或核酸分子或生物材料在构建L-缬氨酸工程菌的应用；
(c2)上述mreC突变体或核酸分子或生物材料在L-缬氨酸生产中的应用。
本发明还提供了一种L-缬氨酸工程菌的构建方法，包括下述步骤：向微生物中导入上述核酸分子。
其中，微生物为现有技术中任何具有生产L-缬氨酸性能且产L-缬氨酸途径中涉及杆状决定蛋白mreC的微生物；大肠杆菌因遗传背景清晰、基因操作工具方便、底物利用广泛、易培养，并且生长快速等优点，已经成为代谢工程改造中最主要的模式菌株之一，并实现了利用工程菌株生产L-丙氨酸、D-乳酸和丁二酸等化合物的产业化，故优选为大肠杆菌，所述大肠杆菌可以是野生型的，也可以是不影响生产L-缬氨酸目的的任何突变类型的；大肠杆菌MG1655被认为是生物安全的微生物菌株，故更优选为大肠杆菌MG1655。
在厌氧发酵时，葡萄糖的代谢主要是通过糖酵解途径，1mol葡萄糖代谢生产2mol丙酮酸的同时，会生成2molATP和2molNADH，导致厌氧条件下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。
为解决厌氧发酵时还原力平衡问题，以实现在厌氧条件下高效生产L-缬氨酸，本发明还对上述重组微生物进行下述(d1)-(d2)任一种改造：
(d1)向上述重组微生物中导入NADH依赖型乙酰羟基酸还原异构酶基因KARI和NADH依赖型亮氨酸脱氢酶基因leuDH，使得酶活性增强；
(d2)向上述重组微生物中导入NADPH依赖型乙酰羟基酸还原异构酶基因ilvC、和导入NADH依赖型亮氨酸脱氢酶基因leuDH、和激活转氢酶PntAB的活性、和激活NAD激酶YfjB的活性，使得酶活性增强。
所述“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于所述微生物中。
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控制下整合入微生物的基因组中。
在一个实施方式中，将包含酶编码基因和调控元件序列的质粒导入所述微生物中。
所述“激活”可以通过本领域已知的任何方式，例如将待激活的酶编码基因置于合适的调控元件控制下，以使得所述酶基因的表达增强。
调控元件可通过已知的基因工程方法插入目标基因的上游。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基因编码序列上游，以增强目标基因表达的强度。
所述调控元件可选自M1-93人工调控元件、M1-37人工调控元件、M1-46人工调控元件或RBS5人工调控元件。
在一个实施例中，M1-93人工调控元件调控乙酰乳酸合成酶基因ilvBN、乙酰乳酸合成酶基因ilvGM、亮氨酸脱氢酶编码基因leuDH、二羟酸脱水酶编码基因ilvD、转氢酶编码基因pntAB。
在一个实施例中，M1-37人工调控元件调控NAD激酶编码基因yfjB。
在一个实施例中，M1-46人工调控元件调控NADPH依赖型乙酰羟基酸还原异构酶编码基因ilvC。
在一个实施例中，RBS5人工调控元件NADH依赖型乙酰羟基酸还原异构酶编码基因kari。
更进一步地，还包括对重组微生物进行以下(1)-(6)的改造：
(1)敲除乳酸脱氢酶基因ldhA；
(2)敲除磷酸乙酰转移酶基因pta和乙酸激酶基因ackA；
(3)敲除醇脱氢酶基因adhE；
(4)敲除富马酸还原酶基因frd和丙酮酸甲酸裂解酶基因pflB；
(5)敲除甲基乙二醛合酶基因mgsA；
(6)增强乙酰乳酸合成酶AHAS和二羟酸脱水酶ilvD的活性。
本领域技术人员能够理解，可以用现有技术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。
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。
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：
(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 Wanner2000，Proc NatlAcad Sci USA 97：6640-6645)转化至出发菌株，然后将DNA片段I转至带有pKD46的出发菌株；经菌落PCR验证正确的单菌落命名为重组菌1；
(3)DNA片段II的制备：以出发菌株的基因组DNA为模板，用扩增引物2扩增出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转化至重组菌1，经菌落PCR验证正确的单菌落命名为重组菌2。
所述“替换”可以本领域技术人员已知的方式，例如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。
进一步地，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)-(5)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，以leuDH基因替换微生物内源性的frd基因来实现第(4)项的敲除；在一个实施方式中，以ilvD基因替换微生物内源性的pflB基因来实现第(4)项的敲除。
在一个实施方式中，以ilvC基因替换微生物内源性的mgsA基因来实现第(5)项的敲除。
其中，所述乙酰乳酸合成酶AHAS包括ilvBN、ilvGM和ilvIH。
优选地，所述ilvIH的活性通过解除缬氨酸对ilvH的反馈抑制得到增强；更优选地，在ilvH基因中引入突变以解除L-缬氨酸的反馈抑制。
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AHAS II)，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，所述二羟酸脱水酶ilvD的活性通过向微生物中导入ilvD基因而增强。
本发明还提供了利用上述构建方法得到的L-缬氨酸工程菌。
在一个实施方式中，所述L-缬氨酸工程菌是在基因组中引入mreC突变点(在野生型mreC基因编码区第449位引入点突变，将C变成T，相应的第150位氨基酸从脯氨酸(Proline，P)变成亮氨酸(Leucine，L))所得的重组大肠杆菌。
在一个实施方式中，所述L-缬氨酸工程菌是在基因组中引入上述mreC突变点，并导入NADH依赖型乙酰羟基酸还原异构酶基因KARI和NADH依赖型亮氨酸脱氢酶基因leuDH所得的重组大肠杆菌。
在一个实施方式中，所述L-缬氨酸工程菌是在基因组中引入上述mreC突变点，并导入NADPH依赖型乙酰羟基酸还原异构酶基因ilvC和NADH依赖型亮氨酸脱氢酶基因leuDH、激活转氢酶PntAB和NAD激酶YfjB的活性所得的重组大肠杆菌。
本发明还提供了上述L-缬氨酸工程菌在L-缬氨酸生产中的应用。
其中，L-缬氨酸生产为好氧发酵培养、微需氧发酵培养或厌氧发酵培养，优选为厌氧发酵培养。
本发明还提供了一种生产L-缬氨酸的方法，包括下述步骤：
(1)发酵培养上述L-缬氨酸工程菌；
(2)分离并收获L-缬氨酸。
其中，发酵培养为好氧、微需氧或厌氧发酵培养，优选为厌氧发酵培养。
在一个实施方式中，所述厌氧发酵培养包括下述步骤：
(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℃，振荡培养，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)：
大量元素：葡萄糖20g/L，NH 4H 2PO 40.87g/L、(NH 4) 2HPO 42.63g/L、MgSO 4·7H 2O 0.18g/L、甜菜碱-HCl 0.15g/L。
微量元素：FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L、H 3BO 3 0.05μg/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。
与现有技术相比，本发明具有下述有益技术效果：本发明通过在微生物基因组中引入mreC的点突变，有效改善了L-缬氨酸工程菌发酵能力，提高了其发酵生产L-缬氨酸的产量，细胞生物量也显著提高。另外通过L-缬氨酸厌氧发酵途径中相关酶基因的敲除和导入，有效解决了厌氧代谢中还原力不平衡问题，获得了L-缬氨酸生产过程中适宜的碳代谢流和还原力平衡控制，通过多基因的调控，协调配合形成适宜L-缬氨酸发酵的代谢途径，实现了在微生物中一步法厌氧高水平发酵生产L-缬氨酸，且降低了生产成本，具有重大的应用价值。
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是L-缬氨酸的合成途径。
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；所用的试剂和材料等，如无特殊说明，均可从商业途径获得。
大肠杆菌因遗传背景清晰、基因操作工具方便、底物利用广泛、易培养，并且生长快速等优点，已经成为代谢工程改造中最主要的模式菌株之一，并实现了利用工程菌株生产L-丙氨酸、D-乳酸和丁二酸等化合物的产业化。大肠杆菌MGl655被认为是生物安全的微生物菌株，使用该菌株构建的细胞工厂生产的化合物将在医药、食品等领域具有极大的应用潜力。本发明申请人通过前期菌株驯化筛选出一批能高效生产L-缬氨酸的重组菌株，并通过对出发菌株和这批重组菌株分别进行基因组测序，发现部分重组菌株的基因组中编码EcolC_0457杆 状决定蛋白的mreC基因发生了点突变。因此，在微生物基因组中引入mreC基因点突变将对L-缬氨酸生产菌株的构建及L-缬氨酸高效发酵生产具有重要意义。下述实施例中，以大肠杆菌MG1655为出发菌，提供了可以显著改善工程菌株(大肠杆菌MG1655经改造所得生产菌)生长和L-缬氨酸产量的基因组突变(mreC的点突变)。mreC基因编码EcolC_0457杆状决定蛋白，通过在大肠杆菌MG1655经改造所得生产菌的基因组中引入该点突变，可以显著提高L-缬氨酸工程菌株的生产能力和生物量。
本研究中使用的菌株和质粒详见表1，所用引物详见表2。
表1本发明使用的菌株和质粒
Figure PCTCN2022143630-appb-000001
Figure PCTCN2022143630-appb-000002
表2本发明使用的引物及序列信息
Figure PCTCN2022143630-appb-000003
Figure PCTCN2022143630-appb-000004
Figure PCTCN2022143630-appb-000005
Figure PCTCN2022143630-appb-000006
Figure PCTCN2022143630-appb-000007
Figure PCTCN2022143630-appb-000008
Figure PCTCN2022143630-appb-000009
实施例1：MG1655菌株中乳酸脱氢酶基因ldhA的敲除
从野生型大肠杆菌MG1655菌株出发，采用两步同源重组的方法敲除乳酸脱氢酶基因ldhA，具体步骤如下：
1.1、重组菌株SvalM001的构建
1)以pXZ-CS质粒DNA(Tan，et al.，Appl Environ Microbiol，2013，79：4838-4844)为模板，使用引物ldhA-cs-up/ldhA-cs-dwon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10mM)1μl、DNA模板20ng、引物(10μM)各2μl、Phusion High-Fidelity DNA聚合酶(2.5U/μl)0.5μl、蒸馏水33.5μl，总体积为50μl。
扩增条件为：98℃预变性2min(1个循环)；98℃变性10s、56℃退火10s、72℃延伸2min(30个循环)；72℃延伸10min(1个循环)。
2)将DNA片段I电转至野生型大肠杆菌MG1655，获得SvalM001菌株
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野生型大肠杆菌MG1655(记作pKD46-MG1655)，然后将DNA片段I电转至带有pKD46的大肠杆菌MG1655(pKD46-MG1655)，获得重组菌株SvalM001。
电转条件为：首先准备带有pKD46质粒的大肠杆菌MG1655(记作pKD46-MG1655)的电转化感受态细胞(感受态细胞的制备方法参阅文献：Dower et al.，1988，Nucleic Acids Res16：6127-6145)；然后将50μl pKD46-MG1655感受态细胞置于冰上，加入50ng DNA片段I，冰上放置2min，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液体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2h。孵育结束后，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，所用引物为XZ-ldhA-up/XZ-ldhA-down，正确的菌落扩增产物为3448bp的片段，挑选一个正确的单菌落，命名为SvalM001。
1.2、重组菌株SvalM002的构建
1)以野生型大肠杆菌MG1655的基因组DNA为模板，采用同步骤1.1的扩增体系和扩增条件，用引物XZ-ldhA-up/ldhA-del-down扩增出476bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至重组菌株SvalM001，获得SvalM002菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至SvalM001(记作pKD46-SvalM001)，然后将DNA片段II电转至pKD46-SvalM001，获得SvalM002菌株。
电转条件为：首先准备pKD46-SvalM001的电转化感受态细胞；然后将50μl pKD46-SvalM001感受态细胞置于冰上，加入50ng DNA片段II，冰上放置2min，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lml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物XZ-ldhA-up/XZ-ldhA-down进行菌落PCR验证，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SvalM002。
实施例2：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
从重组菌株SvalM002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：
2.1、重组菌株SvalM003的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM002菌株，获得SvalM003菌株
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SvalM002(记作pKD46-SvalM002)，然后将DNA片段I电转至pKD46-SvalM002。
电转条件和步骤同实施例1步骤1.1中第2)步的电转条件。电击后迅速将lml LB液体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2h。然后取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固体平板上，30℃过 夜培养后，挑选单菌落，使用引物XZ-ackA-up/XZ-pta-down进行菌落PCR验证，正确的PCR产物应该3351bp，挑选一个正确的单菌落，命名为SvalM003。
2.2、重组菌株SvalM004的构建
1)以野生型大肠杆菌MG1655的基因组DNA为模板，采用同步骤1.1的扩增体系和扩增条件，用引物XZ-ackA-up/ackA-del-down扩增出371bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM003，获得SvalM004菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM003(记作pKD46-SvalM003)，然后将DNA片段II电转至pKD46-SvalM003，获得SvalM004菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物XZ-ackA-up/XZ-pta-down进行菌落PCR验证，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SvalM004。
实施例3：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：
3.1、重组菌株SvalM005的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM004菌株，获得SvalM005菌株
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SvalM004(记作pKD46-SvalM004)，然后将DNA片段I电转至pKD46-SvalM004。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物ilvB pro-YZup/ilvB pro-YZdown进行菌落PCR验证，正确的菌落扩增产物为2996bp的片段，挑选一个正确的单菌落，将其命名为SvalM005。
3.2、重组菌株SvalM006的构建
1)以M1-93(Lu，et al.，Appl Microbiol Biotechnol，2012，93：2455-2462)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，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM005，获得SvalM006菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM005(记作pKD46-SvalM005)，然后将DNA片段II电转至pKD46-SvalM005，获得SvalM006菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物ilvB pro-YZup/ilvB pro-YZdown进行菌落PCR验证，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SvalM006。
实施例4：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：
4.1、重组菌株SvalM007菌株的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM006菌株，获得SvalM007菌株
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SvalM006(记作pKD46-SvalM006)，然后将DNA片段I电转至pKD46-SvalM006。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物ilvG pro-YZup/ilvG p-YZdown进行菌落PCR验证，正确的菌落扩增产物为2993bp的片段，挑选一个正确的单菌落，将其命名为SvalM007。
4.2、重组菌株SvalM008的构建
1)以M1-93(Lu，et al.，Appl Microbiol Biotechnol，2012，93：2455-2462)的基因组DNA为模板，用引物ilvG pro-up/ilvG pro-down扩增出188bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM007，获得SvalM008菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM007(记作pKD46-SvalM007)，然后将DNA片段II电转至pKD46-SvalM007，获得SvalM008菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物ilvG pro-YZup/ilvG p-YZdown进行菌落PCR验证，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SvalM008。
实施例5：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：
5.1、重组菌株SvalM009的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM008菌株，获得SvalM009菌株
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SvalM008(记作pKD46-SvalM008)，然后将DNA片段I电转至pKD46-SvalM008。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物ilvH*-mutYZ-up/ilvH*-mut-down进行菌落PCR验证，正确的菌落扩增产物为3165bp的片段，挑选一个正确的单菌落，将其命名为SvalM009。
5.2、重组菌株SvalM010的构建
1)以野生型大肠杆菌MG1655的DNA为模板，用引物ilvH*-mut-up/ilvH*-mut-down扩增出467bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM009，获得SvalM010菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM009(记作pKD46-SvalM009)，然后将DNA片段II电转至pKD46-SvalM009，获得SvalM010菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物ilvH*-mutYZ-up/ilvH*-mut-down进行菌落PCR验证，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SvalM010。
实施例6：NADH依赖型乙酰羟基酸还原异构酶编码基因kari在醇脱氢酶基因adhE位点的整合
乙酰羟基酸还原异构酶编码基因kari是根据文献报道(Brinkmann-Chen，S.，Cahn，J.K.B.&Arnold，F.H.Uncovering rare NADH-preferring ketol-acid reductoisomerases.Metab Eng 26，17-22，doi：10.1016/j.ymben.2014.08.003(2014).)的来自Thermacetogenium phaeum菌株的kari序列并经过密码子优化后通过全基因合成获得，合成时在kari基因前加上RBS5人工调控元件用于启动kari基因的表达，插入pUC57载体，构建获得质粒pUC57-RBS5-kari(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RBS5人工调控元件和kari基因一起通过两步同源重组的方法整合到SvalM010菌株中醇脱氢酶编码基因adhE位点。具体步骤如下：
6.1、重组菌株SvalM011的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM010菌株，获得SvalM011菌株
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SvalM010(记作pKD46-SvalM0010)，然后将DNA片段I电转至pKD46-SvalM010。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物XZ-adhE-up/XZ-adhE-down进行菌落PCR验证，正确的菌落扩增产物为3167bp的片段，挑选一个正确的单菌落，将其命名为SvalM011。
6.2、重组菌株SvalM012的构建
1)以pUC57-RBS5-kari质粒DNA为模板，用引物adhE-RBS5-up/adhE-kari-down扩增出1188bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM011，获得SvalM012菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM011(记作pKD46-SvalM011)，然后将DNA片段II电转至pKD46-SvalM011，获得SvalM012菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物XZ-adhE-up/XZ-adhE-down进行菌落PCR验证，正确的菌落扩增产物为1636bp的片段，挑选一个正确的单菌落，将其命名为SvalM012。
实施例7：二羟酸脱水酶编码基因ilvD的整合
从SvalM012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和控制ilvD表达的启动子RBSL4一起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：
7.1、重组菌株SvalM013的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM012菌株，获得SvalM013菌株
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SvalM012(记作pKD46-SvalM0012)，然后将DNA片段I电转至pKD46-SvalM012。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物XZ-pflB-up600/XZ-pflB-down进行菌落PCR验证，正确的菌落扩增产物为3675bp的片段，挑选一个正确的单菌落，将其命名为SvalM013。
7.2、重组菌株SvalM014的构建
1)以大肠杆菌MGl655的基因组DNA为模板，用引物ilvD-RBSL4-up/ilvD-pflB-down扩增出1922bp的片段1。以M1-93(Lu，et al.，ApplMicrobiolBiotechnol，2012，93：2455-2462)的基因组DNA为模板，用引物RBSL4-pflB-up/RBSL4-ilvD-down扩增出159bp的DNA片段2。使用同实施例1中所述PCR方法，分别加入20ng片段1和片段2，用引物RBSL4-pflB-up/ilvD-pflB-down进行融合PCR，获得2040bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM013，获得SvalM014菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM013(记作pKD46-SvalM013)，然后将DNA片段II电转至pKD46-SvalM013，获得SvalM014菌株。
电转条件和步骤同实施例1中步骤1.2中第2)步的电转条件。电击后迅速将1ml LB液体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4h。然后将菌液转移至含有10％蔗糖的没有氯化钠的氯化钠的LB液体培养基(250ml烧瓶中装50ml培养基)，培养24h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。使用引物XZ-pflB-up600/XZ-pflB-down进行菌落PCR验证，正确的菌落扩增产物为2996bp的片段，挑选一个正确的单菌落，将其命名为SvalM014。
实施例8：亮氨酸脱氢酶编码基因leuDH的克隆与整合
亮氨酸脱氢酶基因leuDH是根据文献报道(Ohshima，T.et.al，Properties of crystalline leucine dehydrogenase from Bacillus sphaericus.The Journal of biological chemistry253，5719-5725(1978))的来自Lysinibacillus sphaericus IFO 3525菌株的leuDH序列并经过密码子优化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基因一起通过两步同源重组的方法整合到SvalM014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：
8.1、重组菌株SvalM015的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM014菌株，获得SvalM015菌株
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SvalM014(记作pKD46-SvalM0014)，然后将DNA片段I电转至pKD46-SvalM014。
电转条件和步骤同实施例1中步骤1.1中第2)步的电转条件。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物XZ-frd-up/XZ-frd-down进行菌落PCR验证，正确的菌落扩增产物为3493bp的片段，挑选一个正确的单菌落，将其命名为SvalM015。
8.2、重组菌株SvalM016的构建
1)以pUC57-M1-93-leuDH质粒DNA为模板，用引物frd-M93-up/ffd-leuDH-down扩增出1283bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至菌株SvalM015，获得SvalM016菌株
将DNA片段II用于第二次同源重组：首先将pKD46质粒通过电转化法转化至菌株SvalM013(记作pKD46-SvalM013)，然后将DNA片段II电转至pKD46-SvalM013，获得SvalM014菌株。
电转条件和步骤同实施例1中步骤1.2的第2)步。使用引物XZ-pflB-up600/XZ-pflB-down进行菌落PCR验证，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SvalM016。
实施例9：甲基乙二醛合酶编码基因mgsA的敲除和NADPH依赖型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SvalM016出发，通过两步同源重组的方法进行甲基乙二醛合酶编码基因mgsA的敲除和NADPH依赖型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。具体步骤包括：
9.1、重组菌株SvalM017的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将DNA片段I电转至SvalM016菌株，获得SvalM017菌株
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SvalM016(记作pKD46-SvalM0016)，然后将DNA片段I电转至带有pKD46的大肠杆菌SvalM016(pKD46-SvalM016)。
电转条件和步骤同实施例1中步骤1.1的第2)步。电击后迅速将lml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物XZ-mgsA-up/XZ-mgsA-down进行菌落PCR验证，正确的菌落扩增产物为3646bp的片段，挑选一个正确的单菌落，将其命名为SvalM017。
9.2、重组菌株SvalM018的构建
1)以野生型大肠杆菌MG1655的DNA为模板，用引物XZ-mgsA-up/mgsA-del-down扩增出566bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至带有pKD46的菌株SvalM017(记作pKD46-SvalM017)，获得SvalM018菌株。电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物XZ-mgsA-up/XZ-mgsA-down进行菌落PCR验证，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SvalM018。
9.3、重组菌株SvalM019的构建
1)以野生型大肠杆菌MGl655的DNA为模板，用引物ilvC-M46-up/ilvC-mgsA-down扩增出1547bp的片段1。以M1-46(Lu，et al.，Appl Microbiol Biotechnol，2012，93：2455-2462)的基因组DNA为模板，使用引物M46-mgsA-up/M46-ilvC-down扩增出160bp的片段2。使用同实施例1中所述PCR方法，分别加入20ng片段1和片段2，用引物M46-mgsA-up/ilvC-mgsA-down进行融合PCR，获得1664bp的DNA片段II，用于第二次同源重组。
2)将DNA片段II电转至带有pKD46的菌株SvalM017，获得SvalM019菌株。电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物XZ-mgsA-up/XZ-mgsA-down进行菌落PCR验证，正确的菌落扩增产物为259lbp的片段，挑选一个正确的单菌落，将其命名为SvalM019。
实施例10：转氢酶基因pntAB的调控
从SvalM019出发，使用人工调控元件调控pntAB基因的表达从而激活转氢酶PntAB的 活性合。具体步骤包括：
10.1、重组菌株SvalM020的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，使用引物pntAB-cs-up/pntAB-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。
2)将pKD46质粒通过电转化法转化至SvalM019(记作pKD46-SvalM0019)，将DNA片段I电转至菌株pKD46-SvalM0019，获得SvalM020菌株。
电转条件和步骤同实施例1中步骤1.1的第2)步。电击后迅速将lml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物pntAB-YZ-up/pntAB-YZ-down进行菌落PCR验证，正确的菌落扩增产物为3459bp的片段，挑选一个正确的单菌落，将其命名为SvalM020。
10.2、重组菌株SvalM021的构建
1)以M1-93的基因组DNA为模板，用引物pntAB-P-up/pntAB-M93-down扩增出188bp的DNA片段II，用于第二次同源重组。
2)将pKD46质粒通过电转化法转化至SvalM020(记作pKD46-SvalM020)，将DNA片段II电转至菌株pKD46-SvalM020，获得SvalM021菌株。
电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物pntAB-YZ-up/pntAB-YZ-down进行菌落PCR验证，正确的菌落扩增产物为928bp的片段，挑选一个正确的单菌落，将其命名为SvalM021。
实施例11：NAD激酶编码基因yfjB的调控
从SvalM021出发，使用人工调控元件调控NAD激酶基因yfjB的表达从而激活NAD激酶YfjB的活性，使转氢酶和NAD激酶一起实现辅因子供给平衡。具体步骤包括：
11.1、重组菌株SvalM022的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，使用引物yfjb-cs-up/yfjb-cs-down扩增出2719bp的DNA片段I，用于第一步同源重组。
2)将pKD46质粒通过电转化法转化至SvalM021(记作pKD46-SvalM0021)，将DNA片段I电转至菌株pKD46-SvalM0021，获得SvalM022菌株。
电转条件和步骤同实施例1中步骤1.1的第2)步。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物yfjb-YZ-up/yfjb-YZ-down进行菌落PCR验证，正确的菌落扩增产物为3542bp的片段，挑选一个正确的单菌落，将其命名为SvalM022。
11.2、重组菌株SvalM023的构建
1)以M1-37(Lu，et al.，Appl Microbiol Biotechnol，2012，93：2455-2462)的基因组DNA为模板，用引物yfjb-P-up/yfjb-M37-down扩增出188bp的DNA片段II，用于第二次同源重组。
2)将pKD46质粒通过电转化法转化至SvalM022(记作pKD46-SvalM022)，将DNA片段II电转至菌株pKD46-SvalM022，获得SvalM023菌株。
电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物yfjb-YZ-up/yfjb-YZ-down进行菌落PCR验证，正确的菌落扩增产物为1011bp的片段，挑选一个正确的单菌落，将其命名为SvalM023。
实施例12：醇脱氢酶基因adhE位点的敲除
从SvalM023出发，通过两步同源重组的方法敲除醇脱氢酶基因adhE和整合在该位点的kari基因。具体步骤包括：
12.1、重组菌株SvalM024的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，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。
2)将pKD46质粒通过电转化法转化至SvalM023(记作pKD46-SvalM023)，将DNA片段I电转至菌株pKD46-SvalM023，获得SvalM024菌株。
电转条件和步骤同实施例1中步骤1.1的第2)步。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物XZ-adhE-up/XZ-adhE-down进行菌落PCR验证，正确的菌落扩增产物为3167bp的片段，挑选一个正确的单菌落，将其命名为SvalM024。
12.2、重组菌株SvalM025的构建
1)以野生型大肠杆菌MG1655的DNA为模板，用引物XZ-adhE-up/adhE-del-down扩增出271bp的DNA片段II，用于第二次同源重组。
2)将pKD46质粒通过电转化法转化至SvalM024(记作pKD46-SvalM024)，将DNA片段II电转至pKD46-SvalM024，获得SvalM025菌株。
电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物XZ-adhE-up/XZ-adhE-down进行菌落PCR验证，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SvalM025。
实施例13：基因组mreC点突变的引入
从SvalM018菌株出发，通过两步同源重组的方法在SEQ IDNO：1所示野生型mreC基因 编码区第449位引入点突变，将C变成T(突变后核苷酸序列如SEQ ID NO：4所示)，相应的其编码的SEQ ID NO：2所示EcolC_0457杆状决定蛋白的第150位氨基酸从脯氨酸(Proline，P)变成亮氨酸(Leucine，L)(突变后氨基酸序列如SEQ ID NO：3所示)，获得菌株SvalM027。具体步骤如下：
13.1、重组菌株SvalM026的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，使用引物cat-mreC-up/SacB-mreC-down扩增出2719bp的DNA片段I，用于第一步同源重组。
2)将pKD46质粒通过电转化法转化至SvalM018(记作pKD46-SvalM018)，将DNA片段I电转至菌株pKD46-SvalM018，获得SvalM026菌株。
电转条件和步骤同实施例1中步骤1.1的第2)步。电击后迅速将1ml LB液体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2h。然后取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固体平板上，30℃过夜培养后，挑选单菌落，使用引物mreC-YZ490-up/mreC-YZ885-down进行菌落PCR验证，正确的菌落扩增产物为3994bp的片段，挑选一个正确的单菌落，将其命名为SvalM026。
13.2、重组菌株SvalM027的构建
1)以野生型大肠杆菌MG1655的DNA为模板，用引物mreC-up/mreCmut-down扩增出552bp的DNA片段II，用于第二次同源重组。
2)将pKD46质粒通过电转化法转化至SvalM026(记作pKD46-SvalM026)，将DNA片段II电转至菌株pKD46-SvalM026，获得SvalM027菌株。
其中，电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物mreC-YZ490-up/mreC-YZ885-down进行菌落PCR验证，正确的菌落扩增产物为1375bp的片段，挑选一个正确的单菌落，将其命名为SvalM027。
从SvalM025菌株出发，使用同SvalM027相同的方法在SEQ.IDNO：1所示野生型mreC基因编码区第449位引入点突变，将C变成T(突变后核苷酸序列如SEQ ID NO：4所示)，相应的其编码的SEQ ID NO：2所示EcolC_0457杆状决定蛋白的第150位氨基酸从脯氨酸(Proline，P)变成亮氨酸(Leucine，L)(突变后氨基酸序列如SEQ ID NO：3所示)，获得重组菌株SvalM029。具体步骤如下：
13.3、重组菌株SvalM028的构建
1)以pXZ-CS质粒DNA为模板，采用同实施例1中步骤1.1的扩增体系和扩增条件，使用引物cat-mreC-up/SacB-mreC-down扩增出2719bp的DNA片段I，用于第一步同源重组。
2)将pKD46质粒通过电转化法转化至SvalM025(记作pKD46-SvalM025)，将DNA片段I电转至菌株pKD46-SvalM025，获得SvalM028菌株。
其中，电转条件和步骤同实施例1中步骤1.1的第2)步。使用引物mreC-YZ490-up/mreC-YZ885-down进行菌落PCR验证，正确的菌落扩增产物为3994bp的片段，挑选一个正确的单菌落，将其命名为SvalM028。
13.4、重组菌株SvalM029的构建
1)以野生型大肠杆菌MG1655的DNA为模板，用引物mreC-up/mreCmut-down扩增出552bp的DNA片段II，用于第二次同源重组。
2)将pKD46质粒通过电转化法转化至SvalM028(记作pKD46-SvalM028)，将DNA片段II电转至菌株pKD46-SvalM028，获得SvalM029菌株。
其中，电转条件和步骤同实施例1中步骤1.2的第2)步，使用引物mreC-YZ490-up/mreC-YZ885-down进行菌落PCR验证，正确的菌落扩增产物为1375bp的片段，挑选一个正确的单菌落，将其命名为SvalM029。
实施例14：重组菌株发酵生产L-缬氨酸
种子培养基由以下成分组成(溶剂为水)：
大量元素：葡萄糖20g/L，NH 4H 2PO 4 0.87g/L、(NH 4) 2HPO 4 2.63g/L、MgSO 4·7H 2O 0.18g/L、甜菜碱-HCl 0.15g/L。
微量元素：FeCl 3·6H 2O 1.5μg/L、CoCl 2·6H 2O 0.1μg/L、CuCl 2·2H 2O 0.1μg/L、ZnCl 2 0.1μg/L、Na 2MoO 4·2H 2O 0.1μg/L、MnCl 2·4H 2O 0.2μg/L，H 3BO 3 0.05μg/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。
SvalM029菌株厌氧发酵生产L-缬氨酸包括以下步骤：
(1)种子培养：将LB固体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12h，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中，37℃，150rpm，发酵72h，得到发酵液。
其中，使用的中和剂为7M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72h的发酵液组分进行测定。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。
采用上述同样发酵方法，分别利用SvalM018菌株、SvalM027菌株、SvalM025菌株进行厌氧发酵生产L-缬氨酸及分析检测，结果如表3所示。
表3重组菌株厌氧发酵生产缬氨酸
Figure PCTCN2022143630-appb-000010
由表3可见，对于使用NADH依赖型KARI和NADH依赖型LeuDH相结合生产缬氨酸的工程菌(重组菌株SvalM018)，引入基因组点突变后(重组菌株SvalM027)能够使缬氨酸产量提高291％；而对于使用NADPH依赖型IlvC、NADH依赖型LeuDH、转氢酶PntAB和NAD激酶YfjB相结合生产缬氨酸的工程菌(重组菌株SvalM025)，引入基因组点突变后(重组菌株SvalM029)能够使缬氨酸产量提高325％。综上可见，在基因组中引入mreC的点突变可以有效提高缬氨酸工程菌的生产能力，同时细胞的生物量也有显著提高。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。

Claims (14)

  1. 一种mreC突变体，其为由SEQ ID NO：3所示氨基酸序列组成的蛋白质。
  2. 根据权利要求1所述的mreC突变体，其特征在于，所述mreC突变体为SEQ ID NO：2所示的杆状决定蛋白氨基酸序列第150位的脯氨酸突变为亮氨酸后所得的蛋白质。
  3. 编码权利要求1所述mreC突变体的核酸分子。
  4. 根据权利要求3所述的核酸分子，其特征在于：所述核酸分子为(a1)或(a2)所述的DNA分子：
    (a1)编码区是SEQ ID NO：4所示的DNA分子；
    (a2)核苷酸序列是SEQ ID NO：4所示的DNA分子；
    所述SEQ ID NO：4所示的DNA分子为SEQ.IDNO：1所示野生型mreC基因的编码区核苷酸序列的第449位位置上C突变为T。
  5. 一种生物材料，其为下述(b1)-(b3)中任一种：
    (b1)含有权利要求3或4所述核酸分子的基因表达盒；
    (b2)含有权利要求3或4所述核酸分子的重组载体；
    (b3)含有权利要求3或4所述核酸分子的重组微生物。
  6. 下述(c1)-(c2)中的任一种应用：
    (c1)权利要求1或2所述mreC突变体或权利要求3或4所述核酸分子或权利要求5所述生物材料在构建L-缬氨酸工程菌的应用；
    (c2)权利要求1或2所述mreC突变体或权利要求3或4所述核酸分子或权利要求5所述生物材料在L-缬氨酸生产中的应用。
  7. 一种L-缬氨酸工程菌的构建方法，其特征在于，包括下述步骤：向微生物中导入权利要求3或4所述核酸分子。
  8. 根据权利要求7所述的构建方法，其特征在于：还包括进行下述(d1)或(d2)的改造：
    (d1)向权利要求7所述重组微生物中导入NADH依赖型乙酰羟基酸还原异构酶基因KARI和NADH依赖型亮氨酸脱氢酶基因leuDH，使得酶活性增强；
    (d2)向权利要求7所述重组微生物中导入NADPH依赖型乙酰羟基酸还原异构酶基因ilvC、和导入NADH依赖型亮氨酸脱氢酶基因leuDH、和激活转氢酶PntAB的活性、和激活NAD激酶YfjB的活性，使得酶活性增强。
  9. 根据权利要求8所述的构建方法，其特征在于：还包括对重组微生物进行以下(1)-(6)的改造：
    (1)敲除乳酸脱氢酶基因ldhA；
    (2)敲除磷酸乙酰转移酶基因pta和乙酸激酶基因ackA；
    (3)敲除醇脱氢酶基因adhE；
    (4)敲除富马酸还原酶基因frd和丙酮酸甲酸裂解酶基因pflB；
    (5)敲除甲基乙二醛合酶基因mgsA；
    (6)增强乙酰乳酸合成酶AHAS和二羟酸脱水酶ilvD的活性。
  10. 根据权利要求9所述的构建方法，其特征在于：所述微生物为大肠杆菌；优选地，所述微生物为大肠杆菌MG1655。
  11. 根据权利要求9所述的构建方法，其特征在于：所述AHAS包括ilvBN、ilvGM和ilvIH；
    优选地，所述ilvIH的活性通过解除缬氨酸对ilvH的反馈抑制得到增强；
    更优选地，所述ilvIH的活性通过突变ilvH基因得到增强。
  12. 利用权利要求7-11任一项所述构建方法得到的L-缬氨酸工程菌。
  13. 权利要求12所述的L-缬氨酸工程菌在L-缬氨酸生产中的应用。
  14. 一种生产L-缬氨酸的方法，包括下述步骤：
    (1)发酵培养权利要求12所述的L-缬氨酸工程菌；
    (2)分离并收获L-缬氨酸。
PCT/CN2022/143630 2022-06-22 2022-12-29 一种mreC突变体及其在L-缬氨酸发酵生产中的应用 WO2023246071A1 (zh)

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