Recombinant microorganism for producing alpha-ketoisovaleric acid as well as preparation method and application thereof

Abstract

The invention discloses a recombinant microorganism for producing alpha-ketoisovaleric acid as well as a preparation method and application of the recombinant microorganism. The recombinant microorganism for producing alpha-ketoisovaleric acid, disclosed by the invention, has the following characteristics: the recombinant microorganism contains a substance capable of expressing an acetolactate synthase gene and / or a substance capable of promoting the expression of the acetolactate synthase gene and / or a substance capable of improving the activity or content of acetolactate synthase; the composition contains a substance capable of expressing an acetohydroxy acid reductoisomerase gene and / or a substance capable of promoting the expression of the acetohydroxy acid reductoisomerase gene and / or a substance capable of improving the activity or content of acetohydroxy acid reductoisomerase; the composition contains a substance capable of expressing a dihydroxy acid dehydratase gene and / or a substance capable of promoting the expression of the dihydroxy acid dehydratase gene and / or a substance capable of improving the activity or content of the dihydroxy acid dehydratase. Experiments prove that the recombinant microorganism disclosed by the invention can be used for producing alpha-ketoisovaleric acid by utilizing glucose and has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant microorganism for producing alpha-ketoisovaleric acid, and a preparation method and application thereof. Background Art

[0002] As an important keto acid, α-ketoisovaleric acid is widely used in the fields of medicine, chemical industry and feed. It is a key precursor for the synthesis of important compounds such as vitamin B5, branched-chain amino acids, isobutanol, etc. In recent years, with the rapid development of biomedicine and other fields, the market size of α-ketoisovaleric acid has increased year by year. At present, the production methods of α-ketoisovaleric acid include chemical synthesis, enzymatic conversion and biological fermentation. Among them, chemical synthesis involves a series of chemical reactions, which are highly polluting, complex to separate and have high production costs. Compared with chemical synthesis, the enzymatic conversion method has the advantages of mild reaction conditions, few by-products and high conversion efficiency, but it requires valine as a substrate, and the high production cost is still the main limiting factor.

[0003] Microbial fermentation can directly produce α-ketoisovaleric acid using glucose as raw material. Compared with chemical synthesis and enzyme conversion, microbial fermentation has mild reaction conditions, less pollution, renewable raw materials, and most importantly, greatly reduced production costs. At present, there have been many reports on α-ketoisovaleric acid production strains internationally. Darwin et al. used Escherichia coli W as the starting bacteria and obtained a cell factory that can produce 2-ketoisovaleric acid using renewable industrial waste as raw materials through system metabolic engineering. It can produce 3.22g / L of 2-ketoisovaleric acid in batch fermentation.

[0004] Although there have been some reports on the production of α-ketoisovaleric acid by fermentation, the research on the production of α-ketoisovaleric acid by fermentation is still in its infancy, and there are no reports of engineered strains that can achieve industrial production. The main problem with the currently reported engineered strains is that the fermentation process is relatively complicated, and it is difficult for strains to meet both high yield and high conversion rate. Therefore, creating new engineered strains that are suitable for industrial production is of great significance for achieving efficient production of α-ketoisovaleric acid through microbial fermentation with renewable resources. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to prepare alpha-ketoisovaleric acid.

[0006] In order to solve the above technical problems, the present invention first provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli comprises at least one of the following characteristics:

[0007] A1) containing a substance capable of expressing the acetolactate synthase gene and / or a substance promoting the expression of the acetolactate synthase gene and / or a substance increasing the activity or content of acetolactate synthase;

[0008] A2) containing a substance capable of expressing an acetohydroxy acid reductoisomerase gene and / or a substance promoting the expression of an acetohydroxy acid reductoisomerase gene and / or a substance increasing the activity or content of acetohydroxy acid reductoisomerase;

[0009] A3) Contains a substance capable of expressing a dihydroxy-acid dehydratase gene and / or a substance promoting the expression of a dihydroxy-acid dehydratase gene and / or a substance increasing the activity or content of dihydroxy-acid dehydratase.

[0010] In the above recombinant Escherichia coli, the acetolactate synthase may be derived from Bacillus subtilis or Escherichia coli.

[0011] The acetohydroxy acid reductoisomerase may be derived from Thermacetogeniumphaeum.

[0012] The dihydroxy-acid dehydratase may be derived from Escherichia coli.

[0013] In the above-mentioned recombinant Escherichia coli, the acetolactate synthase gene may be alsS gene, ilvB gene, ilvN gene, ilvG gene and / or ilvM gene, and the acetolactate synthase is a protein encoded by alsS gene, ilvB gene, ilvN gene, ilvG gene and / or ilvM gene.

[0014] The acetohydroxy acid reductoisomerase gene may be a kari gene, and the acetohydroxy acid reductoisomerase is a protein encoded by the kari gene.

[0015] The dihydroxy-acid dehydratase gene may be an ilvD gene, and the dihydroxy-acid dehydratase is a protein encoded by the ilvD gene.

[0016] Among them, the two proteins expressed by the ilvB gene and the ilvN gene form acetolactate synthase I, and the two proteins expressed by the ilvG gene and the ilvM gene form acetolactate synthase II.

[0017] In one embodiment of the present invention, the alsS gene is shown in positions 89-1804 of SEQ ID No. 1 in the sequence listing.

[0018] In one embodiment of the present invention, the sequence of the ilvB gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_0028". The sequence of the ilvN gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_0029".

[0019] In one embodiment of the present invention, the sequence of the ilvG gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_4233". The sequence of the ilvM gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_4234".

[0020] In one embodiment of the present invention, the kari gene is shown in positions 90-1088 of SEQ ID No. 5 in the sequence listing.

[0021] In one embodiment of the present invention, the ilvD gene is shown in positions 90-1940 of SEQ ID No. 7 in the sequence listing.

[0022] In the above-mentioned recombinant Escherichia coli, in A1), the substance capable of expressing the acetolactate synthase gene may be an alsS gene expression cassette; the substance that increases the activity or content of acetolactate synthase may be the alsS gene expression cassette and / or a regulatory element that increases the expression amount of the acetolactate synthase gene.

[0023] In A2), the substance containing the gene capable of expressing acetohydroxyacid reductoisomerase may be a kari gene expression cassette.

[0024] In A3), the substance containing a gene capable of expressing a dihydroxy-acid dehydratase may be an ilvD gene expression cassette.

[0025] In the above recombinant Escherichia coli, in A1), the promoter of the alsS gene expression cassette may be an M1-93 promoter or an RBS4 promoter, and the M1-93 promoter can drive the expression of the alsS gene; the M1-93 promoter is any one of the following:

[0026] a1) the nucleotide sequence of one chain is a DNA molecule shown in positions 1 to 88 of SEQ ID No. 1 in the sequence list;

[0027] a2) a DNA molecule that has more than 80% identity with the DNA molecule of a1) and has the function of the M1-93 promoter;

[0028] The RBS4 promoter is any of the following:

[0029] d1) a DNA molecule having a nucleotide sequence of one chain as shown in positions 1 to 89 of SEQ ID No. 9 in the sequence listing;

[0030] d2) A DNA molecule that has more than 80% identity with the DNA molecule of d1) and has RBS4 promoter function.

[0031] In one embodiment of the present invention, the alsS gene expression cassette is shown as SEQ ID No. 1 in the sequence listing.

[0032] In another embodiment of the present invention, the alsS gene expression cassette is shown as SEQ ID No. 9 in the sequence listing.

[0033] In the above-mentioned recombinant Escherichia coli, in A1), the regulatory element for increasing the expression level of the acetolactate synthase gene may be the M1-93 promoter, and the M1-93 promoter can drive the expression of the acetolactate synthase gene.

[0034] In the above-mentioned recombinant Escherichia coli, in A2), the promoter of the kari gene expression cassette may be an RBSL5 promoter, and the RBSL5 promoter can drive the expression of the kari gene, and the RBSL5 promoter is any one of the following:

[0035] b1) a DNA molecule having a nucleotide sequence of one chain as shown in positions 1 to 89 of SEQ ID No. 5 in the sequence list;

[0036] b2) A DNA molecule that has more than 80% identity with the DNA molecule of b1) and has RBSL5 promoter function.

[0037] In one embodiment of the present invention, the kari gene expression cassette is shown as SEQ ID No. 5 in the sequence listing.

[0038] In the above-mentioned recombinant Escherichia coli, in A3), the promoter of the ilvD gene expression cassette may be an RBSL4 promoter, and the RBSL4 promoter can drive the expression of the ilvD gene. The RBSL4 promoter is any one of the following:

[0039] c1) a DNA molecule having a nucleotide sequence of one chain as shown in positions 1 to 89 of SEQ ID No. 7 in the sequence list;

[0040] c2) A DNA molecule that has more than 80% identity with the DNA molecule of c1) and has RBSL4 promoter function.

[0041] In one embodiment of the present invention, the ilvD gene expression cassette is shown as SEQ ID No. 7 in the sequence listing.

[0042] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0043] Specifically, more than 80% identity may be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97%, 98%, 99% or more identity.

[0044] The above-mentioned recombinant Escherichia coli may also include any one, any two, any three or four of the following characteristics:

[0045] A4) does not contain phosphate acetyltransferase gene or contains substances that inhibit the expression of phosphate acetyltransferase gene or contains substances that reduce the content or activity of phosphate acetyltransferase;

[0046] A5) does not contain acetate kinase gene or contains substances that inhibit the expression of acetate kinase gene or contains substances that reduce the content or activity of acetate kinase;

[0047] A6) does not contain methylglyoxal synthase gene or contains substances that inhibit the expression of methylglyoxal synthase gene or contains substances that reduce the content or activity of methylglyoxal synthase;

[0048] A7) does not contain fumarate reductase gene or contains a substance that inhibits the expression of fumarate reductase gene or contains a substance that reduces the content or activity of fumarate reductase.

[0049] The phosphate acetyltransferase gene (encoding the phosphate acetyltransferase) may be a pta gene. In one embodiment of the present invention, the sequence of the pta gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_1355".

[0050] The acetate kinase gene (encoding the acetate kinase) may be an ackA gene. In one embodiment of the present invention, the sequence of the ackA gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_1356".

[0051] The methylglyoxal synthase gene (encoding the methylglyoxal synthase) may be a mgsA gene. In one embodiment of the present invention, the sequence of the mgsA gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC8739, complete genome, 10-FEB-2014), locus_tag="EcolC_2633".

[0052] The fumarate reductase gene (encoding the fumarate reductase) may be a frdB gene and / or a frdC gene. In one embodiment of the present invention, the sequence of the frdB gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_3907"; the sequence of the frdC gene is as follows: GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_3858".

[0053] The above-mentioned recombinant Escherichia coli may also include the following characteristics: it can express ilvH mutant protein, and compared with the ilvH protein, the amino acid residue at position 14 of the ilvH mutant protein is mutated from a glycine residue to an aspartic acid residue, and the amino acid residue at position 17 is mutated from a serine residue to a phenylalanine residue.

[0054] In one embodiment of the present invention, the ilvH mutant protein is shown in SEQ ID No. 4 in the sequence listing. The ilvH mutant protein is encoded by the ilvH mutant gene shown in SEQ ID No. 3.

[0055] The present invention also provides Escherichia coli SKIV007, whose deposit number in the General Microbiological Center of China National Microbiological Culture Collection Committee is CGMCC No.27832.

[0056] The present invention also provides a method for preparing recombinant Escherichia coli, the method comprising transforming the starting Escherichia coli as follows to obtain:

[0057] B1) expressing acetolactate synthase gene and / or promoting acetolactate synthase gene expression and / or increasing acetolactate synthase activity or content;

[0058] B2) expressing an acetohydroxy acid reductoisomerase gene and / or promoting the expression of an acetohydroxy acid reductoisomerase gene and / or increasing the activity or content of acetohydroxy acid reductoisomerase;

[0059] B3) expressing a dihydroxy-acid dehydratase gene and / or promoting the expression of a dihydroxy-acid dehydratase gene and / or increasing the activity or content of dihydroxy-acid dehydratase.

[0060] In the above method, the acetolactate synthase may be derived from Bacillus subtilis or Escherichia coli.

[0061] The acetohydroxy acid reductoisomerase may be derived from Thermacetogeniumphaeum;

[0062] The dihydroxy-acid dehydratase may be derived from Escherichia coli.

[0063] In the above method, the acetolactate synthase gene may be alsS gene, ilvB gene, ilvN gene, ilvG gene and / or ilvM gene, and the acetolactate synthase is a protein encoded by alsS gene, ilvB gene, ilvN gene, ilvG gene and / or ilvM gene.

[0064] The acetohydroxy acid reductoisomerase gene may be a kari gene, and the acetohydroxy acid reductoisomerase is a protein encoded by the kari gene.

[0065] The dihydroxy-acid dehydratase gene may be an ilvD gene, and the dihydroxy-acid dehydratase is a protein encoded by the ilvD gene.

[0066] In the above method B1), the expression of the acetolactate synthase gene can be achieved by introducing the alsS gene expression cassette into the starting Escherichia coli, and the increase in the activity or content of acetolactate synthase can be achieved by replacing the promoters of the ilvB gene, ilvN gene, ilvG gene and ilvM gene with the regulatory element that increases the expression amount of the acetolactate synthase gene.

[0067] The above method B2) can be achieved by introducing the kari gene expression cassette into the starting Escherichia coli.

[0068] The above method B3) can be achieved by introducing the ilvD gene expression cassette into the starting Escherichia coli.

[0069] The above method may further comprise performing any one, any two, any three or four of the following modifications on the starting Escherichia coli:

[0070] B4) knocking out the phosphate acetyltransferase gene or inhibiting the expression of the phosphate acetyltransferase gene or reducing the phosphate acetyltransferase content or activity;

[0071] B5) knocking out the acetate kinase gene or inhibiting the expression of the acetate kinase gene or reducing the content or activity of the acetate kinase;

[0072] B6) knocking out the methylglyoxal synthase gene or inhibiting the expression of the methylglyoxal synthase gene or reducing the methylglyoxal synthase content or activity;

[0073] B7) knocking out the fumarate reductase gene or inhibiting the expression of the fumarate reductase gene or reducing the content or activity of the fumarate reductase.

[0074] The method may further comprise modifying the starting Escherichia coli as follows: mutating the codon encoding the 14th glycine residue of the ilvH gene into a codon encoding an aspartic acid residue, and mutating the codon encoding the 17th serine residue into a codon encoding a phenylalanine residue.

[0075] The present invention also provides a method for preparing α-ketoisovaleric acid, which comprises: culturing the recombinant Escherichia coli or the Escherichia coli SKIV007 to prepare α-ketoisovaleric acid.

[0076] The above method for preparing α-ketoisovaleric acid can be carried out in a medium and under conditions that enable Escherichia coli to grow.

[0077] In one embodiment of the present invention, the culture medium is a fermentation medium, which consists of a solvent and a solute, the solvent is water, and the solute and its concentration in the fermentation medium are as follows: glucose 80 g / L, NH4H2PO4 0.87 g / L, (NH4)2HPO4 2.63 g / L, MgSO4·7H2O 0.18 g / L, betaine-HCl 0.15 g / L, FeCl3·6H2O 1.5 μg / L, CoCl2·6H2O 0.1 μg / L, CuCl2·2H2O 0.1 μg / L, ZnCl2 0.1 μg / L, Na2MoO4·2H2O 0.1 μg / L, MnCl2·4H2O 0.2 μg / L, H3BO3 0.05 μg / L.

[0078] In another embodiment of the present invention, the culture medium is a fermentation medium, which consists of a solvent and a solute, the solvent is water, and the solutes and their concentrations in the fermentation medium are as follows: glucose 105 g / L, NH4H2PO4 0.87 g / L, (NH4)2HPO4 2.63 g / L, MgSO4·7H2O 0.18 g / L, betaine-HCl 0.15 g / L, FeCl3·6H2O 1.5 μg / L, CoCl2·6H2O 0.1 μg / L, CuCl2·2H2O 0.1 μg / L, ZnCl2 0.1 μg / L, Na2MoO4·2H2O 0.1 μg / L, MnCl2·4H2O 0.2 μg / L, H3BO3 0.05 μg / L.

[0079] The present invention also provides a product, which contains (or its active ingredient is) the recombinant Escherichia coli or the Escherichia coli SKIV007.

[0080] The product can be used to produce α-ketoisovaleric acid.

[0081] The present invention also provides any of the following applications of the recombinant Escherichia coli or the Escherichia coli SKIV007 or the product:

[0082] P1) produces α-ketoisovaleric acid;

[0083] P2) Preparation of products for producing α-ketoisovaleric acid.

[0084] In the present invention, each expression cassette can be realized by inserting the expression cassette of the corresponding gene into Escherichia coli. The insertion position of each gene in Escherichia coli can be a site that does not affect the function of Escherichia coli, such as the tdcD site of the propionate kinase encoding gene, the tdcE site of the formate acetyltransferase gene, the adhE site of the alcohol dehydrogenase gene, the pflB site of the pyruvate formate lyase gene, the ldhA site of the lactate dehydrogenase gene, etc.

[0085] In one embodiment of the present invention, the alsS gene expression cassette is inserted into the tdcDE site and the lactate dehydrogenase gene ldhA site of Escherichia coli;

[0086] The kari gene expression cassette is inserted into the alcohol dehydrogenase gene adhE site of Escherichia coli;

[0087] The ilvD gene expression cassette is inserted into the pyruvate formate lyase gene pflB site of Escherichia coli.

[0088] Experiments have shown that the recombinant Escherichia coli of the present invention can produce α-ketoisovaleric acid using glucose, the α-ketoisovaleric acid yield can reach 58 g / L, and the sugar-acid conversion rate can reach 0.6 g / g. The recombinant Escherichia coli of the present invention and the preparation method thereof have good application prospects.

[0089] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0090] Description of biological material deposit

[0091] Abbreviation of depository unit: CGMCC

[0092] Name of depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0093] Address of the depository: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101

[0094] Storage date: July 7, 2023

[0095] CGMCC Registration Number: CGMCC No.27832

[0096] Classification name: Escherichia coli

[0097] Strain ID: SKIV007 DETAILED DESCRIPTION

[0098] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated at least three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0099] In the following examples, Escherichia coli ATCC 8739 is a wild-type Escherichia coli strain; M1-93 is an Escherichia coli strain modified from ATCC 8739 and containing artificial regulatory elements, as described in “Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462”.

[0100] Table 1. Primers used in the present invention

[0101]

[0102]

[0103]

[0104] Hereinafter, two primers separated by " / " are used to form a corresponding primer pair for amplifying the target fragment.

[0105] Example 1: Insertion of the acetolactate synthase gene alsS into ATCC 8739 chromosomal DNA

[0106] Starting from Escherichia coli ATCC 8739, the acetolactate synthase gene alsS from Bacillus subtilis 168 (from ATCC, No. 23857) and the promoter for alsS expression (i.e., alsS expression cassette) were inserted into the propionate kinase encoding gene tdcD and formate acetyltransferase encoding gene tdcE sites on the chromosome using a two-step homologous recombination method. The specific steps are as follows:

[0107] (1) Using pXZ-CS (Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844) plasmid DNA as a template, primers tdcDE-incs-up / tdcDE-incs-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination.

[0108] The amplification system was: Phusion 5X buffer (New England Biolabs) 10 μl, dNTP (10 mM each) 1 μl, DNA template 20 ng, primers (10 μM) 2 μl each, Phusion High-Fidelity DNA polymerase (2.5 U / μl) 0.5 μl, and distilled water was added to 50 μl.

[0109] Amplification conditions were as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, and extension at 72°C for 2 minutes (30 cycles); and extension at 72°C for 10 minutes (1 cycle).

[0110] The DNA fragment I obtained above was used for the first homologous recombination: first, the pKD46 plasmid (CGSC Escherichia coli Collection Center of Yale University, USA, CGSC#7739) was transformed into Escherichia coli ATCC 8739 by electroporation, and then the DNA fragment I was electroporated into Escherichia coli ATCC 8739 carrying pKD46.

[0111] The electroporation conditions are as follows: first prepare electroporation competent cells of Escherichia coli ATCC 8739 carrying the pKD46 plasmid (preparation method according to Dower et al., 1988, Nucleic Acids Res 16: 6127-6145); place 50 μl of competent cells on ice, add 50 ng of DNA fragment I, place on ice for 2 minutes, and transfer to a 0.2 cm Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, the electroporation parameter is 2.5 kV. After electroporation, quickly transfer 1 ml of LB culture medium to the electroporation cup, pipette 5 times, and then transfer to a test tube, incubate at 75 rpm, 30°C for 2 hours. Take 200 μl of bacterial solution and apply it on LB plate containing ampicillin (final concentration of 100 μg / ml) and chloramphenicol (final concentration of 34 μg / ml). After overnight culture at 30°C, select a single colony for PCR verification. The primers used are XZ-tdcDE-up / XZ-tdcDE-down. The amplification product of the correct colony is a fragment of 3615 bp. Select a single colony with the correct sequence and name it S HMBA 001.

[0112] (2) Insert the alsS gene and promoter together into S HMBA 001 genome.

[0113] First, wild-type Bacillus subtilis 168 genomic DNA was used as a template and primers alsS-M93-up / alsS-tdcDE-down were used to amplify a 1796 bp DNA fragment II. The amplification conditions and system were the same as those described in (1).

[0114] In the second step, the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) was used as a template and primers M93-tdcDE-up / M93-alsS-down were used to amplify the 160 bp DNA fragment III. The amplification conditions and system were the same as (1).

[0115] In the third step, the same amplification conditions and system as in (1) were used for fusion PCR, except that the DNA template in (1) was replaced with 20 ng fragment II and 20 ng fragment III, and the primers used were M93-tdcDE-up / alsS-tdcDE-down. The amplified band was 1914 bp, and the fusion DNA fragment I (i.e., alsS expression cassette) for the second step of homologous recombination was obtained after gel excision and recovery. The fusion DNA fragment I was electroporated into the S-containing pKD46. HMBA 001 strain. The electroporation conditions were as follows: First, prepare S HMBA 001 electroporation competent cells; 50 μl competent cells were placed on ice, 50 ng fusion DNA fragment I was added, placed on ice for 2 minutes, and transferred to a 0.2 cm Bio-Rad electroporation cup. MicroPulser (Bio-Rad) electroporator was used, and the electroporation parameters were 2.5 kV. After electroporation, 1 ml of LB culture medium was quickly transferred to the electroporation cup, and after blowing 5 times, it was transferred to a test tube, 75 rpm, and incubated at 30°C for 4 hours. The bacterial liquid was transferred to LB liquid culture medium without sodium chloride containing 10% sucrose (50 ml culture medium in a 250 ml flask), and after culturing for 24 hours, it was streaked on LB solid culture medium without sodium chloride containing 6% sucrose. After PCR verification, the primers used were XZ-tdcDE-up / XZ-tdcDE-down, and the correct colony amplification product was a 2810 bp fragment. A single colony with the correct sequence was selected and named S HMBA 002.

[0116] S HMBA002 is a recombinant bacterium obtained by integrating the alsS expression cassette (nucleotide sequence is SEQ ID No. 1 in the sequence list) into the propionate kinase encoding gene tdcD and the formate acetyltransferase encoding gene tdcE sites of Escherichia coli ATCC 8739. In SEQ ID No. 1, positions 89-1804 are the nucleotide sequence of the acetolactate synthase gene alsS, encoding the alsS protein shown in SEQ ID No. 2; positions 1-88 in SEQ ID No. 1 are the nucleotide sequence of the M1-93 promoter.

[0117] Example 2. Regulation of acetolactate synthase gene ilvBN

[0118] The expression of the acetolactate synthase gene ilvBN of Escherichia coli is regulated by using an artificial regulatory element through a two-step homologous recombination method. The specific steps are as follows:

[0119] (1) Using pXZ-CS plasmid DNA as a template, primers CS-ilvB-up / CS-ilvB-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0120] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 002, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA 002, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers ilvB-YZ-up / ilvB-YZ-down. The correct PCR product was 2996 bp. A single colony with the correct sequence was selected and named S HMBA 003.

[0121] (2) Using the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) as a template, primers M93-ilvB-up\M93-ilvB-down were used to amplify a 188 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S HMBA003, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were ilvB-YZ-up / ilvB-YZ-down, and the correct colony amplification product was a 465 bp fragment. A single colony with the correct sequence was selected and named S HMBA 004.

[0122] S HMBA 004 is to replace the M1-93 promoter (nucleotide sequence is 1-88 in SEQ ID No. 1 in the sequence list) with the Escherichia coli S HMBA The recombinant bacteria were obtained by expressing the promoter of the acetolactate synthase gene ilvB (GenBank: CP000946.1 (Escherichiacoli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_0028") and ilvN (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_0029") of 002. In the recombinant bacteria, the M1-93 promoter can drive the expression of the ilvB gene and the ilvN gene.

[0123] Example 3, Regulation of acetolactate synthase gene ilvGM

[0124] The expression of the acetolactate synthase gene ilvGM of Escherichia coli is regulated by the artificial regulatory element M1-93 promoter through a two-step homologous recombination method. The specific steps are as follows:

[0125] (1) Using pXZ-CS plasmid DNA as a template, primers CS-ilvG-up / CS-ilvG-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0126] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 004, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA004, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers ilvG-YZ-up / ilvG-YZ-down. The correct PCR product was 2993 bp. A single colony with the correct sequence was selected and named S HMBA 005.

[0127] (2) Using the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) as a template, primers M93-ilvG-up / M93-ilvG-down were used to amplify a 188 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S HMBA 005, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were ilvG-YZ-up / ilvG-YZ-down, and the correct colony amplification product was a 462 bp fragment. A single colony with the correct sequence was selected and named S HMBA 006.

[0128] S HMBA 006 is to replace the M1-93 promoter (nucleotide sequence is 1-88 in SEQ ID No. 1 in the sequence list) with the Escherichia coli S HMBA The recombinant bacteria were obtained by combining the promoter of the acetolactate synthase gene ilvG (GenBank: CP000946.1 (Escherichiacoli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_4233") of 004 ​​and the promoter of ilvM (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_4234"), in which the M1-93 promoter can drive the expression of the ilvGM gene.

[0129] Example 4: Mutation of the acetolactate synthase gene ilvH

[0130] A mutation was introduced into the ilvH gene to relieve the feedback inhibition of L-valine by a two-step homologous recombination method. The specific steps are as follows:

[0131] (1) Using pXZ-CS plasmid DNA as a template, primers CS-ilvH-up / CS-ilvH-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0132] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 006, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA 006, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers ilvH-mutYZ-up / ilvH-mut-down. The correct PCR product was 3165 bp. A correct single colony was selected and named S HMBA 007.

[0133] (2) Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, the primers ilvH-mut-up / ilvH-mut-down were used to amplify a 467 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S HMBA 007, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were ilvH-mutYZ-up / ilvH-mut-down, and the correct colony amplification product was a 619 bp fragment. A single colony with the correct sequence was selected and named S HMBA 008.

[0134] S HMBA 008 is S HMBA The recombinant bacteria obtained by mutating the 40-42 positions of the acetolactate synthase gene ilvH in 006 from ggc to gac, and the 49-51 positions from tcc to ttc, the sequence of the mutated ilvH gene (denoted as ilvHm gene) is SEQ ID No.3 in the sequence table, encoding the ilvHm protein shown in SEQ ID No.4 (which is obtained by mutation of G at position 14 to D and S at position 17 to F of the ilvH protein).

[0135] Example 5: Integration of the acetohydroxyacid reductoisomerase gene kari

[0136] (1) Acquisition of acetohydroxyacid reductoisomerases. According to the reported literature (Brinkmann-Chen, S., Cahn, JKB & Arnold. FH, Uncovering rare NADH-preferring ketol-acid reductoisomerases, Metab Eng, 2014, 26: 17-22, doi: 10.1016 / j.ymben.2014.08.003), the kari gene (90-1088 of SEQ ID No. 5, encoding the kari protein shown in SEQ ID No. 6) was obtained by codon optimization and chemical synthesis by Nanjing GenScript Biotechnology Co., Ltd., and the artificial regulatory element RBSL5 promoter (1-89 of SEQ ID No. 5) was added in front of the kari gene. The obtained DNA fragment was recorded as the expression cassette RBSL5-kari (SEQ ID No. 5 in the sequence list). ID No.5), RBSL5-kari was synthesized and connected to pUC57 plasmid of GenScript to obtain pUC57-RBSL5-kari. Then, the RBSL5-kari expression cassette was integrated into S HMBA The adhE locus of the alcohol dehydrogenase encoding gene in strain 008.

[0137] (2) Using pXZ-CS plasmid DNA as a template, primers CS-adhE-up / CS-adhE-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0138] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 008, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA 008, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-adhE-up / XZ-adhE-down. The correct PCR product was 3167 bp. A single colony with the correct sequence was selected and named S HMBA 009.

[0139] (3) Using pUC57-RBSL5-kari plasmid DNA as a template, primers RBSL5-adhE-up / kari-adhE-down were used to amplify a 1188 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S HMBA 009, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were XZ-adhE-up / XZ-adhE-down, and the correct colony amplification product was a 1636 bp fragment. A single colony with the correct sequence was selected and named S HMBA 010.

[0140] S HMBA 010 is to integrate the RBSL5-kari expression cassette into S HMBA The recombinant bacteria were obtained by removing the adhE site of the alcohol dehydrogenase encoding gene in the 008 strain.

[0141] Example 6: Integration of the dihydroxyacid dehydratase gene ilvD into the pyruvate formate lyase gene pflB site

[0142] From S HMBA Starting from 010, the expression cassette of the dihydroxyacid dehydratase encoding gene ilvD from Escherichia coli was integrated into the pyruvate formate lyase encoding gene pflB site by a two-step homologous recombination method. The specific steps are as follows:

[0143] (1) Using pXZ-CS plasmid DNA as a template, primers CS-pflB-up / CS-pflB-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1.

[0144] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 010, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA 010, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-pflB-up600 / XZ-pflB-down. The correct PCR product was 3675 bp. A single colony with the correct sequence was selected and named S HMBA 011.

[0145] (2) Using the genomic DNA of Escherichia coli MG1655 (from ATCC, No. 700926) as a template, the 1922 bp DNA fragment II was amplified using the primers ilvD-up / ilvD-pflB-down.

[0146] Using the genomic DNA of M1-93 (Lu, et al., Appl Microbiol Biotechnol, 2012, 93: 2455-2462) as a template, the 159 bp DNA fragment III was amplified using primers RBSL4-pflB-up\RBSL4-ilvD-down.

[0147] The same fusion PCR method as described in step (2) of Example 1 was used to perform fusion PCR with fragment II and fragment III of this example as templates to obtain a DNA fragment containing the upstream and downstream homologous arms of pflB, the artificial promoter RBSL4 and the ilvD gene after fusion. The fusion PCR primers used were RBSL4-pflB-up\ilvD-pflB-down. The fragment was used for electroporation into S HMBA 011. The electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR. The primers used were XZ-pflB-up600 / XZ-pflB-down. The correct colony amplification product was a 2996 bp fragment. A single colony with the correct sequence was selected and named S HMBA 012.

[0148] S HMBA 012 is to integrate the dihydroxyacid dehydratase encoding gene expression cassette (whose sequence is SEQ ID No.7 in the sequence list) into the Escherichia coli S HMBA The recombinant bacteria were obtained by adding the pflB site of the pyruvate formate lyase encoding gene of 010. In SEQ ID No.7, positions 1-89 are the sequence of the RBSL4 promoter, and positions 90-1940 are the nucleotides of the ilvD gene, encoding the ilvD protein shown in SEQ ID No.8.

[0149] Example 7: Integration of the acetolactate synthase gene alsS into the lactate dehydrogenase gene ldhA site in ATCC 8739 strain

[0150] The RBS4-alsS expression cassette was integrated into the ldhA site on the chromosome by a two-step homologous recombination method.

[0151] (1) Using pXZ-CS plasmid DNA as a template, primers CS-ldhA-up / CS-ldhA-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0152] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. HMBA 012, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 HMBA 012, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-ldhA-up / XZ-ldhA-down. The correct PCR product was 3402 bp. A single colony with the correct sequence was selected and named S HMBA 013.

[0153] (2) Using the same method as described in Example 1 (2), wild-type Bacillus subtilis 168 genomic DNA was used as a template, and primers alsS-RBSL-up / alsS-ldhA-down were used to amplify the 1795 bp alsS gene, and the DNA of M1-93 was used as a template and primers RBS4-ldhA-up / RBS4-alsS-down were used to amplify the RBS4 fragment, and primers RBS4-ldhA-up and alsS-ldhA-down were used to obtain a DNA fragment for integrating the alsS gene and promoter into the ldhA site (i.e., RBS4-alsS expression cassette) by fusion PCR amplification. The fusion fragment was used for the second step of homologous recombination. The RBS4-alsS expression cassette was electroporated into the strain S HMBA 013, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were XZ-ldhA-up / XZ-ldhA-down, and the correct colony amplification product was a 2595 bp fragment. A single colony with the correct sequence was selected and named S KIV 001.

[0154] S KIV 001 is the integration of the RBS4-alsS expression cassette (nucleotide sequence is SEQ ID No. 9 in the sequence list) into the Escherichia coli S HMBAThe recombinant bacteria were obtained by purifying the lactate dehydrogenase gene ldhA site of Escherichia coli ATCC8739 (GenBank: CP000946.1 (Escherichia coli ATCC8739, complete genome, 10-FEB-2014), locus_tag="EcolC_2275"). In SEQ ID No.9, positions 89-1804 are the nucleotide sequence of the acetolactate synthase gene alsS, encoding the alsS protein shown in SEQ ID No.2; positions 1-89 in SEQ ID No.9 are the nucleotide sequence of the RBS4 promoter.

[0155] Embodiment 8, S KIV Fermentation of 001

[0156] The seed culture medium consists of the following ingredients (the solvent is water): glucose 20 g / L, NH4H2PO4 0.87 g / L, (NH4)2HPO4 2.63 g / L, MgSO4·7H2O 0.18 g / L, betaine-HCl 0.15 g / L, FeCl3·6H2O 1.5 μg / L, CoCl2·6H2O 0.1 μg / L, CuCl2·2H2O 0.1 μg / L, ZnCl2 0.1 μg / L, Na2MoO4·2H2O 0.1 μg / L, MnCl2·4H2O 0.2 μg / L, and H3BO3 0.05 μg / L.

[0157] Fermentation medium: Except for the glucose concentration and seed medium, the other components and concentrations are the same. The glucose concentration in the fermentation medium is 80g / L.

[0158] Fermentation steps:

[0159] (1) Seed culture: fresh S KIV The 001 monoclone was inoculated into a test tube containing 4 ml of seed culture medium and cultured overnight at 37°C, 250 rpm. Then, the culture was transferred to a 250 ml Erlenmeyer flask containing 30 ml of seed culture medium at a 2% (V / V) inoculation volume and cultured at 37°C, 250 rpm for 12 hours to obtain a seed culture solution for inoculation of the fermentation medium.

[0160] (2) Fermentation culture: The volume of the fermentation medium in 5L is 3L, and it is sterilized at 115℃ for 25min. The seed liquid is inoculated into the fermentation medium at an inoculum amount of a final concentration of OD550=0.2, cultured at 37℃, with a stirring speed of 300rpm and a ventilation volume of 0.2vvm. The fermentation liquid is obtained after 48h of culture. The fermentation liquid is all the substances in the fermenter. During the fermentation process, the neutralizer 0.4M sodium hydroxide is used to control the pH of the fermenter at 7.0. No gas is passed during the culture process.

[0161] Analytical method: Agilent-1260 high performance liquid chromatograph was used to determine the components in the fermentation broth. The concentrations of glucose and organic acids in the fermentation broth were determined using Biorad's Aminex HPX-87H organic acid analysis column.

[0162] The results showed that: KIV 001 can produce 34 g / L of α-ketoisovaleric acid, and the sugar-acid conversion rate is 0.48 g / g.

[0163] Example 9: Knockout of the phosphate acetyltransferase gene pta and the acetate kinase gene ackA

[0164] From S KIV Starting from 001, the gene encoding the phosphate acetyltransferase pta and the gene encoding the acetate kinase ackA of Escherichia coli were knocked out by a two-step homologous recombination method. The specific steps are as follows:

[0165] (1) Using pXZ-CS plasmid DNA as a template, primers CS-ackA-up / CS-pta-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0166] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. KIV 001, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 KIV 001, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-ackA-up / XZ-pta-down. The correct PCR product was 3351 bp. A single colony with the correct sequence was selected and named S KIV 002.

[0167] (2) Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, primers XZ-ackA-up / ackA-del-down were used to amplify a 371 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S KIV002, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were XZ-ackA-up / XZ-pta-down, and the correct colony amplification product was a 732 bp fragment. A single colony with the correct sequence was selected and named S KIV 003.

[0168] S KIV 003 is S KIV The recombinant bacteria were obtained by knocking out the phosphate acetyltransferase gene pta (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_1355") and the acetate kinase encoding gene ackA (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_1356") of 001.

[0169] Example 10, knockout of methylglyoxal synthase gene mgsA

[0170] From S KIV Starting from 003, the methylglyoxal synthase gene mgsA of Escherichia coli was knocked out by a two-step homologous recombination method. The specific steps are as follows:

[0171] (1) Using pXZ-CS plasmid DNA as a template, primers CS-mgsA-up / CS-mgsA-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0172] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. KIV 003, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 KIV 003, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-mgsA-up / XZ-mgsA-down. The correct PCR product was 3646 bp. A single colony with the correct sequence was selected and named S KIV 004.

[0173] (2) Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, primers XZ-mgsA-up / mgsA-del-down were used to amplify a 566 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S KIV 004, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were XZ-mgsA-up / XZ-mgsA-down, and the correct colony amplification product was a 1027 bp fragment. A single colony with the correct sequence was selected and named S KIV 005.

[0174] S KIV 005 is S KIV The recombinant bacteria were obtained by knocking out the methylglyoxal synthase encoding gene mgsA (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag="EcolC_2633") of 003.

[0175] Example 11: Knockout of the fumarate reductase encoding gene frd

[0176] From S KIV Starting from 005, the fumarate reductase gene frd (including frdB and frdC) of Escherichia coli was knocked out by a two-step homologous recombination method. The specific steps are as follows:

[0177] (1) Using pXZ-CS plasmid DNA as a template, primers CS-frd-up / CS-frd-down were used to amplify a 2719 bp DNA fragment I for the first step of homologous recombination. The amplification system and amplification conditions were the same as those described in Example 1 (1).

[0178] DNA fragment I was used for the first homologous recombination: first, the pKD46 plasmid was transformed into Escherichia coli S by electroporation. KIV 005, and then the DNA fragment I was electroporated into Escherichia coli S carrying pKD46 KIV 005, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (1). A single colony was selected for PCR verification using primers XZ-frd-up / XZ-frd-down. The correct PCR product was 3234 bp. A single colony with the correct sequence was selected and named S KIV 006.

[0179] (2) Using the DNA of wild-type Escherichia coli ATCC 8739 as a template, primers XZ-frd-up / frd-del-down were used to amplify a 270 bp DNA fragment II. DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain S KIV 006, the electroporation conditions and steps were the same as those described in Example 1 for the integration of alsS at the tdcDE site (2). The clones were verified by colony PCR, the primers used were XZ-frd-up / XZ-frd-down, and the correct colony amplification product was a 615 bp fragment. A single colony with the correct sequence was selected and named SKIV007.

[0180] SKIV007 is S KIV The recombinant strain was obtained by knocking out the fumarate reductase encoding gene frdB (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_3907") and frdC (GenBank: CP000946.1 (Escherichia coli ATCC 8739, complete genome, 10-FEB-2014), locus_tag = "EcolC_3858") of SKIV007. SKIV007 was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number CGMCC No. 27832.

[0181] Example 12: Fermentation of recombinant bacteria

[0182] In this example, SKIV007 was used to ferment and produce α-ketoisovaleric acid. The method was the same as that in Example 8. The seed culture medium and fermentation medium used were the same as those in Example 8, except that the glucose concentration in the fermentation medium was 105 g / L.

[0183] The fermentation conditions and analysis methods were the same as in Example 8.

[0184] The results showed that SKIV007 could produce 58g / L of α-ketoisovaleric acid, with a sugar-acid conversion rate of 0.6g / g, reaching 94% of the theoretical conversion rate. The above results show that SKIV007 can well achieve one-step fermentation production of α-ketoisovaleric acid.

[0185] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A recombinant Escherichia coli comprising at least one of the following characteristics: A1) containing a substance capable of expressing the acetolactate synthase gene and / or a substance promoting the expression of the acetolactate synthase gene and / or a substance increasing the activity or content of acetolactate synthase; A2) containing a substance capable of expressing an acetohydroxy acid reductoisomerase gene and / or a substance promoting the expression of an acetohydroxy acid reductoisomerase gene and / or a substance increasing the activity or content of acetohydroxy acid reductoisomerase; A3) Contains a substance capable of expressing a dihydroxy-acid dehydratase gene and / or a substance promoting the expression of a dihydroxy-acid dehydratase gene and / or a substance increasing the activity or content of dihydroxy-acid dehydratase.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The acetolactate synthase is derived from Bacillus subtilis or Escherichia coli; And / or, the acetohydroxyacid reductoisomerase is derived from Thermacetogeniumphaeum; And / or, the dihydroxy-acid dehydratase is derived from Escherichia coli.

3. The recombinant Escherichia coli according to claim 1 or 2, characterized in that: The acetolactate synthase gene is an alsS gene, an ilvB gene, an ilvN gene, an ilvG gene and / or an ilvM gene, and the acetolactate synthase is a protein encoded by the alsS gene, the ilvB gene, the ilvN gene, the ilvG gene and / or the ilvM gene; And / or, the acetohydroxy acid reductoisomerase gene is the kari gene, and the acetohydroxy acid reductoisomerase is a protein encoded by the kari gene; And / or, the dihydroxy-acid dehydratase gene is the ilvD gene, and the dihydroxy-acid dehydratase is a protein encoded by the ilvD gene.

4. The recombinant Escherichia coli according to any one of claims 1 to 3, characterized in that: A1), the substance capable of expressing the acetolactate synthase gene is an alsS gene expression cassette; the substance that increases the activity or content of acetolactate synthase is the alsS gene expression cassette and / or a regulatory element that increases the expression amount of the acetolactate synthase gene; A2), wherein the substance containing the gene capable of expressing acetohydroxyacid reductoisomerase is a kari gene expression cassette; A3), the substance containing the gene capable of expressing dihydroxy-acid dehydratase is an ilvD gene expression cassette.

5. The recombinant Escherichia coli according to any one of claims 1 to 4, characterized in that: The recombinant Escherichia coli also includes any one, any two, any three or four of the following characteristics: A4) does not contain phosphate acetyltransferase gene or contains substances that inhibit the expression of phosphate acetyltransferase gene or contains substances that reduce the content or activity of phosphate acetyltransferase; A5) does not contain acetate kinase gene or contains substances that inhibit the expression of acetate kinase gene or contains substances that reduce the content or activity of acetate kinase; A6) does not contain methylglyoxal synthase gene or contains substances that inhibit the expression of methylglyoxal synthase gene or contains substances that reduce the content or activity of methylglyoxal synthase; A7) does not contain fumarate reductase gene or contains a substance that inhibits the expression of fumarate reductase gene or contains a substance that reduces the content or activity of fumarate reductase.

6. Escherichia coli SKIV007, whose deposit number at the General Microbiological Center of China Culture Collection Administration is CGMCC No.27832.

7. A method for preparing a recombinant Escherichia coli, comprising transforming a starting Escherichia coli as follows: B1) expressing acetolactate synthase gene and / or promoting acetolactate synthase gene expression and / or increasing acetolactate synthase activity or content; B2) expressing an acetohydroxy acid reductoisomerase gene and / or promoting the expression of an acetohydroxy acid reductoisomerase gene and / or increasing the activity or content of acetohydroxy acid reductoisomerase; B3) expressing a dihydroxy-acid dehydratase gene and / or promoting the expression of a dihydroxy-acid dehydratase gene and / or increasing the activity or content of dihydroxy-acid dehydratase.

8. The method according to claim 7, characterized in that: The acetolactate synthase is derived from Bacillus subtilis or Escherichia coli; And / or, the acetohydroxyacid reductoisomerase is derived from Thermacetogeniumphaeum; And / or, the dihydroxy-acid dehydratase is derived from Escherichia coli.

9. The method according to claim 7 or 8, characterized in that: The acetolactate synthase gene is an alsS gene, an ilvB gene, an ilvN gene, an ilvG gene and / or an ilvM gene, and the acetolactate synthase is a protein encoded by the alsS gene, the ilvB gene, the ilvN gene, the ilvG gene and / or the ilvM gene; And / or, the acetohydroxy acid reductoisomerase gene is the kari gene, and the acetohydroxy acid reductoisomerase is a protein encoded by the kari gene; And / or, the dihydroxy-acid dehydratase gene is the ilvD gene, and the dihydroxy-acid dehydratase is a protein encoded by the ilvD gene.

10. The method according to any one of claims 7 to 9, characterized in that: B1), the expression of the acetolactate synthase gene is achieved by introducing the alsS gene expression cassette of claim 4 into the starting Escherichia coli, and the increase in the activity or content of the acetolactate synthase is achieved by replacing the promoters of the ilvB gene, ilvN gene, ilvG gene and ilvM gene with the regulatory element for increasing the expression amount of the acetolactate synthase gene of claim 4; and / or, B2) by introducing the kari gene expression cassette of claim 4 into the starting Escherichia coli; And / or, B3) is achieved by introducing the ilvD gene expression cassette according to claim 4 into the starting Escherichia coli.

11. The method according to any one of claims 7 to 10, characterized in that: The method further comprises performing any one, any two, any three or four of the following modifications on the starting Escherichia coli: B4) knocking out the phosphate acetyltransferase gene or inhibiting the expression of the phosphate acetyltransferase gene or reducing the content or activity of phosphate acetyltransferase; B5) knocking out the acetate kinase gene or inhibiting the expression of the acetate kinase gene or reducing the content or activity of acetate kinase; B6) knocking out the methylglyoxal synthase gene or inhibiting the expression of the methylglyoxal synthase gene or reducing the content or activity of methylglyoxal synthase; B7) Knocking out the fumarate reductase gene or inhibiting the expression of the fumarate reductase gene or reducing the content or activity of fumarate reductase.

12. A method for preparing α-ketoisovaleric acid, comprising: Cultivate the recombinant Escherichia coli according to any one of claims 1 to 5 or the Escherichia coli SKIV007 according to claim 6 to prepare α-ketoisovaleric acid.

13. A product comprising the recombinant Escherichia coli according to any one of claims 1 to 5 or the Escherichia coli SKIV007 according to claim 6.

14. Any of the following uses of the recombinant Escherichia coli of any one of claims 1 to 5, the Escherichia coli SKIV007 of claim 6, or the product of claim 13: P1) produces α-ketoisovaleric acid; P2) Preparation of products for producing α-ketoisovaleric acid.

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