Engineered strain for synthesizing l-isoleucine on basis of methylmalate pathway and use

Abstract

A method for improving the yield of synthesized L-isoleucine on the basis of a methylmalate pathway and an engineered strain. The method comprises the following steps: overexpressing a methylmalate synthase gene; and expressing a coding gene for a carboxylic acid transporter DcuD, DauA or DcuA. The carboxylic acid transporters DcuD, DauA and DcuA have the function of methylmalate uptake proteins, can effectively relieve the inhibitory effect of glucose on methylmalate utilization during the fermentation of the engineered strain, and improve the L-isoleucine synthesis ability based on the methylmalate pathway.

Description

An engineered strain for synthesizing L-isoleucine based on a methylmalonic acid pathway and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of engineered strains, in particular to an engineered strain for synthesizing L-isoleucine based on a methylmalonic acid pathway and application thereof. BACKGROUND

[0002] L-isoleucine belongs to branched-chain amino acids and is a synthetic material for hormones, enzymes and other important substances in human and animal bodies. As one of the essential amino acids, L-isoleucine cannot be synthesized by humans and most livestock and poultry animals, and therefore has a huge market demand in the fields of feed, food, medicine and health care.

[0003] Microbial fermentation method is the main method for industrial scale production of L-isoleucine at present due to its low production cost and environmentally friendly fermentation conditions. The methylmalonic acid pathway (also known as the pyruvate pathway) is one of the pathways for synthesizing L-isoleucine. The methylmalonic acid pathway has shorter synthesis steps and simpler regulation mode, and is a very potential L-isoleucine biosynthesis pathway. However, the engineered strain for producing L-isoleucine by using the methylmalonic acid pathway will produce a large amount of methylmalonic acid when fermented in a culture medium with glucose as the carbon source. The strain itself will quickly excrete excessive methylmalonic acid outside the cell. When glucose is present, the extracellular methylmalonic acid cannot enter the cell for metabolism due to carbon metabolic inhibition effect, resulting in accumulation of extracellular methylmalonic acid, and the efficiency of synthesizing L-isoleucine by using the methylmalonic acid pathway is reduced. SUMMARY

[0004] The purpose of the present application is to provide an engineered strain for synthesizing L-isoleucine based on a methylmalonic acid pathway.

[0005] Another purpose of the present application is to provide the application of the above-mentioned engineered strain for synthesizing L-isoleucine based on a methylmalonic acid pathway.

[0006] The technical solution of the present application has the following advantages:

[0007] 1. The engineered strain for producing L-isoleucine by using the methylmalonic acid pathway (also known as pyruvate pathway) produces a large amount of methylmalonic acid when fermented in a culture medium with glucose as the carbon source. The strain itself can quickly excrete excess methylmalonic acid outside the cell. However, when glucose is present, the extracellular methylmalonic acid cannot re-enter the cell for metabolism due to the carbon metabolic inhibition effect, resulting in the accumulation of extracellular methylmalonic acid and a decrease in the efficiency of synthesizing L-isoleucine by using the methylmalonic acid pathway. The present application determines that carboxylate transporters DcuD, DauA, and DcuA have the function of methylmalonic acid absorption protein, which can effectively eliminate the inhibition of glucose on the utilization of methylmalonic acid during fermentation of the engineered strain, and significantly improve the L-isoleucine synthesis capacity based on the methylmalonic acid pathway.

[0008] 2. The methylmalonic acid pathway synthesizes L-isoleucine using pyruvate and acetyl-CoA as precursors, so a strain with sufficient supply of pyruvate and acetyl-CoA is suitable as a chassis strain for constructing the methylmalonic acid pathway. According to the technical solution of the present application, the synthesis amount and synthesis efficiency of methylmalonic acid are the highest when Escherichia coli BW25113 is used as the chassis strain, which is significantly better than other commonly used Escherichia coli strains. Moreover, by overexpressing the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the phosphotransacetylase gene pta of Escherichia coli to introduce a non-oxidative glycolytic pathway, the supply of intracellular acetyl-CoA is increased, and test data show that the L-isoleucine yield of the obtained engineered strain is significantly improved.

[0009] 3. The three synthesis steps from pyruvate and acetyl-CoA to alpha-ketobutyric acid in the methylmalonic acid pathway are the key points that distinguish it from the traditional threonine pathway, and isopropylmalate isomerase LeuCD and 3-isopropylmalate dehydrogenase LeuB also participate in the leucine synthesis pathway. Therefore, LeuCD and LeuB enzymes that can efficiently convert methylmalonic acid to alpha-ketobutyric acid are the key to synthesizing L-isoleucine by using the methylmalonic acid pathway. According to the technical solution of the present application, the isopropylmalate isomerase GsLeuCD from Geobacillus sp. WCH70 strain and the 3-isopropylmalate dehydrogenase AfLeuB from Archaeoglobus fulgidus have significant advantages compared to enzymes from other sources.

[0010] 4. According to the technical solution of the present application, when the copy number of cimA3.7 integrated on the genome is 3 times that of GsleuCD and the copy number of AfleuB integrated on the genome is 5 times that of GsleuCD, the synthesis efficiency of L-isoleucine is the highest.

[0011] 5. Knocking out branched-chain amino acid absorption protein genes brnQ, livJ, livK and overexpressing branched-chain amino acid exoprotein genes ygaZ, ygaH and regulatory protein gene lrp can reduce intracellular L-isoleucine accumulation and significantly improve L-isoleucine yield.

[0012] 6. Since the methylmalonic acid pathway synthesizes L-isoleucine from pyruvate and acetyl-CoA, and the isopropylmalate isomerase LeuCD, 3-isopropylmalate dehydrogenase LeuB and leucine dehydrogenase LeuDH in the pathway are also involved in leucine biosynthesis, the present application solves the problem of pyruvate and acetyl-CoA supply from the perspective of selecting a chassis cell and introducing a non-oxidative glycolysis (NOG) pathway, and obtains more suitable catalytic enzymes for the methylmalonic acid pathway from the perspective of selecting key enzymes LeuCD, LeuB and LeuDH. In order to further reduce the accumulation of intermediate metabolites and L-isoleucine in the cell, the present application optimizes the expression intensity of the key genes cimA, leuCD and leuB relative to each other and the L-isoleucine transport system, and ultimately greatly improves the synthesis capacity of L-isoleucine. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 shows a comparison of isoleucine biosynthesis pathways based on the threonine pathway and the methylmalonic acid pathway;

[0014] Figure 2 shows the shake flask fermentation results of the starting strain BW25113 and the engineered strains BW1 and BW2 in M9+Glu medium provided in Example 1 of the present application;

[0015] Figure 3 shows the shake flask fermentation results of the engineered strain BW1 in M9+Glu+Ile medium provided in Example 1 of the present application;

[0016] Figure 4 shows the shake flask fermentation results of the engineered strain BW1 in M9+CM medium provided in Example 1 of the present application;

[0017] Figure 5 shows the shake flask fermentation results of the engineered strain BW1 in M9+Glu+CM medium provided in Example 1 of the present application;

[0018] Figure 6 shows the shake flask fermentation results of the engineered strains BW3-BW12 in M9+Glu+CM medium provided in Example 2 of the present application.

[0019] Figure 7 is the shake flask fermentation results of the engineered E. coli strains JQ1, JQ2, JQ3 and JQ4 for producing methylmalonic acid provided in Example 5 of the present application;

[0020] Figure 8 is the shake flask fermentation results of the engineered E. coli strains JQ5-JQ10 provided in Example 6 of the present application;

[0021] Figure 9 is a shake flask fermentation result of E. coli engineered strains ILE-3 and ILE-4 provided in Example 7 of the present application;

[0022] Figure 10 is a fermentation result of E. coli engineered strains ILE-5 to ILE-11 provided in Example 8 and Example 9 of the present application in a 3L fermenter;

[0023] Figure 11 is a fermentation result of E. coli engineered strains ILE-12 to ILE-15 provided in Example 10 of the present application in a 3L fermenter;

[0024] Figure 12 is a fermentation result of E. coli engineered strain ILE-13 provided in Example 11 of the present application in a 10L fermenter. DETAILED DESCRIPTION

[0025] The technical solutions of the present application are described in further detail below through specific embodiments, but the scope or embodiments of the present application are not limited.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used are commercially available unless otherwise specified.

[0027] Table 1 is a list of E. coli engineered strains constructed in Example 1 to Example 4 of the present application

[0028] Table 2 is a list of E. coli engineered strains constructed in Example 5 to Example 12 of the present application

[0029] As shown in Table 2 above, the specific embodiments of the present application further improve the production of L-isoleucine of E. coli by the following measures:

[0030] First, different wild-type E. coli strains including BW25113, W3110, MG1655 and W (ATCC9637) strains are screened by using plasmid P1 (P J23100 -cimA3.7) as the chassis strains for constructing the methylmalonic acid pathway, wherein the methylmalonic acid production capacity of the selected E. coli BW25113 chassis strain is superior to other strains;

[0031] Second, different combinations of plasmids P2-P8 containing leuCD, leuB, leudh genes from different sources were expressed in E. coli BW25113 as a chassis strain, among which the isopropyl malate isomerase gene GsleuCD from Geobacillus sp. WCH70 strain, the 3-isopropyl malate dehydrogenase gene AfleuB from Archaeoglobus fulgidus, and the leucine dehydrogenase gene LsleuDH from Lysinibacillus sphaericus were screened out, which significantly improved the L-isoleucine synthesis ability of E. coli based on the methylmalonic acid pathway;

[0032] Third, the utilization efficiency of methylmalonic acid was improved by overexpressing the methylmalonic acid absorption protein gene dcuD.

[0033] Fourth, by integrating the branched-chain amino acid excretion protein gene ygaZH and the regulatory protein gene lrp into the branched-chain amino acid absorption protein gene brnQ, livJ, and livK sites, the excretion of L-isoleucine was improved.

[0034] Fifth, by integrating the expression cassette of methylmalonic acid pathway related genes into the hypothetical protein gene yjip and gapC sites, overexpressing the genes cimA3.7, GsleuCD, and AfleuB in the yjip site, and overexpressing the genes ilvIH*, ilvC*, ilvD, LsleuDH, and dcuD in the gapC site, a plasmid-free L-isoleucine producing strain was constructed.

[0035] Sixth, by integrating an expression cassette containing the methylmalonic acid synthase gene cimA3.7 and the 3-isopropyl malate dehydrogenase gene AfleuB into the formate acetyltransferase gene pflB and the lactate dehydrogenase gene ldhA sites, the carbon metabolic flow of the methylmalonic acid pathway was increased, and the ability to synthesize L-isoleucine was improved.

[0036] Seventh, by integrating one copy of the 3-isopropyl malate dehydrogenase gene AfleuB expression cassette into the acetaldehyde dehydrogenase gene adhE and the hypothetical protein gene ycjV sites, the carbon metabolic flow of the methylmalonic acid pathway was increased, and the ability to synthesize L-isoleucine was improved.

[0037] Eighth, by integrating the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the phosphotransacetylase gene pta from E. coli into the phosphotransacetylase gene ackA site, the supply of acetyl-CoA was increased, thereby increasing the carbon metabolic flow of the methylmalonic acid pathway and further improving the yield of synthesized L-isoleucine.

[0038] The present application relates to the following protein or nucleotide sequences:

[0039] SEQ ID NO: 1 (DcuD)

[0040] SEQ ID NO: 2 (DauA)

[0041] SEQ ID NO: 3 (DcuA)

[0042] The amino acid sequence of threonine dehydratase, SEQ ID NO: 4

[0043] The amino acid sequence of methylmalate synthase is shown as SEQ ID NO. 5.

[0044] SEQ ID NO. 5

[0045] The nucleotide sequence of the methylmalate synthase gene is shown as SEQ ID NO: 6.

[0046] SEQ ID NO: 6

[0047] The amino acid sequence of LeuC is shown as SEQ ID NO: 7, SEQ ID NO: 7:

[0048] LeuD, the amino acid sequence of which is shown as SEQ ID NO: 8, SEQ ID NO: 8:

[0049] LeuB, the amino acid sequence of which is shown as SEQ ID NO: 9, SEQ ID NO: 9

[0050] Ilvl, the amino acid sequence of which is shown as SEQ ID NO: 10, SEQ ID NO: 10:

[0051] IlvH, the amino acid sequence of which is shown as SEQ ID NO: 11, SEQ ID NO: 11

[0052] IlvC, the amino acid sequence of which is shown as SEQ ID NO: 12, SEQ ID NO: 12:

[0053] IlvD, the amino acid sequence of which is shown as SEQ ID NO: 13, SEQ ID NO: 13:

[0054] IlvE, the amino acid sequence of which is shown as SEQ ID NO: 14, SEQ ID NO: 14:

[0055] SatP, SEQ ID NO: 15:

[0056] TtdT, SEQ ID NO: 16:

[0057] DctA, SEQ ID NO: 17:

[0058] DcuC, SEQ ID NO: 18:

[0059] CitT, SEQ ID NO: 19:

[0060] DcuB, SEQ ID NO: 20:

[0061] YbhI, SEQ ID NO: 21:

[0062] The nucleotide sequence of the branched-chain amino acid absorption protein gene brnQ is shown as SEQ ID NO: 22, SEQ ID NO: 22: brnQ:

[0063] The nucleotide sequence of the branched-chain amino acid absorption protein gene livJ is shown as SEQ ID NO: 23.SEQ ID NO: 23: livJ:

[0064] The nucleotide sequence of the branched-chain amino acid absorption protein gene livK is shown as SEQ ID NO: 24, SEQ ID NO: 24: livK:

[0065] The nucleotide sequence of the branched-chain amino acid excretion protein gene ygaZ is shown as SEQ ID NO: 25, SEQ ID NO: 25: ygaZ:

[0066] The nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown in SEQ ID NO: 26, SEQ ID NO: 26: ygaH:

[0067] The nucleotide sequence of the regulatory protein gene lrp is shown in SEQ ID NO: 27, SEQ ID NO: 27: lrp

[0068] The nucleotide sequence of the bifunctional phosphoketolase gene Bafxpk is shown in SEQ ID NO: 28, SEQ ID NO: 28: Bafxpk:

[0069] The nucleotide sequence of the phosphotransacetylase gene pta is shown in SEQ ID NO: 29, SEQ ID NO: 29: pta:

[0070] Example 1 Construction of an Escherichia coli engineering strain for synthesizing L-isoleucine through a methylmalonate pathway

[0071] As shown in FIG. 1, the biosynthesis of L-isoleucine in Escherichia coli is mainly through the threonine pathway. Knocking out the threonine dehydratase (TD) encoding gene ilvA, whose amino acid sequence is shown in SEQ ID NO: 4, results in an Escherichia coli engineering strain BW1 (Table 1) that is unable to synthesize L-isoleucine. On this basis, overexpression of the methylmalonate synthase gene cimA can establish an L-isoleucine synthesis pathway based on the methylmalonate pathway in Escherichia coli. The specific steps are as follows:

[0072] I. Construction of an L-isoleucine auxotrophic Escherichia coli engineering strain BW1

[0073] Using Escherichia coli BW25113 as the starting strain, the ilvA gene was knocked out using the CRISPR-associated transposases gene editing method, specifically as follows:

[0074] (1) Preparation of Escherichia coli electrotransformation competent cells

[0075] Inoculate 300 μL of glycerol-preserved Escherichia coli BW25113 into 100 mL of LB medium and incubate at 37°C in a shaking incubator overnight. Transfer 1 / 1000 of the inoculum into 100 mL of fresh LB medium and incubate at 37°C in a 200 rpm shaking incubator until the OD 6000.6-0.8. The well-cultured bacteria liquid was placed on ice for 30 min, and then the bacteria were collected by centrifugation (4°C, 6,000 rpm, 6 min), the cells were resuspended with 50 mL of pre-cooled double distilled water, and then the cells were collected by centrifugation again under the same conditions and resuspended with double distilled water. The cells were resuspended with 50 mL of pre-cooled 10% glycerol by centrifugation as above. Finally, the bacteria were collected by centrifugation at 4°C, 6,000 rpm for 10 min, the cells were resuspended with 0.6 mL of pre-cooled 10% glycerol, 100 μL was dispensed into each tube, and the cells were stored in a -80°C refrigerator.

[0076] (2) E. coli electroporation and transformant screening

[0077] After mixing 500 ng of plasmid pTetQCas (containing tniQ and cas876 genes), 500 ng of plasmid pRE57-tns-Ter (containing tnsABC genes and right end-terminator-left end sequence), and 500 ng of plasmid pUC-spacer-ilvA, the mixture was added to BW25113 electroporation competent cells, mixed by blowing, transferred to a 1 mm electroporation cup, and the three plasmids were transformed into E. coli BW25113 using an electroporation instrument. Then, 1 mL of sterile antibiotic-free LB liquid medium was added, and the recovered cells were cultured at 37°C for 2 h. After centrifugation, the cells were spread on LB plates containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin, and cultured in a 37°C incubator overnight. After the appearance of colonies, single colonies were picked with sterile toothpicks for PCR verification to confirm that the three plasmids were successfully transformed into E. coli BW25113 cells.

[0078] (3) Construction of engineered strain BW1

[0079] The BW25113 transformant containing the three plasmids pTetQCas, pRE57-tns-Ter, and pUC-spacer-ilvA was inoculated into LB liquid medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin, and cultured at 37°C with shaking. When the OD 600 When the OD reached about 0.6, 100 ng / mL anhydrotetracycline was added for induction to express the CRISPR-associated transposases gene editing system-related proteins, and the induction was performed at 37°C overnight. The induced culture was gradient-diluted and spread on LB plates containing the three antibiotics and anhydrotetracycline inducer, and cultured at 37°C for 16 h. After the appearance of single colonies, PCR verification was performed, and the PCR product band of the ilvA gene successfully knocked out mutant strain was 841 bp larger than that of the wild type. The positive clones were inoculated into LB liquid medium without antibiotics.

[0080] To discard the gene editing tool plasmid in the obtained engineering strain, the positive clone (containing three gene editing plasmids) was inoculated into 50 mL LB liquid medium without antibiotics for subculture, and the culture conditions were 45°C and 200 rpm. After 6 passages, the bacterial solution was gradiently diluted, spread on an antibiotic-free LB plate, and incubated at 37°C overnight. After the clones on the plate appeared, single colonies were randomly picked with a sterile toothpick and spotted on four LB plates containing no antibiotic and kanamycin, ampicillin and streptomycin, respectively. The single colonies on the four plates corresponded one by one, and were incubated at 37°C overnight. The single colony that could only grow on the antibiotic-free LB plate was selected, and the ilvA gene knockout was verified again by clone PCR. The positive clone was inoculated into 50 mL LB liquid medium to obtain the E. coli BW1 engineering strain.

[0081] II. Construction of E. coli engineering strain BW2 for synthesizing L-isoleucine through the methylmalonic acid pathway

[0082] On the basis of the engineering strain BW1, the methylmalonic acid synthase gene cimA was overexpressed to establish an L-isoleucine synthesis pathway based on the methylmalonic acid pathway, specifically as follows:

[0083] (1) Construction of cimA gene overexpression plasmid T3-cimA

[0084] The methylmalonic acid synthase gene mutant cimA3.7 derived from M. jannaschii was artificially synthesized after codon optimization (the nucleotide sequence is shown as SEQ ID NO: 6), and then the expression cassette was assembled into plasmid pEASY-T3 to obtain plasmid T3-cimA. J23100 The expression of the gene was controlled by the promoter P

[0085] The J 23100 -cimA3.7 fragment was obtained by extension PCR amplification using the artificially synthesized cimA3.7 gene fragment as a template. The obtained fusion fragment was ligated with the pEASY-T3 vector, and was chemically transformed into E. coli Trans1 competent cells after heat shock. After recovery at 37°C for 1 h, the cells were spread on an LB plate containing 100 μg / mL ampicillin and incubated for 16 h. After the colonies appeared, positive clones were screened and verified by PCR and sequencing to obtain plasmid T3-cimA.

[0086] (2) Construction of engineering strain BW2

[0087] Using the methods of preparation of E. coli electrotransformation competent cells, electrotransformation and transformant screening shown above, the engineering strain BW1 was prepared into electrotransformation competent cells, and the constructed plasmid T3-cimA was transformed into BW1 cells to obtain the engineering strain BW2.

[0088] III. Shake flask fermentation experiment of the starting strain BW25113 and the engineering strains BW1 and BW2

[0089] The starting strain BW25113 and the engineering strains BW1 and BW2 were inoculated into 50 mL LB liquid medium (100 μg / mL ampicillin was added to the culture solution of BW2) respectively, and cultured at 37°C, 200 rpm overnight as seed liquid. The cell density of the seed liquid was determined, and inoculated into 1 L flask containing 200 mL fermentation medium according to a certain proportion, so that the initial OD 600 was maintained at about 0.05, and the fermentation was carried out in a 37°C, 200 rpm shaker. The fermentation medium is divided into four kinds, which are fermentation medium M9+Glu: Na2HPO4 6.78 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, glucose 4 g / L; fermentation medium M9+Glu+Ile: Na2HPO4 6.78 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, glucose 4 g / L, L-isoleucine 20 mg / L; fermentation medium M9+CM: Na2HPO4 6.78 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, methyl malate 4 g / L; fermentation medium M9+Glu+CM: Na2HPO4 6.78 g / L, KH2PO4 3.0 g / L, NaCl 0.5 g / L, NH4Cl 1.0 g / L, MgSO4 0.241 g / L, CaCl2 0.011 g / L, glucose 4 g / L, methyl malate 4 g / L. The fermentation was carried out for 48 h, and the cell density (OD 600) and the glucose and methylmalate content in the fermentation broth. As shown in Figure 2, the BW25113 wild strain grew well in the basic medium with glucose as the sole carbon source (M9+Glu), while the engineered strain BW1 with ilvA gene knocked out could not grow normally, but it recovered normal growth and glucose consumption after adding a certain amount of isoleucine to the medium (M9+Glu+Ile) (Figure 3), indicating that BW1 is an isoleucine auxotrophic strain. The engineered strain BW2 obtained by overexpressing the cimA gene in the BW1 strain can grow normally in the basic medium without the addition of isoleucine (M9+Glu) (Figure 2), indicating that the expression of the cimA gene can establish an L-isoleucine synthesis pathway based on the methylmalate pathway in E. coli, so that the growth of the BW2 strain no longer requires the additional addition of L-isoleucine.

[0090] When the BW1 engineered strain was cultured in the basic medium with methylmalate as the sole carbon source (M9+CM), the BW1 strain grew slowly after a growth lag phase of about 10 hours, and methylmalate was also partially consumed, indicating that methylmalate can be used as a carbon source for the growth of the strain, and also as a precursor for the synthesis of L-isoleucine through the methylmalate pathway (Figure 4). However, when the fermentation medium used M9+Glu+CM, the growth of the BW1 strain was greatly inhibited, and neither glucose nor methylmalate was consumed (Figure 5). Combining the above results indicates that due to the presence of carbon metabolic inhibition effect, when glucose is present in the medium, methylmalate cannot be transported into the cell for metabolism, thereby causing the BW1 strain to be unable to grow in the M9+Glu+CM medium.

[0091] Example 2. Overexpression of methylmalate transporter protein to relieve the inhibition of glucose on methylmalate utilization

[0092] The expression of the cimA gene in the cells of the engineered strain BW1 enables the strain to grow normally in the M9+Glu basic medium, indicating that the methylmalate synthesized by CimA can be normally utilized in the cells and L-isoleucine is synthesized, while the addition of methylmalate outside the BW1 cells will be inhibited by glucose and the strain cannot grow. Therefore, it can be concluded that the inhibition of glucose on methylmalate utilization mainly manifests in the inhibition of the absorption of methylmalate from the outside of the cells to the inside of the cells, and the overexpression of the methylmalate transporter protein can relieve the inhibition of glucose on methylmalate utilization. The specific steps are as follows:

[0093] I. Construction of methylmalate transporter protein gene overexpression engineered strains BW3-BW12

[0094] Overexpression of the methylmalate transporter protein gene in the engineered strain BW1 to relieve the inhibition of glucose on methylmalate utilization, specifically:

[0095] (1) Construction of methylmalonate transporter gene overexpression plasmid

[0096] There are few reports on methylmalonate transporters at present. Whether the transporters of C3-C7 binary and ternary carboxylic acids have transport activity on methylmalonate was studied, and 10 known binary and ternary carboxylic acid transporters, DcuD, DauA, DcuA, SatP (the amino acid sequence thereof is shown as SEQ ID NO: 15), TtdT (the amino acid sequence thereof is shown as SEQ ID NO: 16), DctA (the amino acid sequence thereof is shown as SEQ ID NO: 17), DcuC (the amino acid sequence thereof is shown as SEQ ID NO: 18), CitT (the amino acid sequence thereof is shown as SEQ ID NO: 19), DcuB (the amino acid sequence thereof is shown as SEQ ID NO: 20), and YbhI (the amino acid sequence thereof is shown as SEQ ID NO: 21), were overexpressed, and the promoters P J23100 The expression of the genes corresponding to these transporters was controlled. The corresponding gene fragments were amplified by PCR using the E. coli BW25113 genome as a template, and the expression cassettes of the 10 transporter genes were assembled into plasmid pEASY-T3 using the plasmid construction method shown in Example 1 to obtain plasmids T3-DcuD, T3-DauA, T3-DcuA, T3-SatP, T3-TtdT, T3-DctA, T3-DcuC, T3-CitT, T3-DcuB, and T3-YbhI.

[0097] (2) Construction of engineering strains BW3-BW12

[0098] The engineering strain BW1 was prepared into electrotransformation competent cells using the preparation of electrotransformation competent cells, electrotransformation, and transformant screening method of E. coli shown in Example 1, and the plasmids T3-DcuD, T3-DauA, T3-DcuA, T3-SatP, T3-TtdT, T3-DctA, T3-DcuC, T3-CitT, T3-DcuB, and T3-YbhI constructed were transformed into BW1 cells, respectively, to obtain engineering strains BW3-BW12 (Table 1).

[0099] II. Shake flask fermentation experiment of engineering strains BW3-BW12

[0100] The fermentation experiment of engineering strains BW3-BW12 was performed using the fermentation medium M9+Glu+CM by the shake flask fermentation method shown in Example 1. The fermentation was performed for 48 h, and the cell density (OD 600). As shown in Figure 6, the engineering strains BW3-BW12 all grew to different degrees at 48h, while the corresponding BW1 strains could not grow in M9+Glu+CM medium (Figure 5), indicating that the overexpression of the 10 binary or ternary carboxylic acid transporters could all relieve the inhibition of glucose on the utilization of methylmalate to some extent. Among them, the three transporters DcuD, DauA and DcuA had better absorption effect on methylmalate, which could accumulate the biomass of the cells in a shorter time.

[0101] Example 3: Improving the synthesis ability of L-isoleucine by strengthening the methylmalate pathway

[0102] Example 1 proves that overexpression of the cimA gene can establish an L-isoleucine synthesis pathway based on the methylmalate pathway in E. coli. Therefore, in order to further improve the carbon flux of L-isoleucine synthesis, in addition to the cimA gene, other methylmalate pathway related genes and methylmalate absorption protein genes are further overexpressed to improve the synthesis ability of the engineering strain L-isoleucine. In addition to methylmalate synthase CimA, the methylmalate pathway related catalytic enzymes also include isopropylmalate isomerase (IPMI), 3-isopropylmalate dehydrogenase (IPMD), acetylhydroxy acid synthase (AHAS), acetylhydroxy acid reduction isomerase (AHAIR), dihydroxy acid dehydrase (DHAD), branched chain amino acid amino transferase (BCAT), leucine dehydrogenase (LeuDH) and valine dehydrogenase (ValDH). Among them, AHAS has multiple types, including IlvIH, IlvBN and IlvGM, all of which catalyze the same reaction (Figure 1); BCAT, LeuDH and ValDH three enzymes respectively with glutamate (BCAT) or NH4 +(LeuDH and ValDH) as the last step of L-isoleucine synthesis (Figure 1). Therefore, the present application overexpresses the IPMI (leuCD), IPMD (leuB), AHAS (ilvIH), AHAIR (ilvC), DHAD (ilvD) and BCAT (ilvE) genes derived from E. coli to strengthen the methylmalonic acid pathway. Among them, LeuCD includes subunit LeuC and LeuD, LeuC, the amino acid sequence of which is shown as SEQ ID NO: 7; LeuD, the amino acid sequence of which is shown as SEQ ID NO: 8; LeuB, the amino acid sequence of which is shown as SEQ ID NO: 9; IlvIH includes subunit IlvI and subunit IlvH, the amino acid sequence of IlvI is shown as SEQ ID NO: 10; the amino acid sequence of IlvH is shown as SEQ ID NO: 11; the amino acid sequence of IlvC is shown as SEQ ID NO: 12; the amino acid sequence of IlvD is shown as SEQ ID NO: 13; the amino acid sequence of IlvE is shown as SEQ ID NO: 14.

[0103] The specific steps are as follows:

[0104] I. Construction of methylmalonic acid pathway enhanced strains BW13-1 and BW13-2

[0105] On the basis of the engineering strain BW1 and the wild strain BW25113, the methylmalonic acid pathway related genes are overexpressed to improve the carbon flux of L-isoleucine synthesis, specifically:

[0106] (1) Construction of methylmalonic acid pathway related gene overexpression plasmid

[0107] As shown in Figure 1, in addition to the cimA gene, the methylmalonic acid pathway related genes also include IPMI (leuCD), IPMD (leuB), AHAS (ilvIH), AHAIR (ilvC), DHAD (ilvD) and BCAT (ilvE) genes, therefore CimA3.7 and the LeuC (the amino acid sequence of which is shown as SEQ ID NO: 7), LeuD (the amino acid sequence of which is shown as SEQ ID NO: 8), LeuB (the amino acid sequence of which is shown as SEQ ID NO: 9), IlvI (the amino acid sequence of which is shown as SEQ ID NO: 10), IlvH (the amino acid sequence of which is shown as SEQ ID NO: 11), IlvC (the amino acid sequence of which is shown as SEQ ID NO: 12), IlvD (the amino acid sequence of which is shown as SEQ ID NO: 13) and IlvE (the amino acid sequence of which is shown as SEQ ID NO: 14) enzyme proteins derived from E. coli are overexpressed, and the promoter PJ23100 The expression of genes corresponding to these enzyme proteins was controlled. The corresponding gene fragments were amplified by PCR using the E. coli BW25113 genome as a template, and the expression cassettes of cimA, leuC, leuD and leuB were sequentially assembled into plasmid pRSFDuet-1 using the plasmid construction method shown in Example 1 to obtain plasmid pRSFDuet-1-cimA-leuC-leuD-leuB; the expression cassettes of ilvIH, ilvC, ilvD and ilvE were sequentially assembled into plasmid pETDuet-1 to obtain plasmid pETDuet-1-ilvIH-ilvC-ilvD-ilvE.

[0108] (2) Construction of engineered strains BW13-1 and BW13-2

[0109] Using the methods for preparation of electrotransformation competent cells, electrotransformation and transformant screening shown in Example 1, the engineered strain BW1 and the wild-type strain BW25113 were prepared into electrotransformation competent cells, and the constructed plasmids pRSFDuet-1-cimA-leuC-leuD-leuB and pETDuet-1-ilvIH-ilvC-ilvD-ilvE were transformed into BW1 and BW25113 cells, and the transformants were screened using LB plates containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, and verified by colony PCR, and finally the engineered strains BW13-1 and BW13-2 were obtained (Table 1).

[0110] II. Shake flask fermentation experiments of wild-type strain BW25113, engineered strains BW2, BW13-1 and BW13-2

[0111] The wild-type strain BW25113, the engineered strain BW2 (added with 100 μg / mL ampicillin), the engineered strain BW13-1 (added with 50 μg / mL kanamycin and 100 μg / mL ampicillin) and the engineered strain BW13-2 (added with 50 μg / mL kanamycin and 100 μg / mL ampicillin) were inoculated into 50 mL of LB liquid medium, respectively, and cultured at 37°C and 200 rpm overnight to serve as seed liquid. The cell density of the seed liquid was determined, and according to a certain proportion, it was inoculated into a 1L triangular flask containing 200 mL of fermentation medium, so that the initial OD600 was about 0.1. The fermentation was carried out at 37°C and 200 rpm, and the OD600 was measured at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 and 100 h, and the cell density was determined. 600The fermentation was carried out at 37°C, 200 rpm in a shaker. The fermentation medium was as follows: glucose 20 g / L, KH2PO4 13.3 g / L, (NH4)2HPO4 4 g / L, MgSO4·7H2O 1.2 g / L, citric acid monohydrate 1.7 g / L, EDTA·2Na 1.68 mg / L, CoCl2·6H2O 0.5 mg / L, MnCl2·4H2O 3 mg / L, CuCl2·2H2O 0.3 mg / L, H3BO3 0.6 mg / L, Na2MoO4·2H2O 0.5 mg / L, Zn(CH3COO)2·2H2O 2.6 mg / L, Fe-citrate 20 mg / L, VB1 0.9 mg / L. The fermentation was carried out for 48 h. The contents of methylmalonic acid and isoleucine in the fermentation broth were determined at the end of fermentation, and the results are shown in Table 3.

[0112] Table 3. Results of shake flask fermentation of wild strain BW25113, engineering strain BW2, BW13-1 and BW13-2

[0113] As disclosed in the data in Table 3 above, the L-isoleucine produced by the wild strain BW25113 during fermentation could only meet the needs of strain growth, and no accumulation of L-isoleucine was detected in the fermentation broth. There was no methylmalonic acid synthesis related enzyme in BW25113, so no accumulation of methylmalonic acid was detected in the fermentation broth. The engineering strain BW2 produced a large amount of methylmalonic acid, reaching 3.26 g / L, while the yield of L-isoleucine was low, only 0.034 g / L. After further overexpression of other related genes of the methylmalonic acid pathway based on overexpression of the cimA gene, the L-isoleucine yield of the engineering strains BW13-1 and BW13-2 was improved to a certain extent, but was still at a low level, only 0.076 and 0.079 g / L, while the methylmalonic acid (2.80 and 2.95 g / L) was still largely unconverted into L-isoleucine. At the same time, the fermentation results of the wild strain BW25113 and the engineering strains BW13-1 and BW13-2 showed that the ilvA gene of BW25113 had little effect on the accumulation of L-isoleucine in the fermentation broth.

[0114] Example 4. Improvement of L-isoleucine synthesis capacity based on methylmalonic acid pathway by expressing methylmalonic acid transporters

[0115] As shown in Example 1, the extracellular methylmalate cannot be utilized by E. coli when glucose is present in the culture medium. The results of Example 3 show that a large amount of methylmalate produced by E. coli expressing the cimA gene is rapidly excreted outside the cell, and the extracellular methylmalate cannot be transported into the cell again, resulting in a lack of isoleucine synthesis precursors, which leads to a low level of L-isoleucine production. As shown in Example 2, the inhibition of methylmalate utilization by glucose can be eliminated by expressing the methylmalate transporter protein, and thus overexpression of the methylmalate transporter protein genes DcuD, DauA and DcuA can effectively improve the L-isoleucine synthesis efficiency based on the methylmalate pathway. The specific steps are as follows:

[0116] I. Construction of methylmalate transporter protein gene DcuD, DauA and DcuA overexpression engineering strain BW14-BW19

[0117] On the basis of engineering strains BW13-1 and BW13-2, methylmalate transporter proteins are further overexpressed to improve the L-isoleucine synthesis efficiency, and the specific steps are as follows:

[0118] (1) Construction of methylmalate transporter protein gene overexpression plasmid

[0119] As shown in Example 2, the methylmalate transporter protein genes DcuD, DauA and DcuA can more effectively eliminate the inhibition of methylmalate utilization by glucose. Therefore, these three genes are selected for overexpression, and the expression of these genes is controlled by the promoter P J23100 The corresponding gene fragments are amplified by PCR using the E. coli BW25113 genome as a template, and the expression cassettes of DcuD, DauA and DcuA are assembled onto the plasmid pETDuet-1-ilvIH-ilvC-ilvD-ilvE, respectively, by using the plasmid construction method shown in Example 1, to obtain the plasmids pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DcuD, pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DauA and pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DcuA.

[0120] (2) Construction of engineering strains BW14-BW19

[0121] Using the methods of preparation of electrotransformation competent cells, electrotransformation and transformant screening shown in Example 1, the engineering strain BW1 and wild type strain BW25113 were prepared into electrotransformation competent cells, and the constructed plasmid pRSFDuet-1-cimA-leuC-leuD-leuB was transformed into BW1 and BW25113 cells, and the transformants were screened using LB plates containing 50 μg / mL kanamycin, to obtain strains BW1 (pRSFDuet-1-cimA-leuC-leuD-leuB) and BW25113 (pRSFDuet-1-cimA-leuC-leuD-leuB). Using the same methods of preparation of electrotransformation competent cells and electrotransformation, the engineering strains BW1 (pRSFDuet-1-cimA-leuC-leuD-leuB) and BW25113 (pRSFDuet-1-cimA-leuC-leuD-leuB) were prepared into electrotransformation competent cells, and the constructed plasmids pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DcuD, pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DauA and pETDuet-1-ilvIH-ilvC-ilvD-ilvE-DcuA were transformed into BW1 (pRSFDuet-1-cimA-leuC-leuD-leuB) and BW25113 (pRSFDuet-1-cimA-leuC-leuD-leuB) engineering strains, respectively, and the transformants were screened using LB plates containing 50 μg / mL kanamycin and 100 μg / mL ampicillin, and verified by colony PCR, to finally obtain engineering strains BW14-BW19 (Table 1).

[0122] II. Shake flask fermentation experiment of engineering strains BW14-BW19

[0123] Using the shake flask fermentation method and fermentation medium shown in Example 3, fermentation experiments of engineering strains BW14-BW19 were carried out. Fermentation was carried out for 48 h in total, and the contents of methylmalonic acid and isoleucine in the fermentation broth were determined at the end of fermentation, and the results are shown in Table 3.

[0124] Table 4. Shake flask fermentation results of engineering strains BW14-BW19

[0125]

[0126] Based on the data disclosed in Tables 3 and 4, after further overexpressing the methylmalonate transporter protein genes DcuD, DauA and DcuA on the basis of the engineering strain BW13-1, the utilization rate of methylmalonate was significantly improved, and the methylmalonate yield in the fermentation broth of the engineering strains BW14, BW15 and BW16 was 0.64, 0.76 and 0.95 g / L, respectively, which was reduced by about 77.1%, 72.9% and 66.1% compared with BW13-1. Moreover, the synthesis efficiency of L-isoleucine was also greatly improved, and the L-isoleucine yield in the fermentation broth of the engineering strains BW14, BW15 and BW16 reached 0.43, 0.39 and 0.32 g / L, respectively, which was increased by about 4.7, 4.1 and 3.2 times compared with BW13-1. The fermentation results of the engineering strains BW17, BW18 and BW19 were similar to those of BW13-2.

[0127] The above results show that the methylmalonate transporter protein provided in the present application can significantly improve the utilization efficiency of methylmalonate and the L-isoleucine synthesis ability based on the methylmalonate pathway.

[0128] Example 5 Screening of Escherichia coli chassis strains suitable for constructing a methylmalonate pathway

[0129] Methylmalonate is the first key intermediate metabolite for synthesizing L-isoleucine through the methylmalonate pathway, and the synthesis precursor of methylmalonate is pyruvic acid and acetyl-CoA. Therefore, a microbial chassis capable of providing sufficient pyruvic acid and acetyl-CoA and efficiently synthesizing methylmalonate is crucial for producing L-isoleucine through the methylmalonate pathway. As shown in Table 2, different wild-type Escherichia coli strains including BW25113, W3110, MG1655 and W (ATCC 9637) were screened as chassis strains for constructing a methylmalonate pathway using plasmid P1 (P J23100 -cimA3.7), and the results showed that the methylmalonate synthesis efficiency of Escherichia coli BW25113 was the highest. The specific steps are as follows:

[0130] I. Construction of cimA gene overexpression plasmid P1

[0131] The primer was designed to amplify the high-activity mutant gene cimA3.7 of methylmalonate synthase derived from Methanococcus jannaschii, and then the expression of the gene was controlled by using the promoter P J23100 The expression cassette was finally assembled into plasmid pEASY-T3 to obtain plasmid P1; the specific construction method is as follows:

[0132] J23100-cimA3.7 fragment was obtained by extension PCR amplification using the synthetic cimA3.7 gene fragment as a template. The obtained fusion fragment was linked with pEASY-T3 vector, and was chemically transformed into E. coli Trans1 competent cells after heat shock. After recovery at 37°C for 1 h, the cells were spread on LB plates containing 100 μg / mL ampicillin and cultured for 16 h. After the appearance of colonies, positive clones were screened and verified by PCR and sequencing to obtain plasmid P1.

[0133] II. Construction of strains JQ1, JQ2, JQ3, and JQ4

[0134] (1) Preparation of E. coli electrotransformation competent cells

[0135] Inoculate 300 μL of E. coli glycerol-preserved bacteria into 100 mL of LB medium and culture overnight at 37°C in a shaking incubator. Transfer the bacteria into 100 mL of fresh LB medium at a seeding amount of 1 / 1000 and culture at 37°C in a 200 rpm shaking incubator until the OD 600 of the culture reaches 0.6-0.8. After the culture is placed on ice for 30 min, centrifuge the bacteria (4°C, 6,000 rpm, 6 min) and resuspend the cells in 50 mL of pre-cooled double-distilled water. Centrifuge the cells again under the same conditions and resuspend them in double-distilled water. Centrifuge the cells as above and resuspend them in 50 mL of pre-cooled 10% glycerol. Finally, centrifuge the cells at 4°C and 6,000 rpm for 10 min, resuspend the cells in 0.6 mL of pre-cooled 10% glycerol, and distribute 100 μL of the cells in each tube. Store the cells in a -80°C refrigerator.

[0136] (2) E. coli electrotransformation and transformant screening

[0137] Add 500 ng of plasmid to E. coli electrotransformation competent cells, mix well by blowing, and transfer into a 1 mm electrotransformation cup. Use an electrotransformation instrument to transform the plasmid into E. coli, and then add 1 mL of sterile and antibiotic-free LB liquid medium. Incubate at 37°C for 2 h, centrifuge the recovered cells, and spread them on LB plates containing the antibiotic corresponding to the electrotransformation plasmid. Incubate in a 37°C constant-temperature incubator overnight. After the appearance of colonies, use a sterile toothpick to pick single colonies for PCR verification to determine whether the plasmid has been successfully transformed into E. coli cells

[0138] (3) Construction of strains JQ1, JQ2, JQ3, and JQ4

[0139] The E. coli wild strains including BW25113, W3110, MG1655 and W (ATCC 9637) were prepared into electrotransformation competent cells, and the constructed plasmid P1 was transformed into the E. coli BW25113, W3110, MG1655 and W (ATCC 9637) strain cells to obtain corresponding engineering strains JQ1, JQ2, JQ3 and JQ4, respectively.

[0140] III. Shake flask fermentation experiment of engineering strains JQ1, JQ2, JQ3 and JQ4

[0141] The above engineering strains JQ1, JQ2, JQ3 and JQ4 were inoculated into 40 mL LB liquid medium, respectively, and 100 μg / mL ampicillin was added to the culture solution, which was cultured overnight at 37°C, 200 rpm as seed liquid. The bacterial density of the seed liquid was determined, and according to a certain proportion, it was inoculated into 100 mL triangular flask containing 50 mL fermentation medium, so that the initial OD 600 was maintained at about 0.05, and the fermentation was carried out in a 37°C, 200 rpm shaker. The fermentation medium formula was as follows: 5 g / L yeast extract, 20 g / L glucose, 14 g / L KH2PO4, 4 g / L (NH4)2HPO4, 0.6 g / L MgSO4·7H2O, 2 g / L Na2SO4, 1.8 g / L citric acid, and trace elements (13 mg / L Zn (CH3COO)2·2H2O, 10 mg / L ferric citrate, 8.4 mg / L EDTA, 3 mg / L H3BO3, 2.5 mg / L Na2MoO4·2H2O, 2.5 mg / L CoCl2·6H2O, 1.5 mg / L CuCl2·2H2O, 0.9 mg / L VB1, 0.15 mg / L MnCl2·4H2O). The fermentation was carried out for 48 h, and the bacterial density (OD 600 ) and the content of glucose and methylmalate in the fermentation broth were determined at regular intervals. As shown in Figure 7, the methylmalate yield and yield of strain JQ1 reached 1.96 g / L and 0.20 g / g glucose, respectively, and the OD 600 was 2.43. Strain JQ4 grew best among the four tested strains, but its methylmalate yield and yield were only 0.98 g / L and 0.08 g / g glucose, respectively, which were 50% and 60% lower than those of JQ1. W3110 and MG1655 are two E. coli chassis strains most commonly used for amino acid production. However, the methylmalate yield and yield of JQ2 and JQ3 were also much lower than those of JQ1. The results showed that E. coli BW25113 was a superior chassis strain for producing L-isoleucine through the methylmalate pathway. However, no L-isoleucine production was detected in the fermentation broth.

[0142] Example 6: Screening of isopropylmalate isomerase (LeuCD), 3-isopropylmalate dehydrogenase (LeuB), and leucine dehydrogenase (LeuDH) from different sources to improve L-isoleucine production in E. coli based on methylmalonic acid pathway

[0143] E. coli BW25113 possesses most of the genes of the methylmalonic acid pathway except cimA. However, no L-isoleucine production was detected in the fermentation broth of strain JQ1 carrying the cimA3.7 expression plasmid, which might be due to 1) the expression of other methylmalonic acid pathway genes in E. coli is regulated; 2) LeuCD, LeuB, and IlvE from E. coli mainly function in leucine synthesis under natural conditions, and thus these enzymes might have low activity in catalyzing the reactions related to the methylmalonic acid pathway. Therefore, E. coli BW25113 was used as the chassis strain, and the changes in L-isoleucine production after expression of methylmalonic acid pathway genes from different sources were used to screen the key genes. The isopropylmalate isomerase gene GsleuCD from Geobacillus sp. WCH70, the 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobus fulgidus, and the leucine dehydrogenase gene LsleuDH from Lysinibacillus sphaericus were selected to significantly improve the L-isoleucine production in E. coli based on the methylmalonic acid pathway. The specific steps are as follows:

[0144] I. Construction of expression plasmids P2-P8 containing key genes from different sources

[0145] (1) Construction of plasmid P2

[0146] The primers were designed to amplify the cimA3.7 gene, the leuCD gene from E. coli, and the leuB gene from E. coli, respectively. Each gene was placed under the control of the arabinose-inducible promoter P araBAD The expression cassette was finally assembled into plasmid pRSF-Dute-1 to obtain plasmid P2. The specific construction method is as follows:

[0147] The P araBAD -cimA, P araBAD -leuC, P araBAD -leuD, and P araBADThe obtained fusion fragment is recovered and purified by agarose gel electrophoresis, and is connected with the pRSF-Dute-1 vector, and is chemically transformed into E. coli Trans1 competent cells by heat shock, and is cultured on an LB plate containing 50 μg / mL kanamycin at 37°C for 16 hours, and after the appearance of colonies, positive clones are screened and verified by PCR and sequencing, and the plasmid P2 is obtained.

[0148] (2) Construction of plasmid P3

[0149] The primers are designed to amplify the anti-feedback inhibition mutant acetohydroxy acid synthase gene ilvIH* from E. coli, the NADH preference acetohydroxy acid reductoisomerase gene ilvC* from Corynebacterium glutamicum, the dihydroxy acid dehydratase gene ilvD from E. coli, and the branched-chain amino acid transaminase gene ilvE from E. coli, and each gene is induced by an arabinose inducible promoter P araBAD The expression of the genes is controlled, and finally the expression cassette is assembled on the plasmid pET-Dute-1 to obtain the plasmid P3; the specific construction method is as follows:

[0150] The E. coli genome and the Corynebacterium glutamicum genome are used as templates, and P araBAD -ilvIH*, P araBAD -ilvC*, P araBAD -ilvD, P araBAD -ilvE fragments are amplified by extension PCR, and the obtained fusion fragments are connected with the pET-Dute-1 vector, and are chemically transformed into E. coli Trans1 competent cells by heat shock, and are cultured on an LB plate containing 100 μg / mL ampicillin at 37°C for 16 hours, and after the appearance of colonies, positive clones are screened and verified by PCR and sequencing, and the plasmid P3 is obtained.

[0151] (3) Construction of plasmid P4

[0152] The leuCD gene and the leuB gene in the plasmid P2 are replaced by the isopropyl malate isomerase gene GsleuCD from Geobacillus sp. WCH70 and the 3-isopropyl malate dehydrogenase gene LbleuB from Leadbetterella byssophila, respectively; the specific construction method is as follows:

[0153] The GsleuCD and LbleuB genes are obtained by artificial synthesis after codon optimization, and other construction methods are the same as the plasmid P2 construction steps.

[0154] (4) Construction of plasmid P5

[0155] The 3-isopropylmalate dehydrogenase gene LbleuB from Leadbetterella byssophila in plasmid P4 is replaced by the 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobus fulgidus. The specific construction method is as follows:

[0156] The AfleuB gene is obtained by artificial synthesis after codon optimization, and other construction methods are the same as the P4 plasmid construction steps.

[0157] (5) Construction of plasmid P6

[0158] The branched-chain amino acid transaminase gene ilvE from Escherichia coli in plasmid P3 is replaced by the leucine dehydrogenase gene LsleuDH from Lysinibacillus sphaericus. The specific construction method is as follows:

[0159] The LsleuDH gene is obtained by artificial synthesis after codon optimization, and other construction methods are the same as the P3 plasmid construction steps.

[0160] (6) Construction of plasmid P7

[0161] The branched-chain amino acid transaminase gene ilvE from Escherichia coli in plasmid P3 is replaced by the leucine dehydrogenase gene GsleuDH from Geobacillus stearothermophilus. The specific construction method is as follows:

[0162] The GsleuDH gene is obtained by artificial synthesis after codon optimization, and other construction methods are the same as the P3 plasmid construction steps.

[0163] (7) Construction of plasmid P8

[0164] The branched-chain amino acid transaminase gene ilvE from Escherichia coli in plasmid P3 is replaced by the leucine dehydrogenase gene HhleuDH from Halobacillus halophilus. The specific construction method is as follows:

[0165] The HhleuDH gene is obtained by artificial synthesis after codon optimization, and other construction methods are the same as the P3 plasmid construction steps.

[0166] II. Construction of engineering strains JQ5-JQ10 carrying plasmids P2-P8

[0167] The E. coli BW25113 wild strain was prepared into electrotransformation competent cells, and the constructed plasmids P2-P8 were transformed into the E. coli BW25113 strain in the combinations shown in Table 2 to obtain the engineering strains JQ5 (containing P2 and P3 plasmids), JQ6 (containing P4 and P3 plasmids), JQ7 (containing P5 and P3 plasmids), JQ8 (containing P5 and P6 plasmids), JQ9 (containing P5 and P7 plasmids), and JQ10 (containing P5 and P8 plasmids).

[0168] III. Shake flask fermentation experiment of the engineering strains JQ5-JQ10

[0169] The engineering strains JQ5-JQ10 were inoculated into 40 mL LB liquid medium, respectively, and 100 μg / mL ampicillin and 50 μg / mL kanamycin were added to the culture solution, which was cultured overnight at 37°C and 200 rpm as seed liquid. The cell density of the seed liquid was determined, and according to a certain proportion, it was inoculated into a 1L triangular flask containing 100 mL fermentation medium, so that the initial OD 600 was maintained at about 0.05, and the fermentation was carried out in a 37°C, 200 rpm shaker, and the OD 600 was determined. 600 When the OD 600 value reached 0.8-1.0, 2 g / L of arabinose was added to induce gene expression, and the induced cells were cultured for 12 hours and collected by centrifugation. The obtained cells were resuspended in fresh fermentation medium, and the fermentation was carried out at 37°C and 200 rpm for 24 hours. Among them, the fermentation medium formula is as follows:

[0170] The fermentation medium formula is the same as that described in Example 1.

[0171] The cell density (OD 600) and the content of methylmalonic acid and isoleucine in the fermentation broth. As shown in Figure 8, the shake flask fermentation results showed that the methylmalonic acid production of JQ5 reached 2.39 g / L, but no accumulation of L-isoleucine was detected. As shown in Figure 8, the growth of JQ6 and JQ7 strains was similar to that of JQ5, while JQ6 and JQ7 produced 0.03 and 0.06 g / L of L-isoleucine, respectively (Figure 8), indicating that the GsleuCD, LbleuB and AfleuB genes are suitable for constructing a methylmalonic acid pathway. Although the methylmalonic acid production of JQ9 was higher than that of JQ7, the L-isoleucine production of JQ9 and JQ10 was comparable to or lower than that of JQ7 (Figure 8). However, the L-isoleucine production of JQ8 reached 0.10 g / L, which was about 66.7% higher than that of JQ7, indicating that LsleuDH was more efficient in L-isoleucine synthesis. The results proved that the selected isopropylmalate isomerase gene GsleuCD from Geobacillus sp. WCH70 strain, 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobus fulgidus, and leucine dehydrogenase gene LsleuDH from Lysinibacillus sphaericus significantly improved the L-isoleucine synthesis capacity of Escherichia coli based on the methylmalonic acid pathway.

[0172] Example 7. Improving L-isoleucine production by optimizing branched-chain amino acid and methylmalonic acid transport systems

[0173] By improving the excretion capacity of L-isoleucine in the chassis cell, the L-isoleucine synthesis capacity of the strain can be improved by reducing the intracellular concentration of L-isoleucine. In addition, since a large amount of methylmalonic acid still exists in the fermentation broth of the JQ8 strain, improving the utilization capacity of methylmalonic acid is also a key to improving the L-isoleucine synthesis capacity based on the methylmalonic acid pathway. Therefore, knocking out branched-chain amino acid absorption protein genes brnQ (SEQ ID NO: 22), livJ (SEQ ID NO: 23) and livK (SEQ ID NO: 24), overexpressing branched-chain amino acid excretion protein genes ygaZH (SEQ ID NO: 25, SEQ ID NO: 22) and regulatory protein genes lrp (SEQ ID NO: 27), and overexpressing methylmalonic acid absorption protein genes dcuD can improve the L-isoleucine production capacity. I. Construction of Escherichia coli ILE-1 strain

[0174] As shown in Table 2, using *E. coli* BW25113 as the starting strain, the branched-chain amino acid absorption protein genes *brnQ*, *livJ*, and *livK* were knocked out using the CRISPR-associated transposases gene editing method. Specifically, the following steps were taken: Electrotransformation competent cells were prepared; the pKMV-gRNA-brnQ / livJ / livK plasmid containing *brnQ*, *livJ*, and *livK* site gRNAs, along with the pTetQCas-BsaI-tns and pRE57 plasmids, were sequentially electrotransformed into wild-type competent *E. coli* BW25113 cells. Positive transformants were screened using LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and dehydrotetracycline resistance. Transformants were validated by PCR and sequenced using primers. The selected positive transformants were then cultured at 42°C using LB to remove the plasmids containing the three resistances, ultimately yielding the *E. coli* ILE-1 strain with the *brnQ*, *livJ*, and *livK* genes knocked out.

[0175] II. Construction of Escherichia coli ILE-2 strain

[0176] As shown in Table 2, using ILE1 as the starting strain, the branched-chain amino acid exoprotein gene ygaZH and the regulatory protein gene lrp were integrated into the livK site using the CRISPR-associated transposases gene editing method. Specifically:

[0177] The pKMV-gRNA-livK plasmid containing livK site gRNA, the pTetQCas-BsaI-tns plasmid, and the pRE57-ygaZH / lrp plasmid containing ygaZH and lrp gene expression cassettes were transformed into strain ILE1. The strain was screened by antibiotics and tetracycline induction to obtain engineered strain ILE-2 with ygaZH and lrp expression cassettes integrated into the genome (Table 2).

[0178] III. Construction of Escherichia coli ILE-3 and ILE-4 strains

[0179] (1) Construction of plasmid P9

[0180] Using the E. coli genome as a template, P was obtained by extension PCR amplification. araBAD The -dcuD fragment was purified by agarose gel electrophoresis and ligated into plasmid P6. The fusion fragment was then transformed into E. coli Trans1 competent cells by heat shock. After recovery at 37°C with shaking for 1 h, the cells were plated on LB agar plates containing 100 μg / mL ampicillin and cultured for 16 h. After colonies appeared, positive clones were screened and verified by PCR and sequencing to obtain plasmid P9.

[0181] (2) Construction of E. coli ILE-3 and ILE-4 strains

[0182] The P9 plasmid and the P5 plasmid constructed in Example 6 were electrotransformed into the E. coli ILE-1 or ILE-2 strain competent cells, and positive transformants were screened by LB kanamycin and ampicillin resistant plates for LB liquid culture. The transformants were verified by PCR using primer pairs, and the E. coli ILE-3 and ILE-4 strains were obtained (Table 2).

[0183] The above E. coli ILE-3 and ILE-4 were inoculated into 40 mL of LB medium, respectively, and cultured at 37°C, 200 r / min overnight as the first-stage seed liquid. The seed liquid was inoculated into 40 mL of LB medium and cultured at 37°C, and when the OD 600 of the culture reached 0.6-0.8, 2 g / L of L-arabinose was added to the culture medium to induce gene expression. Subsequently, the induced cells were cultured for 12 hours and collected by centrifugation. The obtained cells were resuspended in fresh fermentation medium, and the OD 600 was determined. The working volume was 100 mL, and the conditions were set at 37°C and 200 rpm for 24 hours. The composition of the fermentation medium was as follows:

[0184] The fermentation medium formula was the same as that described in Example 5.

[0185] The cell density (OD 600 ) and the content of methylmalonic acid and isoleucine in the fermentation broth were determined by sampling at regular time intervals. As shown in Figure 9, the L-isoleucine production of ILE-3 and ILE-4 reached 0.32 g / L and 0.41 g / L, respectively, which was a substantial improvement compared to JQ8. The results showed that by knocking out the branched-chain amino acid absorption protein genes brnQ, livJ, livK, and overexpressing the branched-chain amino acid exoprotein gene ygaZH, the regulatory protein gene lrp, and the methylmalonic acid absorption protein gene dcuD, the synthesis of isoleucine was significantly improved.

[0186] Example 8 Construction of plasmid-free E. coli engineering strain for producing L-isoleucine

[0187] Since the expression of methylmalonic acid pathway-related genes on the plasmid will bring a great burden to the cell metabolism of the host strain. Therefore, the expression modules on the plasmids P5 and P9 were integrated into the chromosome of the ILE-2 strain to construct a plasmid-free L-isoleucine production strain.

[0188] I. Construction of E. coli ILE-5 strain

[0189] (1) Construction of pRE57-ILE-5-1 plasmid

[0190] With P5 plasmid as template, the primer was designed to amplify cimA3.7, GsleuCD, AfleuB gene expression module, which was integrated into linearized plasmid pRE57 digested by restriction endonuclease SpeI, PacI by Gibson assembly. The recombination system was transformed into E. coli Trans1-T1 competent cells, and LB ampicillin resistant plate was used for screening. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and PCR verification was performed using primer pairs. The successfully constructed plasmid was named pRE57-ILE-5-1.

[0191] (2) Construction of pRE57-ILE-5-2 plasmid

[0192] With P9 plasmid as template, the primer was designed to amplify ilvIH*, ilvC*, ilvD, LsleuDH, dcuD gene expression module, which was integrated into linearized plasmid pRE57 digested by restriction endonuclease SpeI, PacI by Gibson assembly. The recombination system was transformed into E. coli Trans1-T1 competent cells, and LB ampicillin resistant plate was used for screening. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and PCR verification was performed using primer pairs. The successfully constructed plasmid was named pRE57-ILE-5-2.

[0193] (3) Construction of E. coli ILE-5 strain

[0194] pKMV-gRNA-yjip plasmid and pTetQCas-BsaI-tns and pRE57-ILE-5-1 plasmid were successively electroporated into E. coli ILE-2 competent cells, and LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline were used for positive transformant screening. Primer pairs were used for PCR verification of transformants to screen positive transformants and sequencing, and the plasmid containing the three resistances was removed by 42℃, LB culture. Then pKMV-gRNA-gapC plasmid and pTetQCas-BsaI-tns and pRE57-ILE-5-2 plasmid were transformed into the just obtained engineering strain competent cells, and the same method was used to screen the positive engineering strain, and finally the E. coli ILE-5 strain was obtained (Table 2).

[0195] II. Construction of E. coli ILE-6 strain

[0196] pETDuet-1-P araBAD -dcuD expression module was electroporated into E. coli ILE-5 competent cells, and LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline were used for positive transformant screening. Primer pairs were used for PCR verification of transformants to screen positive transformants and sequencing, and the plasmid containing the three resistances was removed by 42℃, LB culture. Then pETDuet-1-P araBADThe -dcuD plasmid was transformed into Escherichia coli strain ILE-5, and Escherichia coli strain ILE-6 was obtained by antibiotic screening (Table 2), increasing the utilization capacity of methylmalic acid. The specific method is as follows:

[0197] (1) Construct a system with P araBAD pETDuet-1-P of the -dcuD expression module araBAD -dcuD plasmid

[0198] Using P9 plasmid as a template, primers were designed to amplify P. araBAD The -dcuD expression module was integrated into the plasmid pETDuet-1 via Gibson assembly. The recombinant system was transformed into *E. coli* Trans1-T1 competent cells and selected using LB ampicillin resistance plates. Positive clones were picked and cultured in LB liquid medium. The plasmid was extracted and validated by PCR using primers. The successfully constructed plasmid was named pETDuet-1-P. araBAD -dcuD.

[0199] (2) Construction of Escherichia coli ILE-6 strain

[0200] pETDuet-1-P araBAD The -dcuD plasmid was electroporated into competent cells of *E. coli* strain ILE-5. Positive transformants were screened using LB ampicillin-resistant plates and then cultured in LB liquid medium. The transformants were validated by PCR using primers, yielding *E. coli* strain ILE-6.

[0201] III. Construction of Escherichia coli ILE-7 strain

[0202] Will have P araBAD -GsleuCD expression module pRSFDuet-1-P araBAD The GsleuCD plasmid was transformed into Escherichia coli strain ILE-5, and Escherichia coli strain ILE-7 was obtained through antibiotic screening (Table 2), increasing the production capacity of L-isoleucine. The specific method is as follows:

[0203] (1) Construct a system with P araBAD -GsleuCD expression module pRSFDuet-1-P araBAD -GsleuCD plasmid

[0204] Using P5 plasmid as a template, primers were designed to amplify P5 plasmid. araBAD-GsleuCD expression module was integrated into plasmid pRSFDuet-1 by Gibson assembly. The recombination system was transformed into E. coli Trans1-T1 competent cells, and LB kanamycin-resistant plates were used for screening. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and verified by PCR using primers, and the successfully constructed plasmid was named pRSFDuet-1-P araBAD -GsleuCD.

[0205] (2) Construction of E. coli ILE-7 strain

[0206] The pRSFDuet-1-P araBAD -GsleuCD plasmid was electroporated into E. coli ILE-5 strain competent cells, and positive transformants were screened by LB kanamycin-resistant plates and subjected to LB liquid culture. The transformants were verified by PCR using primers, and the E. coli ILE-7 strain was obtained.

[0207] Second, fermentation experiments of E. coli ILE-5, ILE-6 and ILE-7 strains

[0208] The obtained E. coli ILE-5, ILE-6 and ILE-7 strains were subjected to fermentation in a 3L fermenter, and the production capacity of E. coli engineering strains to synthesize L-isoleucine through the methylmalonic acid pathway was improved by using fed-batch fermentation. The specific method is as follows:

[0209] First, the E. coli ILE-5, ILE-6 and ILE-7 strains were inoculated into 40 mL of LB medium and incubated at 37°C, 200 r / min overnight as the first-stage seed liquid. The first-stage seed liquid was inoculated into 1L of LB medium and incubated at 37°C, 200 r / min for 24 h as the second-stage seed liquid. The OD 600 of the second-stage seed liquid was measured and inoculated into 1L of medium (3L fermenter) to make the initial OD 600 about 0.3. The formula of the medium is as follows:

[0210] The formula of the fermentation medium is the same as that described in Example 1.

[0211] The fermentation process temperature was maintained at 37°C, the pH was maintained at 6.8-7.0 by automatic ammonia water supplement, and the motor speed was coupled to control the dissolved oxygen level at 10%-20%. When the initial glucose was consumed, the glucose concentration was controlled below 10 g / L by feeding 700 g / L glucose through a peristaltic pump. The fermentation was carried out for 48 h, and the culture medium was sampled every 3-6 h. The concentration of L-isoleucine in the culture medium was separated and determined by a Shimadzu AJS-01 amino acid analyzer using a high-performance liquid chromatography ultraviolet detector. As shown in FIG. 10, the strain ILE-5 produced 5.2 g / L of L-isoleucine and 10.2 g / L of methylmalate (L-isoleucine / methylmalate: 0.51). Notably, the methylmalate production of ILE-5 was much higher than the L-isoleucine production, indicating that the utilization efficiency of methylmalate in ILE-5 was still low. The strain ILE-6 produced 4.9 g / L of L-isoleucine and 8.9 g / L of methylmalate (L-isoleucine / methylmalate: 0.55), which was slightly lower than ILE-5. Meanwhile, the L-isoleucine and methylmalate production of the strain ILE-7 reached 3.2 g / L and 38.9 g / L (L-isoleucine / methylmalate: 0.08), respectively. The results showed that the L-isoleucine production efficiency of the plasmid-free L-isoleucine strain ILE-5 was significantly improved under the fermentation conditions of fed-batch, pH and dissolved oxygen level control in the fermenter compared with the shake flask fermentation, but there was still a large amount of methylmalate, and further overexpression of GsleuCD and dcuD did not improve the utilization efficiency of methylmalate.

[0212] Example 9 Further improving the L-isoleucine production capacity by overexpressing cimA3.7 and AfleuB genes

[0213] The overexpression copy number of the cimA3.7 and AfleuB genes on the genome was optimized to improve the L-isoleucine production capacity.

[0214] I. Construction of E. coli ILE-8 strain

[0215] In the pflB and ldhA sites of the E. coli ILE-5 strain, one copy of the P araBAD -cimA3.7-AfleuB expression cassette was integrated to obtain the ILE-8 strain (Table 2). The specific method is as follows:

[0216] (1) Construction of pRE57-cimA3.7-AfleuB plasmid with cimA3.7 and AfleuB gene expression cassette

[0217] P5 plasmid as a template, primers were designed to amplify P araBAD -cimA3.7 and ParaBAD -AfleuB expression module, after each fragment is connected by overlap PCR, integrated into plasmid pRE57 by Gibson assembly. The recombination system is transformed into E. coli Trans1-T1 competent cells, and LB ampicillin resistant plates are used for screening. Positive clones are picked and cultured in LB liquid. The plasmid is extracted and PCR verification is performed using primers. The successfully constructed plasmid is named pRE57-cimA3.7-AfleuB.

[0218] (2) Construction of E. coli ILE-8 strain

[0219] The pKMV-gRNA-pflB-ldhA plasmid and the pTetQCas-BsaI-tns and pRE57-cimA3.7-AfleuB plasmid are successively electroporated into E. coli ILE-5 competent cells, and positive transformants are screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline. The transformants are screened by PCR verification using primers and sequencing, and the plasmid containing the three resistances is removed by 42°C, LB culture, and finally the E. coli ILE-8 strain is obtained.

[0220] II. Construction of E. coli ILE-9 strain

[0221] In the adhE and ycjV sites of the E. coli ILE-8 strain, one copy of P araBAD -AfleuB expression cassette, to obtain the ILE-9 strain (Table 2). The specific method is as follows:

[0222] (1) Construction of pRE57-AfleuB plasmid with AfleuB gene expression cassette

[0223] P5 plasmid as template, designed primers to amplify P araBAD -AfleuB expression module, integrated into plasmid pRE57 by Gibson assembly. The recombination system is transformed into E. coli Trans1-T1 competent cells, and LB ampicillin resistant plates are used for screening. Positive clones are picked and cultured in LB liquid. The plasmid is extracted and PCR verification is performed using primers. The successfully constructed plasmid is named pRE57-AfleuB.

[0224] (2) Construction of E. coli ILE-9 strain

[0225] The pKMV-gRNA-adhE-ycjV plasmid and the pTetQCas-BsaI-tns and pRE57-AfleuB plasmids are successively electroporated into the E. coli ILE-8 competent cells, and positive transformants are screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline. The transformants are verified by PCR using primers, and sequenced. The plasmids containing the three resistances are removed by 42°C LB culture, and finally the E. coli ILE-9 strain is obtained.

[0226] III. Construction of the E. coli ILE-10 strain

[0227] In the lafU and ydeU sites of the E. coli ILE-9 strain, one copy of the P araBAD -AfleuB expression cassette is integrated respectively, and the ILE-10 strain is obtained (Table 2). The specific method is as follows:

[0228] The pKMV-gRNA-lafU-ydeU plasmid and the pTetQCas-BsaI-tns and pRE57-AfleuB plasmids are successively electroporated into the E. coli ILE-9 competent cells, and positive transformants are screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline. The transformants are verified by PCR using primers, and sequenced. The plasmids containing the three resistances are removed by 42°C LB culture, and finally the E. coli ILE-10 strain is obtained.

[0229] IV. Construction of the E. coli ILE-11 strain

[0230] In the yciQ and yjiV sites of the E. coli ILE-9 strain, one copy of the P araBAD -cimA3.7 expression cassette is integrated respectively, and the ILE-11 strain is obtained (Table 2). The specific method is as follows:

[0231] (1) Construction of the pRE57-cimA3.7 plasmid with the cimA3.7 gene expression cassette

[0232] The P5 plasmid is used as a template, and primers are designed to amplify P araBAD -cimA3.7 expression module, which is integrated into the plasmid pRE57 by Gibson assembly. The recombination system is transformed into the E. coli Trans1-T1 competent cells, and screened using LB ampicillin resistance plates. Positive clones are picked and subjected to LB liquid culture. The plasmid is extracted and verified by PCR using primers, and the successfully constructed plasmid is named pRE57-cimA3.7.

[0233] (2) Construction of the E. coli ILE-11 strain

[0234] The pKMV-gRNA-yciQ-yjiV plasmid and pTetQCas-BsaI-tns and pRE57-cimA3.7 plasmids were successively electroporated into E. coli ILE-9 competent cells, and positive transformants were screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline. The transformants were screened by PCR verification and sequencing, and the plasmids containing the three resistances were removed by 42℃ LB culture, and finally the E. coli ILE-11 strain was obtained.

[0235] II. Fermentation experiments of E. coli ILE-8, ILE-9, ILE-10 and ILE-11 strains

[0236] The constructed E. coli ILE-8, ILE-9, ILE-10 and ILE-11 strains were fermented in a 3L fermenter, and the specific method was as follows:

[0237] The fermentation medium formula was the same as that described in Example 1.

[0238] The fermentation method was the same as that described in Example 4.

[0239] The fermentation product detection method was the same as that described in Example 4.

[0240] As shown in Figure 10, the results show that after increasing two copies of P araBAD -cimA3.7-AfleuB expression cassette on the ILE-5 strain, the L-isoleucine yield of the obtained ILE-8 strain reached 20.6g / L, which was about 3 times higher than that of ILE-5. After increasing two or four copies of P araBAD -AfleuB expression cassette on the ILE-8 strain, the L-isoleucine yield of the obtained ILE-9 and ILE-10 strains reached 31.1g / L and 24.9g / L, respectively, indicating that a total of five copies of AfleuB had met the demand, and excessive copies would significantly reduce the L-isoleucine yield. After increasing two copies of P araBAD -cimA3.7 expression cassette on the ILE-9 strain, the methylmalonic acid yield of the obtained ILE-11 strain was greatly improved by 31.7%, while the L-isoleucine yield was only slightly improved by 6.1%. It was indicated that a total of three copies of cimA3.7 had met the demand. Therefore, when the copy number of cimA3.7 and AfleuB integrated on the genome was 3 times and 5 times of GsleuCD, respectively, the L-isoleucine yield was optimal.

[0241] Example 10. Improving L-isoleucine production capacity by introducing non-oxidative glycolytic pathway

[0242] The sufficient supply of acetyl-CoA is essential for the biosynthesis of methylmalonic acid. However, the main source of acetyl-CoA in E. coli is derived from the decarboxylation of pyruvate, which leads to carbon loss and thus reduces the conversion rate. Compared with the classic glycolytic pathway, the non-oxidative glycolytic pathway can efficiently produce acetyl-CoA from glucose in fewer synthetic steps and without releasing CO2, which is conducive to the production of L-isoleucine. Therefore, the non-oxidative glycolytic pathway was introduced into the ILE-9 strain by overexpressing the bifunctional phosphoketolase gene Bafxpk (SEQ ID NO: 28) from Bifidobacterium adolescentis and the phosphotransacetylase gene pta (SEQ ID NO: 29) of E. coli itself on the genome.

[0243] I. Construction of E. coli ILE-12 strain

[0244] The ackA gene in the E. coli ILE-9 strain was knocked out to reduce the synthesis of acetyl-CoA to acetic acid, and the E. coli ILE-12 strain was obtained (Table 2). The specific method is as follows:

[0245] The pKMV-gRNA-ackA plasmid with the ackA site gRNA and the pTetQCas-BsaI-tns and pRE57 plasmids were successively electroporated into the E. coli ILE-9 competent cells, and the positive transformants were screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance and dehydrated tetracycline. The positive transformants were screened by PCR verification of the transformants and sequencing, and the ackA gene knockout ILE-12 strain was finally obtained by 42°C, LB culture to remove the plasmid containing the three resistances.

[0246] II. Construction of E. coli ILE-13 strain

[0247] The ackA site of the E. coli ILE-12 strain was overexpressed with the P araBAD -Bafxpk-pta module to obtain the ILE-13 strain (Table 2). The specific method is as follows:

[0248] (1) Construction of pRE57-Bafxpk-pta plasmid with Bafxpk and pta gene expression cassette

[0249] The bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis was artificially synthesized after codon optimization, and the Bafxpk synthesis fragment and E. coli genome were used as templates to design amplification primers to amplify P araBAD -Bafxpk, P araBADThe pta expression module was integrated into plasmid pRE57 by Gibson assembly after the fragments were connected by overlap PCR. The recombination system was transformed into E. coli Transl-Tl competent cells, and LB ampicillin-resistant plates were used for screening. Positive clones were picked and cultured in LB liquid medium. The plasmid was extracted and verified by PCR using primers, and the successfully constructed plasmid was named pRE57-Bafxpk-pta.

[0250] (2) Construction of E. coli ILE-13 strain

[0251] The pKMV-gRNA-ackA plasmid and the pTetQCas-Bsal-tns and pRE57-Bafxpk-pta plasmids were successively electroporated into E. coli ILE-12 competent cells, and positive transformants were screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and dehydrated tetracycline. The transformants were verified by PCR using primers and sequenced, and the plasmids containing the three resistances were removed by 42°C LB culture, and finally the E. coli ILE-13 strain was obtained.

[0252] III. Construction of E. coli ILE-14 strain

[0253] On the basis of the E. coli ILE-13 strain, a copy of P araBAD -Bafxpk-pta module was integrated to obtain the ILE-14 strain (Table 2). The specific method is as follows:

[0254] The pKMV-gRNA-poxB plasmid and the pTetQCas-Bsal-tns and pRE57-Bafxpk-pta plasmids were successively electroporated into E. coli ILE-13 competent cells, and positive transformants were screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and dehydrated tetracycline. The transformants were verified by PCR using primers and sequenced, and the plasmids containing the three resistances were removed by 42°C LB culture, and finally the E. coli ILE-14 strain was obtained.

[0255] IV. Construction of E. coli ILE-15 strain

[0256] The EutD gene in E. coli can perform a similar function to the pta gene, so a copy of P araBAD -Bafxpk-eutD module was overexpressed at the ackA site of the E. coli ILE-12 strain to obtain the ILE-15 strain (Table 2), and the effects of overexpressing pta (ILE13 strain) and eutD (ILE15 strain) on L-isoleucine production were compared. The specific method is as follows:

[0257] (1) Construct pRE57-Bafxpk-eutD plasmid with Bafxpk, eutD gene expression cassette

[0258] The Bafxpk synthetic fragment and the E. coli genome were used as templates to design and amplify primers to obtain P araBAD -Bafxpk, P araBAD -eutD expression module. After the fragments were connected by overlap PCR, they were integrated into the plasmid pRE57 by Gibson assembly. The recombination system was transformed into E. coli Trans1-T1 competent cells, and LB ampicillin-resistant plates were used for screening. Positive clones were picked and subjected to LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-Bafxpk-eutD.

[0259] (2) Construction of E. coli ILE-15 strain

[0260] The pKMV-gRNA-ackA plasmid and the pTetQCas-BsaI-tns and pRE57-Bafxpk-eutD plasmids were sequentially electroporated into E. coli ILE-12 competent cells. Positive transformants were induced and screened by LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and dehydrated tetracycline. The transformants were subjected to PCR verification using primers, and the positive transformants were sequenced. The plasmids containing the three resistances were removed by 42°C LB culture, and finally the E. coli ILE-15 strain was obtained.

[0261] Five, fermentation experiment of E. coli ILE-12 / 13 / 14 / 15 strains

[0262] The constructed ILE-12, ILE-13, ILE-14, and ILE-15 strains were fermented in a 3L fermenter, and the specific method was as follows:

[0263] The fermentation medium formula was the same as that described in Example 1.

[0264] The fermentation method was the same as that described in Example 4.

[0265] The fermentation product detection method was the same as that described in Example 4.

[0266] As shown in Figure 11, the L-isoleucine production of ILE-12 was 43.9 g / L, and the L-isoleucine production of ILE-13 was further improved to 50.7 g / L, indicating that knocking out ackA and introducing non-oxidative glycolysis pathway could significantly improve the L-isoleucine production. However, the L-isoleucine production of ILE-14 strain decreased sharply to 24.2 g / L, indicating that overexpression of Bafxpk and pta was not conducive to the production of L-isoleucine. The L-isoleucine production of ILE-15 strain decreased by 66.3% compared with ILE-13, which indicated that the eutD gene was not a suitable choice for improving the L-isoleucine production.

[0267] Example 11 Evaluation of L-isoleucine production level of ILE-13 strain in 10 L fermenter

[0268] The larger fermenter is more accurate in the control of fermentation conditions, and thus is more conducive to the display of production level. Therefore, the constructed ILE-13 strain was subjected to fermentation test in a 10 L fermenter, and the specific method was as follows:

[0269] The fermentation medium formula was the same as that described in Example 1.

[0270] The fermentation method was the same as that described in Example 4.

[0271] The fermentation product detection method was the same as that described in Example 4.

[0272] As shown in Figure 12, the growth of ILE-13 reached the plateau phase at about 20 hours, and the maximum OD 600 value was 86.7 (Figure 12). Finally, the ILE-13 strain produced 56.6 g / L of L-isoleucine, and the conversion rate and production intensity were 0.21 g / g of glucose and 1.66 g / L / h, respectively, showing the great potential of the methylmalonate pathway in the industrial production of L-isoleucine.

[0273] The above-described examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as limiting the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

An engineered strain for synthesizing L-isoleucine based on a methylmalonic acid pathway, characterized in that, The engineered strain is an Escherichia coli strain obtained by the following modifications: overexpressing a methylmalate synthase gene; expressing a carboxylate transporter DcuD, DauA or DcuA encoding gene, wherein the amino acid sequences of the transporters DcuD, DauA and DcuA are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. The engineered strain for synthesizing L-isoleucine based on the methylmalonic acid pathway according to claim 1, characterized in that, The amino acid sequence of the methylmalate synthase is shown in SEQ ID NO.

5. The engineered strain for synthesizing L-isoleucine based on the methylmalonic acid pathway according to claim 1, characterized in that, Further, the engineered strain further expresses a coding gene of a methylmalate pathway related enzyme, which includes one or more of the following enzymes: isopropylmalate isomerase, 3-isopropylmalate dehydrogenase, acetohydroxy acid synthase, acetohydroxy acid reductoisomerase, dihydroxy acid dehydratase, branched-chain amino acid aminotransferase, leucine dehydrogenase or valine dehydrogenase. The engineered strain for synthesizing L-isoleucine based on a methylmalate pathway according to claim 3, wherein the isopropylmalate isomerase includes isopropylmalate isomerase IPMI or isopropylmalate isomerase LeuCD, wherein the isopropylmalate isomerase LeuCD includes a LeuC subunit and a LeuD subunit; the 3-isopropylmalate dehydrogenase includes 3-isopropylmalate dehydrogenase IPMD or 3-isopropylmalate dehydrogenase LeuB; the acetohydroxy acid synthase includes acetohydroxy acid synthase AHAS or acetohydroxy acid synthase IlvIH; the acetohydroxy acid reductoisomerase includes acetohydroxy acid reductoisomerase AHAIR or acetohydroxy acid reductoisomerase IlvC; the dihydroxy acid dehydratase includes dihydroxy acid dehydratase DHAD or dihydroxy acid dehydratase IlvD; the branched-chain amino acid aminotransferase includes dihydroxy acid dehydratase BCAT or dihydroxy acid dehydratase IlvE; the leucine dehydrogenase includes a LeuD subunit and a LeuH subunit. The engineered strain for synthesizing L-isoleucine based on the methylmalonic acid pathway according to claim 4, characterized in that, the acetohydroxy acid synthase IlvIH includes an IlvI subunit and an IlvH* subunit. The engineered strain for synthesis of L-isoleucine based on the methylmalonic acid pathway according to claim 1, characterized in that, The engineered strain is an Escherichia coli strain obtained by the following modifications: overexpressing a methylmalate synthase gene, an isopropylmalate isomerase gene, a 3-isopropylmalate dehydrogenase gene, and a leucine dehydrogenase gene; expressing a coding gene of a carboxylate transporter DcuD; knocking out branched-chain amino acid absorption protein genes brnQ, livJ, livK; overexpressing branched-chain amino acid excretion protein genes ygaZ, ygaH and regulatory protein gene lrp on the genome; overexpressing bifunctional phosphoketolase gene Bafxpk and Escherichia coli's own phosphotransacetylase gene pta on the genome. The engineered strain for synthesis of L-isoleucine based on the methylmalonic acid pathway according to claim 1 or 6, characterized in that, The chassis strain of the mutant Escherichia coli engineered strain is Escherichia coli BW25113. The engineered strain for synthesizing L-isoleucine based on the methylmalonic acid pathway according to claim 6, characterized in that, The copy number of the methylmalate synthase high-activity mutant gene cimA3.7 integrated on the genome is 3 times that of the isopropylmalate isomerase gene GsleuCD, and the copy number of the 3-isopropylmalate dehydrogenase gene AfleuB integrated on the genome is 5 times that of the isopropylmalate isomerase gene GsleuCD. The engineered strain for synthesis of L-isoleucine based on the methylmalonic acid pathway according to claim 1 or 6, characterized in that, The gene overexpressed in the engineered strain is integrated into the chromosome of the strain. A method for preparing L-isoleucine by fermentation, characterized by, The method comprises the step of preparing L-isoleucine by fermenting the engineered strain for synthesizing L-isoleucine based on the methylmalonic acid pathway according to any one of claims 1 to 11.

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