Improved engineering strain for producing L-isoleucine through methyl malic acid way as well as construction method and application of improved engineering strain

Abstract

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an Escherichia coli engineering strain that produces L-isoleucine through a methylmalate pathway, a construction method thereof and an application thereof. Background Art

[0002] L-isoleucine is one of the three essential branched-chain amino acids (BCAAs). Microbial fermentation is considered to be the most promising method for industrial-scale production of L-isoleucine due to its low production cost and environmentally friendly fermentation conditions.

[0003] Microbial production of L-isoleucine primarily relies on the threonine pathway, which uses threonine as a precursor and α-ketobutyrate to produce L-isoleucine. However, due to the length and complexity of the threonine pathway and the presence of complex feedback regulation mechanisms, L-isoleucine production has remained at a low level.

[0004] In addition to the threonine pathway, microorganisms have evolved several alternative pathways for L-isoleucine biosynthesis. The most common of these is the methylmalate pathway (also known as the pyruvate pathway). In this pathway, L-isoleucine biosynthesis proceeds from pyruvate and acetyl-CoA in just seven steps, five fewer than the threonine pathway. Furthermore, only two enzymes (methylmalate synthase and acetohydroxyacid synthase) are subject to feedback inhibition during the synthesis of L-isoleucine via the methylmalate pathway, far fewer than in the threonine pathway. Therefore, compared to the threonine pathway, the methylmalate pathway is considered a more efficient L-isoleucine biosynthetic pathway due to its simpler synthesis route and simpler feedback control mechanism. However, few studies have systematically explored the utilization of the methylmalate pathway for the efficient production of L-isoleucine. Summary of the Invention

[0005] The object of the present invention is to provide an improved engineered strain for producing L-isoleucine via the methylmalate pathway.

[0006] Another object of the present invention is to provide an improved method for constructing an engineered strain for producing L-isoleucine via the methylmalate pathway.

[0007] Another object of the present application is to provide a method for producing L-isoleucine by fermentation.

[0008] According to the improved engineered strain for producing L-isoleucine via the methylmalate pathway of the present invention, the engineered strain has the following characteristics:

[0009] Overexpressing on the genome the genes of a methylmalate synthase hyperactive mutant gene cimA3.7 from Methanococcusjannaschii, an isopropylmalate isomerase gene GsleuCD from Geobacillus sp.WCH70 strain, a 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobusfulgidus, and a leucine dehydrogenase gene LsleuDH from Lysinibacillussphaericus, wherein the copy number of the methylmalate synthase hyperactive 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;

[0010] Overexpression of the methylmalate uptake protein gene dcuD on the genome;

[0011] Knockout of branched-chain amino acid uptake protein genes brnQ, livJ, and livK;

[0012] Overexpression of branched-chain amino acid exoprotein genes ygaZ, ygaH and regulatory protein gene lrp on the genome;

[0013] The non-oxidative glycolysis pathway was introduced by overexpressing the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the phosphotransacetylase gene pta from Escherichia coli.

[0014] The nucleotide sequence of the methylmalate synthase high-activity mutant gene cimA3.7 is shown in SEQ ID NO: 1; the isopropylmalate isomerase gene GsleuCD comprises two subunit genes, wherein the nucleotide sequence of the isopropylmalate isomerase large subunit gene GsleuC is shown in SEQ ID NO: 2, and the nucleotide sequence of the isopropylmalate isomerase small subunit gene GsleuD is shown in SEQ ID NO: 3; the nucleotide sequence of the 3-isopropylmalate dehydrogenase gene AfleuB is shown in SEQ ID NO: 4; the nucleotide sequence of the leucine dehydrogenase subunit gene Lsleudh is shown in SEQ ID NO: 5; the nucleotide sequence of the methylmalate uptake protein gene dcuD is shown in SEQ ID NO: 6; the nucleotide sequence of the branched-chain amino acid uptake protein gene brnQ is shown in SEQ ID NO: 7; the nucleotide sequence of the branched-chain amino acid uptake protein gene livJ is shown in SEQ ID NO: 8; and the nucleotide sequence of the branched-chain amino acid uptake protein gene livK is shown in SEQ ID NO: NO: 9; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO: 10; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown in SEQ ID NO: 11; the nucleotide sequence of the regulatory protein gene lrp is shown in SEQ ID NO: 12; the nucleotide sequence of the bifunctional phosphoketolase gene Bafxpk is shown in SEQ ID NO: 13; and the nucleotide sequence of the phosphotransacetylase gene pta is shown in SEQ ID NO: 14.

[0015] According to the Escherichia coli engineered strain for producing L-isoleucine through the methylmalate pathway of the present invention, the Escherichia coli chassis strain is Escherichia coli BW25113.

[0016] According to the improved engineered Escherichia coli strain of the present invention for producing L-isoleucine via the methylmalate pathway, genes encoding enzymes related to the methylmalate pathway are further expressed in Escherichia coli BW25113, wherein the genes encoding enzymes related to the methylmalate pathway are one or more genes selected from the following genes:

[0017] Anti-feedback inhibition mutant acetohydroxyacid synthase gene, NADH-preferring acetohydroxyacid reductoisomerase gene, and dihydroxyacid dehydratase gene.

[0018] Among them, the anti-feedback inhibition mutant acetohydroxyacid synthase gene ilvIH* is derived from Escherichia coli, including the ilvI subunit and the ilvH* subunit; the NADH-preferring acetohydroxyacid reductoisomerase gene ilvC* is derived from Corynebacterium glutamicum; and the dihydroxyacid dehydratase gene ilvD is derived from Escherichia coli.

[0019] According to a specific embodiment of the present invention, the nucleotide sequence of the ilvI subunit is shown in SEQ ID NO: 15; the nucleotide sequence of the ilvH* subunit is shown in SEQ ID NO: 16; the nucleotide sequence of ilvC* is shown in SEQ ID NO: 17; and the nucleotide sequence of ilvD is shown in SEQ ID NO: 18.

[0020] The improved method for constructing an engineered Escherichia coli strain that produces L-isoleucine via the methylmalate pathway according to the present invention comprises the following steps:

[0021] The high-activity mutant gene cimA3.7 of methylmalate synthase from Methanococcusjannaschii, the isopropylmalate isomerase gene GsleuCD from Geobacillus sp.WCH70, the 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobusfulgidus, and the leucine dehydrogenase gene LsleuDH from Lysinibacillus sphaericus were overexpressed in the genome of an Escherichia coli chassis strain. The copy number of cimA3.7 integrated in the genome was 3 times that of GsleuCD, and the copy number of AfleuB integrated in the genome was 5 times that of GsleuCD.

[0022] Overexpression of the methylmalate uptake protein gene dcuD on the genome;

[0023] Knockout of branched-chain amino acid uptake protein genes brnQ, livJ, and livK;

[0024] Overexpression of branched-chain amino acid exoprotein genes ygaZ, ygaH and regulatory protein gene lrp on the genome;

[0025] The non-oxidative glycolysis pathway was introduced by overexpressing the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the phosphotransacetylase gene pta from Escherichia coli.

[0026] The nucleotide sequence of the methylmalate synthase high-activity mutant gene cimA3.7 is shown in SEQ ID NO: 1; the isopropylmalate isomerase gene GsleuCD comprises two subunit genes, wherein the nucleotide sequence of the isopropylmalate isomerase large subunit gene GsleuC is shown in SEQ ID NO: 2, and the nucleotide sequence of the isopropylmalate isomerase small subunit gene GsleuD is shown in SEQ ID NO: 3; the nucleotide sequence of the 3-isopropylmalate dehydrogenase gene AfleuB is shown in SEQ ID NO: 4; the nucleotide sequence of the leucine dehydrogenase subunit gene Lsleudh is shown in SEQ ID NO: 5; the nucleotide sequence of the methylmalate uptake protein gene dcuD is shown in SEQ ID NO: 6; the nucleotide sequence of the branched-chain amino acid uptake protein gene brnQ is shown in SEQ ID NO: 7; the nucleotide sequence of the branched-chain amino acid uptake protein gene livJ is shown in SEQ ID NO: 8; and the nucleotide sequence of the branched-chain amino acid uptake protein gene livK is shown in SEQ ID NO: NO: 9; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO: 10; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown in SEQ ID NO: 11; the nucleotide sequence of the regulatory protein gene lrp is shown in SEQ ID NO: 12; the nucleotide sequence of the bifunctional phosphoketolase gene Bafxpk is shown in SEQ ID NO: 13; and the nucleotide sequence of the phosphotransacetylase gene pta is shown in SEQ ID NO: 14.

[0027] According to the improved method for constructing an engineered Escherichia coli strain that produces L-isoleucine through the methylmalate pathway of the present invention, the method further comprises the step of expressing genes encoding enzymes related to the methylmalate pathway in Escherichia coli BW25113, wherein the genes encoding enzymes related to the methylmalate pathway are one or more of the following genes:

[0028] Anti-feedback inhibition mutant acetohydroxyacid synthase gene, NADH-preferring acetohydroxyacid reductoisomerase gene, and dihydroxyacid dehydratase gene.

[0029] Among them, the anti-feedback inhibition mutant acetohydroxyacid synthase gene ilvIH* is derived from Escherichia coli, including the ilvI subunit and the ilvH* subunit; the NADH-preferring acetohydroxyacid reductoisomerase gene ilvC* is derived from Corynebacterium glutamicum; and the dihydroxyacid dehydratase gene ilvD is derived from Escherichia coli.

[0030] According to a specific embodiment of the present invention, the nucleotide sequence of the ilvI subunit is shown in SEQ ID NO: 15; the nucleotide sequence of the ilvH* subunit is shown in SEQ ID NO: 16; the nucleotide sequence of ilvC* is shown in SEQ ID NO: 17; and the nucleotide sequence of ilvD is shown in SEQ ID NO: 18.

[0031] Advantages of the technical solution of the present invention:

[0032] 1. The methylmalate pathway uses pyruvate and acetyl-CoA as precursors to synthesize L-isoleucine. Therefore, strains with sufficient pyruvate and acetyl-CoA supplies are suitable as base strains for constructing the methylmalate pathway. According to the technical solution of the present invention, the synthesis amount and efficiency of methylmalate were the highest when Escherichia coli BW25113 was used as the base strain, significantly outperforming other commonly used E. coli strains. Furthermore, by overexpressing the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the E. coli native phosphotransacetylase gene pta, a non-oxidative glycolysis pathway was introduced to increase the intracellular supply of acetyl-CoA. Experimental data showed that the resulting engineered strain significantly increased L-isoleucine production.

[0033] 2. The three synthesis steps from pyruvate and acetyl-CoA to α-ketobutyrate in the methylmalate pathway are key points that distinguish it from the traditional threonine pathway, among which isopropylmalate isomerase LeuCD and 3-isopropylmalate dehydrogenase LeuB are also involved in the leucine synthesis pathway. Therefore, the LeuCD and LeuB enzymes that can efficiently convert methylmalate to α-ketobutyrate are the key to synthesizing L-isoleucine through the methylmalate pathway. According to the technical solution of the present invention, the selected isopropylmalate isomerase GsLeuCD from the Geobacillus sp.WCH70 strain and the 3-isopropylmalate dehydrogenase AfLeuB from the Archaeoglobus fulgidus source have significant advantages over enzymes from other sources.

[0034] 3. According to the technical solution of the present invention, the L-isoleucine synthesis efficiency is highest when the copy number of cimA3.7 integrated into the genome is 3 times that of GsleuCD and the copy number of AfleuB integrated into the genome is 5 times that of GsleuCD.

[0035] 4. Knocking out the branched-chain amino acid absorption protein genes brnQ, livJ, and livK and overexpressing the branched-chain amino acid excretion protein genes ygaZ, ygaH, and the regulatory protein gene lrp can reduce intracellular L-isoleucine accumulation and significantly increase L-isoleucine production.

[0036] 5. CN 118853722B discloses a method and engineered strain for increasing the production of L-isoleucine synthesis based on the methylmalate pathway. The method identifies carboxylate transporters DcuD, DauA, and DcuA as methylmalate uptake proteins, effectively relieving the inhibitory effect of glucose on methylmalate utilization during fermentation by the engineered strain, and significantly improving the L-isoleucine synthesis capacity based on the methylmalate pathway. Since the methylmalate pathway uses pyruvate and acetyl-CoA as precursors to synthesize L-isoleucine, and isopropylmalate isomerase LeuCD, 3-isopropylmalate dehydrogenase LeuB and leucine dehydrogenase LeuDH in the pathway also participate in leucine biosynthesis, the present application solves the problem of pyruvate and acetyl-CoA supply from the perspective of chassis cell selection and introduction of the non-oxidative glycolysis (NOG) pathway, and obtains a catalytic enzyme that is more suitable for the methylmalate pathway from the perspective of selecting the 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 and the L-isoleucine transport system, ultimately greatly improving the L-isoleucine synthesis capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The shake flask fermentation results of methylmalic acid production by the E. coli engineered strains JQ1, JQ2, JQ3 and JQ4 provided in Example 1 of the present invention are as follows;

[0038] Figure 2 The shake flask fermentation results of the E. coli engineered strains JQ5-JQ10 provided in Example 2 of the present invention are shown;

[0039] Figure 3 The shake flask fermentation results of the E. coli engineered strains ILE-3 and ILE-4 provided in Example 3 of the present invention are shown;

[0040] Figure 4 The fermentation results of the E. coli engineered strains ILE-5 to ILE-11 provided in Examples 4 and 5 of the present invention in a 3 L fermenter;

[0041] Figure 5 The fermentation results of the E. coli engineered strains ILE-12 to ILE-15 provided in Example 6 of the present invention in a 3 L fermentor;

[0042] Figure 6 This is the fermentation result of the Escherichia coli engineered strain ILE-13 provided in Example 7 of the present invention in a 10 L fermentor. DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used are all commercially available.

[0044] Table 1 List of Escherichia coli engineered strains constructed in various embodiments of the present invention

[0045]

[0046]

[0047]

[0048] As shown in Table 1 above, the specific examples of the present application improve the production of L-isoleucine in Escherichia coli by the following measures:

[0049] First, using plasmid P1 (P J23100 -cimA3.7) screening different wild-type Escherichia coli strains including BW25113, W3110, MG1655 and W (ATCC9637) as chassis strains for constructing the methylmalate pathway, among which the selected Escherichia coli BW25113 chassis strain had a better methylmalate production capacity than other strains;

[0050] Second, using Escherichia coli BW25113 as the chassis strain to express different combinations of plasmids P2-P8 containing leuCD, leuB, and leudh genes from different sources, among which 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 screened out, significantly improving the L-isoleucine biosynthesis capacity of E. coli based on the methylmalate pathway;

[0051] Third, the utilization efficiency of methylmalate was improved by overexpressing the methylmalate uptake protein gene dcuD;

[0052] Fourth, by integrating the branched-chain amino acid exocytic protein gene ygaZH and the regulatory protein gene lrp into the branched-chain amino acid uptake protein gene brnQ, livJ, and livK loci, the exocytosis of L-isoleucine was enhanced;

[0053] Fifth, by integrating expression cassettes of genes related to the methylmalate pathway into the hypothetical protein gene yjip and gapC sites, overexpressing cimA3.7, GsleuCD, and AfleuB genes at the yjip site and overexpressing ilvIH*, ilvC*, ilvD, LsleuDH, and dcuD genes at the gapC site, a plasmid-free strain for L-isoleucine production was constructed;

[0054] Sixth, by integrating an expression cassette containing the methylmalate synthase gene cimA3.7 and the 3-isopropylmalate dehydrogenase gene AfleuB into the formate acetyltransferase gene pflB and the lactate dehydrogenase gene ldhA loci, the carbon metabolic flux of the methylmalate pathway was increased, thereby improving the ability to synthesize L-isoleucine;

[0055] Seventh, by integrating a copy of the 3-isopropylmalate dehydrogenase gene AfleuB expression cassette into the aldehyde dehydrogenase gene adhE and the hypothetical protein gene ycjV loci, the carbon metabolic flow of the methylmalate pathway was increased, thereby improving the ability to synthesize L-isoleucine;

[0056] Eighth, by integrating the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the phosphotransacetylase gene pta from Escherichia coli into the ackA site of the phosphate acetyltransferase gene, the supply of acetyl-CoA was increased, thereby increasing the carbon metabolic flow of the methylmalate pathway and further improving the production of synthesized L-isoleucine.

[0057] Example 1 Screening of Escherichia coli chassis strains suitable for constructing the methylmalate pathway

[0058] Methylmalate is the first key intermediate metabolite in the synthesis of L-isoleucine through the methylmalate pathway, and the precursors of methylmalate synthesis are pyruvate and acetyl-CoA. Therefore, a microbial chassis that can provide sufficient pyruvate and acetyl-CoA and efficiently synthesize methylmalate is crucial for the production of L-isoleucine through the methylmalate pathway. As shown in Table 1, using plasmid P1 (P J23100 Using a 3.7-cimA assay, different wild-type E. coli strains, including BW25113, W3110, MG1655, and W (ATCC9637), were screened as chassis strains for constructing the methylmalate pathway. The results showed that E. coli BW25113 had the highest efficiency in methylmalate synthesis. The specific steps are as follows:

[0059] 1. Construction of cimA gene overexpression plasmid P1

[0060] Primers were designed to amplify the high-activity mutant gene cimA3.7 of methylmalate synthase from Methanococcusjannaschii, and then promoter P was used to amplify the high-activity mutant gene cimA3.7. J23100 Control its expression and finally assemble the expression cassette into plasmid pEASY-T3 to obtain plasmid P1; the specific construction method is as follows:

[0061] Using the artificially synthesized cimA3.7 gene fragment as a template, the J23100-cimA3.7 fragment was obtained by extension PCR amplification. The obtained fusion fragment was ligated with the pEASY-T3 vector by agarose gel electrophoresis and recovery and then chemically transformed into E. coli Trans1 competent cells by heat shock. After shaking and resuscitation at 37°C for 1 hour, the cells were coated on LB plates containing 100 μg / mL ampicillin and cultured for 16 hours. After colonies appeared, positive clones were screened and verified by PCR and sequencing to obtain plasmid P1.

[0062] 2. Construction of strains JQ1, JQ2, JQ3, and JQ4

[0063] (1) Preparation of Escherichia coli electroporation competent cells

[0064] Inoculate 300 μL of E. coli glycerol stock into 100 mL of LB medium and culture it in a shaking incubator at 37°C overnight. Transfer one thousandth of the inoculum into 100 mL of fresh LB medium and culture it in a shaking incubator at 200 rpm at 37°C until the OD 600 Reach 0.6-0.8. Place the cultured bacterial solution on ice for 30 minutes, then centrifuge to collect the cells (4°C, 6,000 rpm, 6 minutes), resuspend the cells with 50 mL of pre-cooled double-distilled water, centrifuge again under the same conditions to collect the cells and resuspend the cells with double-distilled water. Centrifuge as above and resuspend the cells with 50 mL of pre-cooled 10% glycerol. Finally, centrifuge at 4°C, 6,000 rpm for 10 minutes to collect the cells, add 0.6 mL of pre-cooled 10% glycerol to suspend the cells, aliquot 100 μL per tube, and store in a -80°C refrigerator.

[0065] (2) Electrotransformation of Escherichia coli and screening of transformants

[0066] Add 500ng of plasmid to the E. coli electroporation competent cells, pipette to mix, transfer to a 1mm electroporation cuvette, use an electroporator to transform the plasmid into E. coli, then add 1mL of sterile, antibiotic-free LB liquid medium, and recover at 37℃ for 2h. After recovery, collect the cells by centrifugation and spread them on LB plates containing the antibiotics corresponding to the electroporated plasmid, and culture them in a 37℃ constant temperature incubator overnight. After colonies appear, use a sterile toothpick to pick single clones for PCR verification to confirm that the plasmids have been successfully transformed into E. coli cells.

[0067] (3) Construction of strains JQ1, JQ2, JQ3, and JQ4

[0068] Wild strains of Escherichia coli including BW25113, W3110, MG1655 and W (ATCC9637) were prepared as electroporation competent cells, and the above-constructed plasmid P1 was transformed into Escherichia coli BW25113, W3110, MG1655 and W (ATCC9637) strain cells to obtain the corresponding engineered strains JQ1, JQ2, JQ3 and JQ4, respectively.

[0069] 3. Shake flask fermentation experiments of engineered strains JQ1, JQ2, JQ3, and JQ4

[0070] The engineered strains JQ1, JQ2, JQ3, and JQ4 were inoculated into 40 mL of LB liquid medium, 100 μg / mL of ampicillin was added to the culture medium, and cultured overnight at 37°C and 200 rpm to serve as seed liquid. The cell density of the seed liquid was determined and the cells were inoculated into 100 mL of Erlenmeyer flask containing 50 mL of fermentation medium according to a certain ratio to achieve an initial OD of 0. 600 The fermentation temperature was maintained at about 0.05, and the fermentation was carried out at 37°C and 200 rpm in a shaking incubator. 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 V B1, 0.15 mg / L MnCl2·4H2O). The fermentation was carried out for a total of 48 hours, and samples were taken regularly to determine the cell density (OD 600 ) and the glucose and methylmalic acid content in the fermentation broth. Figure 1 As shown in Figure 2, the methylmalic acid production and yield of strain JQ1 reached 1.96 g / L and 0.20 g / g glucose, respectively, and the OD 600was 2.43. Strain JQ4 showed the best growth performance among the four tested strains, but its methylmalate production and yield were only 0.98 g / L and 0.08 g / g glucose, 50% and 60% lower than JQ1, respectively. W3110 and MG1655 are two of the most commonly used E. coli chassis strains for amino acid production. However, the methylmalate production and yield of JQ2 and JQ3 were also much lower than JQ1. The results indicate that E. coli BW25113 is a superior chassis strain for producing L-isoleucine via the methylmalate pathway. However, no L-isoleucine production was detected in the fermentation broth.

[0071] Example 2 Improving the L-isoleucine synthesis ability of Escherichia coli based on the methylmalate pathway by screening isopropylmalate isomerase (LeuCD), 3-isopropylmalate dehydrogenase (LeuB), and leucine dehydrogenase (LeuDH) from different sources

[0072] Escherichia coli BW25113 possesses most of the methylmalate pathway genes, except cimA. However, no L-isoleucine production was detected in the fermentation broth of strain JQ1 carrying the cimA3.7 expression plasmid. Possible reasons include: 1) the expression of other methylmalate pathway genes is regulated in E. coli; and 2) E. coli-derived enzymes such as LeuCD, LeuB, and IlvE primarily function in leucine synthesis under natural conditions, resulting in low activity in methylmalate pathway-related reactions. Therefore, using E. coli BW25113 as a chassis strain, we screened for key genes by expressing methylmalate pathway genes from different sources and observing changes in L-isoleucine production. 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 can significantly improve the L-isoleucine biosynthesis capacity of Escherichia coli based on the methylmalate pathway. The specific steps are as follows:

[0073] 1. Construction of expression plasmids P2 to P8 containing key genes from different sources

[0074] (1) Construction of plasmid P2

[0075] Primers were designed to amplify the methylmalate synthase high-activity mutant gene cimA3.7, the leuCD gene from Escherichia coli, and the leuB gene from Escherichia coli, respectively. The nucleotide sequence of the large subunit gene leuC of the isopropylmalate isomerase from Escherichia coli is shown in SEQ ID NO: 19; the nucleotide sequence of the small subunit gene leuD of the isopropylmalate isomerase from Escherichia coli is shown in SEQ ID NO: 20; and the nucleotide sequence of the 3-isopropylmalate dehydrogenase gene leuB from Escherichia coli is shown in SEQ ID NO: 21. Each gene was expressed via an arabinose-inducible promoter P araBAD Control its expression, and finally assemble the expression cassette into plasmid pRSF-Dute-1 to obtain plasmid P2; the specific construction method is as follows:

[0076] The artificially synthesized cimA3.7 gene fragment and the Escherichia coli genome were used as templates to obtain P araBAD -cimA、P araBAD -leuC, P araBAD -leuD、P araBAD -leuB fragment was purified by agarose gel electrophoresis and recovery, and the obtained fusion fragment was ligated with the pRSF-Dute-1 vector. It was then transformed into E. coli Trans1 competent cells by heat shock and chemical transformation. After shaking and recovery at 37°C for 1 hour, it was coated on LB plates containing 50 μg / mL kanamycin and cultured for 16 hours. After colonies appeared, positive clones were screened and verified by PCR and sequencing to obtain plasmid P2.

[0077] (2) Construction of plasmid P3

[0078] Primers were designed to amplify the feedback inhibition-resistant mutant acetohydroxyacid synthase gene ilvIH* from Escherichia coli, the NADH-preferring acetohydroxyacid reductoisomerase gene ilvC* from Corynebacterium glutamicum, the dihydroxyacid dehydratase gene ilvD from Escherichia coli, and the branched-chain amino acid transaminase gene ilvE from Escherichia coli, respectively. The nucleotide sequence of the branched-chain amino acid transaminase gene ilvE from Escherichia coli is shown in SEQ ID NO: 22. Each gene is expressed via an arabinose-inducible promoter P araBAD Control its expression, and finally assemble the expression cassette into plasmid pET-Dute-1 to obtain plasmid P3; the specific construction method is as follows:

[0079] Using the Escherichia coli genome and the Corynebacterium glutamicum genome as templates, P araBAD -ilvIH*、P araBAD -ilvC*、P araBAD -ilvD、P araBAD-ilvE fragment was purified by agarose gel electrophoresis and recovery, and the obtained fusion fragment was ligated with the pET-Dute-1 vector. It was then transformed into E. coli Trans1 competent cells by heat shock chemical transformation. After shaking and recovery at 37°C for 1 hour, it was coated on an LB plate containing 100 μg / mL ampicillin and cultured for 16 hours. After colonies appeared, positive clones were screened and verified by PCR and sequencing to obtain plasmid P3.

[0080] (3) Construction of plasmid P4

[0081] The leuCD gene and leuB gene in plasmid P2 were replaced with the isopropylmalate isomerase gene GsleuCD from Geobacillus sp. WCH70 and the 3-isopropylmalate dehydrogenase gene LbleuB from Leadbetterella byssophila, respectively. The nucleotide sequence of the 3-isopropylmalate dehydrogenase gene LbleuB from Leadbetterella byssophila is shown in SEQ ID NO: 23. The specific construction method is as follows:

[0082] The GsleuCD and LbleuB genes were obtained by artificial synthesis after codon optimization, and the other construction methods were the same as the P2 plasmid construction steps.

[0083] (4) Construction of plasmid P5

[0084] The 3-isopropylmalate dehydrogenase gene LbleuB from Leadbetterella byssophila in plasmid P4 was replaced with the 3-isopropylmalate dehydrogenase gene AfleuB from Archaeoglobus fulgidus; the specific construction method is as follows:

[0085] The AfleuB gene was obtained by artificial synthesis after codon optimization, and the other construction methods were the same as the P4 plasmid construction steps.

[0086] (5) Construction of plasmid P6

[0087] The E. coli-derived branched-chain amino acid transaminase gene ilvE in plasmid P3 was replaced with the Lysinibacillus sphaericus-derived leucine dehydrogenase gene LsleuDH; the specific construction method is as follows:

[0088] The LsleuDH gene was obtained by artificial synthesis after codon optimization, and the other construction methods were the same as the P3 plasmid construction steps.

[0089] (6) Construction of plasmid P7

[0090] The E. coli-derived branched-chain amino acid transaminase gene ilvE in plasmid P3 was replaced with the Geobacillus stearothermophilus-derived leucine dehydrogenase gene GsleuDH, wherein the nucleotide sequence of the Geobacillus stearothermophilus-derived GsleuDH gene is shown in SEQ ID NO: 24; the specific construction method is as follows:

[0091] The GsleuDH gene was obtained by artificial synthesis after codon optimization, and the other construction methods were the same as the P3 plasmid construction steps.

[0092] (7) Construction of plasmid P8

[0093] The E. coli-derived branched-chain amino acid transaminase gene ilvE in plasmid P3 was replaced with the Halobacillus halophilus-derived leucine dehydrogenase gene HhleuDH, wherein the nucleotide sequence of the Halobacillus halophilus-derived leucine dehydrogenase gene HhleuDH is shown in SEQ ID NO: 25. The specific construction method is as follows:

[0094] The HhleuDH gene was obtained by artificial synthesis after codon optimization, and the other construction methods were the same as the P3 plasmid construction steps.

[0095] 2. Construction of engineered strains JQ5-JQ10 carrying plasmids P2-P8

[0096] The wild strain of Escherichia coli BW25113 was prepared as electroporation competent cells, and the constructed plasmids P2 to P8 were transformed into the Escherichia coli BW25113 strain in the combination shown in Table 1 to obtain engineered 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).

[0097] 3. Shake flask fermentation experiments of engineered strains JQ5-JQ10

[0098] The engineered strains JQ5 to JQ10 were inoculated into 40 mL of LB liquid medium, 100 μg / mL ampicillin and 50 μg / mL kanamycin were added to the culture medium, and cultured overnight at 37°C and 200 rpm to serve as seed liquid. The cell density of the seed liquid was determined and the cells were inoculated into 1 L triangular flasks containing 100 mL of fermentation medium according to a certain ratio to make the initial OD 600 The OD value was kept at about 0.05, and the fermentation was carried out in a shaking incubator at 37°C and 200 rpm.600 , when OD 600 When the value reaches 0.8-1.0, 2 g / L of arabinose is added to induce gene expression. The induced cells are cultured for another 12 hours and collected by centrifugation. The obtained cells are resuspended in fresh fermentation medium and fermented at 37°C with a shaking speed of 200 rpm for 24 hours. The fermentation medium formula is:

[0099] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0100] Take samples regularly to measure the cell density (OD 600 ) and the content of methylmalic acid and isoleucine in the fermentation broth. Figure 2 As shown in the shake flask fermentation results, the methylmalic acid production of JQ5 reached 2.39 g / L, but the accumulation of L-isoleucine was still not detected. Figure 2 As shown, 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 2 ), indicating that the GsleuCD, LbleuB, and AfleuB genes are suitable for constructing the methylmalate pathway. Although the methylmalate 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 2 However, JQ8 achieved an L-isoleucine yield of 0.10 g / L, an increase of approximately 66.7% compared to JQ7, indicating that LsleuDH is more efficient in L-isoleucine biosynthesis. The results demonstrated that the selected 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 significantly enhanced E. coli's ability to biosynthesize L-isoleucine via the methylmalate pathway.

[0101] Example 3 Increasing L-isoleucine production by optimizing branched-chain amino acids and methylmalate transport systems

[0102] By increasing the excretion of L-isoleucine from the chassis cells and thereby reducing the intracellular concentration of L-isoleucine, the strain's L-isoleucine biosynthesis capacity can be enhanced. Furthermore, since a large amount of methylmalate is still present in the fermentation broth of the JQ8 strain, improving its ability to utilize methylmalate is also key to enhancing L-isoleucine biosynthesis via the methylmalate pathway. Therefore, knocking out the branched-chain amino acid uptake protein genes brnQ, livJ, and livK, overexpressing the branched-chain amino acid excretion protein gene ygaZH and the regulatory protein gene lrp, and overexpressing the methylmalate uptake protein gene dcuD can improve L-isoleucine production.

[0103] 1. Construction of Escherichia coli ILE-1 strain

[0104] As shown in Table 1, using Escherichia coli BW25113 as the starting strain, the CRISPR-associated transposases gene editing method was used to knock out the branched-chain amino acid uptake protein genes brnQ, livJ, and livK. Specifically, electroporation competent cells were prepared; the pKMV-gRNA-brnQ / livJ / livK plasmid carrying gRNAs for the brnQ, livJ, and livK sites, as well as the pTetQCas-BsaI-tns and pRE57 plasmids, were sequentially electroporated into competent E. coli BW25113 wild-type strains. Positive transformants were selected by induction on LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. Positive transformants were screened and then cultured in LB at 42°C to remove the plasmids containing the three resistance genes. Finally, the E. coli ILE-1 strain with brnQ, livJ, and livK genes knocked out in the genome was obtained.

[0105] 2. Construction of Escherichia coli ILE-2 strain

[0106] As shown in Table 1, using ILE1 as the starting strain, the CRISPR-associated transposases gene editing method was used to integrate the branched-chain amino acid exoprotein gene ygaZH and the regulatory protein gene lrp at the livK locus, specifically:

[0107] The pKMV-gRNA-livK plasmid carrying the livK site gRNA, the pTetQCas-BsaI-tns plasmid, and the pRE57-ygaZH / lrp plasmid containing the ygaZH and lrp gene expression cassettes were transformed into the ILE1 strain. Strain screening was completed using antibiotic selection and dehydrated tetracycline induction to obtain the engineered strain ILE-2 with the ygaZH and lrp expression cassettes integrated into the genome (Table 1).

[0108] 3. Construction of E. coli ILE-3 and ILE-4 strains

[0109] (1) Construction of plasmid P9

[0110] Using the Escherichia coli genome as a template, P araBAD -dcuD fragment was purified by agarose gel electrophoresis and recovery, and the obtained fusion fragment was ligated with plasmid P6. It was transformed into E. coli Trans1 competent cells by heat shock and chemical transformation. After shaking and recovery at 37°C for 1 hour, it was coated on LB plates containing 100 μg / mL ampicillin and cultured for 16 hours. After colonies appeared, PCR and sequencing were used to screen and verify positive clones to obtain plasmid P9.

[0111] (2) Construction of Escherichia coli ILE-3 and ILE-4 strains

[0112] The P9 plasmid and the P5 plasmid constructed in Example 2 were electroporated into competent E. coli ILE-1 or ILE-2 strains. Positive transformants were screened on LB plates with kanamycin and ampicillin resistance and then cultured in LB liquid culture. PCR verification of the transformants was performed using primers to obtain E. coli ILE-3 and ILE-4 strains (Table 1).

[0113] The above-mentioned E. coli ILE-3 and ILE-4 were inoculated into 40 mL LB medium respectively and cultured at 37°C and 200 r / min overnight to serve as the first-stage seed solution. The seed solution was inoculated into 40 mL LB medium and cultured at 37°C. When the OD 600 When the OD value reached 0.6-0.8, 2 g / L L-arabinose was added to the culture medium to induce gene expression. Subsequently, the induced cells were cultured for another 12 hours and collected by centrifugation. The obtained cells were resuspended in fresh fermentation medium and the OD value was measured. 600 The working volume was 100 mL, the conditions were set at 37°C and 200 rpm for 24 hours. The composition of the fermentation medium was:

[0114] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0115] Take samples regularly to measure the cell density (OD 600 ) and the content of methylmalic acid and isoleucine in the fermentation broth. Figure 3As shown, L-isoleucine production in ILE-3 and ILE-4 reached 0.32 g / L and 0.41 g / L, respectively, a significant increase compared to JQ8. The results showed that knocking out the branched-chain amino acid uptake protein genes brnQ, livJ, and livK, and overexpressing the branched-chain amino acid exocytosis protein gene ygaZH, the regulatory protein gene lrp, and the methylmalate uptake protein gene dcuD significantly increased isoleucine synthesis.

[0116] Example 4 Construction of a plasmid-free engineered Escherichia coli strain producing L-isoleucine

[0117] Because plasmid-based expression of genes involved in the methylmalate pathway can significantly burden the host strain's cellular metabolism, the expression modules on plasmids P5 and P9 were integrated into the chromosome of the ILE-2 strain to construct a plasmid-free L-isoleucine-producing strain.

[0118] 1. Construction of Escherichia coli ILE-5 strain

[0119] (1) Construction of pRE57-ILE-5-1 plasmid

[0120] Using the P5 plasmid as a template, primers were designed to amplify the cimA3.7, GsleuCD, and AfleuB gene expression modules. These were then integrated into the linearized plasmid pRE57, which had been double-digested with the restriction endonucleases SpeI and PacI, by Gibson assembly. The recombinant system was transformed into competent Escherichia coli Trans1-T1 cells and selected using LB ampicillin resistance plates. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using the primer pair. The successfully constructed plasmid was named pRE57-ILE-5-1.

[0121] (2) Construction of pRE57-ILE-5-2 plasmid

[0122] Using the P9 plasmid as a template, primers were designed to amplify the ilvIH*, ilvC*, ilvD, LsleuDH, and dcuD gene expression modules. These were then integrated into the linearized plasmid pRE57, which had been double-digested with the restriction endonucleases SpeI and PacI, by Gibson assembly. The recombinant system was transformed into competent Escherichia coli Trans1-T1 cells and selected using LB ampicillin-resistant plates. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using the primer pair. The successfully constructed plasmid was named pRE57-ILE-5-2.

[0123] (3) Construction of Escherichia coli ILE-5 strain

[0124] The pKMV-gRNA-yjip plasmid, pTetQCas-BsaI-tns, and pRE57-ILE-5-1 plasmids were electroporated into E. coli ILE-2 competent cells, and positive transformants were screened by induction on LB plates containing kanamycin resistance, ampicillin resistance, streptomycin resistance, and dehydrated tetracycline. Transformants were PCR-verified using primers to screen positive transformants and sequenced. Plasmids containing the three resistances were removed by LB culture at 42°C. The pKMV-gRNA-gapC plasmid, pTetQCas-BsaI-tns, and pRE57-ILE-5-2 plasmids were then transformed into the newly obtained competent strain, and the same method was used to screen positive engineered strains, ultimately obtaining the E. coli ILE-5 strain (Table 1).

[0125] 2. Construction of Escherichia coli ILE-6 strain

[0126] Will have P araBAD -pETDuet-1-P for dcuD expression module araBAD The -dcuD plasmid was transformed into E. coli ILE-5 strain, and antibiotic selection was used to obtain E. coli ILE-6 strain (Table 1), which increased the ability to utilize methylmalate. The specific method is as follows:

[0127] (1) Construct a araBAD -pETDuet-1-P for dcuD expression module araBAD -dcuD plasmid

[0128] Using P9 plasmid as template, primers were designed to amplify P araBAD The -dcuD expression module was integrated into the plasmid pETDuet-1 by Gibson assembly. The recombinant system was transformed into E. coli Trans1-T1 competent cells and screened using LB ampicillin resistance plates. Positive clones were picked and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pETDuet-1-P araBAD -dcuD.

[0129] (2) Construction of Escherichia coli ILE-6 strain

[0130] pETDuet-1-P araBAD The -dcuD plasmid was electroporated into competent E. coli ILE-5 strain, and positive transformants were screened on LB ampicillin-resistant plates and cultured in LB liquid culture. Transformants were verified by PCR using primers to obtain E. coli ILE-6 strain.

[0131] 3. Construction of Escherichia coli ILE-7 strain

[0132] Will have P araBAD -pRSFDuet-1-P containing the GsleuCD expression module araBAD The -GsleuCD plasmid was transformed into E. coli ILE-5 strain, and antibiotic selection was used to obtain E. coli ILE-7 strain (Table 1), which increased the production capacity of L-isoleucine. The specific method is as follows:

[0133] (1) Construct a araBAD -pRSFDuet-1-P containing the GsleuCD expression module araBAD -GsleuCD plasmid

[0134] Using P5 plasmid as template, primers were designed to amplify P araBAD The GsleuCD expression module was integrated into the plasmid pRSFDuet-1 by Gibson assembly. The recombinant system was transformed into Escherichia coli Trans1-T1 competent cells and screened using LB kanamycin resistance plates. Positive clones were picked and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRSFDuet-1-P araBAD -GsleuCD.

[0135] (2) Construction of Escherichia coli ILE-7 strain

[0136] pRSFDuet-1-P araBAD The -GsleuCD plasmid was electroporated into competent E. coli ILE-5 strain, and positive transformants were screened on LB kanamycin-resistant plates and cultured in LB liquid culture. Transformants were verified by PCR using primers to obtain E. coli ILE-7 strain.

[0137] 2. Fermentation experiments with E. coli strains ILE-5, ILE-6, and ILE-7

[0138] The obtained E. coli ILE-5, ILE-6, and ILE-7 strains were fermented in a 3L fermentor. The production capacity of the engineered E. coli strains through the methylmalate pathway for L-isoleucine was improved by using fed-batch fermentation. The specific method is as follows:

[0139] First, E. coli ILE-5, ILE-6, and ILE-7 strains were inoculated into 40 mL of LB medium and cultured overnight at 37°C and 200 rpm to serve as the primary seed solution. The primary seed solution was inoculated into 1 L of LB medium and cultured at 37°C and 200 rpm for 24 h to serve as the secondary seed solution. The OD value of the secondary seed solution was determined. 600 , inoculated into 1L of culture medium (3L fermenter), so that the initial OD 600The formula of the culture medium is about 0.3.

[0140] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0141] During the fermentation process, the temperature was maintained at 37°C, the pH was maintained at 6.8-7.0 by automatically adding ammonia water, and the dissolved oxygen level was controlled at 10%-20% by coupling the motor speed. After the initial glucose was consumed, the glucose concentration was controlled to be lower than 10g / L by feeding with a peristaltic pump, wherein the feed was 700g / L glucose. Samples were taken every 3-6 hours, and the fermentation was carried out for a total of 48 hours. The concentration of L-isoleucine in the culture medium was separated and determined by a high performance liquid chromatography UV detector using the Shimadzu AJS-01 amino acid analysis method package. Figure 4 As shown, strain ILE-5 produced 5.2 g / L of L-isoleucine and 10.2 g / L of methylmalate (L-isoleucine / methylmalate: 0.51). Notably, ILE-5's methylmalate production was much higher than its L-isoleucine production, suggesting that methylmalate utilization efficiency in ILE-5 remains low. Strain ILE-6 produced 4.9 g / L of L-isoleucine and 8.9 g / L of methylmalate (L-isoleucine / methylmalate: 0.55), both of which were slightly lower than those of ILE-5. Meanwhile, strain ILE-7 achieved L-isoleucine and methylmalate production of 3.2 g / L and 38.9 g / L, respectively (L-isoleucine / methylmalate: 0.08). The results showed that the L-isoleucine production efficiency of the plasmid-free L-isoleucine strain ILE-5 was significantly improved compared with shake flask fermentation under the fermentation conditions of fed-batch method and controlled pH and dissolved oxygen level. However, a large amount of methylmalic acid was still present, and further overexpression of GsleuCD and dcuD did not improve the utilization rate of methylmalic acid.

[0142] Example 5 Further improving L-isoleucine production capacity by overexpressing cimA3.7 and AfleuB genes

[0143] The overexpression copy numbers of cimA3.7 and AfleuB genes on the genome were optimized to improve the production capacity of L-isoleucine.

[0144] 1. Construction of Escherichia coli ILE-8 strain

[0145] One copy of P was integrated into each of the pflB and ldhA sites of E. coli ILE-5 strain. araBAD -cimA3.7-AfleuB expression cassette to obtain ILE-8 strain (Table 1). The specific method is as follows:

[0146] (1) Construction of the pRE57-cimA3.7-AfleuB plasmid containing the cimA3.7 and AfleuB gene expression cassettes

[0147] Using P5 plasmid as template, primers were designed to amplify P araBAD -cimA3.7 and P araBAD The AfleuB expression module was constructed by ligating the fragments using overlap PCR and integrating them into the plasmid pRE57 using Gibson assembly. The recombinant system was transformed into competent E. coli Trans1-T1 cells and selected using LB plates with ampicillin resistance. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-cimA3.7-AfleuB.

[0148] (2) Construction of Escherichia coli ILE-8 strain

[0149] The pKMV-gRNA-pflB-ldhA plasmid, along with the pTetQCas-BsaI-tns and pRE57-cimA3.7-AfleuB plasmids, were sequentially electroporated into competent E. coli ILE-5 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the generation of E. coli ILE-8 strains.

[0150] 2. Construction of Escherichia coli ILE-9 strain

[0151] One copy of P was integrated into each of the adhE and ycjV sites of E. coli ILE-8 strain. araBAD -AfleuB expression cassette, obtain ILE-9 strain (Table 1). The specific method is as follows:

[0152] (1) Construction of pRE57-AfleuB plasmid with AfleuB gene expression cassette

[0153] Using P5 plasmid as template, primers were designed to amplify P araBAD The AfleuB expression module was integrated into the plasmid pRE57 by Gibson assembly. The recombinant system was transformed into competent E. coli Trans1-T1 cells and selected using LB plates with ampicillin resistance. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-AfleuB.

[0154] (2) Construction of Escherichia coli ILE-9 strain

[0155] The pKMV-gRNA-adhE-ycjV plasmid, followed by the pTetQCas-BsaI-tns and pRE57-AfleuB plasmids, were sequentially electroporated into competent E. coli ILE-8. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the generation of E. coli ILE-9.

[0156] 3. Construction of Escherichia coli ILE-10 strain

[0157] One copy of P was integrated into each of the lafU and ydeU sites of E. coli ILE-9 strain. araBAD -AfleuB expression cassette, obtain ILE-10 strain (Table 1). The specific method is as follows:

[0158] The pKMV-gRNA-lafU-ydeU plasmid, along with the pTetQCas-BsaI-tns and pRE57-AfleuB plasmids, were sequentially electroporated into competent E. coli ILE-9 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in E. coli ILE-10.

[0159] 4. Construction of Escherichia coli ILE-11 strain

[0160] One copy of P was integrated into each of the yciQ and yjiV sites of E. coli ILE-9 strain. araBAD -cimA3.7 expression cassette to obtain the ILE-11 strain (Table 1). The specific method is as follows:

[0161] (1) Construction of pRE57-cimA3.7 plasmid with cimA3.7 gene expression cassette

[0162] Using P5 plasmid as template, primers were designed to amplify P araBAD The cimA3.7 expression module was integrated into the pRE57 plasmid by Gibson assembly. The recombinant system was transformed into competent Escherichia coli Trans1-T1 cells and selected using LB plates with ampicillin resistance. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-cimA3.7.

[0163] (2) Construction of Escherichia coli ILE-11 strain

[0164] The pKMV-gRNA-yciQ-yjiV plasmid, along with the pTetQCas-BsaI-tns and pRE57-cimA3.7 plasmids, were sequentially electroporated into competent E. coli ILE-9 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the ILE-11 strain of E. coli.

[0165] 2. Fermentation experiments with E. coli strains ILE-8, ILE-9, ILE-10, and ILE-11

[0166] The constructed Escherichia coli ILE-8, ILE-9, ILE-10, and ILE-11 strains were fermented in a 3 L fermenter as follows:

[0167] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0168] The fermentation method is the same as that described in Example 4.

[0169] The fermentation product detection method is the same as the fermentation product detection method described in Example 4.

[0170] like Figure 4 As shown, the results showed that adding two copies of P araBAD After the -cimA3.7-AfleuB expression cassette was added, the L-isoleucine production of the obtained ILE-8 strain reached 20.6 g / L, which was about 3 times higher than that of ILE-5. araBAD After the expression of the AfleuB cassette, the L-isoleucine production of the obtained ILE-9 and ILE-10 strains reached 31.1 g / L and 24.9 g / L, respectively, indicating that a total of five copies of AfleuB can meet the demand, while excessive amounts will significantly reduce the L-isoleucine production. araBAD After the cimA3.7 expression cassette was incorporated into the strain, the resulting ILE-11 strain significantly increased methylmalate production by 31.7%, while L-isoleucine production only increased slightly by 6.1%. This suggests that three copies of cimA3.7 are sufficient for optimal L-isoleucine production. Therefore, L-isoleucine production is optimal when cimA3.7 and AfleuB are integrated into the genome at 3 and 5 times the number of GsleuCD copies, respectively.

[0171] Example 6 Improving L-isoleucine production capacity by introducing a non-oxidative glycolysis pathway

[0172] An adequate supply of acetyl-CoA is crucial for the biosynthesis of methylmalate. However, acetyl-CoA in Escherichia coli is primarily derived from the decarboxylation of pyruvate, a process that results in carbon loss and reduces conversion efficiency. Compared with the classical glycolytic pathway, the non-oxidative glycolytic pathway can efficiently produce acetyl-CoA from glucose using fewer synthetic steps and without releasing CO₂, thus facilitating the production of L-isoleucine. Therefore, a non-oxidative glycolytic pathway was introduced into the ILE-9 strain by overexpressing the bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis and the E. coli native phosphotransacetylase gene pta.

[0173] 1. Construction of Escherichia coli ILE-12 strain

[0174] The ackA gene was knocked out in the E. coli ILE-9 strain to reduce the synthesis of acetyl-CoA into acetic acid, and the E. coli ILE-12 strain was obtained (Table 1). The specific method is as follows:

[0175] The pKMV-gRNA-ackA plasmid carrying the gRNA for the ackA site, along with the pTetQCas-BsaI-tns and pRE57 plasmids, was electroporated into competent E. coli ILE-9. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing at 42°C in LB, ultimately generating an ILE-12 strain with the ackA gene knockout.

[0176] 2. Construction of Escherichia coli ILE-13 strain

[0177] Overexpression of P at the ackA site in Escherichia coli ILE-12 strain araBAD -Bafxpk-pta module, obtain ILE-13 strain (Table 1). The specific method is as follows:

[0178] (1) Construction of pRE57-Bafxpk-pta plasmid with Bafxpk and pta gene expression cassettes

[0179] The bifunctional phosphoketolase gene Bafxpk from Bifidobacterium adolescentis was artificially synthesized after codon optimization. Using the synthetic fragment of Bafxpk and the Escherichia coli genome as templates, amplification primers were designed to amplify P araBAD -Bafxpk, ParaBAD The pta expression module was constructed by ligating the fragments using overlap PCR and integrating them into the pRE57 plasmid using Gibson assembly. The recombinant system was transformed into competent E. coli Trans1-T1 cells and selected using LB plates for ampicillin resistance. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-Bafxpk-pta.

[0180] (2) Construction of Escherichia coli ILE-13 strain

[0181] The pKMV-gRNA-ackA plasmid, followed by the pTetQCas-BsaI-tns and pRE57-Bafxpk-pta plasmids, were sequentially electroporated into competent E. coli ILE-12 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the generation of E. coli ILE-13 strains.

[0182] 3. Construction of Escherichia coli ILE-14 strain

[0183] In E. coli ILE-13 strain, a copy of P was integrated into araBAD -Bafxpk-pta module, obtain ILE-14 strain (Table 1). The specific method is as follows:

[0184] The pKMV-gRNA-poxB plasmid, followed by the pTetQCas-BsaI-tns and pRE57-Bafxpk-pta plasmids, were sequentially electroporated into competent E. coli ILE-13 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the generation of E. coli ILE-14 strains.

[0185] 4. Construction of Escherichia coli ILE-15 strain

[0186] The eutD gene in E. coli can perform a similar function to the pta gene, so the P was overexpressed at the ackA site in the E. coli ILE-12 strain. araBAD -Bafxpk-eutD module, the ILE-15 strain was obtained (Table 1), and the effects of overexpression of pta (ILE13 strain) and eutD (ILE15 strain) on L-isoleucine production were compared. The specific method is as follows:

[0187] (1) Construction of pRE57-Bafxpk-eutD plasmid with Bafxpk and eutD gene expression cassettes

[0188] Using the Bafxpk synthetic fragment and the E. coli genome as templates, primers were designed to amplify the P araBAD -Bafxpk, P araBAD The eutD expression module was constructed by ligating the fragments using overlap PCR and integrating them into the plasmid pRE57 using Gibson assembly. The recombinant system was transformed into competent E. coli Trans1-T1 cells and selected using LB plates with ampicillin resistance. Positive colonies were selected and cultured in LB liquid culture. The plasmid was extracted and verified by PCR using primers. The successfully constructed plasmid was named pRE57-Bafxpk-eutD.

[0189] (2) Construction of Escherichia coli ILE-15 strain

[0190] The pKMV-gRNA-ackA plasmid, followed by the pTetQCas-BsaI-tns and pRE57-Bafxpk-eutD plasmids, were sequentially electroporated into competent E. coli ILE-12 cells. Positive transformants were screened using LB plates containing resistance markers for kanamycin, ampicillin, and streptomycin, as well as anhydrotetracycline. Transformants were verified by PCR using primers and sequenced. The plasmids containing the three resistance markers were removed by culturing in LB at 42°C, ultimately resulting in the generation of E. coli ILE-15 strains.

[0191] 5. Fermentation experiments with Escherichia coli ILE-12 / 13 / 14 / 15 strains

[0192] The constructed ILE-12, ILE-13, ILE-14, and ILE-15 strains were fermented in a 3 L fermenter as follows:

[0193] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0194] The fermentation method is the same as that described in Example 4.

[0195] The fermentation product detection method is the same as the fermentation product detection method described in Example 4.

[0196] like Figure 5As shown, L-isoleucine production in ILE-12 was 43.9 g / L, and this was further increased to 50.7 g / L in ILE-13, demonstrating that knocking out ackA and introducing a non-oxidative glycolysis pathway significantly increased L-isoleucine production. However, L-isoleucine production in ILE-14 dropped sharply to 24.2 g / L, indicating that overexpression of Bafxpk and pta is detrimental to L-isoleucine production. L-isoleucine production in ILE-15 decreased by 66.3% compared to ILE-13, suggesting that the eutD gene is not a suitable option for increasing L-isoleucine production.

[0197] Example 7 Evaluation of L-isoleucine production level of ILE-13 strain in a 10 L fermenter

[0198] Larger fermenters allow for more precise control of fermentation conditions and are therefore more conducive to demonstrating production levels. Therefore, the constructed ILE-13 strain was fermented in a 10L fermenter using the following method:

[0199] The fermentation medium formula is the same as the fermentation medium formula described in Example 1.

[0200] The fermentation method is the same as that described in Example 4.

[0201] The fermentation product detection method is the same as the fermentation product detection method described in Example 4.

[0202] like Figure 6 As shown, the growth of ILE-13 reached a plateau at approximately 20 hours, with a maximum OD 600 The value is 86.7( Figure 6 Finally, the ILE-13 strain produced 56.6 g / L of L-isoleucine, with a conversion rate and production intensity of 0.21 g / g glucose and 1.66 g / L / h, respectively, demonstrating the great potential of the methylmalate pathway in the industrial production of L-isoleucine.

[0203] The above embodiments are only used to understand the technical solutions of the present application and do not limit the scope of protection of the present application.

Claims

1. An improved engineered Escherichia coli strain for producing L-isoleucine via the methylmalate pathway, characterized in that: The engineered strain is a mutant Escherichia coli engineered strain having the following characteristics: Knockout of branched-chain amino acid uptake protein genes brnQ, livJ, and livK; Overexpression of branched-chain amino acid exoprotein genes ygaZ, ygaH and regulatory protein gene lrp on the genome; Overexpression of the methylmalate synthase hyperactive mutant gene cimA3.7, the isopropylmalate isomerase gene GsleuCD, the 3-isopropylmalate dehydrogenase gene AfleuB, and the leucine dehydrogenase gene LsleuDH on the genome; Overexpression of the methylmalate uptake protein gene dcuD on the genome; Overexpression of the bifunctional phosphoketolase gene Bafxpk and the Escherichia coli phosphotransacetylase gene pta on the genome. The nucleotide sequence of the methylmalate synthase high-activity mutant gene cimA3.7 is shown in SEQ ID NO: 1; the isopropylmalate isomerase gene GsleuCD comprises two subunit genes, wherein the nucleotide sequence of the isopropylmalate isomerase large subunit gene GsleuC is shown in SEQ ID NO: 2, and the nucleotide sequence of the isopropylmalate isomerase small subunit gene GsleuD is shown in SEQ ID NO: 3; the nucleotide sequence of the 3-isopropylmalate dehydrogenase gene AfleuB is shown in SEQ ID NO: 4; the nucleotide sequence of the leucine dehydrogenase subunit gene Lsleudh is shown in SEQ ID NO: 5; the nucleotide sequence of the methylmalate uptake protein gene dcuD is shown in SEQ ID NO: 6; the nucleotide sequence of the branched-chain amino acid uptake protein gene brnQ is shown in SEQ ID NO: 7; the nucleotide sequence of the branched-chain amino acid uptake protein gene livJ is shown in SEQ ID NO: 8; and the nucleotide sequence of the branched-chain amino acid uptake protein gene livK is shown in SEQ ID NO: NO: 9; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO: 10; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown in SEQ ID NO: 11; the nucleotide sequence of the regulatory protein gene lrp is shown in SEQ ID NO: 12; the nucleotide sequence of the bifunctional phosphoketolase gene Bafxpk is shown in SEQ ID NO: 13; and the nucleotide sequence of the phosphotransacetylase gene pta is shown in SEQ ID NO:

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2. The improved Escherichia coli engineered strain for producing L-isoleucine via the methylmalate pathway according to claim 1, characterized in that: The chassis strain of the mutant Escherichia coli engineering strain is Escherichia coli BW25113.

3. The improved Escherichia coli engineered strain for producing L-isoleucine via the methylmalate pathway according to claim 1, characterized in that: in, The copy number of the methylmalate synthase high-activity mutant gene cimA3.7 integrated in the genome is 3 times that of the isopropylmalate isomerase gene GsleuCD, and the copy number of the 3-isopropylmalate dehydrogenase gene AfleuB integrated in the genome is 5 times that of the isopropylmalate isomerase gene GsleuCD.

4. The improved Escherichia coli engineered strain for producing L-isoleucine via the methylmalate pathway according to claim 1, characterized in that: Furthermore, the strain also expresses genes encoding enzymes related to the methylmalate pathway, and the genes encoding enzymes related to the methylmalate pathway are one or more genes selected from the following genes: Anti-feedback inhibition mutant acetohydroxyacid synthase gene ilvIH*, NADH-preferring acetohydroxyacid reductoisomerase gene ilvC*, and dihydroxyacid dehydratase gene ilvD.

5. The improved Escherichia coli engineered strain for producing L-isoleucine via the methylmalate pathway according to claim 4, characterized in that: The anti-feedback inhibition mutant acetohydroxyacid synthase gene includes an ilvI subunit and an ilvH* subunit, wherein the nucleotide sequence of the ilvI subunit of the acetohydroxyacid synthase gene is shown in SEQ ID NO: 15, and the nucleotide sequence of the ilvH* subunit of the acetohydroxyacid synthase gene is shown in SEQ ID NO: 16; the nucleotide sequence of the ilvC* acetohydroxyacid reductoisomerase gene is shown in SEQ ID NO: 17; and the nucleotide sequence of the dihydroxyacid dehydratase gene ilvD is shown in SEQ ID NO:

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6. The improved engineered Escherichia coli strain for producing L-isoleucine via the methylmalate pathway according to any one of claims 1 to 5, characterized in that: The gene overexpressed in the engineered strain is integrated into the chromosome of the strain.

7. An improved method for constructing an engineered Escherichia coli strain that produces L-isoleucine via the methylmalate pathway, characterized in that: The method comprises the following steps: Knockout of branched-chain amino acid uptake protein genes brnQ, livJ, and livK; Overexpression of branched-chain amino acid exoprotein genes ygaZ, ygaH and regulatory protein gene lrp on the genome; Overexpression of the methylmalate synthase hyperactive mutant gene cimA3.7, the isopropylmalate isomerase gene GsleuCD, the 3-isopropylmalate dehydrogenase gene AfleuB, and the leucine dehydrogenase gene LsleuDH on the genome; Overexpression of the methylmalate uptake protein gene dcuD on the genome; Overexpression of the bifunctional phosphoketolase gene Bafxpk and the Escherichia coli phosphotransacetylase gene pta on the genome. The nucleotide sequence of the methylmalate synthase high-activity mutant gene cimA3.7 is shown in SEQ ID NO: 1; the isopropylmalate isomerase gene GsleuCD comprises two subunit genes, wherein the nucleotide sequence of the isopropylmalate isomerase large subunit gene GsleuC is shown in SEQ ID NO: 2, and the nucleotide sequence of the isopropylmalate isomerase small subunit gene GsleuD is shown in SEQ ID NO: 3; the nucleotide sequence of the 3-isopropylmalate dehydrogenase gene AfleuB is shown in SEQ ID NO: 4; the nucleotide sequence of the leucine dehydrogenase subunit gene Lsleudh is shown in SEQ ID NO: 5; the nucleotide sequence of the methylmalate uptake protein gene dcuD is shown in SEQ ID NO: 6; the nucleotide sequence of the branched-chain amino acid uptake protein gene brnQ is shown in SEQ ID NO: 7; the nucleotide sequence of the branched-chain amino acid uptake protein gene livJ is shown in SEQ ID NO: 8; and the nucleotide sequence of the branched-chain amino acid uptake protein gene livK is shown in SEQ ID NO: NO: 9; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaZ is shown in SEQ ID NO: 10; the nucleotide sequence of the branched-chain amino acid exoprotein gene ygaH is shown in SEQ ID NO: 11; the nucleotide sequence of the regulatory protein gene lrp is shown in SEQ ID NO: 12; the nucleotide sequence of the bifunctional phosphoketolase gene Bafxpk is shown in SEQ ID NO: 13; and the nucleotide sequence of the phosphotransacetylase gene pta is shown in SEQ ID NO:

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8. The improved method for constructing an engineered Escherichia coli strain capable of producing L-isoleucine via the methylmalate pathway according to claim 7, characterized in that: The method further comprises the step of expressing a gene encoding an enzyme related to the methylmalate pathway in the E. coli chassis strain, wherein the gene encoding the enzyme related to the methylmalate pathway is selected from one or more of the following genes: Anti-feedback inhibition mutant acetohydroxyacid synthase gene, NADH-preferring acetohydroxyacid reductoisomerase gene, and dihydroxyacid dehydratase gene.

9. The improved method for constructing an engineered Escherichia coli strain capable of producing L-isoleucine via the methylmalate pathway according to claim 8, characterized in that: The anti-feedback inhibition mutant acetohydroxyacid synthase gene includes an ilvI subunit and an ilvH* subunit, wherein the nucleotide sequence of the ilvI subunit of the acetohydroxyacid synthase gene is shown in SEQ ID NO: 15, and the nucleotide sequence of the ilvH* subunit of the acetohydroxyacid synthase gene is shown in SEQ ID NO: 16; the nucleotide sequence of the ilvC* acetohydroxyacid reductoisomerase gene is shown in SEQ ID NO: 17; and the nucleotide sequence of the dihydroxyacid dehydratase gene ilvD is shown in SEQ ID NO:

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10. A method for preparing L-isoleucine by fermentation, characterized in that: The method comprises the step of fermenting the improved engineered strain for synthesizing L-isoleucine based on the methylmalate pathway according to claim 1 in a fermenter.

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