L-isoleucine producing strain, construction method and application thereof

Abstract

This invention provides an L-isoleucine-producing strain, its construction method, and its application. The strain was obtained by modifying the chassis strain *Escherichia coli* XX12 using metabolic engineering methods, and lacks... ldhA The genes adhE and pflB were upregulated, aspC, pykF, pntAB, ppK, ppc, and ilvA were also upregulated. fbr ,ilvIH fbr The transcriptional level of the ygaZH gene was reduced, the transcriptional level of the leuA gene was downregulated, and the bcd gene from B. subtilis 168 and the ppnK and cysK genes from Corynebacterium glutamicum ATCC 13032 were expressed heterologously. No resistance substances were added, and it had good L-isoleucine synthesis ability. It used glucose as a carbon source, had a short fermentation cycle, and had good economic benefits and industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and fermentation engineering technology, and in particular to an L-isoleucine producing strain, its construction method, and its application. Background Technology

[0002] Isoleucine is an essential branched-chain amino acid with wide applications in medicine, food, feed, and health products. With the increasing demand for healthy nutrition, the market demand for isoleucine continues to expand. Currently, isoleucine is mainly produced industrially through microbial fermentation. Compared to traditional chemical synthesis routes, this method has advantages such as being environmentally friendly, lower cost, and having a wide range of raw material sources, making it the mainstream production method.

[0003] During the microbial synthesis of isoleucine, *Escherichia coli* (E. coli) Escherichia coli Natural bacterial strains are widely used as engineered bacterial substrates due to their rapid growth, clear metabolic pathways, and ease of genetic manipulation. However, natural strains have limited ability to synthesize isoleucine due to various factors such as feedback inhibition, insufficient precursor substances, and competition for metabolic pathways. Therefore, developing efficient isoleucine-producing strains is an important research direction at present.

[0004] In recent years, advancements in synthetic biology and metabolic engineering have provided new insights into modifying microbial metabolic networks and enhancing the synthesis of target products. Researchers typically improve isoleucine synthesis efficiency by knocking out competing pathways, overexpressing key enzyme genes, and introducing feedback inhibition resistance mutations. A strain of L-isoleucine-producing bacteria with high acid production efficiency and good genetic stability has been constructed in CN117925666B. However, due to limitations in the supply of precursors and cofactors, there is still room for further improvement in yield and sugar-acid conversion rate. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an L-isoleucine producing strain.

[0006] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned L-isoleucine producing strain.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned L-isoleucine producing strain.

[0008] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0009] An L-isoleucine-producing strain was obtained by modifying the chassis strain *Escherichia coli* XX12 (CN117925666B) using metabolic engineering methods, and it was found to contain... ldhAThe genes adhE and pflB were upregulated, aspC, pykF, pntAB, ppK, ppc, and ilvA were also upregulated. fbr ,ilvIH fbr The transcriptional levels of the ygaZH gene were reduced, the transcriptional level of the leuA gene was downregulated, and the bcd gene from B. subtilis 168 and the ppnK and cysK genes from Corynebacterium glutamicum ATCC 13032 were expressed heterologously.

[0010] Preferably, the above-mentioned L-isoleucine-producing strain uses P at the ycjV and fucK gene loci on the Escherichia coli XX12 genome. trc Promoter overexpression of aspartate transaminase aspC gene (NCBI ID: 945553); P at yncK pseudogene site trc Promoter overexpression of the pyruvate kinase pykF gene (NCBI ID: 946179); P at the yjiK pseudogene site BBa_J23104 Promoter overexpression of the phosphoenolpyruvate carboxylase ppc gene (NCBI ID: 948457); using P BBa_J23116 Promoter weakening of the leuA gene for 2-isopropylmalate synthase (NCBI ID: 947465); knockout of D-lactate dehydrogenase. ldhA Gene knockout (NCBI ID: 946315); knockout of aldehyde dehydrogenase / alcohol dehydrogenase adhE gene (NCBI ID: 945837); knockout of formate C-acetyltransferase pflB gene (NCBI ID: 945514); use P at rph, fhiA pseudogene loci. trc Promoter overexpression of threonine dehydrogenase ilvA fbr Gene (NCBI ID: 948287); P was used at ylbE, yncI, and molR pseudogene loci. trc Promoter overexpression of acetolactate synthase ilvIH fbr Gene (NCBI ID: 947267); P was used at the nmpC, gapC, and yfaS pseudogene loci. trc Promoter overexpression was performed on the leucine dehydrogenase bcd gene derived from B. subtilis 168 (NCBI ID: 938670); P was used at the insB pseudogene site. trcThe promoter overexpressed the pyridine nucleotide transhydrogenase pntAB gene (pntA NCBI ID: 946628, pntB NCBI ID: 946144); and the NAD derived from Corynebacterium glutamicum ATCC 13032 was overexpressed at the yjgX pseudogene site. + The kinase ppnK gene (GenBank: HE802067.1); P was used at the ydeU pseudogene locus. trc Promoter overexpression of the polyphosphate kinase ppk gene (NCBI ID: 946179); P was used at the ydbA pseudogene site. trc Promoter overexpression was performed on the cysK gene (NCBI ID: 1020508) of *Corynebacterium glutamicum* ATCC 13032; P was used at the ychG and yjiP pseudogene sites. trc The promoter overexpressed the L-isoleucine ectotransporter ygaZH gene (ygaZ NCBI ID: 945093, ygaH NCBI ID: 945111).

[0011] Preferably, in the above-mentioned L-isoleucine-producing strain, the nucleotide sequence of the aspC gene is shown in SEQ ID NO. 4; the nucleotide sequence of the pykF gene is shown in SEQ ID NO. 5; the nucleotide sequence of the ppc gene is shown in SEQ ID NO. 6; the nucleotide sequence of the leuA gene is shown in SEQ ID NO. 7; the nucleotide sequence of the ldhA gene is shown in SEQ ID NO. 8; the nucleotide sequence of the adhE gene is shown in SEQ ID NO. 9; the nucleotide sequence of the pflB gene is shown in SEQ ID NO. 10; and the ilvA gene... fbr The nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing; ilvIH fbr The nucleotide sequences of the gene clusters are shown in SEQ ID NO. 12; the nucleotide sequence of the bcd gene is shown in SEQ ID NO. 13; the nucleotide sequence of the pntAB gene cluster is shown in SEQ ID NO. 14; the nucleotide sequence of the ppnK gene is shown in SEQ ID NO. 15; the nucleotide sequence of the ppk gene is shown in SEQ ID NO. 16; the nucleotide sequence of the cysK gene is shown in SEQ ID NO. 17; and the nucleotide sequence of the ygaZH gene is shown in SEQ ID NO. 18.

[0012] Preferably, the above-mentioned L-isoleucine-producing strain, wherein P trc The nucleotide sequence of the promoter is shown in SEQ ID NO. 1; the P BBa_J23104 The nucleotide sequence of the promoter is shown in SEQ ID NO.2 of the sequence listing; the P BBa_J23116 The nucleotide sequence of the promoter is shown in the sequence listing SEQ ID NO.3.

[0013] The construction method of the above-mentioned L-isoleucine-producing strain is based on Escherichia coli XX12 (CN117925666B) and involves directional modification, divided into the following 5 modules:

[0014] (1) Module to increase the accumulation of precursor ketobutyrate: Increase the transcription level of the aspartate transaminase aspC gene, causing carbon metabolism to flow into aspartate, thereby increasing the accumulation of precursor ketobutyrate; increase the threonine dehydrogenase ilvA fbr At the gene transcription level, ketobutyrate accumulates;

[0015] (2) Module to reduce pyruvate consumption: Knock out D-lactate dehydrogenase ldhA Genes that prevent the reduction of pyruvate to lactate enhance carbon efficiency; knocking out the acetaldehyde dehydrogenase / alcohol dehydrogenase adhE gene blocks the ethanol pathway and prevents NADH waste; knocking out the formate C-acetyltransferase pflB gene reduces the metabolism of pyruvate to formate.

[0016] (3) NADPH recycling module: Increases the transcription level of the pyridine nucleotide transhydrogenase pntAB gene, thereby increasing the supply of NADPH; increases the introduction of NADPH from Corynebacterium glutamicum ATCC 13032. + The kinase ppnK gene promotes the conversion of NADH to NADPH; increasing the transcription level of the polyphosphokinase ppk gene increases ATP supply and promotes NADPH conversion. + Kinase expression;

[0017] (4) Balance the precursors ketobutyrate and pyruvate modules: increase the transcription level of the pyruvate kinase pykF gene to avoid excessive carbon metabolism flowing into ketobutyrate, which would lead to an imbalance between the two precursors; increase the transcription level of the phosphoenolpyruvate carboxylase ppc gene to reduce the waste of carbon metabolism and ensure sufficient accumulation of ketobutyrate precursors.

[0018] (5) L-Isoleucine production module: weakens the transcription level of the 2-isopropylmalate synthase leuA gene, reduces the negative feedback of leucine to isoleucine metabolism; increases acetyllactate synthase ilvIH fbrThe transcriptional level of genes is improved to fully utilize pyruvate and avoid the accumulation of the byproduct valine; the transcriptional level of the leucine dehydrogenase bcd gene introduced from B. subtilis 168 is increased to enhance isoleucine production; the transcriptional level of the cysteine ​​synthase cysK gene introduced from Corynebacterium glutamicum ATCC13032 is increased to enhance the transcriptional level of key enzymes in the L-isoleucine synthesis pathway; and the transcriptional level of the L-isoleucine ectotransporter ygaZH gene is increased to enhance the transport of isoleucine from intracellular to extracellular space and avoid excessive intracellular concentration that inhibits metabolism.

[0019] Preferably, the method for constructing the above-mentioned L-isoleucine-producing strain includes the following specific steps:

[0020] (1) Using Escherichia coli XX12 as the chassis strain, P was used at the ycjV gene locus. trc The aspartate transaminase aspC gene was overexpressed in the promoter to obtain strain XX13;

[0021] (2) Using strain XX13 as the chassis strain, P was used at the fucK gene locus. trc The aspartate transaminase aspC gene was overexpressed via the promoter to obtain strain XX14.

[0022] (3) Using strain XX14 as the chassis strain, P was used at the yncK pseudogene locus. trc The pyruvate kinase pykF gene was overexpressed in the promoter to obtain strain XX15;

[0023] (4) Using strain XX15 as the chassis strain, P was used at the yjiK pseudogene locus. BBa_J23104 The promoter overexpression of the phosphoenolpyruvate carboxylase ppc gene yielded strain XX16.

[0024] (5) Using strain XX16 as the chassis strain, P was used. BBa_J23116 The promoter weakened the leuA gene of 2-isopropylmalate synthase, resulting in strain XX17;

[0025] (6) Using strain XX17 as the chassis strain, the D-lactate dehydrogenase ldhA gene was knocked out to obtain strain XX18;

[0026] (7) Using strain XX18 as the chassis strain, the acetaldehyde dehydrogenase / alcohol dehydrogenase adhE gene was knocked out to obtain strain XX19;

[0027] (8) Using strain XX19 as the chassis strain, the formate C-acetyltransferase pflB gene was knocked out to obtain strain XX20;

[0028] (9) Using strain XX20 as the chassis strain, P was used at the rph pseudogene locus. trc Promoter overexpression of threonine dehydrogenase ilvA fbr Genes were used to obtain strain XX21;

[0029] (10) Using strain XX21 as the chassis strain, P was used at the fhiA pseudogene locus. trc Promoter overexpression of threonine dehydrogenase ilvA fbr Genes were used to obtain strain XX22;

[0030] (11) Using strain XX22 as the chassis strain, P was used at the ylbE pseudogene locus. trc Promoter overexpression of acetolactate synthase ilvIH fbr Genes were used to obtain strain XX23;

[0031] (12) Using strain XX23 as the chassis strain, P was used at the yncI pseudogene locus. trc Promoter overexpression of acetolactate synthase ilvIH fbr Genes were used to obtain strain XX24;

[0032] (13) Using strain XX24 as the chassis strain, P was used at the molR pseudogene locus. trc Promoter overexpression of acetolactate synthase ilvIH fbr Genes were used to obtain strain XX25;

[0033] (14) Using strain XX25 at the nmpC pseudogene site, P trc Promoter overexpression was performed on the leucine dehydrogenase bcd gene derived from B. subtilis 168, resulting in strain XX26.

[0034] (15) Using strain XX26 at the gapC pseudogene site, P trc Promoter overexpression was performed on the leucine dehydrogenase bcd gene derived from B. subtilis 168, resulting in strain XX27;

[0035] (16) Using strain XX27 at the yfaS pseudogene locus with P trc Promoter overexpression was performed on the leucine dehydrogenase bcd gene derived from B. subtilis 168, resulting in strain XX28;

[0036] (17) Using strain XX28 at the insB pseudogene site, P trc Overexpression of the pyridine nucleotide transhydrogenase pntAB gene via the promoter yielded strain XX29;

[0037] (18) Overexpression of NAD from Corynebacterium glutamicum ATCC 13032 at the yjgX pseudogene locus by strain XX29 + Gene on the kinase ppnK was used to obtain strain XX30;

[0038] (19) Using strain XX30 at the ydeU pseudogene site, P trc By overexpressing the polyphosphokinase ppk gene via the promoter, strain XX31 was obtained.

[0039] (20) Using strain XX31 at the ydbA pseudogene site, P trc Promoter overexpression of the cysK gene, derived from Corynebacterium glutamicum ATCC 13032, yielded strain XX32;

[0040] (21) Using strain XX31 at the ychG pseudogene locus, P trc strain XX33 was obtained by overexpressing the L-isoleucine ectotransporter ygaZH gene via promoter overexpression.

[0041] (22) Using strain XX31 at the yjiP pseudogene locus, P trc The promoter overexpression of the L-isoleucine ectotransporter ygaZH gene yielded strain XX34.

[0042] The above ilvA fbr The gene underwent point mutations: base position 1339 changed from C to T, base position 1341 changed from G to T, base position 1351 changed from C to G, and base position 1352 changed from T to C. This resulted in amino acid residue 447 changing from leucine to phenylalanine, and amino acid residue 451 changing from leucine to alanine. (The above ilvIH) fbr The gene underwent a point mutation, where the 41st base changed from G to A and the 50th base changed from C to T, resulting in the 14th amino acid residue changing from glycine to aspartic acid and the 17th amino acid residue changing from serine to phenylalanine.

[0043] Application of the above-mentioned L-isoleucine-producing strains in the fermentation production of L-isoleucine.

[0044] Preferably, in the above application, L-isoleucine is synthesized using glucose as a substrate in a mechanically stirred fermenter, and the specific steps are as follows:

[0045] (1) Activation of strain: The L-isoleucine producing strain was streaked onto the activation slant, incubated at 37℃ for 12-15h, and passaged once;

[0046] (2) Seed culture: The culture temperature is 34-36℃, the initial stirring speed is 200 rpm, and the pH of the culture is maintained at 6.7±0.2 by automatically adding 25% ammonia solution. The dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring speed or aeration rate, depending on the OD. 600nm A score of 20 indicates the maturity of the seed culture medium.

[0047] (3) Fermentation culture: The inoculum amount is 30%, the culture temperature is 36℃, the culture pH is maintained at 6.7±0.2 by automatically adding 25% ammonia solution, the dissolved oxygen value of the culture medium is maintained at 60% by adjusting the stirring speed or ventilation, and the glucose concentration in the tank is controlled at ≤0.5g / L by adding 80% glucose solution.

[0048] Preferably, in the above application, the slant culture medium used in step (1) is: glucose 1g / L, peptone 5g / L, potassium dihydrogen phosphate 0.5g / L, yeast extract 5g / L, sodium chloride 5g / L, magnesium sulfate heptahydrate 0.2g / L, agar powder 25g / L, the remainder being water, pH 7.0.

[0049] Preferably, in the above application, the seed culture medium used in step (2) is: glucose 20 g / L, yeast powder 3 g / L, peptone 3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 3 g / L, MgSO4·7H2O 2 g / L, citric acid 2 g / L, glutamic acid 3 g / L, methionine 0.5 g / L, lysine 1 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, MgSO4·7H2O 10 mg / L, and the remainder is water.

[0050] Preferably, in the above application, the fermentation medium used in step (3) is: yeast powder 4 g / L, peptone 3 g / L, (NH4)2SO4 4 g / L, K2HPO4·3H2O 4 g / L, MgSO4·7H2O 2 g / L, glutamic acid 4 g / L, methionine 0.3 g / L, citric acid 3 g / L, lysine 2 g / L, ammonium sulfate 4 g / L, MnSO4·7H2O 10 mg / L, FeSO4·7H2O 30 mg / L, corn steep liquor 20 ml / L, and the remainder is water.

[0051] All of the above-mentioned culture media can be prepared using standard methods.

[0052] Beneficial effects:

[0053] The aforementioned L-isoleucine-producing strains are plasmid-free, genetically stable, and exhibit high acid production efficiency, enabling efficient de novo synthesis of L-isoleucine. This is achieved by: increasing the transcriptional level of the pyruvate kinase (pykF) gene to prevent excessive carbon metabolism from flowing into ketobutyrate, thus avoiding an imbalance between the two precursors; increasing the transcriptional level of the phosphoenolpyruvate carboxylase (ppc) gene to reduce carbon metabolic waste and ensure sufficient ketobutyrate precursor accumulation; weakening the transcriptional level of the 2-isopropylmalate synthase (leuA) gene to reduce the negative feedback of leucine on isoleucine metabolism; and increasing the transcriptional level of acetyllactate synthase (ilvIH). fbr The transcription level of the gene is improved to fully utilize pyruvate and avoid the accumulation of the byproduct valine. The transcription level of the leucine dehydrogenase bcd gene introduced from B. subtilis 168 is increased to reduce NADPH consumption and enhance isoleucine production. The transcription level of the cysteine ​​synthase cysK gene introduced from Corynebacterium glutamicum ATCC 13032 is also increased to improve the transcription levels of key enzymes in the L-isoleucine synthesis pathway, effectively increasing isoleucine production intensity. Furthermore, the transcription level of the L-isoleucine ectotransporter ygaZH gene is increased to enhance the transport of isoleucine from intracellular to extracellular space, avoiding excessive intracellular concentration that inhibits metabolism. This strain does not require the addition of antibiotics and has excellent L-isoleucine synthesis capacity. When applied to the fermentation production of L-isoleucine, using glucose as a carbon source, the fermentation cycle is short. In a 5L mechanically stirred fermenter, it can produce up to 52.6 g / L of L-isoleucine in 34 hours, demonstrating good economic benefits and industrial application value. Attached Figure Description

[0054] Figure 1 This diagram illustrates the process of modifying the de novo synthesis pathway of L-isoleucine in genetically engineered bacteria. Detailed Implementation

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0056] Unless otherwise specified, the percentage sign "%" used in the examples refers to the mass percentage. The percentage of a solution refers to the number of grams of solute contained in 100 mL. The percentage between liquids refers to the volume ratio of the solution at 25°C.

[0057] like Figure 1 As shown, the strain was modified according to the metabolic pathway of the entire strain modification process to obtain an L-isoleucine-producing strain. The modification process mainly includes the following 5 modules:

[0058] (1) Module to increase the accumulation of precursor ketobutyrate: Increase the transcription level of the aspartate transaminase aspC gene, causing carbon metabolism to flow into aspartate, thereby increasing the accumulation of precursor ketobutyrate; increase the threonine dehydrogenase ilvA fbr At the gene transcription level, ketobutyrate accumulates;

[0059] (2) Module to reduce pyruvate consumption: Knock out D-lactate dehydrogenase ldhA Genes that prevent the reduction of pyruvate to lactate enhance carbon efficiency; knocking out the acetaldehyde dehydrogenase / alcohol dehydrogenase adhE gene blocks the ethanol pathway and prevents NADH waste; knocking out the formate C-acetyltransferase pflB gene reduces the metabolism of pyruvate to formate.

[0060] (3) NADPH recycling module: Increases the transcription level of the pyridine nucleotide transhydrogenase pntAB gene, thereby increasing the supply of NADPH; increases the introduction of NADPH from Corynebacterium glutamicum ATCC 13032. + The kinase ppnK gene promotes the conversion of NADH to NADPH; increasing the transcription level of the polyphosphokinase ppk gene increases ATP supply and promotes NADPH conversion. + Kinase expression;

[0061] (4) Balance the precursors ketobutyrate and pyruvate modules: increase the transcription level of the pyruvate kinase pykF gene to avoid excessive carbon metabolism flowing into ketobutyrate, which would lead to an imbalance between the two precursors; increase the transcription level of the phosphoenolpyruvate carboxylase ppc gene to reduce the waste of carbon metabolism and ensure sufficient accumulation of ketobutyrate precursors.

[0062] (5) L-Isoleucine production module: weakens the transcription level of the 2-isopropylmalate synthase leuA gene, reduces the negative feedback of leucine to isoleucine metabolism; increases acetyllactate synthase ilvIH fbr The transcriptional level of genes is improved to fully utilize pyruvate and avoid the accumulation of the byproduct valine; the transcriptional level of the leucine dehydrogenase bcd gene introduced from B. subtilis 168 ATCC 23857 is increased to enhance isoleucine production; the transcriptional level of the cysteine ​​synthase cysK gene introduced from Corynebacterium glutamicum ATCC 13032 is increased to improve the transcriptional level of key enzymes in the L-isoleucine synthesis pathway; and the transcriptional level of the L-isoleucine ectotransporter ygaZH gene is increased to enhance the transport of isoleucine from the intracellular to the extracellular space and avoid excessive intracellular concentration that inhibits metabolism.

[0063] The gene editing method used is based on the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli Using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). The technical terms used in the following examples, such as gene integration and plasmid construction, are explained in that article. In this invention, "knockout" refers to the inactivation of the target gene, and "introduction" refers to the insertion of a foreign gene into the engineered bacterial genome after linking it with a promoter and terminator.

[0064] E. coli W3110 is E. coli W3110 ATCC 27325, and Escherichia coli XX12 is referred to in CN117925666B. The primers used in the construction of the strains involved are shown in Table 1.

[0065] Table 1 Primers used in strain construction

[0066] Primer name Sequence No. Primer sequence (5'-3') ycjV-US SEQ ID NO. 19 GGAAATCTTTCTCGCCGCCT ycjV-UA SEQ ID NO. 20 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGCCGCTGATCTCCTCAAG ycjV-DS SEQ ID NO. 21 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCATAAGCCCATCGTGATGGG ycjV-DA SEQ ID NO. 22 CTTCGCCATAACCCTCGACG aspC-S SEQ ID NO. 23 GGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGTTTGAGAACATTACCGCCGC aspC-A SEQ ID NO. 24 GACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGCACTGCCACAATCGC pGRB-ycjV-S SEQ ID NO. 25 CAAAGCACGCAATATAGCGAGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-ycjV-A SEQ ID NO. 26 TCGCTATATTGCGTGCTTTGACTAGTATTATACCTAGGACTGAGC fucK-US SEQ ID NO. 27 GCAATGTTATCCGGCTATATTGCAGG fucK-UA SEQ ID NO. 28 ATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCTGTGCGGAGATTAACCGT fucK-DS SEQ ID NO. 29 AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTAGGGGAATTTAACAGCCCGGA fucK-DA SEQ ID NO. 30 TGTAGTGAAAGCGCATTACGGT pGRB-fucK-S SEQ ID NO. 31 TGTCGTGTCAGAACGCTGGCGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-fucK-A SEQ ID NO. 32 GCCAGCGTTCTGACACGACAACTAGTATTATACCTAGGACTGAGC yncK-US SEQ ID NO. 33 CATTTTTCACAACACCCGTGACCT yncK-UA SEQ ID NO. 34 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCATCAGGAAAACACAATGCCTTC yncK-DS SEQ ID NO. 35 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGCCCTGAAGGATGGGGTTTTAC yncK-DA SEQ ID NO. 36 CGCAGCGTAAACCACTGGTTAC pykF-S SEQ ID NO. 37 ATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGAAAAAGACCAAAATTGTTTGCACCA pykF-A SEQ ID NO. 38 caccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttatttgTTACAGGACGTGAACAGATGCGG pGRB-yncK-S SEQ ID NO. 39 AGTCCTAGGTATAATACTAGTAATTAACTATCCTCCCAAACGTTTTAGAGCTAGAA pGRB-yncK-A SEQ ID NO. 40 TTCTAGCTCTAAAACGTTTGGGAGGATAGTTAATTACTAGTATTATACCTAGGACT yjiK-US SEQ ID NO. 41 GAATCTCACCTGCGAATGCC yjiK-UA SEQ ID NO. 42 GCTAGCACAATACCTAGGACTGAGCTAGCTGTCAAACACCATCCTGCTGGCAATAAA yjiK-DS SEQ ID NO. 43 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTTGCGATGGATGCCTCTGGT yjiK-DA SEQ ID NO.44 CCTAAGGTCAGAACCAGCATC ppc-S SEQ ID NO.45 TTGACAGCTAGCTCAGTCCTAGGTATTGTGCTAGCATGAACGAACAATATTCCGCATTGC ppc-A SEQ ID NO.46 TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGCCGGTATTACGCATACCTGC pGRB-yjiK-S SEQ ID NO.47 AGTCCTAGGTATACTAGTCAAGACTCCCCAACCAATTGGTTTTAGAGCTAGAA pGRB-yjiK-A SEQ ID NO.48 TTCTAGCTCTAAAACCAATTGGTTGGGGAGTCTTGACTAGTATTATACCTAGGACT farA-US SEQ ID NO.49 CCGCGAATACTCAATCATCTACAA leuA-UA SEQ ID NO.50 CATAGTCCCTAGGACTGAGCTAGCTGTCAAGCTGGCTTAATGCTGGATGC leuA-DS SEQ ID NO.51 CAGCTAGCTCAGTCCTAGGGACTATGCTAGCATGAGCCAGCAAGTCATTATTTTCGAT farA-DA SEQ ID NO.52 CACGGTGTCCGGAATGTTG pGRB-leuA-S SEQ ID NO.53 AAAAAACCCGCGCCATTGCGGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-leuA-A SEQ ID NO.54 CGCAATGGCGCGGGTTTTTTACTAGTATTATACCTAGGACTGAGC ldhA-US SEQ ID NO.55 AGCAGCGTCAACGGCAC ldhA-UA SEQ ID NO.56 TCCTGGATCACGTCGTTGGATTTATGCTGCCGTCATCGTTTACGAAA ldhA-DS SEQ ID NO.57 TTTCGTAAACGATGACGGCAGCATAAATCCAACGACGTGATCCAGGA ldhA-DA SEQ ID NO.58 GCTGTTCTGGCGTAACAGCAA pGRB-ldhA-S SEQ ID NO.59 AGTCCTAGGTATAACTAGTCCGTGATGCTAACTTCTCTCGTTTTAGAGCTAGAA pGRB-ldhA-A SEQ ID NO.60 TTCTGCTCTAAAACGAGAGAAGTTAGCATCACGGACTAGTATTATACCTAGGACT adhE-US SEQ ID NO.61 ATTACTTGCTTACGCCACCTG adhE-UA SEQ ID NO.62 GAAGCAGACTTCCTGGCGAACTCTTCGACGATACCCATGCCG adhE-DS SEQ ID NO.63 CGGCATGGGTATCGTCGAAGAGAAGCAGACTTCCTGGCGAAC adhE-DA SEQ ID NO.64 GGAAGGTGTTCTGCAAATAGTTGTG pGRB-adhE-S SEQ ID NO.65 AGTCCTAGGTATAATACTGGAAACTCACTTCGAAGAGCGTTTTAGAGCTAGAA pGRB-adhE-A SEQ ID NO.66 TTCTAGCTCTAAAACGCTCTTCGAAGTGAGTTTCCACTAGTATTATACCTAGGACT pflB-US SEQ ID NO.67 GTGGTTTGTTGGTTGGGTTGACATA pflB-UA SEQ ID NO.68 GCGTTCGGAACGATAGAGAAGGTTGTACGCTTTGCAGGAACCTT pflB-DS SEQ ID NO.69 AAGGTTCCTGCAAAGCGTACAACCCTTCTCTATCGTTCCGAACGC pflB-DA SEQ ID NO.70 TGAATGCGACCAATAACTGACATTG pGRB-pflB-S SEQ ID NO.71 AGTCCTAGGTATATACTAGTAACGGTGCTGCAATGTCCTTGTTTTAGAGCTAGAA pGRB-pflB-A SEQ ID NO.72 TTCTAGCTCTAAAACAAGGACATTGCAGCACCGTTACTAGTATTATACCTAGGACT rph-US SEQ ID NO.73 ATAGCGCAGGGTACATTCCACT rph-UA SEQ ID NO.74 GAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACCTTCTTCAATAGAGGCGGTACA rph-DS SEQ ID NO.75 TGCCGCAGAGACCGACATCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAAT rph-DA SEQ ID NO.76 ACAGCGGTTGTGGTGGCA ilvA fbr -S]] SEQ ID NO.77 CATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGGCTGACTCGCAACCC ilvA fbr -A]]> SEQ ID NO.78 AACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGCTAACCCGCCAAAAAGAACCTGA pGRB-rph-S SEQ ID NO.79 AGTCCTAGGTATATACTAGTTGCGACGTGCTTCAGGCTGAGTTTTAGAGCTAGAA pGRB-rph-A SEQ ID NO.80 TTCTAGCTCTAAAACTCAGCCTGAAGCACGTCGCAACTAGTATTATACCTAGGACT fhiA-US SEQ ID NO.81 GGGCAATGGTGTTGATACTGG fhiA-UA SEQ ID NO.82 AATGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAATCGCCAGAATCATCATCCC fhiA-DS SEQ ID NO.83 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCAAGCAGGAGCTGACGGTGT fhiA-DA SEQ ID NO.84 TGCACCAATGCTGGATACTTACA pGRB-fhiA-S SEQ ID NO.85 AGTCCTAGGTATAATACTAGTTGACGTGCGTAACCAGCTGCGTTTTAGAGCTAGAA pGRB-fhiA-A SEQ ID NO.86 TTCTAGCTCTAAAACGCAGCTGGTTACGCACGTCAACTAGTATTATACCTAGGACT ylbe-US SEQ ID NO.87 ACCCAACCTTACCCAACCAG ylbE-UA SEQ ID NO.88 GTGAAATTGTTATCCGCTCAATTCCACATTATACGAGCCGGATGATTAATTGTCAATTGTTCGATAACCGCAGCAT ylbE-DS SEQ ID NO.89 AAAGACTGGGCCTTTCGTTATCTCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGCAAATCGCTGGCGTGCTTTGAA ylbE-DA SEQ ID NO.90 GGCGTAACTCAGCAGGCAG ilvIH fbr -S]] SEQ ID NO.91 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAGATGTTGTCTGGAGCC ilvIH fbr -A]]> SEQ ID NO.92 AAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTCAACGCATTATTTTATCGCCGCG pGRB-ylbE-S SEQ ID NO.93 AGTCCTAGGTATAATACTAGCACTGGCTGGATGTGCAACGTTTTAGGCTAGAA pGRB-ylbE-A SEQ ID NO.94 TTCTAGCTCTAAAACGTTGCACATCCAGCCAGTGTACTAGTTATACCTAGGACT incI-US SEQ ID NO.95 GGGCAACTTCTCGGGTTAGATG IncI-UA SEQ ID NO.96 CCGCTCACAATTCCACACATTATACGAGCCGGATTATTGTCAATGTAGGCGTTAAAGCAAAGATGAAAATCG IncI-DS SEQ ID NO.97 GGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTGCCAGCCGGATAGAAGAGG IncI-DA SEQ ID NO.98 GCAATGACGTCTTTATCATCTGAAGATGG pGRB-syncI-S SEQ ID NO.99 GAGTGCCATGAATCACTGAGTTTGAGCTAGAAAATCACTGAGTTA pGRB-syncI-A SEQ ID NO.100 TCAGTGATTTCATGGCACTCACTAGTATTATACCTAGGACTGAGC molR-US SEQ ID NO.101 GTTGTTTTCTTGCGATTTTGTCTCTCTC molR-UA SEQ ID NO.102 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGAAAGCGGCGATGACTTAACG molR-DS SEQ ID NO.103 CCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCCGAAAAACCAGAAATTGCAC molR-DA SEQ ID NO.104 TTTTGCCCAAGAAGTTCCGTCA pGRB-molR-S SEQ ID NO.105 TGGCATTGCACCTAGCTACTGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-molR-A SEQ ID NO.106 AGTAGCTAGGTGCAATGCCAACTAGTATTATACCTAGGACTGAGC nmpC-US SEQ ID NO.107 GCTACCGAGTATTGAAAACACCAC nmpC-UA SEQ ID NO.108 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAGCCAAGACGGGCATAAGTAGTAT nmpC-DS SEQ ID NO.109 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATTAAAAATGCAGAAGTTTGGGCCG nmpC-DA SEQ ID NO.110 TGATGACTCATGATGAACCCTGTTC bcd-S SEQ ID NO.111 GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAACTTTTTAAATATATGGAGAAATACG bcd-A SEQ ID NO.112 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAACGTCTGCTTAATACACTGTGGC pGRB-nmpC-S SEQ ID NO.113 AGTCCTAGGTATATACTAGTCAGAATTCGGTGGTGACACTGTTTTAGAGCTAGAA pGRB-nmpC-A SEQ ID NO.114 TTCTAGCTCTAAAACAGTGTCACCACCGAATTCTGACTAGTATTATACCTAGGACT gapC-US SEQ ID NO.115 TGGGAAGAAACCACGAAACTC gapC-UA SEQ ID NO.116 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAATGTTTCAGCAGGTAGGCGAGA gapC-DS SEQ ID NO.117 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAATAAAACGGTCGCCTGGTACG gapC-DA SEQ ID NO.118 TTATCCGCCGACATTGCTG pGRB-gapC-S SEQ ID NO.119 AGTCCTAGGTATAATACTAGTAAGTAGCCACCAATCTGGGTTAGTTTTAGAGCTAGAA pGRB-gapC-A SEQ ID NO.120 TTCTAGCTCTAAAACTAACCCAGATTGGTGGCATACTTACTAGTATTATACCTAGGACT yfaS-US SEQ ID NO.121 GAAGCGCCACGAACTGACA yfaS-UA SEQ ID NO.122 GTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGAGATTTGCCGTACCAGTTATCC yfaS-DS SEQ ID NO.123 TTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTGACAAATACAACATATATCGCCAGAACGTTCA yfaS-DA SEQ ID NO.124 TTAACTGCACGCGGGCATTTTTTA pGRB-yfaS-S SEQ ID NO.125 ACCGTTCGAGCCGCAGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-yfaS-A SEQ ID NO.126 CTTGCTGCGGCTCGAACGGTACTAGTATTATACCTAGGACTGAGC insB-US SEQ ID NO.127 GGATGGAGCCTCTGCTTCTGG insB-UA SEQ ID NO.128 TGTGTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAACGGTTGTATGCGCGAGGTTAC insB-DS SEQ ID NO.129 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGGTGGAGTTGCATGACAAGGTC insB-DA SEQ ID NO.130 GGTGGGCGTGCTGAATAAGAAG pntAB-S SEQ ID NO.131 CCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGCGAATTGGCATACCAAGAG pntAB-A SEQ ID NO.132 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACAGAGCTTTCAGGATTGCATCCAC pGRB-insB-S SEQ ID NO.133 GTGCTAAATCACGTCAGCGCGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-insB-A SEQ ID NO.134 GCGCTGACGTGATTTAGCACACTAGTATTATACCTAGGACTGAGC yjgX-US SEQ ID NO.135 GGAAGTCAACGGGTTATGCG yjgX-UA SEQ ID NO.136 GTTATCCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAAATCACCACGAATACCAGAATCGC yjgX-DS SEQ ID NO.137 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACAGTGTCTTCCCTGAGCCG yjgX-DA SEQ ID NO.138 GGCGAAGGATACCATCAAGC ppnK-S SEQ ID NO.139 CGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGACTGCACCCACGAACG ppnK-A SEQ ID NO.140 GTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTACCCCGCTGACCTGGGA pGRB-yjgX-S SEQ ID NO.141 AGTCCTAGGTATACTAGTTCGCGACCACCGTAACTGGCGTTTTAGAGCTAGAA pGRB-yjgX-A SEQ ID NO.142 TTCTAGCTCTAAAACGCCAGTTACGGTGGTCGCGAACTAGTATTATACCTAGGACT ydeU-US SEQ ID NO.143 CAACGATTCCGCGGCGTAT ydeU-UA SEQ ID NO.144 GTGAAATTGTTATCCGCTCAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAACCGTTGACCGTTTGTGCA ydeU-DS SEQ ID NO.145 GACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATACTCAGGGCATGCTGGGA ydeU-DA SEQ ID NO.146 GCGTCGCATAGCGTCATTGA ppK-S SEQ ID NO.147 GGCTCGTATAATGTGTGGAATTTGTGAGCGGATAACAATTTCACAGGAAACAGACCATGGGTCAGGAAAAGCTATACATCGAAA ppK-A SEQ ID NO.148 CAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATTCAGGTTGTTCGAGTGATTTGATGT pGRB-ydeU-S SEQ ID NO.149 AGTCCTAGGTATACTAGTTCAGTTTATCGCCCGGAGGCGTTTTAGAGCTAGAA pGRB-ydeU-A SEQ ID NO.150 TTCTAGCTCTAAAACGCCTCCGGGCGATAAACTGAACTAGTATTATACCTAGGACT ydbA-US SEQ ID NO. 151 CGTGAACTCAGTGAGCAGGAG ydbA-UA SEQ ID NO. 152 TGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAAAGCCGGATGCACTAACCC ydbA-DS SEQ ID NO. 153 AGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGTTGCCCTTGAAGGGGTGA ydbA-DA SEQ ID NO. 154 CCGTTGTTGTTTTGCCGTGG cysK-S SEQ ID NO. 155 GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGATTGGAGCACCACCCGA cysK-A SEQ ID NO. 156 CAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTAGTCGCGGATGTCTTCGTAAAGAA pGRB-ydbA-S SEQ ID NO. 157 TCGAACAAAACCTGATGCCAGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-ydbA-A SEQ ID NO. 158 TGGCATCAGGTTTTGTTCGAACTAGTATTATACCTAGGACTGAGC ychG-US SEQ ID NO. 159 GGGAGCCGTTTTCTTATGCCAC ychG-UA SEQ ID NO. 160 GAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGGCGTTCTGCCGCTTAGTG ychG-DS SEQ ID NO. 161 CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATATCAAAAACCCGTTATCCTGTGAAAC ychG-DA SEQ ID NO. 162 CTGCGTCTTGATCAAGCAGG ygaZH-S SEQ ID NO. 163 GTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGACCATGGAAAGCCCTACTCCACAG ygaZH-A SEQ ID NO. 164 GATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGTTATATAATCGCCATCACTTTCCAGGC pGRB-ychG-S SEQ ID NO.165 CTGGCGTTGATTATGCCCATGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-ychG-A SEQ ID NO.166 ATGGGCATAATCAACGCCAGACTAGTATTATACCTAGGACTGAGC yjiP-US SEQ ID NO.167 GCCATACCGCCAGCAAGAT yjiP-UA SEQ ID NO.168 AATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGATGATTAATTGTCAAGCAGATATTCCCCTTTCCACC yjiP-DS SEQ ID NO.169 AAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATGACGGATGACAAACGCAAAGC yjiP-DA SEQ ID NO.170 AAAGGCGGATTTTTACTGTGGA pGRB-yjiP-S SEQ ID NO.171 GTAGCCGTAGTCCTTACCCCGTTTTAGAGCTAGAAATAGCAAGTTAA pGRB-yjiP-A SEQ ID NO.172 GGGGTAAGGACTACGGCTACACTAGTATTATACCTAGGACTGAGC

[0067] Example 1

[0068] This embodiment aims to illustrate the specific construction steps of strain XX34. In particular, if there are similar gene manipulation methods in the embodiment, they will only be provided once and annotated, without further elaboration.

[0069] (1) Controlling the overexpression of the aspC gene at the ycjV gene locus using the trc promoter: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms and the target gene fragment were obtained by PCR amplification using ycjV-US, ycjV-UA and ycjV-DS, ycjV-DA, aspC-S, aspC-A respectively. Then, using the upstream and downstream homologous arms and the target gene fragment as templates, overlapping fragments were obtained by overlapping PCR amplification using ycjV-US and ycjV-DA as primers. Using pGRB-ycjV-S and pGRB-ycjV-A as primers, gRNA fragments were obtained by annealing and ligated with the pGRB vector to obtain ycjV-pGRB. XX12 electrotransformation competent cells were prepared, and the target fragment and ycjV-pGRB were electrotransformed into competent cells together. Positive transformants were screened to obtain strain XX13.

[0070] (2) Controlling the overexpression of the aspC gene at the fucK pseudogene site using the trc promoter: The operation method is the same as in (1), except that the primers used are fucK-US, fucK-UA and fucK-DS, fucK-DA, aspC-S, aspC-A, pGRB-fucK-S, pGRB-fucK-A, the competent cells are XX13, and the strain obtained is XX14.

[0071] (3) Controlling pykF gene overexpression at the yncK pseudogene site using the trc promoter: The operation method is the same as in (1), except that the primers used are yncK-US, yncK-UA and yncK-DS, yncK-DA, pykF-S, pykF-A, pGRB-yncK-S, pGRB-yncK-A, the competent cells are XX14, and the strain XX15 is obtained.

[0072] (4) Using P at the yjiK pseudogene locus BBa_J23104 Promoter control of ppc gene overexpression: The operation method is the same as in (1), except that the primers used are yjiK-US, yjiK-UA and yjiK-DS, yjiK-DA, ppc-S, ppc-A, pGRB-yjiK-S, pGRB-yjiK-A, the competent cells are XX15, and the strain obtained is XX16.

[0073] (5) Use P BBa_J23116 Promoter weakening of the leuA gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were amplified by PCR using leuA-US, leuA-UA and leuA-DS, leuA-DA, respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were amplified by overlapping PCR using leuA-US and leuA-DA primers. Using pGRB-leuA-S and pGRB-leuA-A primers, gRNA fragments were annealed to obtain gRNA fragments, which were then ligated into the pGRB vector to obtain leuA-pGRB. XX16 electrotransformation competent cells were prepared, and the overlapping fragments and leuA-pGRB were electrotransformed into competent cells together. Positive transformants were screened to obtain strain XX17.

[0074] (6) Knockout of ldhA gene: Using the E. coli W3110 genome as a template, upstream and downstream homologous arms were obtained by PCR amplification using ldhA-US, ldhA-UA and ldhA-DS, ldhA-DA respectively. Then, using the upstream and downstream homologous arms as templates, overlapping fragments were obtained by overlapping PCR amplification using ldhA-US and ldhA-DA as primers. The gRNA fragment was obtained by annealing using pGRB-ldhA-S and pGRB-ldhA-A as primers and then ligated with the pGRB vector to obtain ldhA-pGRB. XX17 electrotransformation competent cells were prepared, and the overlapping fragment and ldhA-pGRB were electrotransformed into competent cells together. Positive transformants were screened to obtain strain XX18.

[0075] (7) Knockout of the adhE gene: The operation method is the same as in (5), except that the primers used are adhE-US, adhE-UA, adhE-DS, adhE-DA, pGRB-adhE-S, and pGRB-adhE-A. The competent cells are XX18, and strain XX19 is obtained.

[0076] (8) Knockout of the pflB gene: The operation method is the same as in (5), except that the primers used are pflB-US, pflB-UA, pflB-DS, pflB-DA, pGRB-pflB-S, and pGRB-pflB-A. The competent cells are XX19, and strain XX20 is obtained.

[0077] (9) Use the trc promoter to control ilvA at the rph pseudogene site. fbr Gene overexpression: It has the same operation method as in (1), except that the primers used are rph-US, rph-UA and rph-DS, rph-DA, ilvA fbr -S、ilvA fbr -A, pGRB-rph-S, pGRB-rph-A, competent cells were used to obtain strain XX21.

[0078] (10) Use the trc promoter to control ilvA at the fhiA pseudogene site. fbr Gene overexpression: It has the same operation method as in (1), except that the primers used are fhiA-US, fhiA-UA and fhiA-DS, fhiA-DA, ilvA fbr -S、ilvA fbr -A, pGRB-fhiA-S, pGRB-fhiA-A, competent cells were XX21, and strain XX22 was obtained.

[0079] (11) Use the trc promoter to control ilvIH at the ylbE pseudogene site. fbr Gene overexpression: It has the same operation method as in (1), except that the primers used are ylbE-US, ylbE-UA and ylbE-DS, ylbE-DA, ilvIH fbr -S、ilvIH fbr -A, pGRB-ylbE-S, pGRB-ylbE-A, competent cells were XX22, and strain XX23 was obtained.

[0080] (12) Use the trc promoter to control ilvIH at the yncI pseudogene site. fbr Gene overexpression: It has the same operation method as in (1), except that the primers used are yncI-US, yncI-UA and yncI-DS, yncI-DA, ilvIH fbr -S、ilvIH fbr -A, pGRB-yncI-S, pGRB-yncI-A, competent cells were XX23, and strain XX24 was obtained.

[0081] (13) Use the trc promoter to control ilvIH at the molR pseudogene site. fbr Gene overexpression: It has the same operation method as in (1), except that the primers used are molR-US, molR-UA and molR-DS, molR-DA, ilvIH fbr -S、ilvIH fbr -A, pGRB-molR-S, pGRB-molR-A, competent cells were XX24, and strain XX25 was obtained.

[0082] (14) Overexpression of the bcd gene derived from B. subtilis 168 was controlled by the trc promoter at the nmpC pseudogene site: The operation method was the same as in (1), except that the genomic template used was B. subtilis 168, the primers used were nmpC-US, nmpC-UA and nmpC-DS, nmpC-DA, bcd-S, bcd-A, pGRB-nmpC-S, pGRB-nmpC-A, the competent cells were XX25, and strain XX26 was obtained.

[0083] (15) Overexpression of the bcd gene derived from B. subtilis 168 was controlled by the trc promoter at the gapC pseudogene site: The operation method was the same as in (1), except that the genomic template used was B. subtilis 168, the primers used were gapC-US, gapC-UA and gapC-DS, gapC-DA, bcd-S, bcd-A, pGRB-gapC-S, pGRB-gapC-A, the competent cells were XX26, and the strain obtained was XX27.

[0084] (16) Overexpression of the bcd gene derived from B. subtilis 168 was controlled by the trc promoter at the yfaS pseudogene site: The operation method was the same as in (1), except that the genomic template used was B. subtilis 168, the primers used were yfaS-US, yfaS-UA and yfaS-DS, yfaS-DA, bcd-S, bcd-A, pGRB-yfaS-S, pGRB-yfaS-A, the competent cells were XX27, and the strain obtained was XX28.

[0085] (17) Controlling pntAB gene overexpression at the insB pseudogene site using the trc promoter: The operation method is the same as in (1), except that the primers used are insB-US, insB-UA and insB-DS, insB-DA, pntAB-S, pntAB-A, pGRB-insB-S, pGRB-insB-A, competent cells are XX28, and strain XX29 is obtained.

[0086] (18) Overexpression of the ppnK gene derived from Corynebacterium glutamicum ATCC 13032 at the yjgX pseudogene site using the trc promoter: The operation method is the same as in (1), except that the genomic template used is Corynebacterium glutamicum ATCC 13032, the primers used are yjgX-US, yjgX-UA and yjgX-DS, yjgX-DA, ppnK-S, ppnK-A, pGRB-yjgX-S, pGRB-yjgX-A, the competent cells are XX29, and strain XX30 is obtained.

[0087] (19) Overexpression of the ppk gene derived from Corynebacterium glutamicum ATCC 13032 was controlled at the ydeU pseudogene site using the trc promoter: The operation method was the same as in (1), except that the genomic template used was Corynebacterium glutamicum ATCC 13032, the primers used were ydeU-US, ydeU-UA and ydeU-DS, ydeU-DA, pppk-S, pppk-A, pGRB-ydeU-S, pGRB-ydeU-A, the competent cells were XX30, and strain XX31 was obtained.

[0088] (20) Overexpression of the cysK gene derived from Corynebacterium glutamicum ATCC 13032 was controlled at the ydbA pseudogene site using the trc promoter: The operation method was the same as in (1), except that the genomic template used was Corynebacterium glutamicum ATCC 13032, the primers used were ydbA-US, ydbA-UA and ydbA-DS, ydbA-DA, cysK-S, cysK-A, pGRB-ydbA-S, pGRB-ydbA-A, the competent cells were XX31, and strain XX32 was obtained.

[0089] (21) Controlling the overexpression of the ygaZH gene at the ychG pseudogene site using the trc promoter: The operation method is the same as in (1), except that the primers used are ychG-US, ychG-UA and ychG-DS, ychG-DA, ygaZH-S, ygaZH-A, pGRB-ychG-S, pGRB-ychG-A, the competent cells are XX32, and the strain XX33 is obtained.

[0090] (22) Controlling the overexpression of the ygaZH gene at the yjiP pseudogene site using the trc promoter: The operation method is the same as in (1), except that the primers used are yjiP-US, yjiP-UA and yjiP-DS, yjiP-DA, ygaZH-S, ygaZH-A, pGRB-yjiP-S, pGRB-yjiP-A, the competent cells are XX33, and the strain obtained is XX34.

[0091] Example 2

[0092] Using strain XX34 as the L-isoleucine production strain, this example aims to illustrate the L-isoleucine production method using this strain. The specific steps are as follows:

[0093] L-Isoleucine-producing strains were streaked onto antibiotic-free activation slants and incubated at 37°C for 15 hours, followed by two subcultures. The activated cells on the slants were then washed with sterile distilled water and transferred to a 5 L mechanically stirred fermenter for seed culture. The culture temperature was 34°C, with an initial stirring speed of 200 rpm. The pH was maintained at 6.7 ± 0.2 by automatic addition of 25% ammonia solution. The dissolved oxygen level was maintained at 35% by adjusting the stirring speed or aeration rate, depending on the OD value. 600nm A value of 20 indicates the seed culture is mature. After the seed culture matures, retain 600 mL of seed culture and immediately add fresh fermentation medium to bring the final fermentation volume to 2 L. Fermentation begins at 36℃. The pH is maintained at 6.7±0.2 by automatically adding 25% ammonia solution, and the dissolved oxygen level is maintained at 60% by adjusting the stirring speed or aeration. The glucose concentration in the tank is controlled to ≤0.5 g / L by adding 80% glucose solution. The fermentation cycle is 34 hours.

[0094] The slant culture medium used consisted of: glucose 1 g / L, peptone 5 g / L, potassium dihydrogen phosphate 0.5 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, magnesium sulfate heptahydrate 0.2 g / L, agar powder 25 g / L, and the remainder being water. The pH was 7.0. The medium was sterilized at 121°C for 20 min and then dispensed into test tubes.

[0095] The seed culture medium used consisted of: glucose 20 g / L, yeast extract 3 g / L, peptone 3 g / L, (NH4)2SO4 1 g / L, K2HPO4·3H2O 3 g / L, MgSO4·7H2O 2 g / L, citric acid 2 g / L, glutamic acid 3 g / L, methionine 0.5 g / L, lysine 1 g / L, ammonium sulfate 1 g / L, FeSO4·7H2O 10 mg / L, MgSO4·7H2O 10 mg / L, and the remainder being water.

[0096] The fermentation medium used consisted of: yeast extract 4 g / L, peptone 3 g / L, (NH4)2SO4 4 g / L, K2HPO4·3H2O 4 g / L, MgSO4·7H2O 2 g / L, glutamic acid 4 g / L, methionine 0.3 g / L, citric acid 3 g / L, lysine 2 g / L, ammonium sulfate 4 g / L, MnSO4·7H2O 10 mg / L, FeSO4·7H2O 30 mg / L, corn steep liquor 20 ml / L, and the remainder being water.

[0097] Example 3

[0098] This experiment aims to verify the three key genes ilvA. fbr Genes, ilvIH fbrThe optimal copy number ratio of the gene and the bcd gene is 3:4:3 (the copy number includes one ilvA and one ilvIH that have been integrated into the original strain XX12).

[0099] Following the method for constructing strain XX34 as described in Example 1, XX19 was used as the starting strain for verification. The strains generated in the verification experiments were only used for verifying the optimal copy number. The strains generated in the experiments are shown in Table 2 (copy number includes one ilvA and one ilvIH already integrated into the original strain):

[0100] Table 2

[0101]

[0102] Using the culture method and culture medium in Example 2, fermentation was carried out in a 5L fermenter for 34 hours. The strains in Table 2 were verified by fermentation, and the results are shown in Table 3 below:

[0103] Table 3

[0104]

[0105] Through comparison of experiments 1 to 5, it was found that by continuously copying ilvA... fbr Genes, ilvIH fbr The production of L-isoleucine showed a continuous upward trend in the genes and bcd gene, but the bacterial cell mass decreased until ilvA. fbr Genes, ilvIH fbr After the fifth copy of the gene and the bcd gene, the yield decreased slightly, but the cell count rebounded, indicating that ilvA... fbr Genes, ilvIH fbr The fifth copy of the gene and the bcd gene did not increase the production of L-isoleucine, and the bacterial cell mass was high while the acid production capacity decreased, indicating that four copies is the maximum number of copies.

[0106] Experiments 6 through 11 were then conducted. The results showed that, between the three-copy and four-copy controls, the L-isoleucine production in Experiment 7 was the highest, but the cell count was relatively low, indicating that the cells had a stronger acid-producing capacity and produced the most acid.

[0107] In summary, when ilvA fbr Genes, ilvIH fbr The optimal copy numbers for the gene and the bcd gene are 3, 4, and 3, respectively.

[0108] Example 4

[0109] This example aims to verify the L-isoleucine production capacity of strain XX34 (the strain with the optimal copy number obtained in Example 3). The culture method and culture medium in Example 2 were used, and the results are shown in Table 4 below:

[0110] Table 4

[0111]

[0112] It can be seen that strain XX34 can produce up to 52.6 g / L of L-isoleucine after 34 hours in a 5 L fermenter.

[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. A strain for producing L-isoleucine, characterized in that: It was obtained by modifying the chassis strain of *Escherichia coli* XX12 using metabolic engineering methods, deleting the ldhA, adhE, and pflB genes, and upregulating aspC, pykF, pntAB, ppk, ppc, and ilvA. fbr ,ilvIH fbr The transcriptional levels of the ygaZH gene were reduced, and the transcriptional level of the leuA gene was downregulated. The bcd gene from B. subtilis 168 and the ppnK and cysK genes from Corynebacterium glutamicum ATCC 13032 were expressed heterologously. Specifically, the aspC, pykF, and ilvA genes were overexpressed using the Ptrc promoter. fbr ,ilvIH fbr The following genes were included: bcd, pntAB, ppk, ygaZH, ppnK, and cysK; the ppc gene was overexpressed using the PBBa_J23104 promoter; the leuA gene was attenuated using the PBBa_J23116 promoter; the nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO.1; the nucleotide sequence of the PBBa_J23104 promoter is shown in SEQ ID NO.2; the nucleotide sequence of the PBBa_J23116 promoter is shown in SEQ ID NO.3; the nucleotide sequence of the aspC gene is shown in SEQ ID NO.4; the nucleotide sequence of the pykF gene is shown in SEQ ID NO.5; the nucleotide sequence of the pntAB gene cluster is shown in SEQ ID NO.14; the nucleotide sequence of the ppk gene is shown in SEQ ID NO.16; the nucleotide sequence of the ppc gene is shown in SEQ ID NO.6; ilvA fbr The nucleotide sequence of the gene is shown in SEQ ID NO.11 of the sequence listing; ilvIH fbr The nucleotide sequences of the gene clusters are shown in SEQ ID NO.12; the nucleotide sequence of the ygaZH gene is shown in SEQ ID NO.18; the nucleotide sequence of the leuA gene is shown in SEQ ID NO.7; the nucleotide sequence of the bcd gene is shown in SEQ ID NO.13; the nucleotide sequence of the ppnK gene is shown in SEQ ID NO.15; and the nucleotide sequence of the cysK gene is shown in SEQ ID NO.

17.

2. The L-isoleucine-producing strain according to claim 1, characterized in that: Overexpression of aspartate transaminase (aspC) at the ycjV and fucK gene sites on the *E. coli* XX12 genome using the Ptrc promoter; overexpression of pyruvate kinase (pykF) at the yncK pseudogene site using the Ptrc promoter; overexpression of phosphoenolpyruvate carboxylase (ppc) at the yjiK pseudogene site using the PBBa_J23104 promoter; attenuation of 2-isopropylmalate synthase (leuA) using the PBBa_J23116 promoter; knockout of D-lactate dehydrogenase (ldhA); knockout of acetaldehyde dehydrogenase / alcohol dehydrogenase (adhE); knockout of formate C-acetyltransferase (pflB); and overexpression of threonine dehydrogenase (ilvA) at the rph and fhiA pseudogene sites using the Ptrc promoter. fbr Gene; overexpression of acetyllactate synthase ilvIH using the Ptrc promoter at the ylbE, yncI, and molR pseudogene sites. fbr Genes: Leucine dehydrogenase (bcd) from *B. subtilis* 168 was overexpressed using the Ptrc promoter at the nmpC, gapC, and yfaS pseudogene sites; pyridine nucleotide transhydrogenase (pntAB) was overexpressed using the Ptrc promoter at the insB pseudogene site; and NAD from *Corynebacterium glutamicum* ATCC13032 was overexpressed using the Ptrc promoter at the yjgX pseudogene site. + The kinase ppnK gene was overexpressed using the Ptrc promoter at the ydeU pseudogene site; the cysteine ​​synthase cysK gene, derived from Corynebacterium glutamicum ATCC13032, was overexpressed using the Ptrc promoter at the ydbA pseudogene site; and the L-isoleucine ectotransporter ygaZH gene was overexpressed using the Ptrc promoter at the ychG and yjiP pseudogene sites.

3. The L-isoleucine-producing strain according to claim 1 or 2, characterized in that: The nucleotide sequence of the ldhA gene is shown in SEQ ID NO.8; the nucleotide sequence of the adhE gene is shown in SEQ ID NO.9; and the nucleotide sequence of the pflB gene is shown in SEQ ID NO.

10.

4. The method for constructing the L-isoleucine-producing strain according to claim 1, characterized in that: The directional modification of Escherichia coli XX12 was carried out, and it was divided into the following 5 modules: (1) Module to increase the accumulation of precursor ketobutyrate: increase the transcription level of aspartate transaminase aspC gene; increase the threonine dehydrogenase ilvA fbr The transcriptional level of genes; (2) Module to reduce pyruvate consumption: knock out D-lactate dehydrogenase ldhA gene; knock out acetaldehyde dehydrogenase / alcohol dehydrogenase adhE gene; knock out formate C-acetyltransferase pflB gene; (3) NADPH recycling module: increases the transcription level of the pyridine nucleotide transhydrogenase pntAB gene; increases the amount of NADPH introduced from Corynebacterium glutamicum ATCC 13032. + kinase ppnK gene; increase the transcription level of polyphosphoric acid kinase ppk gene; (4) Balancing the precursors ketobutyrate and pyruvate modules: increasing the transcriptional level of the pyruvate kinase pykF gene; increasing the transcriptional level of the phosphoenolpyruvate carboxylase ppc gene; increasing the transcriptional level of acetyllactate synthase ilvIH fbr The transcriptional level of genes; (5) L-Isoleucine production module: Reduces the transcription level of the 2-isopropylmalate synthase leuA gene; Increase the transcription level of the leucine dehydrogenase bcd gene introduced from B. subtilis 168; increase the transcription level of the cysteine ​​synthase cysK gene introduced from Corynebacterium glutamicum ATCC 13032; increase the transcription level of the L-isoleucine ectotransporter ygaZH gene.

5. The method for constructing the L-isoleucine-producing strain according to claim 4, characterized in that: The specific steps are as follows: (1) Using Escherichia coli XX12 as the chassis strain, the aspartate transaminase aspC gene was overexpressed at the ycjV gene site using the Ptrc promoter to obtain strain XX13; (2) Using strain XX13 as the chassis strain, the aspartate transaminase aspC gene was overexpressed at the fucK gene site using the Ptrc promoter to obtain strain XX14; (3) Using strain XX14 as the chassis strain, the pyruvate kinase pykF gene was overexpressed at the yncK pseudogene site using the Ptrc promoter to obtain strain XX15; (4) Using strain XX15 as the chassis strain, the phosphoenolpyruvate carboxylase ppc gene was overexpressed at the yjiK pseudogene site using the PBBa_J23104 promoter to obtain strain XX16. (5) Using strain XX16 as the chassis strain, the 2-isopropylmalate synthase leuA gene was weakened using the PBBa_J23116 promoter to obtain strain XX17; (6) Using strain XX17 as the chassis strain, the D-lactate dehydrogenase ldhA gene was knocked out to obtain strain XX18; (7) Using strain XX18 as the chassis strain, the acetaldehyde dehydrogenase / alcohol dehydrogenase adhE gene was knocked out to obtain strain XX19; (8) Using strain XX19 as the chassis strain, the formate C-acetyltransferase pflB gene was knocked out to obtain strain XX20; (9) Using strain XX20 as the chassis strain, threonine dehydrogenase ilvA was overexpressed at the rph pseudogene site using the Ptrc promoter. fbr Genes were used to obtain strain XX21; (10) Using strain XX21 as the chassis strain, threonine dehydrogenase ilvA was overexpressed at the fhiA pseudogene site using the Ptrc promoter. fbr Genes were used to obtain strain XX22; (11) Using strain XX22 as the chassis strain, acetyllactate synthase ilvIH was overexpressed at the ylbE pseudogene site using the Ptrc promoter. fbr Genes were used to obtain strain XX23; (12) Using strain XX23 as the chassis strain, acetyllactate synthase ilvIH was overexpressed at the yncI pseudogene site using the Ptrc promoter. fbr Genes were used to obtain strain XX24; (13) Using strain XX24 as the chassis strain, acetyllactate synthase ilvIH was overexpressed at the molR pseudogene site using the Ptrc promoter. fbr Genes were used to obtain strain XX25; (14) Using the Ptrc promoter, strain XX25 overexpressed the leucine dehydrogenase bcd gene derived from B. subtilis 168 at the nmpC pseudogene site to obtain strain XX26; (15) Using the Ptrc promoter, strain XX26 overexpressed the leucine dehydrogenase bcd gene derived from B. subtilis 168 at the gapC pseudogene site to obtain strain XX27; (16) Using the Ptrc promoter, strain XX27 overexpressed the leucine dehydrogenase bcd gene derived from B. subtilis 168 at the yfaS pseudogene site to obtain strain XX28; (17) Using strain XX28, the pyridine nucleotide transhydrogenase pntAB gene was overexpressed at the insB pseudogene site using the Ptrc promoter to obtain strain XX29; (18) Using strain XX29, NAD derived from Corynebacterium glutamicum ATCC 13032 was overexpressed at the yjgX pseudogene site using the Ptrc promoter. + Gene on the kinase ppnK was used to obtain strain XX30; (19) Using the Ptrc promoter to overexpress the polyphosphoric kinase ppk gene at the ydeU pseudogene site of strain XX30, strain XX31 was obtained; (20) The cysK gene of cysteine ​​synthase derived from Corynebacterium glutamicum ATCC 13032 was overexpressed by strain XX31 at the ydbA pseudogene site using the Ptrc promoter to obtain strain XX32; (21) Using the Ptrc promoter, strain XX31 was overexpressed with the L-isoleucine ectotransporter ygaZH gene at the ychG pseudogene site to obtain strain XX33; (22) The L-isoleucine ectotransporter ygaZH gene was overexpressed by strain XX31 at the yjiP pseudogene site using the Ptrc promoter.

6. The use of the L-isoleucine-producing strain according to any one of claims 1-3 in the fermentation production of L-isoleucine.

7. The application according to claim 6, characterized in that: L-Isoleucine was synthesized using glucose as a substrate in a fermenter. The specific steps are as follows: (1) Activation of strain: The L-isoleucine producing strain was streaked onto the activation slant, incubated at 37℃ for 12-15h, and passaged once; (2) Seed culture: The culture temperature is 34-36℃, the initial stirring speed is 200 rpm, and the culture pH is maintained at 6.7±0.2 by automatic addition of ammonia solution. The dissolved oxygen value of the culture medium is maintained at 35% by adjusting the stirring speed or aeration rate, depending on the OD. 600nm A score of 20 indicates the maturity of the seed culture medium. (3) Fermentation culture: The inoculum amount is 30%, the culture temperature is 36℃, the culture pH is maintained at 6.7±0.2 by automatically adding ammonia solution, the dissolved oxygen value of the culture medium is maintained at 60% by adjusting the stirring speed or ventilation, and the glucose concentration in the tank is controlled at ≤0.5g / L by adding glucose solution.

8. The application according to claim 7, characterized in that: The slant culture medium used in step (1) is: glucose 1g / L, peptone 5g / L, potassium dihydrogen phosphate 0.5g / L, yeast extract 5g / L, sodium chloride 5g / L, magnesium sulfate heptahydrate 0.2g / L, agar powder 25g / L, the remainder being water, pH 7.0; the seed culture medium used in step (2) is: glucose 20g / L, yeast powder 3g / L, peptone 3g / L, (NH4)2SO4 1g / L, K2HPO4·3H2O 3g / L, MgSO4·7H2O 2g / L, citric acid 2g / L, glutamic acid 3g / L, methionine 0.5g / L, lysine 1g / L, ammonium sulfate 1g / L, FeSO4·7H2O 10mg / L, MgSO4·7H2O 10 mg / L, the remainder being water.

9. The application according to claim 7, characterized in that: The fermentation medium used in step (3) is: yeast powder 4g / L, peptone 3g / L, (NH4)2SO4 4g / L, K2HPO4·3H2O 4g / L, MgSO4·7H2O 2g / L, glutamic acid 4g / L, methionine 0.3g / L, citric acid 3g / L, lysine 2g / L, ammonium sulfate 4g / L, MnSO4·7H2O 10mg / L, FeSO4·7H2O 30mg / L, corn steep liquor 20ml / L, and the remainder is water.

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