Genetically engineered bacteria producing L-threonine and its construction method and application

Abstract

The present invention discloses a genetically engineered bacterium producing L-threonine and a construction method and application thereof. The present invention strengthens the biotin synthesis of the L-threonine producing strain by strengthening the bioABFCD gene (including promoter strengthening, RBS sequence strengthening, and multiple copies), relieving the transcriptional repression of the birA gene, and relieving the transcriptional repression of birA while strengthening bioABFCD. The constructed genetically engineered bacterium producing L-threonine can produce up to 20.2 g / L of L-threonine and a sugar-acid conversion rate of 23.8% after fermentation culture.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a genetic engineering bacterium producing L-threonine and a construction method and application thereof. Background Art

[0002] L-threonine is one of the 8 kinds of amino acids necessary for human and animal growth, and is widely used in feed, food additives and preparation of pharmaceutical auxiliary materials. At present, L-threonine is mainly produced by microbial fermentation, and various bacteria can be used for L-threonine production, such as mutants obtained by wild-type induction of Escherichia coli, Corynebacterium, Serratia, etc. as production strains. Specific examples include amino acid analogue-resistant mutants or various nutritional deficiency types such as methionine, lysine, and isoleucine (Japanese Patent Application Publication No. 224684 / 83; Korean Patent Application Publication No. 8022 / 87). However, traditional mutagenesis breeding is difficult to obtain high-yield strains due to slow growth of strains and production of more by-products caused by random mutations.

[0003] With the increasing global demand for threonine, the construction and transformation of high-yield threonine strains is particularly important. In 2003, CJ Co., Ltd. of South Korea applied for Chinese patent CN03811059.8, using Escherichia coli, by deleting the 39bp sequence from -56 to -18 of the threonine operon sequence, the expression of the key gene thrABC for threonine synthesis was enhanced, and the threonine productivity was increased by 22%. Kwang Ho Lee et al. (Kwang Ho Lee et al., Systems metabolic engineering of Escherichia coli for L-threonine production, Mol Syst Biol. 2007; 3: 149) et al. used a system metabolic engineering strategy to remove product feedback inhibition by mutating the genes thrA and lysC encoding aspartate kinases I and III, removing byproducts glycine and isoleucine by knocking out tdh and weakening ilvA, and providing more precursors for threonine synthesis by inactivating competitive pathway genes metA and lysA. The TH28C (pBRThrABCR3) strain finally obtained can produce 82.4 g / L of acid in 50 hours of fermentation, and the sugar-acid conversion rate is 39.3%. CN105543156A obtained the MHZ-0213-3 strain by strengthening thrA*BC and knocking out tdh. The strain has a threonine production of 4.2 g / L and a conversion rate of about 8.9%, and has no plasmid burden. CN106635945A obtained the MHZ-0215-2 strain by strengthening the pntAB gene and heterologously introducing the pyc gene in Escherichia coli. The strain has a threonine yield of 12.4 g / L, a conversion rate of about 16.2%, and no plasmid burden. Summary of the invention

[0004] The purpose of the present invention is to provide a novel genetically engineered bacterium producing L-threonine and a construction method and application thereof.

[0005] The present invention is conceived as follows: the present invention uses Escherichia coli MHZ-0215-2 as the starting strain (see ZL201611250306.8), and according to the metabolic pathway of L-threonine in Escherichia coli and the genetic background of the starting strain MHZ-0215-2, performs relevant modifications on its genome to strengthen the biotin synthesis of the strain. Specifically, it includes the enhancement and overexpression of key genes in the biotin synthesis pathway, for example, the expression of the biotin synthesis operon bioABFCD is enhanced, including replacing the original promoter with a strong promoter, or enhancing the strength of RBS and increasing the copy number of bioABFCD on the genome; relieving the transcriptional repression of the biotin synthesis operon bioABFCD by BirA (DNA binding transcription repressor / biotin-[acetyl-CoA-carboxylase] ligase); in addition, the above two modification methods are combined to synergistically increase the supply of biotin, a cofactor of pyruvate carboxylase (encoded by the pyc gene), so as to further improve the strain's ability to produce threonine.

[0006] In addition, the expression of enzymes related to the threonine synthesis pathway in Corynebacterium glutamicum (mainly pyruvate carboxylase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase) is enhanced, thereby improving the amino acid production capacity of Corynebacterium glutamicum, especially the production capacity of L-threonine; on this basis, the expression of the biotin synthesis birA gene is enhanced to further improve the threonine production capacity.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a genetically engineered bacterium producing L-threonine, wherein the genetically engineered bacterium producing L-threonine is obtained by using genetic engineering means to strengthen the biotin synthesis pathway of the strain.

[0008] The strain is a strain capable of producing threonine, preferably a bacterium in the genus Escherichia or Corynebacterium, more preferably Escherichia coli or Corynebacterium glutamicum.

[0009] The genetically engineered bacteria may be selected from any one of A to D:

[0010] A, a genetically enhanced strain obtained by enhancing the gene bioABFCD encoding the NCBI reference sequence NC_000913.3 (807968..812947) in the strain;

[0011] B, a genetically modified strain obtained by modifying the gene birA encoding the NCBI reference sequence NC_000913.3 (4173082..4174047) in the strain;

[0012] C. A genetically engineered bacterium obtained by enhancing the gene bioABFCD encoding the NCBI reference sequence NC_000913.3 (807968..812947) in the strain and weakening the gene birA encoding the NCBI reference sequence NC_000913.3 (4173082..4174047) in Escherichia coli;

[0013] D. Enhanced expression of birA (NCBI No.: cg0814, Cgl0709, NCgl067) in Corynebacterium glutamicum.

[0014] In the present invention, bioABFCD is an operon, and 5 genes are linked together, and the Gene IDs are 945376, 945370, 945384, 945388 and 945387. Sequence: NC_000913.3 (807968..812947, complement).

[0015] The enhanced approach can be selected from at least one of the following 1) to 3):

[0016] 1) Enhanced by operably linking a strong promoter to a biotin synthesis gene;

[0017] 2) Enhanced by increasing the copy number of biotin synthesis genes on chromosomes;

[0018] 3) Enhanced by increasing the strength of the RBS sequence of the biotin synthesis gene promoter on the chromosome;

[0019] The weakening includes knocking out or reducing the expression of the gene. The weakening method can be selected from at least one of mutagenesis, site-directed mutagenesis, homologous recombination, etc.

[0020] Considering that the enhancement and weakening of gene expression cannot be exhaustively listed through examples, the present invention only provides illustrative examples, and technicians can make adaptive adjustments according to specific circumstances during actual operations. Therefore, the enhancement and weakening referred to in the present invention include but are not limited to the contents disclosed in the embodiments.

[0021] The strong promoter can be selected from Ptac, Plac, Ptrp, Ptrc, Psod and the like.

[0022] The site-directed mutagenesis method can be selected from any one of S1 to S3:

[0023] S1. Perform site-directed mutagenesis on the birA gene, and after the mutation, the 57th amino acid of the protein encoded by the birA gene mutates from G to S;

[0024] S2. Performing site-directed mutagenesis on the birA gene, after which the 58th amino acid of the protein encoded by the birA gene mutates from Y to F;

[0025] S3. Perform site-directed mutagenesis on the birA gene. After the mutation, the 57th amino acid of the protein encoded by the birA gene mutates from G to S, and the 58th amino acid mutates from Y to F.

[0026] Preferably, the Escherichia coli is Escherichia coli MHZ-0215-2 with a deposit number of CGMCC No.13403.

[0027] Preferably, the Corynebacterium glutamicum is a model strain in which the expression of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine synthase and pyruvate carboxylase is enhanced.

[0028] In a second aspect, the present invention provides a method for constructing a genetically engineered bacterium that produces L-threonine, wherein the method is selected from the following schemes I to VII, or an optional combination thereof:

[0029] Scheme I: Using genetic engineering methods to operably link the strong promoter Ptac to the gene bioABFCD in Escherichia coli;

[0030] Scheme II: Use genetic engineering to increase the copy number of the gene bioABFCD in E. coli;

[0031] Scheme III: Use genetic engineering to increase the strength of the RBS sequence of the bioABFCD gene promoter in E. coli;

[0032] Scheme IV: using genetic engineering to perform site-directed mutation on the birA gene in Escherichia coli, so that the 57th amino acid of the protein encoded by the birA gene is mutated from G to S;

[0033] Scheme V: Using genetic engineering methods to perform site-directed mutation on the birA gene in Escherichia coli, the 58th amino acid of the protein encoded by the birA gene mutates from Y to F;

[0034] Scheme VI: Using genetic engineering methods to carry out site-directed mutation of the birA gene in Escherichia coli, after which the 57th amino acid of the protein encoded by the birA gene mutates from G to S and the 58th amino acid mutates from Y to F;

[0035] Scheme VII: Use genetic engineering to operably link the strong promoter Psod to the gene birA in Corynebacterium glutamicum.

[0036] Preferably, the method can be selected from any one of i to vii:

[0037] i. Using genetic engineering methods, the strong promoter Ptac was operably linked to the gene bioABFCD in Escherichia coli, and the gene birA in Escherichia coli was site-directed mutated. After the mutation, the 57th amino acid of the protein encoded by the gene birA was mutated from G to S;

[0038] ii. Using genetic engineering methods, the strong promoter Ptac was operably linked to the gene bioABFCD in Escherichia coli, and the gene birA in Escherichia coli was site-directed mutated. After the mutation, the 58th amino acid of the protein encoded by the gene birA was mutated from Y to F;

[0039] iii. Using genetic engineering methods to increase the copy number of the bioABFCD gene in E. coli, and at the same time performing site-directed mutation on the birA gene in E. coli, the 57th amino acid of the protein encoded by the birA gene was mutated from G to S;

[0040] iv. Using genetic engineering methods to increase the copy number of the bioABFCD gene in E. coli, and at the same time performing site-directed mutation on the birA gene in E. coli, the 58th amino acid of the protein encoded by the birA gene was mutated from Y to F;

[0041] v. Using genetic engineering methods to increase the strength of the RBS sequence of the promoter of the gene bioABFCD in E. coli, and at the same time performing site-directed mutation on the gene birA in E. coli, the 57th amino acid of the protein encoded by the gene birA was mutated from G to S;

[0042] vi. Using genetic engineering methods to increase the strength of the RBS sequence of the promoter of the gene bioABFCD in E. coli, and at the same time performing site-directed mutation on the gene birA in E. coli, the 58th amino acid of the protein encoded by the gene birA was mutated from Y to F;

[0043] vii. The strong promoter Psd is operably linked to birA in Corynebacterium glutamicum by genetic engineering to achieve the purpose of enhancing the expression of birA gene.

[0044] In a third aspect, the present invention provides a genetically engineered bacterium producing L-threonine constructed according to the above method.

[0045] In a fourth aspect, the present invention provides the use of the genetically engineered bacteria, or the genetically engineered bacteria constructed according to the above method, in the fermentation production of L-threonine.

[0046] The present invention only provides some examples of the enhanced expression of genes in the biotin synthesis pathway. Those skilled in the art may make certain changes to enhance the synthesis pathway, including but not limited to relieving feedback inhibition, enhancing the promoter, enhancing the ribosome binding site, increasing the copy number, etc. The above methods can enhance the enhancement of the biotin synthesis pathway and are all within the scope of protection of the present invention.

[0047] In addition, the expression of pyruvate carboxylase, aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine synthase in Corynebacterium glutamicum is enhanced, including but not limited to release of feedback inhibition, promoter enhancement, ribosome binding site enhancement, copy number increase, etc.

[0048] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0049] The present invention strengthens the biotin synthesis of the L-threonine production strain, and the specific means adopted are strengthening the bioABFCD gene (including promoter strengthening, RBS sequence strengthening, and multiple copies), releasing the transcriptional repression of the birA gene, and simultaneously strengthening the bioABFCD and releasing the transcriptional repression of birA. The constructed L-threonine-producing genetically engineered bacteria can produce up to 20.2 g / L of L-threonine and a sugar-acid conversion rate of 23.8% after fermentation culture. The present invention is also applicable to increasing the production and sugar-acid conversion rate of aspartic acid family amino acids such as aspartic acid, lysine, homoserine, methionine, isoleucine, etc. In addition, the present invention is also applicable to other enzymes that use biotin as a cofactor to improve the enzyme activity and thus enhance the synthesis path of amino acids. DETAILED DESCRIPTION

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or the conditions recommended by the manufacturer's instructions.

[0051] The reagents used in the following examples are all commercially available. The parent strain of the high conversion rate threonine production strain provided by the present invention is MHZ-0215-2, which belongs to W3110 (Escherichia). The deposit number of Escherichia coli MHZ-0215-2 is CGMCC No.13403, see ZL201611250306.8; Corynebacterium glutamicum is constructed from a wild strain.

[0052] The primer sequences used in the examples are shown in Table 1.

[0053] Table 1

[0054]

[0055]

[0056] The gene names involved in the present invention are explained as follows:

[0057] bioA: adenosylmethionine-8-amino-7-oxonanoate aminotransferase;

[0058] bioB: biotin synthetase;

[0059] bioF: 8-amino-7-oxononanoate synthase;

[0060] bioC: malonyl acyl carrier protein methyltransferase;

[0061] bioD: dethiobiotin synthetase;

[0062] birA: DNA-binding transcriptional repressor / biotin-[acetyl-CoA-carboxylase] ligase;

[0063] pyc: pyruvate carboxylase;

[0064] Example 1 Preparation of strain MHZ-0215-3 with enhanced bioABFCD gene (promoter enhancement)

[0065] (1) Construction of pTargetF-N20 (tac-bioABFCD) plasmid and Donor DNA-1

[0066] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), select the pTF-sgRNA-F1 / pTF-sgRNA-R1 primer pair to amplify the pTF linear plasmid with N20, assemble the linear plasmid at 37°C using the seamless assembly ClonExpress kit, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (tac-bioABFCD), and perform PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, select bioAtac-UF / bioAtac-UR1, bioAtac-UF / bi oAtac-UR2 primer pair was used to amplify the upstream homology arm containing tac promoter ①; Step 3: Using W3110 genome as template, PtacI-F / PtacI-R primer pair was selected to amplify tac promoter ②; Step 4: Using W3110 genome as template, bioBtac-DF1 / bioBtac-DR and bioBtac-DF2 / bioBtac-DR primer pairs were selected to amplify the downstream homology arm containing tac promoter ③; Step 5: Using ①, ②, ③ as templates, bioAtac-UF / bioBtac-DR primer pair was selected to amplify the up-Ptac-down fragment, also known as Donor DNA-1.

[0067] (2) Preparation of competent cells and electroporation

[0068] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650After the p-value is 0.4, prepare the competent cells for electroporation (refer to "Molecular Cloning III" for the competent cell preparation method). Step 3: The pTargetF-N20 (tac-bioABFCD) plasmid and Donor DNA-1 constructed in (1) are simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0069] (3) Recombination verification

[0070] Step 1: Use the primer pair bioAtac-F / bioBtac-R to perform colony PCR verification on the above single colony; Step 2: Use the primer pair bioAtac-F / bioBtac-R to amplify the target fragment, and send the amplified product for sequencing to verify the integrity of the sequence.

[0071] (4) Construction-related plasmids are lost

[0072] Step 1: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing at 30°C overnight, streak it onto an LB plate containing kanamycin. Step 2: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it at 30°C overnight. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (tac-bioABFCD) plasmid has been lost. Step 3: Pick pTargetF- The positive colonies with N20 (tac-bioABFCD) plasmid loss were inoculated into non-antibiotic LB test tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight; Step 4: Pick single colonies and spot them on LB plates containing kanamycin and LB plates without antibiotics. If they cannot grow on LB plates containing kanamycin but grow on LB plates without antibiotics, it indicates that the pCas plasmid has been lost, and the MHZ-0215-3 (Ptac-bioA / Ptac-BFCD) strain was obtained.

[0073] Example 2 Preparation of bioABFCD gene RBS enhanced strain MHZ-0215-4 (RBS-bioABFCD)

[0074] (1) Construction of pTargetF-N20 (RBS bioABFCD) plasmid and Donor DNA-2

[0075] Step 1: Using pTargetF plasmid as template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), select pTF-sgRNA-F1 / pTF-sgRNA-R1 primer pair to amplify pTF linear plasmid with N20, assemble this linear plasmid at 37°C using seamless assembly ClonExpress kit, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (RBS bioABFCD), and PCR identification and sequencing verification were performed; step 2: using the W3110 genome as a template, the bioA-RBS-UF / bioA-RBS-UR primer pair was selected to amplify the upstream homology arm containing RBS ①; step 3: using the W3110 genome as a template, the bioAp-F / bioAp-R, bioAp-F / bioABp-R, bioAp-F / bioBp-R1, bioAp-F / bioBp-R2 primer pairs were selected to amplify the bioA / bioB promoter containing RBS ②; step 4: using the W3110 genome as a template, the bioB-RBS-DF / bioB-RBS-DR primer pair was selected to amplify the downstream homology arm containing RBS ③; step 5: using ①, ②, ③ as templates, the bioA-RBS-UF / bioB-RBS-DR primer pair was selected to amplify the up-RBS-down fragment, also known as DonorDNA-2.

[0076] (2) Preparation of competent cells and electroporation

[0077] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650After the p-value is 0.4, prepare the competent cells for electroporation (refer to "Molecular Cloning III" for the competent cell preparation method). Step 3: The pTargetF-N20 (RBS bioABFCD) plasmid and Donor DNA-2 constructed in (1) are simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0078] (3) Recombination verification

[0079] Step 1: Use the primer pair bioA-RBS-F / bioB-RBS-R to perform colony PCR verification on the above single colony; Step 2: Amplify the strain identified correctly by PCR with the primer pair bioA-RBS-F / bioB-RBS-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0080] (4) Construction-related plasmids are lost

[0081] Step 1: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 2: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (RBSbioABFCD) plasmid has been lost. Step 3: Pick pTargetF-N20 (RBS The positive colonies with the pCas plasmid lost were inoculated into non-antibiotic LB test tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight. Step 4: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0215-4 (RBS-bioABFCD) strain was obtained.

[0082] Example 3 Preparation of strain MHZ-0215-5 (multi-copy) with enhanced bioABFCD gene

[0083] (1) Construction of pTargetF-N20(IS1::bioABFCD) plasmid and Donor DNA-3

[0084] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), select the pTF-sgRNA-F2 / pTF-sgRNA-R2 primer pair to amplify the pTF linear plasmid with N20, assemble the linear plasmid at 37°C using the seamless assembly ClonExpress kit, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (IS1:: bioABFCD), and perform PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, select the bioABFCD2-UF / bioABFCD2-UR primer pair to amplify the upstream homology arm ①; Step 3: Using the W3110 genome as a template, select Use the bioABFCD2-F1 / bioABFCD2-R1 primer pair to amplify the upper half of the bioABFCD operon ②; Step 4: Using the W3110 genome as a template, select the bioABFCD2-F2 / bioABFCD2-R2 primer pair to amplify the lower half of the bioABFCD operon ③; Step 5: Using the W3110 genome as a template, select the bioABFCD2-DF / bioABFCD2-DR primer pair to amplify the downstream homology arm ④; Step 6: Using ①, ②, ③, and ④ as templates, select the bioABFCD2-UF / bioABFCD2-DR primer pair to amplify the up-bioABFCD2-down fragment, also known as Donor DNA-3.

[0085] (2) Preparation of competent cells and electroporation

[0086] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650After the p-value is 0.4, prepare the competent cells for electroporation (refer to "Molecular Cloning III" for the competent cell preparation method). Step 3: The pTargetF-N20 (IS1:: bioABFCD) plasmid and Donor DNA-3 constructed in (1) are simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0087] (3) Recombination verification

[0088] Step 1: Use the primer pair bioABFCD2-F / bioABFCD2-R to perform colony PCR verification on the above single colony; Step 2: Amplify the strain identified correctly by PCR with the primer pair bioA-RBS-F / bioB-RBS-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0089] (4) Construction-related plasmids are lost

[0090] Step 1: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 2: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (IS1:: bioABFCD) plasmid has been lost. Step 3: Pick pTarget The positive colonies with lost tF-N20 (IS1:: bioABFCD) plasmid were inoculated into antibiotic-free LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight. Step 4: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0215-5 (IS1:: bioABFCD) strain was obtained.

[0091] Example 4 Preparation of strain MHZ-0215-6 (birA point mutation G57S) that relieves transcriptional repression of bioABFCD gene by BirA

[0092] (1) Construction of pTargetF-N20 (birA G57S) plasmid and Donor DNA-4

[0093] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), select the pTF-birA-sgRNA-F / pTF-birA-sgRNA-R primer pair to amplify the pTF linear plasmid with N20, assemble the linear plasmid at 37°C using the seamless assembly ClonExpress kit, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (birA G57S), and PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, the birAG57S-UF / birAG57S-UR primer pair was selected to amplify the upstream homology arm ①; Step 3: Using the W3110 genome as a template, the birAG57S-DF / birAG57S-DR primer pair was selected to amplify the downstream homology arm ②; Step 4: Using ① and ② as templates, the birAG57S-UF / birAG57S-DR primer pair was selected to amplify the up-down fragment, also known as Donor DNA-4.

[0094] (2) Preparation of competent cells and electroporation

[0095] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the competent cells for electroporation (refer to "Molecular Cloning III" for the competent cell preparation method). Step 3: The pTargetF-N20 (birA G57S) plasmid and DonorDNA-4 constructed in (1) are simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0096] (3) Recombination verification

[0097] Step 1: Use the primer pair birAG57S-F1 / birAG57S-R to perform colony PCR verification on the above single colony; Step 2: Amplify the strain correctly identified by PCR with the primer pair birAG57S-F / birAG57S-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0098] (4) Construction-related plasmids are lost

[0099] Step 1: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing at 30°C overnight, streak it onto an LB plate containing kanamycin. Step 2: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it at 30°C overnight. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birAG57S) plasmid has been lost. Step 3: Pick pTargetF-N20 (birA The positive colonies with the pCas plasmid lost were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight. Step 4: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0215-6 (birA G57S) strain was obtained.

[0100] Example 5 Preparation of strain MHZ-0215-7 (birA point mutation Y58F) that relieves transcriptional repression of bioABFCD gene by BirA

[0101] (1) Construction of pTargetF-N20 (birA Y58F) plasmid and Donor DNA-5

[0102] Step 1: Using the pTargetF plasmid as a template (see Multigene Editing in the Escherichiacoli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015), select the pTF-birA-sgRNA-F / pTF-birA-sgRNA-R primer pair to amplify the pTF linear plasmid with N20, assemble the linear plasmid at 37°C using the seamless assembly ClonExpress kit, and then transform Trans1-T1 competent cells to obtain pTargetF-N20 (birA Y58F), and PCR identification and sequencing verification; Step 2: Using the W3110 genome as a template, the birAG57S-UF / birAY58F-UR primer pair was selected to amplify the upstream homology arm ①; Step 3: Using the W3110 genome as a template, the birAY58F-DF / birAG57S-DR primer pair was selected to amplify the downstream homology arm ②; Step 4: Using ① and ② as templates, the birAG57S-UF / birAG57S-DR primer pair was selected to amplify the up-down fragment, also known as Donor DNA-5.

[0103] (2) Preparation of competent cells and electroporation

[0104] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-2 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-2 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the competent cells for electroporation (refer to "Molecular Cloning III" for the competent cell preparation method). Step 3: The pTargetF-N20 (birA Y58F) plasmid and DonorDNA-5 constructed in (1) are simultaneously electroporated into MHZ-0215-2 (pCas) competent cells (electroporation conditions: 2.5 kV, 200 Ω, 25 μF), spread on LB plates containing spectinomycin and kanamycin, and culture at 30°C until single colonies are visible.

[0105] (3) Recombination verification

[0106] Step 1: Use the primer pair birAY58F-F1 / birAG57S-R to perform colony PCR verification on the above single colony; Step 2: Amplify the strain correctly identified by PCR with the primer pair birAG57S-F / birAG57S-R, and send the amplified product for sequencing to verify the integrity of the sequence.

[0107] (4) Construction-related plasmids are lost

[0108] Step 1: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing at 30°C overnight, streak it onto an LB plate containing kanamycin. Step 2: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it at 30°C overnight. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birAY58F) plasmid has been lost. Step 3: Pick pTargetF-N20 (birA The positive colonies with plasmid loss were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight. Step 4: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0215-7 (birA Y58F) strain was obtained.

[0109] Example 6 Preparation of strains with enhanced bioABFCD genes and simultaneous introduction of birA gene point mutations

[0110] According to the method of Examples 1-5, different transformation methods were superimposed on the bioABFCD and birA sites, and a total of 6 transformed bacteria (MHZ-0217-0 to MHZ-0217-5) were obtained.

[0111] (1) Construction of MHZ-0217-0 (Ptac-bioABFCD, birA G57S) strain

[0112] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-3 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-3 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the competent cells for electrotransformation (refer to "Molecular Cloning III" for the competent cell preparation method); Step 3: The pTargetF-N20 (birA G57S) plasmid and Donor DNA-4 constructed in Example 4 are simultaneously electrotransformed into MHZ-0215-3 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAG57S-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA G57S) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA The positive colonies that showed the loss of pCas plasmid G57S) were inoculated into non-antibiotic LB test tubes, cultured at 42°C for 8h, then streaked onto LB plates and cultured at 37°C overnight; Step 9: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0217-0 (Ptac-bioABFCD, birA G57S) strain was obtained.

[0113] (2) Construction of MHZ-0217-1 (Ptac-bioABFCD, birA Y58F) strain

[0114] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-3 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-3 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the electrotransformation competent cells (refer to "Molecular Cloning III" for the preparation method of competent cells); Step 3: The pTargetF-N20 (birA Y58F) plasmid and Donor DNA-5 constructed in Example 5 are simultaneously electrotransformed into MHZ-0215-3 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAY58F-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA Y58F) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA Y58F) positive colonies with plasmid loss were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8h, then streaked onto LB plates and cultured at 37°C overnight; Step 9: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0217-1 (Ptac-bioABFCD, birA Y58F) strain was obtained.

[0115] (3) Construction of MHZ-0217-2 (RBS-bioABFCD, birA G57S) strain

[0116] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-4 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-4 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the electrotransformation competent cells (refer to "Molecular Cloning III" for the preparation method of competent cells); Step 3: The pTargetF-N20 (birAG57S) plasmid and Donor DNA-4 constructed in Example 4 are simultaneously electrotransformed into MHZ-0215-4 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAG57S-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA G57S) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA The positive colonies that showed the loss of pCas plasmid G57S) were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight; Step 9: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0217-2 (RBS-bioABFCD, birA G57S) strain was obtained.

[0117] (4) Construction of MHZ-0217-3 (RBS-bioABFCD, birA Y58F) strain

[0118] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-4 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-4 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the electrotransformation competent cells (refer to "Molecular Cloning III" for the preparation method of competent cells); Step 3: The pTargetF-N20 (birA Y58F) plasmid and Donor DNA-5 constructed in Example 5 are simultaneously electrotransformed into MHZ-0215-4 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAY58F-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA Y58F) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA Y58F) positive colonies with plasmid loss were inoculated into non-antibiotic LB test tubes, cultured at 42°C for 8h, then streaked onto LB plates and cultured at 37°C overnight; Step 9: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0217-3 (RBS-bioABFCD, birA Y58F) strain was obtained.

[0119] (5) Construction of MHZ-0217-4 (IS1::bioABFCD, birA G57S) strain

[0120] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-5 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-5 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650 After the p-value is 0.4, prepare the electrotransformation competent cells (refer to "Molecular Cloning III" for the preparation method of competent cells); Step 3: The pTargetF-N20 (birA G57S) plasmid and Donor DNA-4 constructed in Example 4 are simultaneously electrotransformed into MHZ-0215-5 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAG57S-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA G57S) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA The positive colonies that showed the loss of pCas plasmid were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight. Step 9: Pick a single colony and spot it onto an LB plate containing kanamycin and an LB plate without antibiotics. If it cannot grow on the LB plate containing kanamycin but grows on the LB plate without antibiotics, it indicates that the pCas plasmid is lost, and the MHZ-0217-4 (IS1::bioABFCD,birA G57S) strain is obtained.

[0121] (6) Construction of MHZ-0217-5 (IS1::bioABFCD, birA Y58F) strain

[0122] Step 1: Electroporate the pCas plasmid (see Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al. Appl. Environ Microbiol, 2015) into MHZ-0215-5 competent cells (the transformation method and competent preparation method are both referred to "Molecular Cloning III"); Step 2: Pick a single colony of MHZ-0215-5 (pCas) in a 5 mL LB test tube containing kanamycin and a final concentration of 10 mM arabinose, and culture at 30 ° C 200 r / min until OD 650After the p-value is 0.4, prepare the competent cells for electrotransformation (refer to "Molecular Cloning III" for the competent cell preparation method); Step 3: The pTargetF-N20 (birA Y58F) plasmid and Donor DNA-5 constructed in Example 5 are simultaneously electrotransformed into MHZ-0215-5 (pCas) competent cells (electroporation conditions: 2.5kV, 200Ω, 25μF), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30°C until single colonies are visible. Step 4: Use the primer pair birAY58F-F1 / birAG57S-R to perform colony PCR verification on the above single colonies; Step 5: The strain identified correctly by PCR is amplified with the primer pair birAG57S-F / birAG57S-R, and the amplified product is sent for sequencing to verify the integrity of the sequence. Step 6: Pick a single colony that has been verified by sequencing and inoculate it into a 5 mL LB test tube containing kanamycin and a final concentration of 0.5 mM IPTG. After culturing overnight at 30°C, streak it onto an LB plate containing kanamycin. Step 7: Pick a single colony and spot it onto an LB plate containing kanamycin and spectinomycin and an LB plate containing only kanamycin. Cultivate it overnight at 30°C. If it cannot grow on an LB plate containing kanamycin and spectinomycin but grows on an LB plate containing kanamycin, it indicates that the pTargetF-N20 (birA Y58F) plasmid has been lost. Step 8: Pick pTargetF-N20 (birA Y58F) positive colonies with plasmid loss were inoculated into non-antibiotic LB tubes, cultured at 42°C for 8 h, then streaked onto LB plates and cultured at 37°C overnight; Step 9: single colonies were picked and spotted onto LB plates containing kanamycin and LB plates without antibiotics. If they could not grow on LB plates containing kanamycin but grew on LB plates without antibiotics, it indicated that the pCas plasmid was lost, and the MHZ-0217-5 (IS1::bioABFCD,birA Y58F) strain was obtained.

[0123] The threonine-producing genetically modified strains obtained in Examples 1-6 are shown in Table 2.

[0124] Table 2 Genetically engineered bacteria constructed by the present invention

[0125]

[0126] Example 7 Shake flask fermentation verification of L-threonine-producing genetically engineered bacteria

[0127] Step 1: Take MHZ-0215-2, MHZ-0215-3, MHZ-0215-4, MHZ-0215-5, MHZ-0215-6, MHZ-0215-7, MHZ-0217-0, MHZ-0217-1, MHZ-0217-2, MHZ-0217-3, MHZ-0217-4, and MHZ-0217-5 from the cryopreservation tube, streak on the LB plate for activation, and culture at 37°C for 18-24h; Step 2: Scrape a loop of the bacteria from the plate and inoculate it into a shake flask containing 50mL of seed culture medium (Table 3), culture at 37°C, 90rpm, for about 5 hours, and make the OD 650 Step 3: Transfer 2 mL of seed solution to a shake flask containing 20 mL of fermentation medium (Table 4), and culture on a reciprocating shaker at 37°C and 100 rpm until the residual sugar is exhausted. After the fermentation is completed, measure the sample OD 650 The L-threonine content was determined by HPLC, and the residual sugar content was determined by biosensor method. In order to ensure the reliability of the experiment, the shake flask was repeated 3 times, and the average values ​​of the acid production and conversion rate results are shown in Table 5.

[0128] Table 3 Seed culture medium (g / L)

[0129] Element concentration glucose 25 Corn Syrup 25 Soybean meal hydrolyzate 7.7 Yeast extract 2.5 <![CDATA[KH2PO4]]> 1.4 Magnesium sulfate heptahydrate 0.5 <![CDATA[FeSO4、MnSO4]]> 20mg / L pH 7.0

[0130] Table 4 Fermentation medium (g / L)

[0131] Element concentration glucose 85 Corn Syrup 6 Soybean meal hydrolyzate 7.7 Magnesium sulfate heptahydrate 0.5 <![CDATA[KH2PO4]]> 1.0 Aspartic acid 10 <![CDATA[FeSO4、MnSO4]]> 30mg / L Biotin 50μg Thiamine 500 μg pH 7.2

[0132] Table 5 Comparison of productivity of threonine-producing genetically engineered bacteria

[0133]

[0134]

[0135] Note: * indicates P value < 0.01, indicating that there is a significant difference compared with the control.

[0136] As shown in Table 5, the L-threonine production of the novel genetically engineered Escherichia coli provided by the present invention is higher than that of the respective control strains, wherein the sugar-acid conversion rate of the strain with enhanced expression activity of bioABFCD in the shake flask is between 18-19%, which is 1.6-2.7 percentage points higher than that of the starting strain; the shake flask conversion rate of the strain with birA released from transcriptional repression is also between 18-19%, which is 2.2-3 percentage points higher than that of the starting strain. From the shake flask fermentation results of the two site modifications, it can be concluded that the enhanced expression of the biotin synthesis operon bioABFCD or the release of the transcriptional repression of bioABFCD by BirA can significantly improve the production capacity of L-threonine.

[0137] The strains with two sites modified at the same time had a shake flask fermentation conversion rate between 21-24%, and their L-threonine conversion rate was further improved. Among them, the best performing strain MHZ-0217-4 had a yield of 20.2g / L and a conversion rate of 23.8%, while the starting strain MHZ-0215-2 had an L-threonine yield of 13.8g / L and a conversion rate of 16.2%. The L-threonine yield of MHZ-0217-4 increased by 46.38% compared with the starting strain, and the conversion rate increased by 46.91%. It can be seen that the L-threonine production capacity of the genetically engineered bacteria is significantly better than that of MHZ-0214-2.

[0138] Example 8 Construction of auxiliary plasmids required for genome transformation of Corynebacterium glutamicum

[0139] 1. Plasmid pK18mobsacB-P for enhanced expression of aspartate kinase sod -lysC V1M-T311I

[0140] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream homology arm up was obtained by PCR amplification with the primer pair P21 / P22, and the promoter fragment P was obtained by PCR amplification with the primer pair P23 / P24. sod lysC was obtained by PCR amplification using primer pair P25 / P26. V1M-T311I The downstream homology arm dn was obtained by PCR amplification with the primer pair P27 / P28. sod Fusion PCR was performed as a template to obtain the fragment up-P sod . The P21 / P28 primer pair was used to up-P sod ,lysC V1M -T311I , dn as templates for fusion PCR to obtain the full-length fragment up-P sod -lysC V1M-T311I -dn. pK18mobsacB was digested with BamHI / HindIII. The two were assembled using a seamless cloning kit and transformed into Trans1 T1 competent cells to obtain the recombinant plasmid pK18mobsacB-P sod -lysC V1M-T311I .

[0141] 2. Plasmid pK18mobsacB-P for enhanced expression of aspartate semialdehyde dehydrogenase sod -asd

[0142] The plasmid construction method was the same as above, and the primers used were: P1, P2, P3, P4, P5, and P6.

[0143] 3. Plasmid pK18mobsacB-P for enhanced expression of homoserine dehydrogenase cspB -hom G378E

[0144] The plasmid construction method was the same as above, and the primers used were: P29, P30, P31, P32, P33, P34, P35, and P36.

[0145] 4. Plasmid pK18mobsacB-P for enhanced expression of homoserine kinase cspB -thrB

[0146] The plasmid construction method was the same as above, and the primers used were: P7, P8, P9, P10, P11, and P12.

[0147] 5. Plasmid pK18mobsacB-P for enhanced expression of threonine synthase sod -thrC V1M

[0148] The plasmid construction method was the same as above, and the primers used were: P37, P38, P39, P40, P41, and P42.

[0149] 6. Plasmid pK18mobsacB-P for enhanced expression of pyruvate carboxylase sod -pyc P458S

[0150] The plasmid construction method is the same as above, and the primers used are: P13, P14, P15, P16, P17, P18, P19, P207, and the plasmid pK18mobsacB-P tuf -birA

[0151] The plasmid construction procedures were the same as above, and the primers used were: P43, P44, P45, P46, P47, and P48.

[0152] The primer sequences used in plasmid construction are shown in Table 6.

[0153] Table 6

[0154]

[0155]

[0156] Example 9 Construction of engineered bacteria of Corynebacterium glutamicum

[0157] 1. Construction of strains with enhanced expression of aspartate kinase

[0158] ATCC13032 competent cells were prepared according to the classic method of C. glutamicum (C. glutamicum Handbook, Chart 23). Recombinant plasmid pK18mobsacB-Psod-lysC V1M-T311I The competent cells were transformed by electroporation and the transformants were screened on a selective medium containing 15 mg / L kanamycin, in which the gene of interest was inserted into the chromosome due to homology. The screened transformants were cultured overnight in a normal liquid brain heart infusion medium at 30°C and shaken at 220 rpm on a rotary shaker. During this culture process, the transformants underwent a second recombination, and the vector sequence was removed from the genome by gene exchange. The culture was diluted in a series of gradients (10 -2 Serial dilution up to 10 -4 ), the dilution was spread on a common solid brain heart infusion medium containing 10% sucrose and cultured at 33°C for 48 hours. The strain grown on the sucrose medium did not carry the inserted vector sequence in its genome. The target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was named SMCT039.

[0159] 2. Construction of aspartate semialdehyde dehydrogenase enhanced strain

[0160] The strain construction method was the same as above, and aspartate semialdehyde dehydrogenase was enhanced on SMCT039, and the obtained transformed strain was named SMCT040.

[0161] 3. Construction of homoserine dehydrogenase enhanced strain

[0162] The strain construction method was the same as above, and homoserine dehydrogenase was enhanced on SMCT040, and the obtained transformed strain was named SMCT041.

[0163] 4. Construction of homoserine kinase-enhanced strains

[0164] The strain construction method was the same as above, and homoserine kinase enhancement was performed on SMCT041, and the obtained transformed strain was named SMCT042.

[0165] 5. Construction of threonine synthase enhanced strain

[0166] The strain construction method was the same as above. The threonine synthase enhanced strain was constructed on SMCT042, and the obtained transformed strain was named SMCT043.

[0167] 6. Construction of pyruvate carboxylase enhanced strain

[0168] The strain construction method was the same as above. The pyruvate carboxylase enhanced strain was constructed on SMCT043, and the obtained transformed strain was named SMCT044.

[0169] 7. Construction of strains with enhanced biotin synthesis pathway

[0170] The strain construction method was the same as above, except that the bifunctional biotin ligase / biotin operon repressor birA promoter was replaced on SMCT044, and the resulting transformed strain was named SMCT045.

[0171] The engineered strains are shown in Table 7:

[0172] Table 7

[0173] Strain name genotype SMCT039 <![CDATA[ATCC13032,P sod -C court V1M-T311I ]]> SMCT040 <![CDATA[ATCC13032,P sod -lysC V1M-T311I ,P sod -asd]]> SMCT041 <![CDATA[ATCC13032,P sod -C court V1M-T311I ,P sod -asd,PcspB-hom G378E ]]> SMCT042 <![CDATA[ATCC13032,P sod -lysC V1M-T311I ,P sod -asd,PcspB-hom G378E ,P cspB -thrB]]> SMCT043 <![CDATA[ATCC13032,P sod -lysC V1M-T311I ,P sod -asd,PcspB-hom G378E ,P cspB -thrB,P sod -thrC V1M ]]> SMCT044 <![CDATA[ATCC13032,P sod -lysC V1M-T311I ,P sod -asd,PcspB-hom G378E ,P cspB -thrB,P sod -thrC V1M ,P sod -pyc P458S ]]> SMCT045 <![CDATA[ATCC13032,P sod -lysC V1M-T311I ,P sod -asd,PcspB-hom G378E ,P cspB -thrB,P sod -thrC V1M ,P sod -pyc P458S ,P tuf -birA]]>

[0174] Example 10 Performance verification of L-threonine production by engineered bacteria of Corynebacterium glutamicum

[0175] 1. Culture medium

[0176] Seed activation medium: BHI 3.7%, agar 2%, pH 7.

[0177] Seed culture medium: peptone 5 / L, yeast extract 5g / L, sodium chloride 10g / L, ammonium sulfate 16g / L, urea 8g / L, potassium dihydrogen phosphate 10.4g / L, potassium hydrogen phosphate 21.4g / L, biotin 5mg / L, magnesium sulfate 3g / L. Glucose 50g / L, pH 7.2.

[0178] Fermentation medium: corn steep liquor 50 mL / L, glucose 45 g / L, ammonium sulfate 4 g / L, MOPS 30 g / L, potassium dihydrogen phosphate 10 g / L, urea 20 g / L, biotin 10 mg / L, magnesium sulfate 6 g / L, ferrous sulfate 1 g / L, VB1·HCl 40 mg / L, calcium pantothenate 50 mg / L, nicotinamide 40 mg / L, manganese sulfate 1 g / L, zinc sulfate 20 mg / L, copper sulfate 20 mg / L, pH 7.2.

[0179] 2. Production of L-threonine by Shake Flask Fermentation of Engineered Bacteria

[0180] (1) Seed culture: Select SMCT021, SMCT023, SMCT031, SMCT033, SMCT034, SMCT035, SMCT036, and SMCT037 slant seeds and inoculate them into a 500-mL Erlenmeyer flask containing 20 mL of seed culture medium. Culture them at 30°C and 220 rpm for 16 h.

[0181] (2) Fermentation culture: 2 mL of seed solution was inoculated into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium and cultured at 33 °C and 220 rpm for 24 h.

[0182] (3) 1 mL of the fermentation broth was centrifuged (12000 rpm, 2 min), the supernatant was collected, and L-threonine in the fermentation broth of the engineered bacteria and the control bacteria was detected by HPLC. The results are shown in Table 8:

[0183] Table 8 Comparison of threonine production capacity of Corynebacterium glutamicum

[0184]

[0185] It can be seen that the threonine production of the strain after the biotin synthesis pathway was strengthened increased from 6.4 g / L to 8.3 g / L, an increase of 30%, indicating that after the terminal threonine synthesis pathway was opened, the improvement of biotin synthesis ability can significantly enhance the strain's ability to produce threonine.

[0186] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A genetically engineered bacterium producing L-threonine, characterized in that: The L-threonine-producing genetically engineered bacteria are obtained by genetic engineering by strengthening Escherichia coli ( Escherichia coli ) biotin synthesis pathway to obtain engineered bacteria, By enhancing the gene encoding NCBI reference sequence NC_000913.3 in Escherichia coli bioABFCD , while simultaneously removing the gene encoding the NCBI reference sequence NC_000913.3 in E. coli birA The transcriptional repression of biotin synthesis, the genetically engineered bacteria obtained, The enhanced approach is selected from at least one of the following 1) to 3): 1) By combining a strong promoter with the bioABFCD Genes are operably linked for enhancement; 2) By increasing the amount of bioABFCD The number of copies of the gene is increased; 3) By increasing the amount of bioABFCD The strength of the RBS sequence in the gene promoter is enhanced; Disabling E. coli genes birA Transcriptional repression of biotin synthesis was obtained by site-directed mutagenesis. birA The 57th amino acid of the encoded protein mutated from G to S. The Escherichia coli is Escherichia coli MHZ-0215-2 with a deposit number of CGMCC No.13403.

2. The genetically engineered bacterium according to claim 1, characterized in that: The strong promoter is selected from Ptac, Plac, Ptrp, Ptrc or Psod.

3. A method for constructing a genetically engineered bacterium producing L-threonine, characterized in that: The method comprises: using genetic engineering means to modify the gene in Escherichia coli birA Perform site-directed mutagenesis. birA The 57th amino acid of the encoded protein mutated from G to S. The method is selected from any one of i, iii, and v: i. Use genetic engineering to combine the strong promoter Ptac with the gene in Escherichia coli. bioABFCD Operably linked to the gene in Escherichia coli birA Perform site-directed mutagenesis. birA The 57th amino acid of the encoded protein mutated from G to S; iii. Use genetic engineering to increase the number of genes in Escherichia coli bioABFCD The copy number of genes in Escherichia coli birA Perform site-directed mutagenesis. birA The 57th amino acid of the encoded protein mutated from G to S; v. Use genetic engineering to increase the number of genes in Escherichia coli bioABFCD The strength of the promoter's RBS sequence and the gene birA Perform site-directed mutagenesis. birA The 57th amino acid of the encoded protein mutated from G to S; The Escherichia coli is Escherichia coli MHZ-0215-2 with a deposit number of CGMCC No.13403.

4. A genetically engineered bacterium producing L-threonine constructed according to the method of claim 3.

5. Use of the genetically engineered bacteria according to claim 1 or 2 or the genetically engineered bacteria according to claim 4 in the fermentation production of L-threonine.

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