Method for producing abinogen by co-culture of recombinant corynebacterium glutamicum and bacillus subtilis mixed bacteria system

Abstract

The invention discloses a method for co-culturing a recombinant corynebacterium glutamicum and bacillus subtilis mixed bacterium system to produce abrumogen, which comprises the following steps of: simultaneously inoculating secondary seeds of recombinant corynebacterium glutamicum cgb-8 according to OD600 = 0.6 and secondary seeds of bacillus subtilis CGF26-IV according to OD600 = 0.2 into a culture medium A; the recombinant corynebacterium glutamicum cgb-8 and bacillus subtilis CGF26-IV mixed bacterium system is obtained, fermentation is carried out, and the abrumogen is obtained.According to the method, the metabolic burden of producing abrumogen through single bacillus subtilis is weakened, and a required amino acid precursor is provided for synthesis of abrumogen through bacillus subtilis. According to the method disclosed by the invention, all florae are clear in division of labor, the product-producing bacteria concentrate on working by utilizing a precursor generated by the precursor-synthesizing bacteria, so that the yield of the abelegen is obviously improved, and the yield of the abelegen reaches 1764mg / L or above by adopting the method disclosed by the invention.

Description

Technical Field

[0001] The invention relates to a method for producing fengycin by co-culturing a mixed bacterial system of recombinant Corynebacterium glutamicum and Bacillus subtilis, and belongs to the field of microbial co-culture fermentation. Background Art

[0002] Fengyuansu is one of the cyclic lipopeptide molecules produced by Bacillus. It has a significant inhibitory effect on the growth of filamentous fungi and has great biological control value. The synthesis process is completed under the action of non-ribosomal peptide synthetase (NRPS). The peptide ring in the structure is a ten-membered peptide, and the typical amino acid sequence is: Glu-Orn-Tyr-Thr-Glu-Ala / Val-Pro-Gln-L-Tyr-Ile. Due to its low yield and high production cost, its scope of application is limited. Therefore, people are constantly exploring effective strategies to increase the yield of lipopeptides. At present, research on increasing the yield of Fengyuansu mainly focuses on exogenous regulation and endogenous regulation. Amino acids and fatty acids are important components of lipopeptide structures. Exogenous addition of amino acids (Chen XY, Sun HZ, Qiao B, Miao CH, Hou ZJ, Xu SJ, Xu QM, ChengJS. Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with the recombinant Corynebacterium glutamicum producing high-level proline. Bioresour Technol. 2022, 349: 126863.) can not only significantly increase the lipopeptide level of Bacillus, but also change the ratio of lipopeptide homologues. At the same time, enhancing the synthesis of Fengycin through endogenous regulation is also one of the research hotspots. The transformation of promoters, regulatory factors, metabolic pathways, etc. has further enhanced the synthesis of Fengycin (Gao GR, Hou ZJ, Ding MZ, Bai S, Wei SY, Qiao B, Xu QM, Cheng JS, Yuan YJ. Improved Production of Fengycin in Bacillus subtilis by Integrated Strain Engineering Strategy. ACS synthetic biology. 2022; 11(12): 4065-4076.).

[0003] Amino acids are important precursors for the synthesis of fengycin. Corynebacterium glutamicum is an excellent base strain for the production of amino acids. Corynebacterium glutamicum is used to produce amino acids, providing the required amino acid precursors for Bacillus subtilis to synthesize fengycin. Corynebacterium glutamicum produces amino acids through microbial fermentation, and metabolic engineering is used to transform Corynebacterium glutamicum to produce high-yield specific amino acids. The metabolic network of amino acids is intricate (Vogt M, Krumbach K, Bang WG, van Ooyen J, Noack S, Klein B, Bott M, Eggeling L. The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproductformation. Applied microbiology and biotechnology. 2015 99 (2): 791-800.), and various amino acids affect each other. The biosynthetic pathway of L-isoleucine in Corynebacterium glutamicum is relatively long, and its synthesis pathway overlaps with other amino acids, such as threonine and valine. The production of L-threonine and L-isoleucine by Corynebacterium glutamicum is usually accompanied by L-lysine and L-methionine to compete for carbon flux. In order to reduce the production of byproduct L-lysine, the production of L-threonine can be increased by gathering carbon flow through related gene modification. The key enzyme AHAS in the L-valine flow path is encoded by two genes, ilvB and ilvN (Chen C, Li Y, Hu J, Dong X, Wang X. Metabolic engineering of Corynebacterium glutamicum ATCC13869 for L-valine production. Metabolic engineering. 2015; 29: 66-75.), which also catalyzes similar reactions of L-isoleucine in biosynthesis. Overexpression of related genes can effectively increase the production of L-valine. However, L-proline is not in the same metabolic pathway as them. The gene encoding proline dehydrogenase catalyzes the process of proline synthesis of glutamate. At present, the process of producing related amino acids and their derivatives by biosynthesis still has difficulties, such as low fermentation yield or low sugar acid conversion rate. Therefore, constructing efficient microbial cell factories for producing amino acids remains a major challenge.

[0004] The production of fengyuan using a single strain of Bacillus subtilis has limitations and is prone to potential metabolic burden. Co-culture engineering shows good application prospects in the biosynthesis of complex chemicals (Li X, Zhou Z, Li W, Yan Y, Shen X, Wang J, Sun X, Yuan Q. Design of stable and self-regulated microbial consortia for chemical synthesis. Nature communications. 2022 23; 13(1): 1554.), and the stability of the co-culture system depends on the correlation between the various bacterial communities. The existing artificial mixed bacteria technology for the production of fengyuan does not meet the complete supply of multiple dependent amino acid precursors, and there is currently no report on the co-culture of Corynebacterium glutamicum cgb-8 and Bacillus subtilis CGF26-IV to produce fengyuan. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for producing fengycin by co-culturing a mixed bacterial system of recombinant Corynebacterium glutamicum and Bacillus subtilis.

[0006] The technical solution of the present invention is summarized as follows:

[0007] The method for producing Fengyuan by co-culturing a mixed bacterial system of recombinant Corynebacterium glutamicum and Bacillus subtilis comprises the following steps:

[0008] The secondary seeds of recombinant Corynebacterium glutamicum cgb-8 were cultured according to OD 600 = 0.6 and the secondary seeds of Bacillus subtilis CGF26-IV were measured by OD 600 =0.2 and inoculated into culture medium A at the same time to obtain a mixed bacterial system of recombinant Corynebacterium glutamicum cgb-8 and Bacillus subtilis CGF26-IV, and fermented to obtain Fengyuansu.

[0009] The recombinant Corynebacterium glutamicum cgb-8 was constructed by the following method:

[0010] Homologous recombination technology was used to knock out the pc k gene, ddh gene, lysE gene, ilvA gene, metX gene and putA gene of Corynebacterium glutamicum ATCC13032; the proB gene was mutated, and the ilvBN gene was overexpressed using the promoter Ptrc to obtain the recombinant Corynebacterium glutamicum cgb-8.

[0011] The nucleotide sequence of the pck gene is shown in SEQ ID NO.1; the nucleotide sequence of the ddh gene is shown in SEQ ID NO.2; the nucleotide sequence of the lysE gene is shown in SEQ ID NO.3; the nucleotide sequence of the ilvA gene is shown in SEQ ID NO.4; the nucleotide sequence of the metX gene is shown in SEQ ID NO.5; the nucleotide sequence of the putA gene is shown in SEQ ID NO.6; the nucleotide sequence of the proB gene after mutation is shown in SEQ ID NO.7; the nucleotide sequence of the ilvBN gene is shown in SEQ ID NO.8; the promoter P trc The nucleotide sequence is shown in SEQ ID NO.9.

[0012] Preferably, medium A is composed of the following components in g / L: 40 g of maltodextrin, 10 g of glucose, 30 g of yeast extract, 1.3 g of MgSO4·7H2O, 20 g of Na2HPO4·12H2O, 6.7 g of Na2MoO4·2H2O, 3 g of KH2PO4, 5 g of (NH4)2SO4, 0.02 g of FeSO4·7H2O, 0.02 g of MnSO4 H2O, and 4.5 g of vitamin B1. -4 , Vitamin B7 5×10 -5 , pH 7.0, the balance being deionized water.

[0013] Preferably, the fermentation conditions are: fermentation at 30° C. and 180 rpm for 72 hours.

[0014] Advantages of the present invention:

[0015] The method for producing Fengyuan by co-culturing a mixed bacterial system of recombinant Corynebacterium glutamicum and Bacillus subtilis of the present invention reduces the metabolic burden of producing Fengyuan by a single Bacillus subtilis, and provides the required amino acid precursors for Bacillus subtilis to synthesize Fengyuan. The division of labor between the bacterial groups of the present invention is clear, and the product-producing bacteria use the precursors produced by the precursor-synthesizing bacteria to concentrate on "working", so that the yield of Fengyuan is significantly increased. Using the method of the present invention, the yield of Fengyuan reaches more than 1764 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the amino acid yield of each Corynebacterium glutamicum after fermentation.

[0017] Figure 2 The results are as follows: The co-culture of recombinant Corynebacterium glutamicum cgb-8 and Bacillus subtilis CGF26-IV with an initial inoculation ratio of 0.6:0.2 and the production of fengyrin in pure culture of CGF26-IV. DETAILED DESCRIPTION

[0018] Bacillus subtilis CGF26, deposit number CGMCC No.25313, was deposited on July 15, 2022 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0019] Bacillus subtilis CGF26-IV was constructed using Bacillus subtilis CGF26 as the starting strain according to the following contents.

[0020] For the specific construction, see (Gao GR, Wei SY, Ding MZ, et al. Enhancing fengycin production in the co-culture of Bacillus subtilis and Corynebacterium glutamicum by engineering proline transporter Bioresource technology 2023; 383:129229.doi:10.1016 / j.biortech.2023.129229).

[0021] CRISPER gene editing technology was used to modify Bacillus subtilis CGF26 and construct pJOE-P fbaA -livH plasmid, with P fbaA The promoter overexpressed the livH gene to obtain CGF26-IV.

[0022] The primers involved are as follows:

[0023] livH-sgRNA-F (SEQ ID NO.44), livH-sgRNA-R (SEQ ID NO.45). (Reference website CRISPR RGEN Tools (rgenome.net) Design), annealing and connecting livH-sgRNA-F / R to obtain the connection product livH-sgRNA fragment. livH-UF (SEQ ID NO.46), livH-UR (SEQ ID NO.47); livH-DF (SEQ ID NO.48), livH-DR (SEQ ID NO.49); P fbaA -F (SEQ ID NO.50), P fbaA-R (SEQ ID NO.51); livH-F (SEQ ID NO.52), livH-R (SEQ ID NO.53). The above primers were used for PCR amplification using the B. subtilis CGF26 genome as a template, and then the upstream and downstream homologous arms livH-U and livH-D, P fbaA Promoter and livH fragment.

[0024] The above five fragments were connected with the linearized pJOE (commercial product) to obtain pJOE-P fbaA -livH plasmid, which was transformed into CGF26 competent cells by chemical transformation, and CGF26-IV was obtained using CRISPER gene editing technology.

[0025] The original Corynebacterium glutamicum ATCC13032 was purchased from ATCC, https: / / www.atcc.org / in April 2021.

[0026] The invention transforms the amino acid synthesis network of Corynebacterium glutamicum, and utilizes homologous recombination technology to knock out the pck gene (SEQ ID NO.1) of Corynebacterium glutamicum ATCC13032 (abbreviated as cgb) to obtain recombinant Corynebacterium glutamicum cgb-1; knock out the ddh gene (SEQ ID NO.2) of cgb-1 to obtain recombinant Corynebacterium glutamicum cgb-2; knock out the lysE gene (SEQ ID NO.3) of cgb-2 to obtain recombinant Corynebacterium glutamicum cgb-3; knock out the ilvA gene (SEQ ID NO.4) of cgb-3 to obtain recombinant Corynebacterium glutamicum cgb-4; knock out the metX gene (SEQ ID NO.5) of cgb-4 to obtain recombinant Corynebacterium glutamicum cgb-5; knock out the putA gene (SEQ ID NO.6) of cgb-5 to obtain recombinant Corynebacterium glutamicum cgb-6. IDNO.6), to obtain recombinant Corynebacterium glutamicum cgb-6; site-directed mutagenesis was performed on the base pairs of the proB gene of cgb-6, and the nucleotide sequence of the proB gene after mutation was shown in SEQ ID NO.7, to obtain recombinant Corynebacterium glutamicum cgb-7; using promoter P trc (SEQ ID NO.9) overexpressing the ilvBN gene (SEQ ID NO.8) of cgb-7 to obtain recombinant Corynebacterium glutamicum cgb-8;

[0027] The present invention will be further described below in conjunction with specific embodiments:

[0028] Example 1: Construction of recombinant Corynebacterium glutamicum cgb-1 (C. glutamicum-Δpck)

[0029] 1. Construction of pk18-Δpck plasmid

[0030] (1) Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms pck-U and pck-D of pck were PCR amplified using primers pck-UF (SEQ ID NO.10), pck-UR (SEQ ID NO.11), pck-DF (SEQ ID NO.12), and pck-DR (SEQ ID NO.13), respectively. The pk18mobsacB plasmid (purchased from Fenghai Biological Co., Ltd.) was digested with BamH I and EcoR I to obtain a linearized pk18mobsacB plasmid vector. pck-U, pck-D, and the linearized vector pk18mobsacB were connected using seamless cloning technology and transformed into Escherichia coli DH5α, and the positive transformants were screened on a 25 μg / mL kanamycin resistance plate. The pk18-Δpck plasmid was extracted using a plasmid mini-extraction kit. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0031] (2) PCR reaction system. The total system is 50 μL. Phanta Max Super Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 2 μL, downstream primer 2 μL, template 100 ng, sterile water to 50 μL; PCR reaction program: 95℃ 3 min, 95℃ 15 s, 55℃ 15 s, 72℃ 60 s / kb, 72℃ 5 min, 16℃ insulation, number of cycles: 35.

[0032] (3) Seamless cloning ligation reaction system. The total system is 20 μL. Exnase MultiS 2 μL, 5×CE MultiS Buffer 4 μL, linearized pk18mobsacB plasmid vector 120 ng, pck-U 1 μL, pck-D 1 μL, and sterile water to make up to 20 μL; the ligation reaction conditions are 37°C for 30 min.

[0033] 2. Transformation of pk18-Δpck plasmid

[0034] (1) Preparation of competent cells of Corynebacterium glutamicum ATCC 13032

[0035] Three zones of Corynebacterium glutamicum ATCC 13032 were streaked onto a BHI solid plate and placed in a 30°C incubator until a single colony grew out; a single colony was picked and inoculated into 5.0 mL of BHI liquid culture medium and cultured at 30°C, 220 rpm for 15 h; 1.0 mL of the above bacterial solution was added to 100.0 mL of BHI liquid culture medium and cultured at 30°C, 220 rpm for about 3 h until OD 600 Reach 1.3-1.5; transfer the above bacterial solution to two pre-cooled 50.0mL centrifuge tubes and place them on ice for 15 minutes to stop the growth of the bacteria. Place the 50.0mL centrifuge tube containing the bacterial solution in a 4℃, 5000rpm centrifuge for 20 minutes, and discard the supernatant. Wash the bacteria with 20.0mL pre-cooled ultrapure water, centrifuge at 4℃, 5000rpm for 20 minutes, discard the supernatant, and repeat the wash twice; wash the bacteria with 20.0mL pre-cooled sterilized 10% glycerol aqueous solution (the same below), centrifuge at 4℃, 5000rpm for 20 minutes, discard the supernatant, and repeat the wash twice; finally, resuspend the bacteria with 1.0mL pre-cooled 10% glycerol and mix them evenly, and divide them into pre-cooled 1.5mL centrifuge tubes (100.0μL / tube) to obtain competent cells.

[0036] (2) Electric shock transformation

[0037] Add 10.0 μL of pk18-Δpck plasmid to 100.0 μL of the competent cells above and mix by pipetting. Transfer all to a pre-cooled 1.0 mm electric shock cup, wipe the outer surface of the electric shock cup dry and then perform electric shock (1.8 KV). Take out 1.0 mL of preheated BHI liquid culture and mix it in the electric shock cup after the electric shock is completed. Transfer the mixed solution to a sterile 1.5 mL centrifuge tube, place the centrifuge tube in a 46 ° C water bath for 6 minutes, and then place it in a 30 ° C, 220 rpm shaker for 1 hour. Collect the bacteria by centrifugation at 4200 rpm for 6 minutes, resuspend the bacteria with 100.0 μL of supernatant and evenly apply it on a BHI solid plate containing 25 μg / mL kanamycin resistance, and place the plate in a 30 ° C constant temperature incubator for 40 hours. Positive strains were screened and cultured in BHI liquid medium containing 250 g / L sucrose to screen recombinant strains that lost the sacB gene (the gene in the pk18mobsacB plasmid). The strain that finally lost the sacB screening tag was the recombinant C. glutamicum-Δpck, named cgb-1.

[0038] The nucleotide sequence of the pck gene is shown in SEQ ID NO.1.

[0039] Example 2: Construction of recombinant Corynebacterium glutamicum cgb-2 (C. glutamicum-Δpck-Δddh)

[0040] 1. Construction of pk18-Δddh plasmid

[0041] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms ddh-U and ddh-D of ddh were amplified by PCR using primers ddh-UF (SEQ ID NO.14), ddh-UR (SEQ ID NO.15), ddh-DF (SEQ ID NO.16), and ddh-DR (SEQ ID NO.17), respectively.

[0042] Refer to the method in step 1 of Example 1 to obtain the pk18-Δddh plasmid.

[0043] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0044] 2. Transformation of pk18-Δddh Plasmid

[0045] Refer to the method in step 2(1) of Example 1 to obtain cgb-1 competent cells.

[0046] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh, which was named cgb-2.

[0047] The nucleotide sequence of the ddh gene is shown in SEQ ID NO.2.

[0048] Example 3: Construction of recombinant Corynebacterium glutamicum cgb-3 (C. glutamicum-Δpck-Δddh-ΔlysE)

[0049] 1. Construction of pk18-ΔlysE plasmid

[0050] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms lysE-U and lysE-D of lysE were PCR amplified using primers lysE-UF (SEQ ID NO.18), lysE-UR (SEQ ID NO.19), lysE-DF (SEQ ID NO.20), and lysE-DR (SEQID NO.21), respectively.

[0051] Refer to the method in step 1 of Example 1 to obtain the pk18-ΔlysE plasmid.

[0052] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0053] 2. Transformation of pk18-ΔlysE

[0054] Refer to the method in step 2(1) of Example 1 to obtain cgb-2 competent cells.

[0055] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE, which was named cgb-3.

[0056] The nucleotide sequence of the lysE gene is shown in SEQ ID NO.3;

[0057] Example 4: Construction of recombinant Corynebacterium glutamicum cgb-4 (C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA)

[0058] 1. Construction of pk18-ΔilvA plasmid

[0059] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms ilvA-U and ilvA-D of ilvA were amplified by PCR using primers ilvA-UF (SEQ ID NO.22), ilvA-UR (SEQ ID NO.23), ilvA-DF (SEQ ID NO.24), and ilvA-DR (SEQID NO.25), respectively.

[0060] Refer to the method in step 1 of Example 1 to obtain the pk18-ΔilvA plasmid.

[0061] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0062] 2. Transformation of pk18-ΔilvA plasmid

[0063] Refer to the method in step 2(1) of Example 1 to obtain cgb-3 competent cells.

[0064] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA, which was named cgb-4.

[0065] The nucleotide sequence of the ilvA gene is shown in SEQ ID NO.4;

[0066] Example 5: Construction of recombinant Corynebacterium glutamicum cgb-5 (C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX)

[0067] 1. Construction of pk18-ΔmetX plasmid

[0068] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms of metX, metX-U and metX-D, were amplified by PCR using primers metX-UF (SEQ ID NO.26), metX-UR (SEQ ID NO.27), metX-DF (SEQ ID NO.28) and metX-DR (SEQID NO.29), respectively.

[0069] Refer to the method in step 1 of Example 1 to obtain the pk18-ΔmetX plasmid.

[0070] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0071] 2. Transformation of pk18-ΔmetX

[0072] Refer to the method in step 2(1) of Example 1 to obtain cgb-4 competent cells.

[0073] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX, which was named cgb-5.

[0074] The nucleotide sequence of the metX gene is shown in SEQ ID NO.5;

[0075] Example 6: Construction of recombinant Corynebacterium glutamicum cgb-6 (C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA)

[0076] 1. Construction of pk18-ΔPutA plasmid

[0077] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms of PutA, PutA-U and PutA-D, were PCR amplified using primers PutA-UF (SEQ ID NO.30), PutA-UR (SEQ ID NO.31), PutA-DF (SEQ ID NO.32), and PutA-DR (SEQID NO.33), respectively.

[0078] Refer to the method in step 1 of Example 1 to obtain the pk18-ΔPutA plasmid.

[0079] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0080] 2. Transformation of pk18-ΔPutA

[0081] Refer to the method in step 2(1) of Example 1 to obtain cgb-5 competent cells.

[0082] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA, which was named cgb-6.

[0083] The nucleotide sequence of the putA gene is shown in SEQ ID NO.6;

[0084] Example 7: Construction of recombinant Corynebacterium glutamicum cgb-7 (C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA-proB)

[0085] 1. Construction of pk18-ProB plasmid

[0086] Using the genome of C. glutamicum ATCC 13032 as a template, the upstream and downstream homologous arms ProB-U and ProB-D of ProB were PCR amplified using primers ProB-UF (SEQ ID NO.34), ProB-UR (SEQ ID NO.35), ProB-DF (SEQ ID NO.36), and ProB-DR (SEQID NO.37), respectively.

[0087] Refer to the method in step 1 of Example 1 to obtain the pk18-ProB plasmid.

[0088] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0089] 2. Transformation of pk18-proB

[0090] Refer to the method in step 2(1) of Example 1 to obtain cgb-6 competent cells.

[0091] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA-proB, which was named cgb-7.

[0092] The nucleotide sequence of the mutated proB gene is shown in SEQ ID NO.7;

[0093] Example 8: Recombinant Corynebacterium glutamicum cgb-8 (C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA-proB-P trc -ilvBN)

[0094] 1. Construction of pk18-ilvBN plasmid

[0095] Using the genome of C. glutamicum ATCC 13032 as a template, ilvBN and its upstream and downstream homologous arms ilvBN-U and ilvBN-D (P trc already included in the primer sequences).

[0096] Refer to the method in step 1 of Example 1 to obtain the pk18-ΔilvBN plasmid.

[0097] The PCR reaction system and the seamless cloning and ligation reaction system are shown in Example 1.

[0098] 2. Transformation of pk18-ΔilvBN

[0099] Refer to the method in step 2(1) of Example 1 to obtain cgb-7 competent cells.

[0100] Electroporation was performed according to the method in step 2 (2) of Example 1 to obtain C. glutamicum-Δpck-Δddh-ΔlysE-ΔilvA-ΔmetX-ΔputA-proB-P trc -ilvBN, named cgb-8.

[0101] The nucleotide sequence of the ilvBN gene is shown in SEQ ID NO.8;

[0102] The promoter P trc The nucleotide sequence is shown in SEQ ID NO.9.

[0103] Embodiment 9:

[0104] (1) Medium A, ingredients in g / L: maltodextrin 40, glucose 10, yeast extract 30, MgSO4·7H2O 1.3, Na2HPO4·12H2O 20, Na2MoO4·2H2O 6.7, KH2PO4 3, (NH4)2SO4 5, FeSO4·7H2O 0.02, MnSO4H2O 0.02, vitamin B1 4.5×10 -4 , Vitamin B7 5×10 -5 , pH 7.0, the balance being deionized water.

[0105] (2) Culture medium B, composition (g / L): glucose 30, beef extract 5, peptone 10, yeast extract powder 5, NaCl 5, and the balance is deionized water.

[0106] Example 10

[0107] Amino Acid Production by Recombinant Corynebacterium glutamicum

[0108] (1) Seed culture:

[0109] Corynebacterium glutamicum cgb, cgb-4, cgb-7 and cgb-8 were respectively activated by streaking three areas on a 30 g / L BHI solid plate and cultured at 30°C for 36 h; a single colony was picked and inoculated into a 30 g / L BHI liquid medium and cultured at 30°C for 12 h as primary seeds; the primary seeds were quantified according to OD 600 =0.2 was inoculated into 25 mL of medium A, cultured at 30°C, 200 rpm for 24 h to obtain secondary seeds;

[0110] (2) Pure culture of Corynebacterium glutamicum cgb, cgb-4, cgb-7 and cgb-8: The secondary seeds were respectively cultured according to OD 600 =0.2 was inoculated into fermentation medium A and cultured at 30°C and 180rpm for 72h.

[0111] like Figure 1 As shown, the amino acid production of each Corynebacterium glutamicum after fermentation.

[0112] Embodiment 11

[0113] Application of co-culture of recombinant Corynebacterium glutamicum cgb-8 and Bacillus subtilis CGF26-IV to produce fengycin

[0114] (1) Seed culture:

[0115] The recombinant Corynebacterium glutamicum cgb-8 was streaked into three zones on a 30 g / L BHI solid plate to activate the seeds, and cultured at 30°C for 36 hours; a single colony was picked and inoculated into a 30 g / L BHI liquid medium, and cultured at 30°C for 12 hours as the primary seeds of the recombinant Corynebacterium glutamicum cgb-8; the primary seeds were quantified according to OD 600 =0.2 was inoculated into 25 mL of culture medium A, and cultured at 30°C and 200 rpm for 24 h to obtain the secondary seeds of recombinant Corynebacterium glutamicum cgb-8;

[0116] The Bacillus subtilis CGF26-IV was activated by streaking three zones on an LB solid plate and incubated at 30°C for 12 hours; a single colony was picked and inoculated into an LB liquid test tube and incubated at 30°C for 24 hours as the primary seed of Bacillus subtilis CGF26-IV; the primary seed was measured by OD 600 =0.2 was inoculated into 50 mL of culture medium B, and cultured at 37°C and 200 rpm for 24 h to obtain secondary seeds of Bacillus subtilis CGF26-IV strain;

[0117] (2) Co-cultivation:

[0118] The secondary seeds of recombinant Corynebacterium glutamicum cgb-8 were cultured according to OD 600 = 0.6 and the secondary seeds of Bacillus subtilis CGF26-IV were measured by OD 600 =0.2 and inoculated into medium A at the same time to obtain a mixed bacterial system of recombinant Corynebacterium glutamicum cgb-8 and Bacillus subtilis CGF26-IV, and fermented at 30°C and 180rpm for 72h;

[0119] (3) Pure culture of Bacillus subtilis CGF26-IV: The secondary seeds of CGF26-IV strain were cultured according to OD 600 =0.2 was inoculated into fermentation medium A and cultured at 30°C and 180rpm for 72h.

[0120] like Figure 2 As shown, the fengycin production of pure culture of Bacillus subtilis CGF26-IV and co-culture of Bacillus subtilis CGF26-IV and recombinant Corynebacterium glutamicum cgb-8 was 597 mg / L and 1764 mg / L, respectively.

Claims

1. A method for producing Fengyuan by co-culturing a mixed bacterial system of recombinant Corynebacterium glutamicum and Bacillus subtilis, characterized in that The steps include: The secondary seeds of recombinant Corynebacterium glutamicum cgb-8 were cultured according to OD 600 = 0.6 and the secondary seeds of Bacillus subtilis CGF26-IV were measured by OD 600 =0.2 and inoculated into culture medium A at the same time to obtain a mixed bacterial system of recombinant Corynebacterium glutamicum cgb-8 and Bacillus subtilis C GF26-IV, and fermented to obtain Fengyuansu.

2. The method according to claim 1, characterized in that Recombinant Corynebacterium glutamicum cgb-8 was constructed by the following method: Homologous recombination technology was used to knock out the pc k gene, ddh gene, lysE gene, ilvA gene, metX gene and putA gene of Corynebacterium glutamicum ATCC 13032; the proB gene was mutated, and the ilvBN gene was overexpressed using the promoter Ptrc to obtain the recombinant Corynebacterium glutamicum cgb-8.

3. The method according to claim 2, characterized in that The nucleotide sequence of the pck gene is shown in SEQ ID NO.1; the nucleotide sequence of the ddh gene is shown in SEQ ID NO.2; the nucleotide sequence of the lysE gene is shown in SEQ ID NO.3; the nucleotide sequence of the ilvA gene is shown in SEQ ID NO.4; the nucleotide sequence of the metX gene is shown in SEQ ID NO.5; the nucleotide sequence of the putA gene is shown in SEQ ID NO.6; the nucleotide sequence of the proB gene after mutation is shown in SEQ ID NO.7; the nucleotide sequence of the ilvBN gene is shown in SEQ ID NO.8; the promoter P trc The nucleotide sequence is shown in SEQ ID NO.

9.

4. The method according to claim 1, characterized in that: Medium A consisted of the following ingredients in g / L: maltodextrin 40, glucose 10, yeast extract 30, MgSO4·7H2O 1.3, Na2HPO4·12H2O 20, Na2MoO4·2H2O 6.7, KH2PO4 3, (NH4)2SO4 5, FeSO4·7H2O 0.02, MnSO4 H2O 0.02, vitamin B1 4.5×10 -4 , Vitamin B75×10 -5 , pH 7.0, the balance being deionized water.

5. The method according to claim 1, characterized in that The fermentation conditions are: fermentation at 30° C. and 180 rpm for 72 hours.

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