A genetically engineered bacterium tolerant to high concentration of acetoin and its construction method and application

Abstract

The present invention discloses a genetic engineering bacterium tolerant to high concentration of acetoin and a construction method and application thereof, wherein Bacillus subtilis ( Bacillus subtilis ) 13932 is the original starting strain, knocking out the key gene for acetoin decomposition metabolism bdhA , then biotin transporter bioY and proline transporter work Overexpression was performed to obtain genetically engineered strains with high production and tolerance to high concentrations of acetoin. bioY and work The acetoin production of the genetically engineered strains reached 80.89 g / L and 84.93 g / L, respectively, and the tolerance to acetoin was also enhanced. bioY and work The co-expressed genetically engineered strain significantly improved the growth of the recombinant strain under acetoin stress compared to the starting strain. B.subtilis 13932, the growth rate increased by 29.6%; its acetoin fermentation yield reached 88.76g / L, which was B.subtilis 13932Δ bdhA The genetic engineering bacteria tolerant to high concentration of acetoin constructed by the present invention have important theoretical and practical significance and broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biological fermentation engineering technology, and specifically relates to a genetic engineering bacterium tolerant to high concentration of acetoin and a construction method and application thereof. Background Art

[0002] Acetoin is an important four-carbon platform compound, which is widely used in the food, agriculture, chemical and other industries. In food, it is an extremely important spice variety and can be used to prepare different types of flavors. In addition, it is also used as a food additive, mainly used in the preparation of cream, dairy products, yogurt, etc. In the chemical industry, it is often used in cosmetics and detergents due to its good biocompatibility.

[0003] Since the consumer market of acetoin is mainly in the fields of food and cosmetics, the use of safe and green microbial fermentation has become a research hotspot for the efficient production of acetoin. However, this method has a typical product inhibition effect problem, that is, the excessive accumulation of acetoin will significantly inhibit the normal metabolic process of functional microorganisms; thus limiting the acquisition of higher concentrations of acetoin. Therefore, enhancing stress resistance is considered a necessary means to improve microbial metabolic growth and increase product yield.

[0004] At present, the traditional methods of enhancing microbial stress resistance mainly include adaptive evolution and mutagenesis by physical or chemical means, but these methods generally have many disadvantages, such as long experimental cycle and easy loss of excellent phenotypes. Compared with traditional methods, introducing certain stress resistance elements or overexpressing exogenous resistance structural genes through molecular biology technology is a more direct and effective way. Yunpeng Yang et al. heterologously expressed biotin synthesis genes bioA, bioB, bioD, bioY, etc. from Bacillus subtilis in C. acetobutylicum and optimized their combined expression, which significantly improved the strain's solvent tolerance to acetone, butanol and ethanol (ABE). The currently constructed strain cannot tolerate high concentrations of acetoin, which limits the growth of the strain and the increase in yield. Summary of the invention

[0005] The purpose of the present invention is to provide a genetically engineered bacterium tolerant to high concentrations of acetoin and a construction method and application thereof, which can effectively solve the problems existing in the above-mentioned background technology.

[0006] By metabolic engineering the acetoin synthesis pathway, knocking out the key gene bdhA for acetoin catabolism, and then overexpressing the biotin synthesis metabolism genes related to the fatty acid synthesis pathway and the proline transport system encoding genes, the ability of the strain to ferment and produce acetoin can be improved, and its stress resistance can be enhanced, which can improve the growth of the strain under acetoin stress.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows:

[0008] The invention provides a genetically engineered bacterium tolerant to high concentration of acetoin and a construction method and application thereof. The genetically engineered bacterium is based on Bacillus subtilis as an original starting strain. Firstly, metabolic engineering is performed on the acetoin synthesis pathway to knock out the key gene bdhA for acetoin decomposition metabolism. Then, biotin synthesis metabolism genes bioY, bioD, bioB, bioW, bioFC and proline transport system encoding genes opuAA, opuAB, opuAC and opuE related to the fatty acid synthesis pathway are overexpressed through an autonomously replicating plasmid pHP13 to obtain a genetically engineered strain with high acetoin tolerance and high yield.

[0009] Further preferably, the biotin transporter bioY and the proline transporter opuE are co-expressed to obtain a genetically engineered strain with high yield and tolerance to high concentrations of acetoin.

[0010] Overexpression of key genes is to improve the strain's tolerance to high concentrations of acetoin and further increase acetoin production.

[0011] The original starting strain is Bacillus subtilis CGMCC No.13932.

[0012] The nucleotide sequence of the key gene bdhA for acetoin decomposition metabolism is shown in SEQ ID NO.1.

[0013] The nucleotide sequences of the biotin synthesis metabolism genes bioY, bioD, bioB, bioW, and bioFC related to the fatty acid synthesis pathway are shown in SEQ ID NO.2-SEQ ID NO.6.

[0014] The nucleotide sequences of the proline transport system encoding genes opuAA, opuAB, opuAC and opuE are shown in SEQ ID NO.7-SEQ ID NO.10.

[0015] A method for constructing a genetically engineered bacterium tolerant to high concentrations of acetoin comprises the following steps:

[0016] 1) The bdhA knockout was performed using the B. subtilis 13932 genome as a template and primers bdhA-LF-F / R,

[0017] bdhA-DR-F / R, bdhA-PC-F / R, and bdhA-RF-F / R amplified the upstream homologous arm of gene bdhA respectively

[0018] LF, DR sequence, PC box, bdhA downstream homology arm RF, the four fragments were spliced ​​together using Overlap PCR to obtain the bdhA knockout recombinant fragment.

[0019] 2) After purification, the gene was transformed into B. subtilis 13932 competent cells, and then spread on a resistance plate containing 12 mg / L chloramphenicol. After the positive clones were verified by colony PCR, they were inoculated into non-resistant LB liquid medium and cultured at 37°C and 200 rpm until the OD was equal to 1. The gene was then diluted 100 times and spread on a MGY-Cl solid plate. After overnight culture at 37°C, colony PCR was used to verify whether the gene was knocked out. After preliminary confirmation, sequencing was performed to obtain the recombinant strain B. subtilis 13932ΔbdhA.

[0020] 3) Escherichia coli-Bacillus subtilis shuttle plasmid pHP13 was used as an expression vector, and restriction endonucleases were used

[0021] pHP13 was linearized with PstI and EcoRI for later use.

[0022] 4) Use a genome extraction kit to extract the Bacillus subtilis genome for target gene fragment PCR, and design primer pairs bioB-F / R, bioD-F / R, bioFC-F / R, bioW-F / R, bioY-F / R,

[0023] opuAA-F / R, opuAB-F / R, opuAC-F / R, opuE-F / R amplified genes bioB, bioD,

[0024] After the target gene and vector were connected by one-step cloning method and transferred into E.coli DH5α competent cells, the cells were plated on LB resistance plates containing 12 μg / mL chloramphenicol, positive clones were screened and inoculated into LB liquid medium containing 12 μg / mL chloramphenicol, and recombinant plasmids pHP13-bioB, pHP13-

[0025] bioD, pHP13-bioFC, pHP13-bioW, pHP13-bioY, pHP13-opuAA, pHP13-

[0026] opuAB, pHP13-opuAC, pHP13-opuE, pHP13-bioY-opuE.

[0027] 5) After designing the primer pairs bioY1-F / R and opuE1-F / R to amplify the genes bioY and opuE, the target gene and the vector were connected using the one-step cloning method and transferred into E. coli DH5α competent cells, spread on chloramphenicol LB resistance plates, screen positive clones and inoculate them into chloramphenicol LB liquid culture medium, and obtain the recombinant plasmid pHP13-bioY-opuE after sequencing verification.

[0028] 6) The recombinant plasmids were transformed into the recombinant strain B. subtilis 13932ΔbdhA to obtain genetically engineered bacteria.

[0029] A genetically engineered strain B. subtilis 13932ΔbdhA / pHP13-bioY is obtained by knocking out the key gene bdhA for acetoin decomposition and overexpressing the biotin transporter bioY, thereby obtaining the genetically engineered strain B. subtilis 13932ΔbdhA / pHP13-bioY with high yield and tolerance to high concentration of acetoin.

[0030] A genetically engineered strain B. subtilis 13932ΔbdhA / pHP13-opuE is disclosed, in which the key gene bdhA for acetoin decomposition is knocked out and the proline transporter opuE is overexpressed, so as to obtain the genetically engineered strain B. subtilis 13932ΔbdhA / pHP13-opuE with high yield and tolerance to high concentration of acetoin.

[0031] A genetically engineered strain B. subtilis 13932ΔbdhA / pHP13-bioY-opuE is obtained by knocking out the key gene bdhA for acetoin decomposition metabolism, and co-expressing the biotin transporter bioY and the proline transporter opuE, so as to obtain the genetically engineered strain B. subtilis13932ΔbdhA / pHP13-bioY-opuE with high yield and high concentration of acetoin tolerance, and the strain has further improved tolerance to high concentration of acetoin.

[0032] The genetically engineered bacteria constructed by the present invention can tolerate high concentration of acetoin, can grow normally in a 40g / L acetoin stress culture medium and can further synthesize acetoin.

[0033] The invention provides an application of the genetically engineered strain in the production of acetoin. The application method is as follows: the constructed genetically engineered bacteria are first cultured in a seed culture medium at 32-37°C and 200±20rpm for 12-14h to obtain a seed solution, and then the obtained seed solution is inoculated into an acetoin fermentation culture medium at an inoculation amount of 5-10% (v / v), and fermented at 32-37°C and 200±20rpm / min for 60-84h to obtain a fermentation solution containing acetoin.

[0034] The seed culture medium is: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, distilled water is added to 1000ml, pH is natural, and sterilized at 121°C for 15min.

[0035] The acetoin fermentation medium comprises the following components: 100-120 g / L of glucose, 10-20 g / L of corn steep liquor dry powder, 1-5 g / L of urea, 5-10 g / L of yeast powder, 1-5 g / L of K2HPO4, 1-5 g / L of KH2PO4, 0.5-1.0 g / L of MgSO4, 0.5-1 g / L of CaCl2, and 0.5-1 g / L of ZnSO4. The pH value of the fermentation medium is 6.8-7.2, and the medium is sterilized at 110°C for 10 min.

[0036] The genetically engineered strain constructed by the present invention can tolerate high concentration of acetoin. The constructed genetically engineered bacteria are spread on an LB solid plate, and a loop of colonies are scraped and inoculated into an LB liquid medium to culture overnight at 32-37°C and 200±20rpm / min to obtain a seed solution. The obtained seed solution is then inoculated into a 40g / L acetoin stress medium at a 5% (v / v) inoculation amount, and samples are taken at 0h, 4h, 6h, 8h, and 10h to measure OD600. The results are as follows: Figure 2 As shown in the figure, these genes can improve the growth of Bacillus subtilis under acetoin stress to a certain extent. Among them, bioY and opuE have the best effect, and the effect of co-expressing bioY and opuE is further improved compared with overexpressing bioY and opuE separately.

[0037] Beneficial effects:

[0038] The present invention can improve the ability of strains to produce acetoin by fermentation and improve the growth of strains under acetoin stress by metabolic engineering transformation of the acetoin synthesis pathway, knocking out the key gene bdhA for acetoin decomposition, and then overexpressing the biotin synthesis metabolism gene and the proline transport system encoding gene related to the fatty acid synthesis pathway. The genetically engineered bacteria that tolerate high concentrations of acetoin constructed by the present invention have important theoretical and practical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Comparison of acetoin fermentation between the starting strain and the recombinant strain.

[0040] Figure 2 The effect of overexpression of key genes on the growth of Bacillus subtilis under acetoin stress.

[0041] Figure 3 Comparison of fed-batch fermentation of acetoin by genetically engineered strains in a 7.5L fermenter. DETAILED DESCRIPTION

[0042] The present invention can be better understood by the following examples. Then, it is easy for those skilled in the art to understand that the specific process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0043] The strains involved in the following embodiments are: the starting strain Bacillus subtilis CGMCC No. 13932, which is derived from the General Microbiological Center of the China National Microbiological Culture Collection Administration, with a collection number of CGMCC No. 13932; the strain E. coli DH5α used for expression plasmid construction was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the biotin synthesis metabolism genes bioY, bioD, bioB, bioW, bioFC related to the fatty acid synthesis pathway and the proline transport system encoding genes opuAA, opuAB, opuAC, and opuE are all derived from the strain Bacillus subtilis CGMCC No. 13932.

[0044] The plasmid pHP13 described in the examples was purchased from Wuhan Miaoling Biotechnology Co., Ltd.;

[0045] The PCR enzyme 2×Phanta Max Master Mix and restriction endonucleases PstI and EcoRI used in the examples were purchased from Novartis, and the one-step cloning kit ClonExpress II One Step Cloning Kit was purchased from Takara. The primers were synthesized by General Biosystems (Anhui) Co., Ltd.

[0046] The strains, plasmids and primers involved in the examples are shown in Table 1 and Table 2.

[0047] Table 1 Strains and plasmids

[0048]

[0049]

[0050] Table 2 Primers

[0051]

[0052]

[0053] Example 1 Construction of a genetically engineered bacterium tolerant to high concentrations of acetoin

[0054] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0055] LB liquid medium: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0056] MGY-Cl solid culture medium: glucose 5 g / L, yeast extract 4 g / L, ammonium nitrate 1 g / L, Nacl 0.5 g / L, K2HPO4 1.5 g / L, KH2PO4 0.5 g / L, MgSO4 0.2 g / L, p-Cl-Phe 5 mM, agar 15 g / L, add distilled water to 1000 ml, pH natural, sterilize at 115 ° C for 15 min.

[0057] 1) bdhA knockout: Using the B. subtilis 13932 genome as a template, primer pairs bdhA-LF-F / R,

[0058] bdhA-DR-F / R, bdhA-PC-F / R, and bdhA-RF-F / R amplified the upstream homologous arm of gene bdhA respectively

[0059] LF, DR sequence, PC box, bdhA downstream homology arm RF, use Overlap PCR to splice the four fragments together to obtain the bdhA knockout recombinant fragment; the PCR amplification conditions are: pre-denaturation 95℃

[0060] 5min, denaturation at 95℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 4min, final extension at 72℃ for 5

[0061] The Overlap PCR conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 4 min, final extension at 72°C for 5 min, storage at 4°C for 59 min, and 5 cycles.

[0062] F, bdhA-RF-R PCR, pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 50℃

[0063] 30s, extension at 72℃ for 4min, final extension at 72℃ for 5min, storage at 4℃ for 59min; cycle number 25

[0064] cycles.

[0065] 2) After purification, the cells were transformed into competent cells of B. subtilis 13932, and then spread on a resistance plate containing 12 mg / L chloramphenicol. After the positive clones were verified by colony PCR, they were inoculated into non-resistant LB liquid medium and cultured at 37°C and 200 rpm until the OD was equal to 1. The cells were diluted 100 times and spread on MGY-Cl solid plates. After overnight culture at 37°C, colony PCR was used to verify whether the gene was knocked out. After preliminary confirmation, sequencing was performed to obtain the recombinant strain B. subtilis 13932ΔbdhA;

[0066] 3) Escherichia coli-Bacillus subtilis shuttle plasmid pHP13 was used as an expression vector, and restriction endonucleases were used

[0067] pHP13 was linearized with PstI and EcoRI for later use;

[0068] 4) Use a genome extraction kit to extract the Bacillus subtilis genome for target gene fragment PCR, and design primer pairs bioB-F / R, bioD-F / R, bioFC-F / R, bioW-F / R, bioY-F / R,

[0069] opuAA-F / R, opuAB-F / R, opuAC-F / R, opuE-F / R amplified genes bioB, bioD,

[0070] After the target gene and vector were connected by one-step cloning method and transferred into E.coli DH5α competent cells, the cells were plated on LB resistance plates containing 12 μg / mL chloramphenicol, positive clones were screened and inoculated into LB liquid medium containing 12 μg / mL chloramphenicol, and recombinant plasmids pHP13-bioB, pHP13-

[0071] bioD, pHP13-bioFC, pHP13-bioW, pHP13-bioY, pHP13-opuAA, pHP13-

[0072] opuAB, pHP13-opuAC, pHP13-opuE; PCR conditions were: pre-denaturation at 95°C for 5

[0073] min, denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 1min, and final extension at 72°C for 5

[0074] min, stored at 4℃ for 59 min; cycle number: 30 cycles.

[0075] 5) After designing primer pairs bioY1-F / R and opuE1-F / R to amplify genes bioY and opuE, the target gene and vector were connected using the one-step cloning method and transferred into E. coli DH5α competent cells, spread on chloramphenicol LB resistance plates, screen positive clones and inoculate them into chloramphenicol LB liquid culture medium, and obtain recombinant plasmid pHP13-bioY-opuE after sequencing verification; the PCR conditions were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, final extension at 72°C for 5 min, and storage at 4°C for 59 min; the number of cycles was 30 cycles.

[0076] 6) The recombinant plasmids were transformed into the recombinant strain B. subtilis 13932ΔbdhA to obtain genetically engineered bacteria. The strain names are shown in Table 1.

[0077] Among them, the preparation and transformation steps of B. subtilis 13932 competent cells are as follows:

[0078] (1) Pick a single colony of B. subtilis 13932 and inoculate it into 5 mL of GMI medium and culture it overnight in a constant temperature shaker at 37°C;

[0079] (2) Transfer 2 mL of overnight culture to 5 mL of GMI medium and culture at 37°C in a shaking incubator for 2 to 6 h before measuring OD600. When OD600 is approximately 1.0, transfer 10 mL of bacterial culture to 90 mL of GMII medium and culture at 37°C and 200 rpm in a shaking incubator for 1.5 h.

[0080] (3) Collect the cells by centrifugation at 4°C for 10 min, retain 10 mL of liquid for resuspending the cells, and dispense 500 μL of the bacterial solution into each 1.5 mL centrifuge tube.

[0081] (4) Add the sequencing-verified plasmid to the tube, pipette to mix, and then incubate in a shaker at 37°C and 200 rpm for 2 h;

[0082] (5) After the culture was completed, the cells were collected by centrifugation at 5000 rpm, part of the supernatant was discarded, and 100 μL of the resuspended cells was spread on a plate containing 6 μg / mL erythromycin resistance and cultured at 37°C overnight.

[0083] Example 2 Fermentation production of acetoin by recombinant strain B. subtilis 13932ΔbdhA

[0084] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0085] Seed culture medium (LB liquid medium): peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, add distilled water to 1000 ml, pH natural, sterilize at 121°C for 15 min.

[0086] Acetoin fermentation medium: glucose 100 g / L, corn steep liquor powder 15 g / L, urea 3 g / L, yeast powder 6 g / L, K2HPO4 3 g / L, KH2PO4 3 g / L, MgSO4 0.8 g / L, CaCl2 0.6 g / L, ZnSO4 0.5 g / L, the pH value of the fermentation medium is 6.8-7.2, and it is sterilized at 110°C for 10 min.

[0087] (1) The starting strain B. subtilis 13932 and the recombinant strain B. subtilis 13932ΔbdhA were spread on fresh LB plate culture medium respectively and activated at 37°C overnight.

[0088] (2) Use an inoculation loop to pick a loop of bacterial sludge from the activated plate and inoculate it into 100 mL LB liquid medium (500 mL

[0089] mL Erlenmeyer flask) at 37°C, 200 rpm, and culture overnight for 12 h to obtain seed solution.

[0090] (3) The seed solution was inoculated into 100 mL of acetoin fermentation medium (500 mL Erlenmeyer flask) at a 5% inoculum amount, and cultured at 37° C. and 200 rpm for 84 h to obtain a fermentation liquid containing acetoin.

[0091] (4) Determination of acetoin content: Glucose, organic acids, acetoin and 2,3-butanediol were detected by high performance liquid chromatography. The injection volume was 20 μL, the mobile phase was 5 mM H2SO4, the flow rate was 0.6 mL / min, and the column temperature was 240 °C.

[0092] 65℃, differential detector temperature 45℃, the measurement results are as follows Figure 1 shown.

[0093] like Figure 1 As shown in the figure, in the fermentation of acetoin by Bacillus subtilis, 2,3-butanediol is the main byproduct. The fermentation yield of acetoin by the starting strain B.subtilis 13932 was 28.72 g / L. After knocking out the key gene bdhA for acetoin decomposition, the fermentation yield of acetoin reached 39.56 g / L, which was 37.74% higher than that of the starting strain B.subtilis 13932. The fermentation yield of 2,3-butanediol by the starting strain B.subtilis 13932 was 17.31 g / L, and the fermentation yield of 2,3-butanediol by the recombinant strain B.subtilis 13932ΔbdhA was 9.4 g / L, and the content of byproduct 2,3-butanediol was reduced by 45.66%.

[0094] Example 3 Effect of overexpression of key genes in the recombinant strain B. subtilis 13932ΔbdhA on strain growth under acetoin stress

[0095] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0096] Seed culture medium (LB liquid medium): peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, add distilled water to 1000 ml, pH natural, sterilize at 121°C for 15 min.

[0097] Acetoin stress medium: LB liquid medium containing 40 g / L acetoin, pH 7.0, sterilized at 110°C for 15 min.

[0098] The genetically engineered bacteria obtained in Example 1 were spread on LB solid plates, and one loop of colonies was scraped and inoculated into LB liquid medium for overnight culture at 37°C and 200±20rpm / min to obtain seed solution. The obtained seed solution was then inoculated into 40g / L acetoin stress medium at a 5% (v / v) inoculation rate, and samples were taken at 0h, 4h, 6h, 8h, and 10h to measure OD600. The recombinant strain B.

[0099] subtilis13932 / pHP13 was used as blank control Control-1, and the starting strain B.subtilis13932 was inoculated into LB liquid medium without acetoin as blank control Control-2. Figure 2 shown.

[0100] Under acetoin stress, the recombinant strain B. subtilis 13932ΔbdhA overexpressed the key genes bioY, bioD, bioB, bioW, bioFC in the biotin synthesis process and the genes encoding the proline transport system opuAA, opuAB, opuAC, and opuE. Figure 2 As shown in the figure, these genes can improve the growth of Bacillus subtilis under acetoin stress to a certain extent. Among them, bioY and opuE have the best effect, with the specific growth rates of 0.51, 0.45 and 0.53, 0.48 at 8h and 10h, respectively, which are significantly higher than the control strain control-1 (0.42, 0.36).

[0101] Example 4 Effect of overexpression of key genes in recombinant strain B. subtilis 13932ΔbdhA on acetoin synthesis

[0102] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0103] Seed culture medium (LB liquid medium): peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, add distilled water to 1000 ml, pH natural, sterilize at 121°C for 15 min.

[0104] Acetoin fermentation medium: glucose 110 g / L, corn steep liquor powder 15 g / L, urea 3 g / L, yeast powder 8 g / L, K2HPO4 3 g / L, KH2PO4 3 g / L, MgSO4 0.6 g / L, CaCl2 0.5 g / L, ZnSO4 0.5 g / L, the pH value of the fermentation medium is 6.8-7.2, and it is sterilized at 110°C for 10 min.

[0105] In order to further improve the fermentation yield of acetoin, we conducted a fed-batch fermentation experiment in a 7.5L fermenter. Two genetically engineered bacteria B. subtilis 13932ΔbdhA / pHP13-bioY and B. subtilis 13932ΔbdhA / pHP13-opuE with good tolerance to acetoin were selected to ferment and produce acetoin in a 7.5L fermenter. The stirring speed was 600rpm, the ventilation volume was 2:1, the inoculation amount was 5% (v / v), the culture temperature was 37°C, and the initial glucose concentration was 120g / L. When the glucose was fermented to less than 20g / L, 100mL of glucose (500g / L) and 100mL of yeast powder (60g / L) were supplemented for the first time, the stirring speed was increased to 800rpm, and 100mL of glucose (500g / L) was supplemented for the second time. The pH of the fermentation process was controlled at 6.8. The results are as follows Figure 3 shown.

[0106] like Figure 3 As shown, the acetoin production and sugar conversion rate of the genetically engineered strains B. subtilis 13932ΔbdhA / pHP13-bioY and B. subtilis 13932ΔbdhA / pHP13-opuE were 76.42 g / L, 0.347 g / g and 80.76 g / L, 0.367 g / g, respectively. The acetoin fermentation yield was increased by 20.2% and 27.1% compared with B. subtilis 13932ΔbdhA, respectively.

[0107] Example 5 Effect of co-expression of biotin transporter bioY and proline transporter opuE by recombinant strain B. subtilis 13932ΔbdhA on acetoin synthesis and strain growth

[0108] Plate culture medium: peptone 10g / L, yeast extract 5g / L, Nacl 10g / L, agar 20g / L, add distilled water to 1000ml, pH natural, sterilize at 121℃ for 15min.

[0109] Seed culture medium (LB liquid medium): peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, add distilled water to 1000 ml, pH natural, sterilize at 121°C for 15 min.

[0110] Acetoin stress medium: LB liquid medium containing 40 g / L acetoin, pH 7.0, sterilized at 110°C for 15 min.

[0111] Acetoin fermentation medium: glucose 110 g / L, corn steep liquor powder 15 g / L, urea 3 g / L, yeast powder 8 g / L, K2HPO4 3 g / L, KH2PO4 3 g / L, MgSO4 0.6 g / L, CaCl2 0.5 g / L, ZnSO4 0.5 g / L, the pH value of the fermentation medium is 6.8-7.2, and it is sterilized at 110°C for 10 min.

[0112] The genetically engineered bacteria B. subtilis 13932ΔbdhA / pHP13-bioY-opuE, B. subtilis 13932ΔbdhA / pHP13-bioY and B. subtilis 13932ΔbdhA / pHP13-opuE constructed in Example 1 were spread on LB solid plates, and one loop of colonies was scraped and inoculated into LB liquid medium and cultured overnight at 37°C and 200±20rpm / min to obtain seed liquid. The obtained seed liquid was then inoculated into a 40g / L acetoin stress medium at a 5% (v / v) inoculation amount, and OD600 was measured at 0h, 4h, 6h, 8h, and 10h, respectively. The starting strain B. subtilis13932 was used as a blank control Control. The results are shown in Table 3.

[0113] Table 3 Growth of recombinant strains in acetoin stress medium

[0114]

[0115] The results are shown in Table 3. After co-expression of the biotin transporter bioY and the proline transporter opuE, the tolerance of the recombinant strain B.subtilis 13932ΔbdhA / pHP13-bioY-opuE to acetoin was further improved. The specific growth rates of the three recombinant strains at 8h were 0.57, 0.51 and 0.53, respectively, which were significantly higher than those of the control strain control (0.44). The recombinant strain B.subtilis 13932ΔbdhA / pHP13-bioY-opuE significantly improved the growth of the recombinant strain under acetoin stress, and the specific growth rate was increased by 29.6% compared with the starting strain B.subtilis 13932.

[0116] Genetically engineered bacteria B. subtilis 13932ΔbdhA / pHP13-bioY, B. subtilis 13932ΔbdhA / pHP13-opuE and B. subtilis 13932ΔbdhA / pHP13-bioY-opuE were fermented in a 7.5 L fermentor to produce acetoin, with a stirring speed of 600 rpm, a ventilation volume of 2:1, an inoculation amount of 5% (v / v), a culture temperature of 37°C, an initial glucose concentration of 120 g / L, and when the glucose concentration was lower than 20 g / L, 100 mL of glucose (650 g / L) and 100 mL of yeast powder (60 g / L) were added for the first time, the stirring speed was increased to 800 rpm, and 100 mL of glucose (650 g / L) was added for the second time. The pH value of the fermentation process was controlled at 6.8. Genetically engineered strains B. subtilis 13932ΔbdhA / pHP13-bioY, B. subtilis The acetoin production of 13932ΔbdhA / pHP13-opuE and B. subtilis 13932ΔbdhA / pHP13-bioY-opuE were 80.89 g / L, 84.93 g / L and 88.76 g / L, respectively.

[0117]

[0118]

[0119]

Claims

1. A genetically engineered bacterium tolerant to high concentrations of acetoin, characterized in that: The genetically engineered bacteria are to perform metabolic engineering on the acetoin synthesis pathway of Bacillus subtilis and knock out the key gene for acetoin decomposition metabolism. BnA , and then the biotin synthesis metabolism genes related to the fatty acid synthesis pathway bioY and genes encoding the proline transport system opuE Overexpression is performed to obtain a genetically engineered strain that is tolerant to high concentrations of acetoin and has high production; The Bacillus subtilis is Bacillus subtilis ( Bacillus subtilis )CGMCC No.13932; The key gene for acetoin decomposition BnA The nucleotide sequence is shown in SEQ ID NO.1; Biotin synthesis metabolism genes related to the fatty acid synthesis pathway bioY The nucleotide sequence is shown in SEQ ID NO.2; The proline transport system encoding gene opuE The nucleotide sequence is shown in SEQ ID NO.

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2. A method for constructing a genetically engineered bacterium tolerant to high concentrations of acetoin, characterized in that: The following steps are involved: 1) bdhA Knockout: B. subtilis 13932 genome as template, using primer pair BnA -LF-F / R, BnA -DR-F / R, BnA -PC-F / R, BnA -RF-F / R amplify genes separately BnA Upstream homology arm LF, DR sequence, PC box, BnA The downstream homology arm RF was obtained by splicing the four fragments together using Overlap PCR BnA Knock out the recombinant fragment; 2) After purification, transform into B. subtilis 13932 competent cells, then spread on a chloramphenicol-containing resistance plate, verify the positive clones by colony PCR, inoculate into non-resistant LB liquid medium at 30-37°C, 100-300 rpm until OD is equal to 0.5-2, dilute 100 times and spread on MGY-Cl solid plate, culture at 30-37°C overnight, verify whether the gene is knocked out by colony PCR, and obtain recombinant strains by sequencing after preliminary confirmation B. subtilis 13932Δ BnA ; 3) Escherichia coli-Bacillus subtilis shuttle plasmid pHP13 was used as an expression vector, and restriction endonucleases were used Pst I and Eco Linearize pHP13 with RI and set aside; 4) Use a genome extraction kit to extract the Bacillus subtilis genome for target gene fragment PCR, and design primer pairs bioY -F / R, opuE -F / R amplified genes bioY、opuE Then, the target gene and vector were connected using the one-step cloning method and transferred into E. coli DH5α competent cells were plated on chloramphenicol LB resistance plates, positive clones were screened and inoculated into chloramphenicol LB liquid culture medium, and recombinant plasmid pHP13- bioY 、pHP13- opuE ; 5) Design primer pairs bioY 1-F / R, opuE 1-F / R amplified gene bioY、opuE Then, the target gene and vector were connected using the one-step cloning method and transferred into E. coli DH5α competent cells were plated on chloramphenicol LB resistance plates, positive clones were screened and inoculated into chloramphenicol LB liquid culture medium, and recombinant plasmid pHP13- bioY- opuE; 6) Transform the recombinant plasmid into the recombinant strain B. subtilis 13932Δ BnA In order to obtain genetically engineered bacteria; Gene BnA The nucleotide sequence is shown in SEQ ID NO.1; Gene bioY The nucleotide sequence is shown in SEQ ID NO.2; opuE The nucleotide sequence is shown in SEQ ID NO.

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3. Use of the genetically engineered bacteria according to claim 1 in the production of acetoin.

4. The use according to claim 3, characterized in that: The constructed genetically engineered bacteria are first cultured in a seed culture medium at 32~37°C and 200±20 rpm / min for 12-14 h to obtain a seed solution, and then the obtained seed solution is inoculated into an acetoin fermentation medium at an inoculum amount of 5~10% (v / v), and fermented at 32~37°C and 200±20 rpm / min for 60~84 h to obtain a fermentation solution containing acetoin.

5. The use according to claim 4, characterized in that: The acetoin fermentation medium comprises the following components: 100-120 g / L of glucose, 10-20 g / L of corn steep liquor powder, 1-5 g / L of urea, 5-10 g / L of yeast powder, 1-5 g / L of K2HPO4, 1-5 g / L of KH2PO4, 0.5-1.0 g / L of MgSO4, 0.5-1 g / L of CaCl2, and 0.5-1 g / L of ZnSO4. The pH value of the fermentation medium is 6.8-7.2, and the medium is sterilized at 110°C for 10 min.

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