A genetically engineered bacterial strain with high yield of acetoin and 2,3-butanediol, a construction method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a genetically engineered strain with high yield of acetoin and 2,3-butanediol, a construction method and application. The present application obtains acoR deletion mutant MW03-ΔA by knocking out the regulatory gene acoR of the acetoin dehydrogenase enzyme system, blocks the degradation of AoDHES to acetoin, effectively reduces the production of by-products, thereby facilitating the accumulation of more acetoin. A strong promoter expression vector is further constructed in the MW03-ΔA strain, and the alpha-acetyl lactate decarboxylase gene alsD is connected, and then introduced into the acoR deletion mutant MW03-ΔA of Bacillus licheniformis, so as to overexpress alpha-acetyl lactate decarboxylase, and obtain a genetically engineered strain MW-BL4 with high yield of acetoin and 2,3-butanediol. The present application has the beneficial effect that compared with a single product of the engineered strain, the present application can realize the production of acetoin and 2,3-butanediol and other multiple products by using a single strain, and the yield is considerable, which can significantly reduce the cost of bacterial strains in industrial production and improve the production efficiency.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a genetically engineered strain that produces high levels of acetoin and 2,3-butanediol, its construction method, and its applications. Background Technology

[0002] With the global energy crisis worsening and oil resources gradually depleting, finding alternative chemical production methods has become a crucial issue in the chemical industry. Traditional chemical production relies heavily on petroleum-based feedstocks, which not only puts enormous pressure on the environment but also exacerbates the consumption of petroleum resources. Therefore, developing sustainable production processes that do not depend on petroleum resources is of great significance for achieving environmental friendliness and efficient resource utilization.

[0003] 2,3-Butanediol (2,3-BD) and acetoin are two promising chemicals with wide applications in food, pharmaceuticals, agriculture, and chemical industries. However, traditional production methods for 2,3-butanediol and acetoin rely heavily on fossil resources, which are not only energy-intensive and polluting but also risk resource depletion.

[0004] Microbial fermentation, as an alternative production process, is becoming an important route for producing these high-value chemicals due to its advantages such as low cost, low energy consumption, and environmental friendliness. Producing 2,3-butanediol, acetoin, and other high-value chemicals through microbial fermentation can not only effectively reduce dependence on petroleum resources but also provide strong support for environmental protection and efficient resource utilization, thus possessing significant economic and social value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a genetically engineered strain that produces high levels of acetoin and 2,3-butanediol, its construction method, and its applications.

[0006] This invention obtains the acoR-deficient mutant strain MW03-ΔA by knocking out the positive regulatory gene acoR of the acetoin dehydrogenase system (AoDHES) operon aco. This blocks the degradation of acetoin by AoDHES, effectively reducing the production of byproducts and thus promoting the accumulation of more acetoin. Furthermore, a strong promoter expression vector is constructed in the MW03-ΔA strain, and the gene encoding α-acetolactate decarboxylase alsD (Gene ID: 936857) is ligated into it. This vector is then introduced into the Bacillus licheniformis acoR-deficient mutant MW03-ΔA to overexpress α-acetolactate decarboxylase (ALDC), resulting in a genetically engineered strain MW-BL4 that produces high levels of acetoin and 2,3-butanediol.

[0007] The first aspect of this invention provides a genetically engineered strain that produces high levels of acetoin and 2,3-butanediol. This strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 31, 2025, with accession number CCTCC NO: M 2025653, and is classified as *Bacillus licheniformis* MW-BL4. The second aspect of this invention provides a method for constructing the genetically engineered strain, comprising the following steps:

[0008] Construction of S1 acoR deletion mutant strain MW03-ΔA: Starting with Bacillus licheniformis MW03 (source: Microbial Enzyme Technology Laboratory, School of Bioengineering, Qilu University of Technology), the acoR gene, which regulates acetoin dehydrogenase, was knocked out in Bacillus licheniformis to obtain a mutant strain with acoR deletion, named MW03-ΔA.

[0009] Construction of S2 genetically engineered strain MW-BL4: The target gene α-acetolactate decarboxylase encoding gene alsD was ligated into an expression vector to obtain a recombinant plasmid. The recombinant plasmid was then introduced into the acoR deletion mutant strain MW03-ΔA constructed in S1 to obtain the genetically engineered strain, named MW-BL4.

[0010] Preferably, in S2, the recombinant plasmid is prepared as follows: synthesize the promoter Plaps fragment and the target gene alsD, use primers alsD-F / alsD-F to obtain the target gene fragment Plaps+alsD, and ligate the gene fragment to the expression vector pHY300PLK to obtain the recombinant plasmid pHY300PLK-Plaps-alsD that overexpresses the gene alsD.

[0011] A third aspect of the invention is the use of the genetically engineered strain in the production of 2,3-butanediol and acetoin.

[0012] As a preferred embodiment, the fermentation conditions for the production of 2,3-butanediol and acetoin by the genetically engineered strain are as follows:

[0013] The fermentation temperature was 35-38℃, the pH was 6.2-7.8, the inoculum size of the seed culture was 8%-12%, and the culture was carried out at a constant temperature with a rotation speed of 180-250 r / min. During the fermentation period, sterile glucose solution was added starting from the 12th hour, and then 10 mL of 70 g / L sterile glucose solution was added every 12 hours. The fermentation cycle was 3-5 days.

[0014] As a further preferred embodiment, when the genetically engineered strain produces 2,3-butanediol, the fermentation conditions are: fermentation temperature of 37°C, pH of 6.2-6.7, inoculum size of 10%, and fermentation cycle of 3 days.

[0015] Preferably, when the genetically engineered strain produces acetoin, the fermentation conditions are: fermentation temperature of 37°C, pH of 7.2-7.8, inoculum size of 10%, and fermentation cycle of 5 days.

[0016] The beneficial effects of this invention are as follows:

[0017] By knocking out the positive regulatory gene acoR of the AoDHES operon aco, the degradation of acetoin by AoDHES was blocked, resulting in strain MW03-ΔA. Furthermore, the gene alsD encoding ALDC was overexpressed in strain MW03-ΔA, which directed more carbon flow to the 2,3-BD synthesis pathway, thus constructing the genetically engineered strain MW-BL4.

[0018] The genetically engineered strain MW-BL4 provided by this invention can produce a variety of products such as 2,3-butanediol and acetoin, and the yield is considerable. In industrial production, it can significantly reduce the cost of the strain and improve production efficiency. Attached Figure Description

[0019] Figure 1 The result of constructing the acoR deletion mutant MW03-ΔA;

[0020] Figure 2 This is a schematic diagram illustrating the construction of the recombinant plasmid pHY300PLK-Plaps-alsD.

[0021] Figure 3 Construct and validate nucleic acid electrophoresis images for recombinant strain MW-BL4;

[0022] Figure 4 This is an SDS-PAGE electrophoresis image of the overexpression of the alsD gene in the recombinant strain MW-BL4. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the invention will be further described below with reference to specific embodiments. The advantages and features of the invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the invention.

[0024] Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the protection scope of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

[0026] The following examples illustrate a method for constructing an engineered strain that produces high levels of 2,3-butanediol and acetoin, using Bacillus licheniformis MW03 as an example.

[0027] The culture medium and its components used in this invention are as follows:

[0028] LB liquid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, adjust pH to 7.0, autoclave at 121°C for 20 min.

[0029] LB solid medium: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar, adjust pH to 7.0, autoclave at 121°C for 20 min.

[0030] Fermentation medium: yeast powder 5g / L, corn steep liquor powder 20g / L, urea 5g / L, KH2PO4 6g / L, K2HPO4 14g / L, trisodium citrate 8g / L, glucose 70g / L, adjust pH to 7.0, autoclave at 115℃ for 20min.

[0031] In this invention, the PCR amplification system is shown in Table 1.

[0032] Table 1 50μL PCR amplification system

[0033]

[0034] Example 1

[0035] A genetically engineered strain that produces high levels of acetoin and 2,3-butanediol was constructed as follows:

[0036] Construction of S1 acoR deletion mutant strain MW03-ΔA

[0037] The acoR gene in B. licheniformis MW03 (MW03) was knocked out using CRISPR-Cas9 gene editing technology to obtain a mutant strain with acoR deletion, named MW03-ΔA;

[0038] Using primers acoR-vF / acoR-vR and the MW03 genome as a template, the gene knockout in the two mutant strains was confirmed by PCR. Figure 1 The construction results of the acoR deletion mutant MW03-ΔA are shown, with appendix. Figure 1In the diagram, lane M represents the DNA Marker, lane 1 represents the negative control result for the pJOE8999-acoR plasmid, lane 2 represents the negative control result for the genome, and lanes 3 and 4 represent the results for the correct mutant.

[0039] Appendix Figure 1 As can be seen, the mutation of the acoR gene resulted in its fragment being truncated from 2481bp to 1347bp, and the band is as expected, proving that the acoR gene knockout was successful.

[0040] Construction of S2 genetically engineered strain MW-BL4

[0041] First, the promoter Plaps fragment and the coding gene alsD for the target gene ALDC were synthesized. The target fragment Plaps+alsD was obtained using primers alsD-F / alsD-F, and this gene fragment was ligated into the expression vector pHY300PLK using Vazyme's seamless cloning enzyme. This completed the construction of the recombinant plasmid pHY300PLK-Plaps-alsD, overexpressing the gene alsD. A schematic diagram of the construction of the recombinant plasmid pHY300PLK-Plaps-alsD is attached. Figure 2 As shown;

[0042] Next, the recombinant plasmid pHY300PLK-Plaps-alsD was transformed into competent E. coli DH5α cells, and the plasmid was extracted and sequenced after culture.

[0043] Finally, after confirming that the gene sequencing was correct, the recombinant plasmid was transformed into Bacillus licheniformis MW03-ΔA to complete the construction of the recombinant engineered strain MW-BL4.

[0044] The recombinant engineered bacterial strain MW-BL4 was cultured, and colony PCR was used to verify whether the recombinant plasmid had been transformed into the host bacteria (using primers pHY-F / pHY-R). The results are shown in the attached figure. Figure 3 As shown, attached Figure 3 In the diagram, lane 4 corresponds to the correct recombinant strain, lanes 1-3 are negative controls, lane 1 is an empty vector template band of approximately 250 bp, lane 2 has no amplicon, and lane 3 is the correct fragment band on the vector. Figure 3 As can be seen, all bands are clear and of the correct size, proving that the recombinant plasmid was successfully transformed into the host strain.

[0045] The genetically engineered strain MW-BL4 and the control strain were cultured, and crude protein solutions were extracted and subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 4 As shown, lane 3 is the protein electrophoresis marker, lane 4 is the crude MW03-ΔA protein, and lanes 1-2 are the crude MW-BL4 protein. All of them have obvious bands at 29kDa and are consistent with the ALDC size.

[0046] The genetically engineered strain MW-BL4 obtained in this embodiment was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 31, 2025, with accession number CCTCC No: M 2025653, and classified as Bacillus licheniformis MW-BL4.

[0047] The preparation of electroconversion competent cells of B. licheniformis can be carried out using conventional methods, and the specific steps are as follows:

[0048] B. licheniformis MW03, stored at -80℃, was inoculated onto solid LB medium and incubated at 37℃ for approximately 18 hours. Single colonies obtained from the culture were picked and transferred to 5 mL of LBS medium, and incubated at 200 rpm at 37℃ for 12 hours. 1 mL of the culture was then inoculated onto 50 mL of liquid LBS medium and incubated at 37℃ at 200 rpm until the bacterial growth reached OD. 600 At approximately 0.8–1.2 μL, incubate on ice for 10 min, then transfer to pre-chilled sterile centrifuge tubes. Centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend the cells in pre-chilled solution A, gently mix, add more solution A, incubate on ice for 10 min, then centrifuge at 6000 rpm for 10 min at 4°C. Discard the supernatant, repeat twice, resuspend the cells in pre-chilled solution B, aliquot 80 μL / tube, and store at -80°C for later use.

[0049] The primers and sequences involved in the above embodiments are shown in Table 2 below.

[0050] Table 2 Primers and Sequences

[0051]

[0052]

[0053] Example 2

[0054] The initial strain MW03, the genetically engineered strain MW03-ΔA, and the genetically engineered strain MW-BL4 were used to conduct shake-flask culture of 2,3-butanediol, respectively, as follows:

[0055] (1) Activation of strains: Strains MW03, MW03-ΔA and MW-BL4 were inoculated onto solid LB medium and cultured at 37℃ for 24h.

[0056] (2) Seed culture: Pick a single colony from the LB solid medium in (1) and inoculate it into 50 mL of liquid LB medium. Incubate at 37℃ and 200 r / min for 8 h.

[0057] (3) Shake flask culture: Take 20 mL of the seed liquid from (2) and inoculate it into 200 mL of fermentation medium. The seed liquid inoculation amount is 10%, the pH is 6.5, and the culture is carried out at 37℃ and 200 r / min. During the fermentation period, starting from the 12th hour, 10 mL of 70 g / L sterile glucose solution is added every 12 hours.

[0058] Example 3

[0059] The initial strain MW03, the genetically engineered strain MW03-ΔA, and the genetically engineered strain MW-BL4 were used to conduct shake flask culture of acetoin.

[0060] Unlike Example 2, in (3), the pH is 7.5, the inoculum size is 10%, and the other operations and conditions are the same as in Example 2.

[0061] The methods for determining 2,3-butanediol and acetoin in the fermentation broth of Examples 2-3 are as follows:

[0062] 2,3-Butanediol and acetoin were simultaneously determined by capillary gas chromatography using a Shimadzu GC-2030 gas chromatograph (Japan). The column specifications were: 30 μm length, 0.32 mm inner diameter, and 0.5 μm film thickness. Detection conditions were: column temperature 80 °C, injector and detector temperature 250 °C, injection volume 0.1 μL, and an FID detector.

[0063] After centrifuging to remove the bacterial cells, the fermentation broth was extracted with ethyl acetate in a 1:1 ratio by shaking. The upper extract was filtered through a 0.22 μm organic filter membrane and the sample was analyzed by gas chromatography.

[0064] The test results are shown in Table 3.

[0065] Table 3. Yields (g / L) of 2,3-BD and acetoin produced by different strains.

[0066]

[0067] As shown in Table 3, in the shake-flask fermentation experiment, the yield of 2,3-butanediol by each strain showed an accelerating growth trend after 24 hours of fermentation. Among them, strain MW03-ΔA reached its maximum yield of 2,3-BD at 75.3 g / L after 84 hours of fermentation, while the yield of the original strain MW03 was 73.1 g / L. The 2,3-butanediol productivity of strain MW03-ΔA was 0.3% higher than that of the original strain MW03. With continued fermentation, the content of 2,3-BD gradually decreased, while the yield of acetoin increased significantly. At 108 hours of fermentation, the concentration of acetoin produced by strain MW03-ΔA was the highest at 75.0 g / L, while the acetoin yield of the original strain reached its maximum at 96 hours at 61.6 g / L. The acetoin yield of strain MW03-ΔA was 21.7% higher than that of the original strain.

[0068] Furthermore, after 60 hours of fermentation, the 2,3-BD yield of the genetically engineered strain MW-BL4 reached 121.9 g / L (fermentation intensity of 2.03 g / L·h), while the maximum 2,3-BD yield of the control strain MW03-ΔA was 75.3 g / L (fermentation intensity of 0.9 g / L·h). The 2,3-butanediol production rate of the genetically engineered strain MW-BL4 was 61.9% higher than that of the control strain MW03-ΔA. With continued fermentation, the 2,3-BD content decreased slightly, while the acetoin yield gradually increased. After 108 hours of fermentation, the acetoin concentration produced by strain MW-BL4 was 76.7 g / L, while the acetoin yield of the control strain at 108 hours was 75 g / L. The acetoin yield of the genetically engineered strain MW-BL4 was 0.2% higher than that of the control strain MW03-ΔA and 24.5% higher than that of the original strain MW03.

Claims

1. A genetically engineered strain that produces high levels of acetoin and 2,3-butanediol, characterized in that, The genetically engineered strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, on March 31, 2025, with accession number CCTCC NO: M 2025653, and is classified as Bacillus licheniformis MW-BL4.

2. The use of the genetically engineered strain according to claim 1 in the production of 2,3-butanediol and acetoin.

3. The application as described in claim 2, characterized in that, The fermentation conditions for the production of 2,3-butanediol and acetoin by the genetically engineered strain are as follows: The fermentation temperature was 35-38℃, the pH was 6.2-7.8, the inoculum size of the seed culture was 8%-12%, and the culture was carried out at a constant temperature with a rotation speed of 180-250 r / min. During the fermentation period, sterile glucose solution was added starting from the 12th hour, and then 10 mL of 70 g / L sterile glucose solution was added every 12 hours. The fermentation cycle was 3-5 days.

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