Construction of surfactin-producing bacillus subtilis and anaerobic fermentation method

Abstract

This invention discloses a method for constructing and fermenting a strain of Bacillus subtilis to enhance anaerobic expression and improve surfactant synthesis. First, a chassis strain 158T7P is obtained by integrating and inserting an inducible transcriptional T7 RNA polymerase expression gene into the genome of Bacillus subtilis 158. Further genetic manipulation of this Bacillus subtilis, through systematic substitution of the T7 promoter, yields strain 158T7P-srf, capable of producing surfactant in high-density microaerobic fermentation. Specifically, this involves using homologous recombination to transform the operon of Bacillus subtilis 158... resde The starter and control sfp The promoters for transcription of the gene and the srfABCD operon were replaced with T7 promoters. The obtained Bacillus subtilis 158T7P-Srf was able to achieve high production of surfactant using inexpensive carbon sources such as brown sugar as substrates. In the fermentation experiment, the surfactant production reached a maximum of 36.58 g / L.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and microbial synthesis technology, specifically to a genetic modification method for Bacillus subtilis, and the application of the modified bacteria in the fermentation synthesis of bioactive substances (especially surfactants), which is particularly suitable for high-efficiency fermentation production under anaerobic or microaerobic conditions. Background Technology

[0002] As a food-safe bacterium, it possesses advantages such as a clear genetic background, strong protein secretion capacity, and mature fermentation technology, and is widely used in the synthesis and production of industrial enzymes, antimicrobial peptides, biosurfactants, and other biological products. Bacillus subtilis 158 is a preserved strain capable of efficiently producing surfactants, with the accession number CCTCC AB 2025197. Surfactants, as lipopeptide biosurfactants with excellent emulsifying and antibacterial activities, have extremely high application value in the fields of medicine, food, and environmental protection.

[0003] However, existing Bacillus subtilis expression systems face several technical challenges in practical applications. During high-density growth, insufficient dissolved oxygen leads to decreased growth performance and metabolic synthesis energy, resulting in reduced metabolite yields. Therefore, it is necessary to co-express the multiple gene chains involved in the synthesis of complex metabolites, such as surfactant. However, efficient co-expression of multiple gene clusters is often difficult. For example, surfactant biosynthesis depends on the srfABCD gene cluster (multi-gene synergistic action), and its synthesis efficiency is also regulated by key genes such as the anaerobic expression regulator resDE and the 4-phosphate pantothenic acid thioethylamine transferase gene sfp. Existing expression systems often use Bacillus subtilis' own promoters. These promoters suffer from complex regulatory elements, limited transcriptional strength, and poor coordination of multiple gene expression, preventing the simultaneous and efficient expression of multiple gene clusters and severely limiting the synthesis efficiency of the target product.

[0004] Furthermore, Bacillus subtilis is a facultative anaerobic bacterium with poor adaptability to anaerobic / microaerobic fermentation. Wild-type and existing engineered strains exhibit problems such as metabolic pathway disorders, insufficient energy supply, and inhibited expression of key regulatory genes under anaerobic or microaerobic conditions, leading to a significant decrease or even stagnation in the synthesis of target products, which cannot meet the needs of reducing energy consumption and lowering fermentation costs in industrial production.

[0005] Therefore, developing a novel Bacillus subtilis expression system that can solve the problem of efficient synergistic expression of multiple gene clusters and is adapted to anaerobic / microaerobic fermentation conditions is a technical problem that urgently needs to be solved in this field to achieve efficient production of surfactants. Summary of the Invention

[0006] The purpose of this invention is to provide a novel Bacillus subtilis expression system, which constructs a dedicated chassis bacterium by integrating a genome-integrated T7 RNA polymerase expression system and systematically replaces the promoters of key genes / gene clusters to solve the technical problems of efficient synergistic expression of multiple gene clusters and low efficiency of anaerobic / microaerobic fermentation, thereby achieving the efficient synthesis of target products such as surfactants.

[0007] The technical solution adopted by the present invention to achieve the above objectives is as follows:

[0008] 1) Construction of a novel chassis-type Bacillus subtilis 158T7P:

[0009] Using the screened Bacillus subtilis 158 (accession number: CCTCC AB 2025197) as the original strain, a novel spore-forming Bacillus subtilis 158T7P was constructed by integrating the T7 RNA polymerase expression system into its genome through homologous recombination technology. This spore-forming bacterium retains the advantages of the original strain, such as rapid growth rate and strong product tolerance, while also possessing highly efficient transcriptional capabilities mediated by T7 RNA polymerase.

[0010] 2) Systematic replacement of key gene / gene cluster promoters:

[0011] Genetic modification was performed on *Bacillus subtilis* 158T7P, replacing the original promoters of the following genes / gene clusters in the *Bacillus subtilis* 158T7P genome with promoters specifically recognized by *E. coli* T7 phage RNA polymerase:

[0012] The key gene cluster for surfactant synthesis, srfABCD (key sequence shown in SEQ ID NO:1); the anaerobic expression regulator gene, resDE (expression sequence region shown in SEQ ID NO:2); and the 4-phosphate pantothenic thioethylamine transferase gene, sfp (expression sequence region shown in SEQ ID NO:3).

[0013] Through the above modifications, synchronous and efficient transcription of multiple gene clusters mediated by the T7 promoter was achieved, solving the technical bottleneck of uncoordinated multi-gene expression under traditional promoter regulation.

[0014] 3) Method for producing surfactants by anaerobic fermentation:

[0015] The genetically modified Bacillus subtilis 158T7P-srf strain was used as the production strain. The fermentation medium formula was as follows: brown sugar 10-40 g / L, tryptone 5-30 g / L, monosodium glutamate 5-20 g / L, ammonium sulfate 2-10 g / L, sodium nitrate 0.2-2.0 g / L, ferrous sulfate 0.05-0.2 g / L, disodium hydrogen phosphate 7.5 g / L, and potassium dihydrogen phosphate 2.5 g / L.

[0016] The fermentation process conditions are as follows: the strain is first cultured aerobically to a high density (OD). 600 >100), and then controlled dissolved oxygen levels (by controlling the air flux to less than 5.0 L / h / L) for anaerobic or microaerobic fermentation to achieve efficient synthesis of surfactants. The genetically modified Bacillus subtilis 158T7P-srf described in this invention can be widely used in the field of biosynthesis, including but not limited to the fermentation synthesis of surfactants, and can also be extended to the efficient production of other bioactive substances such as lipopeptides and polyketides that depend on the synergistic expression of multiple gene clusters.

[0017] Compared with existing technologies, it has the following significant technical advantages and novelty:

[0018] The novel and highly adaptable *Bacillus subtilis* strain was selected as the original strain. Compared to traditional *Bacillus subtilis* strains, this strain exhibits stronger surfactant synthesis potential, a faster growth rate, and broader nutritional requirements, laying the foundation for the subsequent construction of a highly efficient expression system. This invention integrates the T7 expression system with the genome of *Bacillus subtilis* 158 to construct a novel *Bacillus subtilis* strain, 158T7P. Utilizing the high specificity and high transcription efficiency of the T7 promoter, the expression intensity of the target gene was significantly enhanced.

[0019] This invention systematically solves the problem of multi-gene cluster expression: by simultaneously replacing the promoters of the srfABCD gene cluster, resDE gene, and sfp gene through the T7 promoter, it achieves synergistic and efficient expression of multiple gene clusters, solves the core technical difficulties of uncoordinated multi-gene regulation and low expression efficiency in traditional expression systems, and provides a new solution for the reconstruction of complex biosynthetic pathways.

[0020] Breakthrough in anaerobic fermentation technology bottlenecks: By mediating the expression of the resDE gene (anaerobic regulatory factor) through the T7 promoter, combined with optimized anaerobic fermentation medium and process, the technical problem of low fermentation efficiency of Bacillus subtilis under anaerobic / micro-aerobic conditions has been solved, reducing energy consumption in the fermentation process (no need for high dissolved oxygen supply) and expanding its industrial application scenarios.

[0021] Significantly improved fermentation efficiency and product yield: The optimized culture medium formula (using brown sugar as a carbon source, combined with nutrients such as monosodium glutamate) works synergistically with the high-efficiency expression system, combined with the high-density pre-culture-anaerobic fermentation process, which increases the fermentation yield of surfactant by more than 10 times compared with the original production strain, and the fermentation process is more stable, making it suitable for industrial-scale production. Attached Figure Description

[0022] Figure 1This is a schematic diagram illustrating the construction and application of Bacillus subtilis 158T7P-srf according to the present invention;

[0023] Figure 2 This is a graph showing the data results for high-density fermentation to produce surfactants. Detailed Implementation

[0024] The technical solution of the present invention will be described in detail below with reference to the embodiments. Operations not specifically described in the embodiments shall be performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents and biological materials used below are commercial products.

[0025] The sources of the biological materials used in the following embodiments are as follows:

[0026] Bacillus subtilis 158 was derived from the inventor's deposited strain, with accession number CCTCC AB 2025197. DNA synthesis and sequencing annotation were performed at Sangon Biotech (Shanghai) Co., Ltd. Common chemical reagents and raw materials used in the examples were purchased from distributors with existing stock; 2×Phanta Master Mix (catalog number P511-01) and ClonExpress®II One Step Cloning Kit (catalog number C115-01) were purchased from Nanjing Novizan Biotechnology Co., Ltd.; erythromycin (catalog number E808819) and D-xylose (catalog number D856756) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0027] Example 1: Construction of recombinant Bacillus subtilis 158T7P-srf

[0028] The *Bacillus subtilis* 158T7P-srf strain was developed through multi-step homologous recombination in the genome of *Bacillus subtilis* strain 158, involving the integration of a T7 RNA polymerase expression sequence at the aprE site. The technical procedures can be referenced in the published results (DOI: 10.1016 / j.enzmictec.2020.109726). While the original published results focused on *Bacillus subtilis* 164, this invention focuses on *Bacillus subtilis* 158. After obtaining 158T7P, further homologous recombination was performed, converting the original promoters of srfABCD, resDE, and sfp into T7 promoters. The replacement of the srfABCD promoter was performed first, followed by resDE and sfp. The principle of homologous operation is consistent. Detailed procedures can be found in invention patents CN114317384A and CN114438001B. This invention uses the srfABCD promoter replacement as an example, described below:

[0029] First, using the synthesized resistance gene DNA fragment ermC-pT7 (SEQ ID NO:4) as a template, ermC-pT7 was amplified; then, using Bacillus subtilis 158 genomic DNA as a template, sfp-U and sfp-D were amplified. Since the 158 genomic sequence involved in this invention is completely identical to that of Bacillus subtilis ATCC 6051 (NCIB 3610), its genomic sequence can be obtained from the NCBI database, and its sequence number is GCF_006088795.1.

[0030] Next, the three fragments were fused using fusion PCR. The fusion PCR method is as follows: The PCR reaction preparation system consisted of 10 μL 2× Phanta Master Mix, with 200 ng of each of the three fragments added, and ddH2O was added to bring the system volume to 20 μL. The first round of PCR was performed under the following conditions: pre-denaturation at 95 ℃ for 5 min, followed by denaturation at 95 ℃ for 30 s, annealing at 60 ℃ for 20 s, and extension at 72 ℃ for 2 min, for a total of 6 cycles. This yielded the fusion fragment sfp-U-ermC-pT7-sfp-D.

[0031] The obtained sfp-U-ermC-pT7-sfp-D was then transformed into Bacillus subtilis 158T7P and evenly spread on a plate containing 10 μg / mL erythromycin resistance, and incubated overnight at 37 ℃. Transformants were identified by colony PCR; positive transformants were recombinant strains where sfp-U-ermC-pT7-sfp-D replaced the original homologous fragment, thus replacing the original promoter with the T7 promoter. Using positive transformant strains as templates, the integrated fragment was amplified using 2 × Phanta Master Mix. The PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Transformants with correct sequencing results were named Bacillus subtilis 158T7P-srf.

[0032] Example 2: High-density fermentation synthesis of surfactants

[0033] The following is the procedure for culturing 158T7P-srf bacteria and performing fed-batch high-density fermentation to produce surfactants. First, the seed culture for fermentation is prepared. The process is as follows: First, the prepared recombinant Bacillus subtilis 158T7P-srf is streaked onto an LB agar plate for activation. After overnight incubation, a single colony is picked and inoculated into a test tube containing 3 mL of LB. The culture is incubated overnight at 37°C with shaking at 200 rpm. 1.0 mL of the overnight culture is then inoculated into a 1000 mL shake flask containing 100 mL of fresh LB and incubated for another 5 h at 37°C and 200 rpm. All 100 mL of seed culture were inoculated into 2 L of initial fermentation medium containing 10 g / L brown sugar, 5 g / L tryptone, 5 g / L monosodium glutamate, 0.2 g / L magnesium sulfate, 0.1 g / L ferrous sulfate, 7.5 g / L disodium hydrogen phosphate, 2.5 g / L potassium dihydrogen phosphate, and 0.5 g / L antifoaming agent. The initial pH was 6.8. Fermentation control parameters were: aeration rate of 5 L / min, turbine rotation of 700 rpm, and temperature of 37℃. After 3 h of fermentation, xylose was added to a final concentration of 1%. Brown sugar, ammonium nitrate, and monosodium glutamate were fed in to maintain the pH of the fermenter at approximately 6.8. Cell growth density (OD) was measured periodically. 600 The yield of surfactants was determined by liquid chromatography; OD 600 Once high density (>100) is reached, dissolved oxygen levels are monitored, and aeration is controlled below 2.0 L / min / L, implementing microaerobic or localized anaerobic fermentation with dissolved oxygen levels below 5. For example... Figure 2 As shown, surfactants showed a significant increase after high-density fermentation, but the amount synthesized tended to stabilize during long-term fermentation. The highest surfactant yield was measured at around 60 hours, which was 36.58 g / L.

[0034] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of these embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the present invention's technical solutions, and any such changes and modifications are within the protection scope of the present invention.

[0035] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A Bacillus subtilis expression system, characterized in that: The novel chassis Bacillus subtilis, 158T7P, was established by integrating a T7 RNA polymerase expression system into the Bacillus subtilis 158 genome.

2. The Bacillus subtilis expression system as described in claim 1, characterized in that: The Bacillus subtilis strain is derived from Bacillus subtilis strain 158 obtained through screening, with accession number CCTCC AB 2025197.

3. The Bacillus subtilis 158T7P as described in claim 1, followed by a series of genetic operations, characterized in that: The promoters of several original genes or operators were replaced with promoters recognized by E. coli T7 phage RNA polymerase, respectively, to express the surfactant gene cluster (srfABCD), the anaerobic exchange factor (desDE), and the 4-phosphate pantothenic acid thioethylamine transferase gene (sfp). The promoter sequences and protein expression sequences are shown in SEQ ID NO:1-3, respectively.

4. The application of the genetically modified Bacillus subtilis strain according to claim 3 in biosynthesis, including but not limited to the fermentation synthesis of surfactants.

5. The method for producing surfactants by fermentation of Bacillus subtilis according to claim 3, characterized in that, The culture medium used contained 10-40 g / L brown sugar, 5-30 g / L tryptone, 5-20 g / L monosodium glutamate, 0.2 g / L magnesium sulfate, 0.1 g / L ferrous sulfate, 7.5 g / L disodium hydrogen phosphate, and 2.5 g / L potassium dihydrogen phosphate. The Bacillus subtilis was cultured aerobically to a high density (OD). 600 After reaching >100), control the dissolved oxygen level (air flux less than 2.0 L / h / L).

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