A method for increasing MK-7 production through farnesyl synthase engineering and fermentation optimization and Bacillus subtilis used therein

Abstract

A method for increasing MK-7 production through farnesyl synthase engineering and fermentation optimization, and the Bacillus subtilis used therein. The Bacillus subtilis BS018 has been deposited with the China Center for Type Culture Collection (CCTCC) on January 2, 2024, with a strain deposit number of CCTCC NO: M2024004. A protein fusion co-expression strategy effectively addresses the problem of insufficient carbon flux for farnesyl pyrophosphate (FPP). This strategy enables heppentenyl pyrophosphate synthase (HepS / T) to efficiently utilize the precursor FPP, thereby improving the synthesis efficiency of the side chain HepPP.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and biotechnology, and particularly relates to a method for increasing MK-7 production through farnesyl synthase engineering and fermentation optimization, and the Bacillus subtilis used therein. Background Art

[0002] Menaquinone heptadenone (MK-7) is an important form of fat-soluble VK2. It is primarily composed of a 2-methyl-1,4-naphthoquinone moiety with a side chain consisting of seven isoprenyl units at the 3-position. Among VK2 homologues, MK-7 has a long half-life in the bloodstream and high bioavailability, and it has the most significant effect on the carboxylation of osteocalcin in humans. Therefore, MK-7 holds great market potential as a safe and effective dietary supplement. The European Food Safety Authority (EFSA) approved MK-7 as a food and fortified food additive in 2008, and China approved its inclusion in its national nutrient fortification catalogue in 2016.

[0003] MK-7 production is expensive due to its low natural abundance and high downstream extraction, separation, and purification costs. Currently, 90% of the world's VK2 raw materials come from Japan, and the price of imported VK2 per kilogram ranges from 2 to 4 million RMB. Currently, chemical synthesis and traditional fermentation methods are commonly used to mass-produce VK2. Chemical synthesis methods often involve complex steps, low yields, low activity, and the production of different cis-isomers and numerous byproducts, which can pollute the environment. Microbial fermentation, on the other hand, offers advantages such as mild conditions and readily available raw materials, resulting in production costs far lower than chemical synthesis. The main strains used to produce MK-7 include Bacillus subtilis, Bacillus natto, and Escherichia coli. Bacillus subtilis has been the most studied due to its well-defined genetic background, strong stress tolerance, and Generally Recognized as Safe (GRAS) status as a food-grade microorganism. Furthermore, Bacillus subtilis possesses a natural MK-7 synthesis pathway. When using glycerol as a substrate, the main pathways involved include the glycerol dissimilation pathway, the classical MK-7 pathway, the shikimate (SA) pathway, the erythritol phosphate (MEP) pathway, the pentose phosphate (PPP) pathway, the glycolytic pathway (EMP), and the tricarboxylic acid (TCA) cycle. Currently, most research focuses on enhancing the accumulation of key precursors and blocking or weakening metabolic flux in competing pathways to increase the accumulation of the target compound, MK-7. Although these strategies have significantly improved microbial production of MK-7, significant progress remains to be made in industrial production. Studies have shown that farnesyl pyrophosphate (FPP), synthesized via the MEP pathway, is not only a precursor for the MK-7 side chain heptapentyl pyrophosphate (HePP), but also a key precursor of the cell wall peptidoglycan undecenyl pyrophosphate (UPP). However, the activity of farnesyl synthase (BSIspA) in B. subtilis is low, at only 0.0012 µmol / min / mg, and is typically tightly controlled by its own regulatory system, resulting in low utilization of the substrate FPP by heptaphosphate synthase (HepS / T). Therefore, how to enable HepS / T to efficiently utilize the competing precursor FPP is a pressing challenge. Studies have shown that the activity of farnesyl synthase (GSIspA) in thermophilic Bacillus licheniformis can reach as high as 4.69 µmol / min / mg, 3908 times that of BSIspA. Summary of the Invention

[0004] The present invention aims to provide a method for increasing the yield of MK-7 through farnesyl synthase engineering and fermentation optimization, and the Bacillus subtilis used therein.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a Bacillus subtilis BS018, wherein the Bacillus subtilis BS018 has been deposited in the China Center for Type Culture Collection (CCTCC) on January 2, 2024, and the strain deposit number is CCTCC NO: M2024004.

[0006] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: use of Bacillus subtilis BS018 in the fermentation preparation of MK-7. The Bacillus subtilis BS018 has been deposited with the China Center for Type Culture Collection (CCTCC) on January 2, 2024, and the strain deposit number is CCTCC NO: M2024004.

[0007] To achieve the above-mentioned and other related objectives, the present invention provides a technical solution: a method for increasing the yield of MK-7 through farnesyl synthase engineering and fermentation optimization, wherein Bacillus subtilis BS018 is inoculated into LB liquid culture medium and cultured to prepare a seed solution; the seed solution is transferred into a fermentation medium, and after culture, a fermentation liquid containing MK-7 is obtained; the Bacillus subtilis BS018 has been deposited with the China Center for Type Culture Collection (CCTCC) on January 2, 2024, and the strain deposit number is CCTCC NO: M2024004.

[0008] The preferred technical solution is: calculated according to the liquid volume of a 500 mL shake flask, the liquid volume in a 500 ml shake flask is 40-60 mL.

[0009] The preferred technical solution is: the initial pH value of the fermentation medium is 6.8-7.2.

[0010] The preferred technical solution is: the inoculation amount is 7-10%.

[0011] The preferred technical solution is: when glucose or sucrose is used as a fast-acting carbon source, the concentration is 20-30 g / L; the added mass is 1-2.5%.

[0012] The preferred technical solution is: the fermentation medium contains 6-8% by mass of soy peptone and 0.3-0.6% by mass of yeast extract.

[0013] The preferred technical solution is: the fermentation medium contains KH2PO4 with a mass content of 0.08-0.12% and MgSO4·7H2O with a mass content of 0.08-0.12%.

[0014] The preferred technical solution is: adding DHNA with a concentration of 0.8-2 mmol / L to the fermentation medium 48 hours after fermentation.

[0015] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0016] The present invention assembled the highly active farnesyl pyrophosphate synthase (GSIspA) from thermophilic Bacillus licheniformis with the strong promoter Phbs and integrated it upstream of the HepS / T encoding operon hepS-menG-hepT-ndk, generating the recombinant strain Bacillus subtilis BS018. Shake flask fermentation assays revealed that Bacillus subtilis BS018 produced the highest MK-7 yield, reaching 91.1 mg / L. Shake flask culture conditions for MK-7 production by Bacillus subtilis BS018 were then optimized using single-factor experiments. The optimized culture medium composition and fermentation conditions were: 2% sucrose, 3% glycerol, 7% soy peptone, 0.4% yeast extract, 0.1% K₂HPO₄, and 0.1% MgSO₄·7H₂O, 1 mmol / L DHNA, and an initial pH of 7.0. With an inoculum size of 8% (v / v), a liquid volume of 50 mL / 500 mL, and a culture temperature of 40°C for 120 h, strain BS018 achieved a yield of 122.0 mg / L of MK-7. In summary, the protein fusion co-expression strategy effectively addressed the issue of insufficient carbon flux to farnesyl pyrophosphate (FPP). This strategy enabled the heptenyl pyrophosphate synthase (HepS / T) to efficiently utilize the precursor FPP, thereby increasing the efficiency of the synthesis of the side chain HepPP. Furthermore, through single-factor experiments, the optimal fermentation conditions for MK-7 synthesis by strain Bacillus subtilis BS018 were identified, and its metabolic patterns during fermentation were explored, providing strong support for further process research. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The effects of fusion expression of IspA and HepS / T from different sources on MK-7 production and cell growth. Figure: (A) Synthesis route of the side chain HepPP in B. subtilis; (B) Schematic diagram of the construction of recombinant strains expressing in-frame fusions of IspA and HepS / T from different sources; (C) Growth curve of the recombinant strains; (D) MK-7 production in the recombinant strains after 120 hours of fermentation. Results are presented as the mean of three replicates, with error bars representing standard deviation.

[0018] Figure 2 The effect of different initial pH on vitamin K2 production.

[0019] Figure 3 The effect of different inoculum amounts on vitamin K2 production.

[0020] Figure 4 The effect of different liquid volumes on vitamin K2 production.

[0021] Figure 5 Effects of different fast-acting carbon sources on vitamin K2 production. (A) Growth curve of BS018 at different glucose and sucrose concentrations; (B) Vitamin K2 production of MK-7 at different glucose and sucrose concentrations. Results are presented as the mean of three replicates, with error bars representing standard deviation.

[0022] Figure 6 Effects of different nitrogen source concentrations on vitamin K2 production. (A) Growth curves of BS018 and MK-7 production in the presence of different soy peptone concentrations; (B) Growth curves of BS018 and MK-7 production in the presence of different yeast extract concentrations. Results are presented as the mean of three replicates, with error bars representing standard deviations.

[0023] Figure 7 Effects of different inorganic ions on vitamin K2 production. (A) Growth curves after 6 days of fermentation with different phosphate concentrations added to the fermentation medium; (B) MK-7 production after 4 and 6 days of fermentation with different phosphate concentrations added to the fermentation medium; (C) Growth and MK-7 production of the strain under different KH2PO4 concentrations; (D) Effect of different MgSO4·7H2O concentrations on MK-7 production. Results are presented as the mean of three replicates, with error bars representing standard deviations.

[0024] Figure 8 Effects of different DHNA concentrations on BS018 biomass and MK-7 yield. (A) Growth curves of BS018 after 6 days of fermentation with different DHNA concentrations added to the fermentation medium; (B) MK-7 yields after 6 days of fermentation with different DHNA concentrations added to the fermentation medium. Results are presented as the mean of three replicates, with error bars representing standard deviations. DETAILED DESCRIPTION

[0025] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in these embodiments.

[0026] See also Figure 1-8 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no technical substantive significance. Any modification of the structure, change in the proportional relationship or adjustment of the size. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0027] Deposit of biological materials:

[0028] Bacillus subtilis BS018 has been deposited with the China Center for Type Culture Collection (CCTCC) on January 2, 2024, with the strain accession number M2024004. The address of the China Center for Type Culture Collection is Wuhan University, Wuhan, China.

[0029] Example 1: A method for increasing MK-7 production through farnesyl synthase engineering and fermentation optimization, and the Bacillus subtilis used therein

[0030] To improve the utilization of the substrate farnesyl pyrophosphate (FPP) by hepS / T, allowing FPP to be directly utilized by HepS / T without requiring transporters, the BSispA and GSispA genes were assembled using strong constitutive promoters and expressed upstream of the hepS-menG-hepT-ndk operon on the chromosome of Bacillus subtilis BS018, generating recombinant strains Bacillus subtilis BS017 and Bacillus subtilis BS018, respectively. Fermentation conditions for MK-7 production by the recombinant strains were then investigated using single-factor experiments, focusing on initial pH, inoculum size, and liquid volume. The carbon and nitrogen source composition of the culture medium, supplemental carbon and nitrogen source composition, and inorganic salts were also optimized, and significant influencing factors were identified.

[0031] 1. Materials and Methods

[0032] (1) Materials and reagents

[0033] Organic reagents used for high-performance liquid chromatography (HPLC) analysis were purchased from Sigma-Aldrich. MK-7 standards were purchased from Fujifilm Wako Pure Chemical Industries, Ltd., Japan. Bacterial genome extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Plasmid extraction kits and other chemicals were purchased from Sangon Biotech (Shanghai) Co., Ltd. High-fidelity DNA polymerase PrimeSTAR HS DNA was purchased from Takara (Dalian, China). MonScript™ 5×RTIII All-in-One Mix and MonAmp™ Fast SYBR® Green qPCR Mix were purchased from Mona Biotechnology Co., Ltd. Sucrose, vitamin E, and reduced glutathione were purchased from Shanghai Sangon Biotech Co., Ltd.

[0034] (2) Strains, plasmids, and culture conditions

[0035] All strains and plasmids used in this example are listed in Table 1. For genetic manipulation experiments, all strains were cultured in LB medium at 37°C and 220 rpm. For MK-7 fermentation production, Bacillus subtilis was first cultured in LB medium at 37°C and 220 rpm with shaking for 12 h. The seed solution was then inoculated at a 10% inoculum into a 500 mL Erlenmeyer flask containing 50 mL of fresh fermentation medium (3% glycerol, 6% soy peptone, 0.5% yeast extract, 0.3% K₂HPO₄, and 0.05% MgSO₄·7H₂O, pH 7.3). The culture was then incubated at 40°C and 250 rpm for 120 h. 1 mL of the fermentation broth was sampled every 6 h, and cell density was measured at 600 nm. Antibiotic working concentrations included kanamycin (50 μg / mL), ampicillin (100 μg / mL), spectinomycin (50 μg / mL), and chloramphenicol (5 μg / mL).

[0036] Table 1: Involved strains and plasmids

[0037]

[0038] (3) DNA assembly method and Bacillus subtilis transformation method

[0039] The assembly of multiple fragments in DNA assembly was performed using overlapping PCR based on homologous sequences, and the successfully constructed multi-gene fragments were sequenced at Shanghai Sangon Biotechnology Co., Ltd. Homologous fragments or plasmids were transformed into B. subtilis by the Sipizizen transformation method.

[0040] (4) Construction of strains for gene promoter replacement and gene integration

[0041] Method for constructing recombinant bacteria for integrating exogenous genes into the genome The site-specific recombination strategy used in this example mainly refers to previous literature reports. First, the kinase encoding gene GsispA of farnesyl synthase (GSIspA) in thermophilic Bacillus licheniformis was artificially synthesized. Using the artificially synthesized GsispA as a template, the primers gsispa3-F / gsispa3-R were used to amplify the GsispA fragment; then, using the B. subtilis genome as a template, the primers heps1-F / heps1-R were used to amplify the upstream homology arm sequence of heps - heps upper, and the primers gsispa4-F / heps4-R were used to amplify the first 1000 bp fragment of heps as the downstream homology arm sequence - heps lower; using the plasmid p7C6P hbs As a template, primers heps2-F / gsispa2-R were used to amplify lox71-cm-lox66 and P hbs The four independent fragments were then connected by overlapping PCR to obtain the fusion fragment - heps + lox71-cm-lox66-P hbs Finally, the purified fusion fragment was transformed into BS016 to allow homologous recombination with the chromosome.

[0042] Colonies were picked from the chloramphenicol-resistant transformation plate and verified by colony PCR using primers heps1-F / gsispa3-R. Positive transformants were identified by a band size of 3400 bp. The pDGC plasmid encoding the recombinase Cre gene was then transferred into these positive transformants. After induction with 0.5 mM IPTG, the cells were plated on plates containing kanamycin resistance. Once colonies emerged, colony PCR was performed using primers heps1-F / gsispa3-R. A band size of approximately 2250 bp indicated that chloramphenicol resistance had been eliminated. Finally, positive transformants carrying the pDGC plasmid were cultured overnight at 50°C and plated on blank LB plates. Transformants were amplified using primers heps1-F / gsispa3-R and, after verification, sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The overexpression procedure for the BSispA gene was similar to that for GsispA.

[0043] Table 2: Primer sequences used for gene modification

[0044]

[0045] (5) Shake flask fermentation and growth curve determination

[0046] Fermentation production of MK-7: B. subtilis was inoculated into LB liquid medium and cultured at 37°C, 220 rpm, for 12 hours to prepare a seed solution. A 10% inoculum of this seed solution was transferred to GSY medium (30 mL / L glycerol, 60 g / L soy peptone, 5 g / L yeast extract, 3 g / L K2HPO4, 0.5 g / L MgSO4·7H2O, pH 7.3) and cultured at 40°C, 250 rpm, for 120 hours. Intermediate samples were taken every 12 hours and the OD was measured. 600 value.

[0047] (6) Optimization of culture conditions

[0048] 1. Optimization of liquid filling volume

[0049] Using fermentation medium as the basis, the effects of inoculum size on the growth and fermentation production of MK-7 by Bacillus subtilis were investigated, with the composition and concentration of the medium remaining constant, along with other conditions. Six different inoculum sizes (50, 60, 70, 80, 90, and 100 mL) were used, with three replicates performed in each group. The final MK-7 yield was used as a reference value to determine the optimal fermentation medium volume.

[0050] 2. Optimization of initial pH

[0051] Using the same fermentation medium composition and concentration, and other conditions, the effect of initial pH on the growth and fermentation production of MK-7 by Bacillus subtilis was investigated. Prior to autoclaving, the pH of the medium was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 using 0.1M NaOH or 0.1M HCl. Three replicates were performed for each group, and the final MK-7 yield was used as a reference value to determine the initial pH of the fermentation medium.

[0052] 3. Optimization of inoculation amount

[0053] Using the fermentation medium as the basis, the effects of inoculum size on the growth and fermentation production of MK-7 by Bacillus subtilis were investigated, with the medium composition and concentration being the same, and other conditions remaining unchanged. Four different inoculum sizes (8%, 10%, 12%, and 15%) were set, with three replicates performed in each group. The final MK-7 yield was used as a reference value to determine the optimal inoculum size for the fermentation medium.

[0054] (7) Single factor optimization of fermentation medium

[0055] First, the optimal composition and concentration of a fast-acting carbon source were screened. Using glucose and sucrose as fast-acting carbon sources, the effects of glucose and sucrose addition (10, 20, 30, and 40 g / L) on cell growth and MK-7 production were investigated. Second, to identify nitrogen source concentrations suitable for BS018 production of MK-7, different concentration gradients of soy peptone (40, 50, 60, 70, and 80 g / L) and yeast extract (4, 5, 6, 7, 8, 9, and 10 g / L) were set up. Biomass and MK-7 yield were measured to determine the optimal addition levels.

[0056] Then, the effects of inorganic salt ions on MK-7 production were investigated. First, the types of phosphates, including K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 (at a concentration of 3 g / L), were optimized. Then, six concentrations of KH2PO4 (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6% (w / v)) were used to explore their optimal concentrations. In addition, in order to determine the effect of Mg 2+ To determine the optimal addition amount, five gradients of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / v) were set for screening. Each experiment was repeated three times and the average value was calculated.

[0057] Finally, the effect of adding the MK-7 precursor DHNA on strain growth and MK-7 production was investigated. DHNA was added to the fermentation medium at different concentrations (1 mmol / L, 2 mmol / L, 3 mmol / L, and 4 mmol / L) at 48 hours of fermentation. Biomass and MK-7 yield were measured to identify the optimal addition level. Each experiment was repeated three times, and the average value was calculated.

[0058] (8) Optimization of the extraction method of MK-7 from fermentation broth

[0059] The main steps of the MK-7 extraction method are as follows:

[0060] 2 mL of bacterial fermentation broth was centrifuged at 9000×g for 6 min, and 1 mL of supernatant was collected as the fermentation supernatant. A mixture of 2 mL of n-hexane and isopropanol (n-hexane:isopropanol = 2:1, v / v) was added, and the mixture was vortexed vigorously for 20 min. Then, 1 mL of n-butanol was added, and the mixture was vortexed vigorously for another 20 min. The mixture was centrifuged at 5000×g for 3 min, and the upper organic phase was filtered with an organic filter membrane (0.22 μm pore size) to obtain the MK-7 extract of the fermentation supernatant.

[0061] The wet cells obtained by the above centrifugation were placed in a -80°C refrigerator for freezing, and then vacuum-freeze-dried. 2 mL of ethanol was added to the dry cells, and ultrasonic-assisted extraction was performed for 10 minutes. The cells were then vigorously vortexed for 20 minutes and centrifuged at 5000 × g for 3 minutes. The organic phase was collected and filtered with an organic filter membrane (0.22 μm pore size) to obtain the intracellular MK-7 extract.

[0062] (9) Analytical methods

[0063] MK-7 yield was determined by high-performance liquid chromatography (HPLC): a UV detector (SPD-16) was used on a reversed-phase C-18 column (VP-ODS, 4.6 mm × 250 mm). The mobile phase consisted of a mixture of methanol and dichloromethane in a 4:1 volume ratio. The flow rate was 1 mL / min, the column temperature was set at 35°C, the detection wavelength was 248 nm, and the injection volume was 20 μL. Within the range of 0–150 mg / L, a good linear relationship between MK-7 concentration and absorption peak area was observed (R 2 =0.999).

[0064] 2. Results and Analysis

[0065] (1) Screening and analysis of farnesyl synthase

[0066] A search of the BRENDA enzyme database revealed that the specific enzyme activity of farnesene synthase (BsIspA) from Bacillus subtilis was only 0.0012 µmol / min / mg, while the specific enzyme activity of farnesene synthase (GsIspA) from thermophilic Bacillus licheniformis was as high as 4.69 µmol / min / mg, which is 3908 times that of BsIspA.

[0067] (2) Screening and fusion co-expression of farnesyl synthase

[0068] Studies have shown that FPP is not only necessary for cell growth, but also a general substrate for the synthesis of terpenoids, and has a direct impact on the yield of target terpenoids. The synthesis pathway of the side chain HepPP of MK-7 is as follows: the MEP pathway provides intermediates DMAPP and IPP, FPP is generated under the catalysis of FPP synthase (IspA), and then HepS / T catalyzes FPP to synthesize HepPP. In addition, the HepS / T encoding genes hepS and hepT exist in the same operon hepS-menG-hepT-ndk. In addition, there is the DMK-7 methyltransferase encoding gene menG, and the nucleoside diphosphate kinase encoding gene ndk, of which ndk has no direct connection with the MK-7 synthesis pathway ( Figure 1 A).

[0069] In order to increase the FPP concentration near HepS / T in strain BS016, a strategy of co-localization of IspA and HepS / T in-frame protein fusion was adopted. In this section, two recombinant bacteria with enhanced FPP flux were constructed. One was to combine the highly active thermophilic Bacillus licheniformis GSispA with the constitutive strong promoter P hbs After ligation, the recombinant strain Bacillus subtilis BS018 was obtained by integrating the upstream site of the operon hepS-menG-hepT-ndk. Another method was to construct the endogenous BSispA in the same way to obtain the recombinant strain BS017 ( Figure 1 The growth curves showed that there was no significant difference in the growth of BS016, BS017, and BS018 in the early stage; however, in the late stable stage, the OD 600 The value is lower than that of BS016 and BS018; BS018 has the best growth condition among the three ( Figure 1 C). It's possible that the successful integration and expression of exogenous GSispA in the genome mitigated the accumulation of toxic intermediates DMAPP and IPP, thereby alleviating the burden on cell growth. MK-7 production assays revealed that after 120 hours of fermentation, the MK-7 yield in Bacillus subtilis BS018 reached 91.1 mg / L, 1.1 times that of strain BS016. However, the MK-7 yield in BS017 decreased compared to BS016. This suggests that endogenous BSispA is subject to feedback inhibition by the host itself ( Figure 1 These results demonstrate that exogenous GsIspA fusion expression with HepS / T is an effective strategy for enhancing MK-7 synthesis. This allows IspA and HepS / T proteins to form a chimera, resulting in excellent kinetics for FPP delivery to HepS / T and avoiding waste caused by free diffusion of intermediates into the cytoplasmic fluid. Therefore, strain BS018, known for its robust MK-7 synthesis, was selected as the final production strain for this study.

[0070] (3) Fermentation condition optimization results and analysis

[0071] 1. Optimization of liquid filling volume

[0072] The growth of Bacillus subtilis requires a certain amount of oxygen, and the volume of liquid will affect the dissolved oxygen. MK-7 fermentation was carried out by filling 500 mL shake flasks with 50, 60, 70, 80, 90, and 100 mL of fermentation medium with an initial pH of 7.0, using an 8% inoculum, and incubating at 40°C, 250 rpm, and shaking for 6 days. The results are shown in Figure 2. Figure 4As shown in the figure, when the liquid volume was 50 mL, the MK-7 yield was 83.45 mg / L, which was significantly higher than that of the other control groups. Therefore, when the liquid volume was 50 mL in a 500 mL shake flask, the most favorable condition for the synthesis of MK-7 was 50 mL.

[0073] 2. Optimization of initial pH

[0074] The pH of the fermentation medium has a significant impact on enzymatic reactions, as well as the charge and permeability of bacterial cell membranes. Product synthesis also requires a certain pH value. However, during microbial growth, the pH value of the environment will change significantly due to metabolism. Therefore, adjusting the appropriate initial pH value of the medium is of great significance for the production of MK-7. B. subtilis was cultured under different initial pH conditions to study the effect of pH on the yield of MK-7 in B. subtilis. Figure 2 It can be seen that too high or too low pH is not conducive to the production of vitamin K2. When the pH is 7.0, the yield of vitamin K2 is the highest, which is 98.49 mg / L. Therefore, 7.0 is selected as the optimal fermentation pH value.

[0075] 3. Optimization of inoculation amount

[0076] Appropriately increasing the inoculum size in the fermentation medium can reduce the time it takes for B. subtilis to reach the stationary phase. However, excessive inoculum size can cause the bacteria to grow too quickly, rapidly consuming nutrients and causing the bacteria to enter the premature death phase, thus affecting the synthesis of the fermentation product. However, too low an inoculum size can prolong the fermentation cycle of B. subtilis. Therefore, the appropriate inoculum size is also a key factor in determining the fermentation performance. While other fermentation conditions remained unchanged, four different inoculum sizes (8%, 10%, 12%, and 15%) were set and fermented under the same conditions. Figure 3 The results showed that when the inoculation amount was 8%, the MK-7 yield was 109.58 mg / L, indicating that an inoculation amount of 8% was most conducive to the production of vitamin K2.

[0077] (4) Culture medium composition optimization results and analysis

[0078] 1. Impact of carbon source

[0079] Optimization of culture medium composition is an effective method to increase MK-7 production. Carbon is the most critical culture medium component because it is the energy source for microorganisms and plays an important role in growth and the production of primary and secondary metabolites. Different concentrations of glucose and sucrose (10, 20, 30, 40 g / L) were used to study their effects on cell growth and MK-7 production. Figure 5As can be seen from the data, when glucose and sucrose were used as carbon sources, the cell density and MK-7 yield were greater when sucrose was used as a fast-acting carbon source than when glucose was used at all concentrations. With increasing sucrose concentration, the cell density initially increased and then decreased, but remained higher than the control group. At a sucrose concentration of 20 g / L, the cell density reached a maximum of 23.96, and the MK-7 yield reached 72.65 mg / L, 7.2% higher than the control group (10 g / L). In contrast, with increasing glucose concentration, the cell density increased significantly, but the increase in MK-7 yield was not significant.

[0080] Different carbon sources enter metabolic pathways differently. Glucose can be directly utilized by microorganisms and enter the metabolic pathway through conversion to glucose-6-phosphate and fructose-6-phosphate under the catalysis of hexokinase. Sucrose, on the other hand, must first be hydrolyzed by sucrase into one molecule of fructose and one molecule of glucose before it can enter the metabolic pathway under the catalysis of the enzyme. Furthermore, different carbon sources are metabolized along different pathways, providing energy and precursors for microbial growth, metabolism, and product synthesis. Therefore, B. subtilis exhibits significant differences in biomass and MK-7 yield when utilizing different carbon sources (glucose and sucrose). This may be due to the following reasons: First, the activities or amounts of enzymes involved in metabolizing different carbon sources into the metabolic pathway differ. Second, when using the disaccharide substrate sucrose as a carbon source, glucose is simultaneously decomposed and utilized, alleviating the inhibitory effect caused by high glucose concentrations.

[0081] In summary, 2% sucrose promoted cell growth and MK-7 synthesis, and 2% sucrose was selected as the fast-acting carbon source in the fermentation medium.

[0082] 2. Impact of nitrogen source

[0083] The optimal nitrogen source for MK-7 production was investigated. A combination of yeast extract and soy peptone was found to be beneficial in promoting MK-7 production. Yeast extract can be used as a nutritional supplement for the production of value-added products because it is readily utilized by bacteria. Therefore, the effects of different yeast extract concentrations (4, 5, 6, 7, 8, 9, and 10 g / L) on cell density and MK-7 production were first investigated to further improve MK-7 production in strain BS018. Figure 6 Figure B shows that when yeast extract was added at a concentration of 9 g / L, the cell density reached a maximum of 38.92, but MK-7 production peaked at 53.07 mg / L at a yeast extract concentration of 4 g / L. When 8 g / L yeast extract was added, MK-7 production dropped sharply, indicating that only an appropriate yeast extract concentration can improve MK-7 production. It should be noted that high yeast extract concentrations impose a heavy metabolic burden on the bacteria and increase osmotic pressure, which is detrimental to their normal physiological metabolism, thereby reducing cell density and MK-7 titer.

[0084] Secondly, the optimal concentration of nitrogen source in the starting medium was studied using 60 g / L soy peptone as the starting nitrogen source. Figure 6 As can be seen from Figure A, when 7% (W / V) soy peptone was used as the nitrogen source, the cell density and MK-7 production of BS018 reached the highest levels of 30.2 and 72 mg / L, respectively. This result suggests that 7% (W / V) soy peptone may be the optimal nitrogen source for cell growth and MK-7 production of strain BS018.

[0085] Therefore, 7% soy peptone and 0.4% yeast extract were the optimal nitrogen sources for strain BS018 to produce MK-7.

[0086] 3. Optimization of metal ions

[0087] Inorganic salts, especially some metal ions, play an indispensable role in the growth of microorganisms. They participate in various metabolic activities in microbial cells and constitute the coenzyme center of various enzyme systems. Keeping other components in the fermentation medium unchanged, K2HPO4, KH2PO4, Na2HPO4, and NaH2PO4 were added as inorganic salts to examine their effects on the fermentation production of vitamin K2. Figure 7 A. Figure 7 As shown in B, the effect of KH2PO4 on MK-7 synthesis is significantly higher than that of the other three inorganic salts, so KH2PO4 was selected for the next concentration optimization experiment. Different concentration gradients of KH2PO4 (0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%) were set to study their effects on Bacillus subtilis biomass and MK-7 production. When the KH2PO4 concentration was 0.1%, the MK-7 production was 53.43 mg / L, which was significantly higher than that of the other control groups ( Figure 7 C), so 0.1% was selected for the next optimization experiment.

[0088] Mg 2+ It is an activator of many enzymes and is widely involved in material metabolism and energy metabolism. Therefore, the effects of 0.1%, 0.2%, 0.3%, 0.4% and 0.5% (w / v) MgSO4·7H2O on MK-7 production were studied. The results showed that when the concentration of MgSO4·7H2O was 0.1%, the MK-7 yield was 122 mg / L ( Figure 7 Therefore, this concentration was selected for subsequent experiments.

[0089] 4. Addition of MK-7 precursor DHNA

[0090] 1,4-Dihydroxy-2-naphthoic acid (DHNA) acts as a precursor for the naphthoquinone ring core of MK, so its addition as an important precursor may promote the fermentation synthesis of MK. At 48 hours of fermentation, different concentrations of DHNA (1 mmol / L, 2 mmol / L, 3 mmol / L, and 4 mmol / L) were added to the fermentation medium to observe the effects of the precursor on the growth of BS018 bacteria and the synthesis of MK-7. The experiment used a normal fermentation medium without any additives as the control group. The results are shown in Figure 2. Figure 8 shown.

[0091] like Figure 8 As shown in A, compared with the control group, the bacterial counts at the four concentrations all showed a trend of increasing first and then decreasing. Among them, when the addition amount was 4 mmol / L, the bacterial count decreased rapidly in the late fermentation period, considering that the addition amount of this concentration had toxicity to bacterial growth. Figure 8 Figure B shows that when the additive concentration is 2 mmol / L, 3 mmol / L, and 4 mmol / L, the yield of synthesized MK-7 is lower than that of the control group. Among them, when the additive amount is 4 mmol / L, the MK-7 yield is the lowest, which is 41.38 mg / L, which is significantly lower than 82.66 mg / L of the control group.

[0092] When 1 mmol / L of DHNA was added, MK-7 yield reached its highest level, reaching 92.36 mg / L, a 10.5% increase compared to the control group. Therefore, the 1 mmol / L addition was selected for subsequent experiments.

[0093] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A Bacillus subtilis BS018, characterized in that: The Bacillus subtilis BS018 has been deposited in the China Center for Type Culture Collection (CCTCC) on January 2, 2024, and the strain deposit number is CCTCC NO: M2024004.

2. The use of Bacillus subtilis BS018 in the fermentation preparation of MK-7 is characterized by: The Bacillus subtilis BS018 has been deposited in the China Center for Type Culture Collection (CCTCC) on January 2, 2024, and the strain deposit number is CCTCC NO: M2024004.

3. A method for increasing MK-7 production through farnesyl synthase engineering and fermentation optimization, characterized in that: Bacillus subtilis BS018 was inoculated into LB liquid culture medium and cultured to prepare a seed solution; the seed solution was transferred into a fermentation medium, and after culture, a fermentation solution containing MK-7 was obtained; the Bacillus subtilis BS018 has been deposited in the China Center for Type Culture Collection (CCTCC) with a deposit date of January 2, 2024, and a strain deposit number of CCTCC NO: M2024004.

4. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: According to the liquid volume of a 500mL shake flask, the liquid volume in a 500mL shake flask is 40-60mL.

5. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: The initial pH of the fermentation medium was 6.8-7.

2.

6. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: The inoculation rate is 7-10%.

7. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: When glucose or sucrose is used as a fast-acting carbon source, the concentration is 20-30 g / L; the added mass is 1-2.5%.

8. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: The fermentation medium contains 6-8% by mass of soybean peptone and 0.3-0.6% by mass of yeast extract.

9. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: The fermentation medium contains KH2PO4 with a mass content of 0.08-0.12% and MgSO4·7H2O with a mass content of 0.08-0.12%.

10. The method for increasing MK-7 production by farnesyl synthase engineering and fermentation optimization according to claim 3, characterized in that: DHNA at a concentration of 0.8-2 mmol / L was added to the fermentation medium at 48 hours of fermentation.

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