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    Synergistic Optimization of Bacillus subtilis for Efficiently Producing Menaquinone-7 (MK-7) by Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Metabolic Engineering

    Greenberg     March 12, 2025 in News - 16 Minutes


    1. Introduction

    Vitamin K2, also known as menaquinone (MK), is primarily produced through microbial metabolism. As a member of the vitamin K2 family, it is considered one of the fourth-generation drugs for osteoporosis treatment, offering significant potential in medical and food applications [1]. Additionally, vitamin K2 plays a crucial role as a key electron carrier in the respiratory chain [2], contributing to both electron transport and blood coagulation [3]. Vitamin K2 is not a single compound, but rather a family of compounds composed of several subtypes. Its chemical structure consists of a methylated naphthoquinone ring and a variable-length isoprenoid side chain. The number of units in the isoprenoid chain ranges from 1 to 14, with each variant denoted as MK-1 to MK-14 based on the number of isoprenoid units [4]. Among these subtypes, MK-7 is particularly notable for its superior bioavailability and longer half-life compared to other forms of vitamin K2 [5].
    Vitamin K2 was initially developed and used in Japan, primarily due to its presence in the traditional Japanese food natto, a fermented soybean dish that contains approximately 800–900 µg of vitamin K2 per 100 g [6]. With ongoing research, it was found that the primary fermentation strains in natto are subtilis, which not only produce fibrinolysin but also generate MK-7. While the extraction of MK-7 from natto remains an important method of obtaining this compound, its low titer poses a challenge for industrial-scale production. In recent years, in order to achieve higher production rates, researchers have identified various strains capable of producing MK-7 [7]. Among the strains, Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis are considered the most promising candidates for MK-7 production. Notably, Bacillus subtilis is regarded as one of the most viable strains due to its rapid growth rate, genetic stability, and availability of extensive genetic modification tools. Therefore, Bacillus subtilis is considered one of the most promising species for large-scale MK-7 production [8]. The main strategies for improving MK-7 production focus on mutagenesis and metabolic engineering. Atmospheric and Room Temperature Plasma (ARTP) is an innovative and efficient biological breeding mutagenesis method. This system generates a plasma jet with a high concentration of reactive particles at atmospheric pressure and room temperature, representing a composite mutagenesis technique. Compared to traditional chemical or ultraviolet (UV) mutagenesis methods, ARTP mutagenesis offers several advantages, including safety, simplicity, and a high rate of positive mutations [9]. The metabolic biosynthesis pathway of MK-7 consists of four modules (Figure 1). In Module II, diphenylamine (DPA) inhibits the enzyme polyprenyl pyrophosphate synthetase in the MEP metabolic pathway, which regulates the synthesis of the isoprene side chain [10]. Additionally, 1,4-dihydroxy-2-naphthoic acid (DHNA) exerts feedback inhibition on DAHP synthase in Module III’s shikimate pathway, an enzyme responsible for the formation of the quinone backbone. 1-Hydroxy-2-naphthoate (HNA), a structural analog of DHNA, is commonly used to screen for DHNA-resistant strains that produce higher titers of MK-7 [11]. Xu et al. applied Atmospheric Pressure Room Temperature plasma (ARTP) mutagenesis and protoplast fusion technology to wild-type Bacillus amyloliquefaciens, obtaining resistant mutant strains with various traits, including resistance to 1-hydroxy-2-naphthoic acid (HNA), sulfanilamide (SG), and menaquinone. The MK-7 titer reached 73.57 mg/L, which is 1.36 times higher than the parental strain [12]. In another study by Xu et al., ARTP mutagenesis was used to generate DPA-resistant mutant strains, resulting in enhanced MK-7 production [13]. MK-7, as a secondary metabolite, is involved in several metabolic pathways, including the glycerol metabolism pathway, the methylerythritol phosphate (MEP) pathway, the shikimate (SA) pathway, and the menaquinone biosynthesis pathway. The metabolic network is complex, involving numerous enzymes. Low expression levels of key enzymes in these pathways are a major factor limiting the MK-7 titer. MA et al. used Bacillus subtilis 168 as a chassis and overexpressed different combinations of rate-limiting enzymes, including Dxs, Dxr, IdI, and MenA, constructing 12 different Bacillus subtilis 168 strains. The best MK-7-producing strain achieved a titer of 50 mg/L, which is 11 times higher than the parental strain [14]. Yang et al. also used Bacillus subtilis 168 as a chassis for modular metabolic engineering design, dividing the MK-7 biosynthesis pathway into four modules. By overexpressing rate-limiting enzymes in different modules and knocking out metabolic branches, they obtained a strain that produced MK-7 with a titer of 69.5 mg/L [15]. Kong et al. overexpressed menA and menD, leading to a five-fold increase in MK-8 production compared to the wild-type Escherichia coli [16]. Although previous studies on mutagenesis and metabolic engineering have led to improvements in MK-7 production, the titers are still insufficient for industrial-scale production.

    The starting strain for this study was a wild-type Bacillus subtilis isolated from a natto fermentation agent, with an initial MK-7 production of 75.18 mg/L, which was designated as L-5. ARTP mutagenesis was performed to randomly mutate the strain, resulting in two MK-7-producing mutant strains with resistance to 1-hydroxy-2-naphthoic acid (HNA) and diphenylamine (DPA), named H-10 and D-15, with MK-7 titers of 175.55 mg/L and 164.49 mg/L, respectively. Subsequently, protoplast fusion technology was applied to fuse the H-10 and D-15 strains, generating the optimal fusion strain R-8, which produced 196.68 mg/L MK-7. Whole-genome sequencing and resequencing were performed on the R-8 strain and its parental L-5 strain, respectively. Subsequent sequence alignment between the two revealed mutations in key genes involved in the MK-7 biosynthesis pathway. Specifically, in the menaquinone biosynthesis pathway, the key enzyme MenD (succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexen-1-carboxylate synthase) showed a mutation at position 249, where serine was replaced by leucine. Another rate-limiting enzyme, MenA (1,4-dihydroxy-2-naphthoic acid-7-isoprenyl transferase), exhibited a mutation at position 196, where serine was replaced by lysine. In the methylerythritol phosphate (MEP) pathway, the rate-limiting enzyme Dxs (1-deoxy-D-xylulose-5-phosphate synthase) had two mutations: asparagine at position 60 was replaced by aspartic acid, and glutamine at position 185 was replaced by histidine. Additionally, another rate-limiting enzyme, Dxr (hydroxyacid reductase isomerase), exhibited a mutation at position 351, where glutamine was replaced by lysine. The wild-type genes from the L-5 strain and the mutated genes from the R-8 strain were overexpressed in the R-8 strain. Overexpression of the menD, menA, Dxs, and Dxr genes from R-8 resulted in MK-7 titers that were 19.65 mg, 20.71 mg, 17.35 mg, and 16.28 mg higher, respectively, compared to the menD, menA, Dxs, and Dxr genes from the wild-type strain L-5. For each gene, overexpression of the mutant alleles resulted in higher titers than the wild-type, suggesting that these mutations in menD, menA, Dxs, and Dxr are beneficial, likely through enhanced enzyme activity or increased expression levels, thereby improving MK-7 production. To further increase the MK-7 titer, the menD, menA, Dxs, and Dxr genes were co-expressed, resulting in a final MK-7 titer of 239.65 mg/L. This study provides theoretical guidance for future modifications of key enzymes in the MK-7 biosynthetic pathway.

    2. Materials and Methods

    2.1. Experimental Materials

    Natto Fermentation Agent Samples: A total of 10 samples from different provinces and manufacturers in China were collected. The manufacturers included the following: T manufacturer (Sweet Kitchen, Beijing, China), H manufacturer (Podikai Health Food Exclusive Online Store, Changzhou, China), S manufacturer (Chuanxiu Flagship Store, Langfang, China), R manufacturer (Hao Ding Food Speciality, Chongqing, China), L manufacturer (Tangdoudou Snack House, Zhaoqing, China), Q manufacturer (Nongjian Flagship Store, Anqing, China), D manufacturer (Yongyuan Food Ferment, Ningde, China), J manufacturer (Yinuan Food Flagship Store, Sanming, China), Y manufacturer (Baisenyou Food Flagship Store, Shanghai, China) and W manufacturer (Runwanxiang Seasoning Shop, Dezhou, China).

    2.2. Strains and Plasmids

    In this study, Escherichia coli JM110 was used for DNA fragment amplification, plasmid construction, and plasmid storage. The strains and plasmids used and constructed in this study are listed in Table 1. The primers used in this study are listed in Supplementary S1.2, Table S1.

    2.3. Media and Cultivation Conditions

    2.3.1. Media

    Basic Isolation Medium (g/L): Glucose 15, Soybean peptone 15, Fibrinogen 5, KH2PO4·12H2O 1, K2HPO4·3H2O 2.5, MgSO4·7H2O 2.5, Streptomycin 0.5, Agar 15, pH 7.0–7.2.

    Luria-Bertani (LB) Medium (g/L): Tryptone 10, Yeast extract 5, NaCl 10, pH 7.0.

    Seed Culture Medium (g/L): Glucose 15, Soybean peptone 15, K2HPO4·3H2O 2.5, KH2PO4 1.5, MgSO4·7H2O 2.5, pH 7.0.

    Fermentation Medium (g/L): Glycerol 50, Soybean peptone 100, NaCl 3, K2HPO4 6, pH 7.0–7.3.

    Regeneration Medium (g/L): Tryptone 10, Yeast extract 5, Beef extract 5, KH2PO4 1.5, K2HPO4·3H2O 4.6, NaCl 40.28, Maleic acid 2.32, MgCl2 1.9, pH 7.0.

    All media, with the exception of the fermentation medium that was subjected to autoclaving at 115 °C for 10 min, were sterilized at 121 °C for 20 min.

    2.3.2. Main Solutions

    Hypertonic Buffer (SMM Buffer): Sucrose 0.5 mol·L−1, Maleic acid 20 mmol·L−1, MgCl2 20 mmol·L−1, pH 6.5.

    Lysozyme Buffer (0.1 mg·mL−1): Dissolve 1 mg of lysozyme (activity ≥ 20,000 U·mg−1) in 10 mL of SMM buffer.

    PEG6000 Buffer: Dissolve 4 g of PEG6000 in SMM buffer and adjust the final volume to 10 mL.

    The sterilization conditions for both the Hypertonic Buffer (SMM Buffer) and the PEG6000 Buffer were autoclaving at 121 °C for 20 min, and the Lysozyme Buffer was sterilized by filtration.

    2.3.3. Cultivation Methods

    Shake Flask Culture: After activation (inoculate 100 µL of the strain preserved in glycerol stock into a 100 mL conical flask containing 10 mL of liquid LB medium), 600 µL of the wild-type Bacillus subtilis was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of LB medium. The culture was incubated at 37 °C with shaking at 220 rpm during the exponential growth phase for subsequent mutagenesis.

    Shake Flask Fermentation: After activation (inoculate 100 µL of the strain preserved in glycerol stock into a 100 mL conical flask containing 10 mL of liquid LB medium), 300 µL of the mutated and selected strain was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of seed culture medium. The culture was incubated at 37 °C with shaking at 220 rpm for 10 h. Then, 600 µL of the seed culture was inoculated into a 250 mL Erlenmeyer flask containing 30 mL of fermentation medium and incubated at 37 °C with shaking at 220 rpm for 6 days before sampling and analysis.

    2.4. Isolation and Screening of Wild-Type Bacillus subtilis for MK-7 Production

    Five grams of each of the ten natto fermentation agent samples mentioned above was dissolved in 20 mL of sterile water and subjected to a 10 min incubation in a water bath at 85 °C. The mixture was then centrifuged at 5000 rpm for 10 min, and the supernatant was collected. After appropriate dilution, the supernatant was plated on initial screening plates. The plates were incubated at 37 °C for 24 h. Strains with larger clear zones were selected for further shake flask fermentation to validate MK-7 production. One high-yield strain was ultimately selected.

    To identify the isolated MK-7-producing strain, we followed the procedures described in the Bergey’s Manual of Determinative Bacteriology [17]. The strain was subjected to morphological, physiological, and biochemical identification. Genomic DNA of the isolated strain was extracted using the Genomic DNA Extraction Kit from Nanjing China Genewiz. The 16S rRNA gene was amplified using universal bacterial primers (F: 5′ GAGAGTTTGATCCTGGCTCAG-3′; R: 5′ CTACGGCTACCTTGTTACGA-3′) via PCR. The PCR conditions were as follows: pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 90 s, and a final extension at 72 °C for 10 min. This cycle was repeated 30 times. The purified PCR products were then sent to Shanghai Sheng gong Biotechnology Co., Ltd. (Shanghai, China) for sequencing.

    2.5. ARTP Mutagenesis

    To initiate ARTP mutagenesis, 1 mL of the parent strain cultured to the exponential phase was centrifuged at 5000 rpm, and the supernatant was discarded. The cell pellet was washed three times with saline solution containing 5% glycerol and resuspended. A 10 μL aliquot of the resuspended cells was then spread on a metal plate for mutagenesis treatment. The ARTP-IIS mutagenesis (Wuxi TMAXTREE Biotechnology Co. Ltd., Wuxi, China) device was used to perform random mutagenesis on the strain. The mutagenesis protocol, based on prior studies [18] with minor modifications, was set as follows: incident power of 100 W, gas flow rate of 10 SLM, and the distance between the sample and the plasma nozzle was maintained at 2 mm. The mutagenesis treatment lasted for 90 s. After mutagenesis, the metal plate was placed in a 1.5 mL centrifuge tube containing 990 μL of physiological saline. The mixture was vortexed for at least one minute to elute the mutagenized cells from the plate into the saline solution. The eluate was then diluted appropriately and spread on LB solid agar plates containing HNA and DPA. The plates were incubated at 37 °C for 24 to 36 h. Colonies with larger diameters were selected, and each colony was subjected to shake flask fermentation validation with three replicates.

    2.6. Protoplast Fusion

    The protoplast fusion method used in this study was adapted from the previous literature with minor modifications [19]. Initially, two parental strains were inoculated into 10 mL of LB liquid medium and cultured overnight at 37 °C. Subsequently, 1 mL of the culture was transferred to 100 mL of LB medium and cultured at 37 °C until the logarithmic phase was reached. The culture was centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The cell pellet was washed twice with SMM solution, and the cells were resuspended in SMM containing 0.1 mg/mL lysozyme to an OD600 of 0.8. The suspension was then incubated at 8 °C for 20 min in a water bath. Under a microscope, the morphological change from rod-shaped to spherical cells was observed to confirm successful protoplast preparation. The successfully prepared protoplasts were washed twice with SMM, centrifuged, and resuspended.

    Next, 2 mL of protoplasts from each of the two parent strains was combined and incubated for 5 min before centrifugation. A total of 1.8 mL of pre-warmed (42 °C) 40% PEG6000 and 0.2 mL of 0.2 mol/L CaCl2 solution were added to resuspend the protoplasts. The mixture was then incubated in a 37 °C water bath for 10 min with gentle shaking. After the incubation, the protoplasts were collected by centrifugation at 4000 rpm for 15 min, washed twice with SMM, and resuspended in 1 mL of SMM. The protoplast suspension was appropriately diluted and spread onto regeneration medium plates. The plates were incubated at 37 °C for 12 to 24 h.

    Finally, colonies that grew on the regeneration medium were harvested by washing the plate with SMM solution and then transferred onto selective plates containing HNA and DPA for screening protoplast fusion strains resistant to both HNA and DPA. Colonies with larger diameters were selected, and each colony was subjected to shake flask fermentation validation with three replicates.

    2.7. Whole-Genome Sequencing of Wild-Type Bacillus subtilis

    The successfully fused resistant mutant strains were inoculated into 10 mL of liquid LB medium and cultured at 37 °C with shaking at 200 rpm until the exponential growth phase was reached. The cells were collected by centrifugation at 3000 rpm for 5–10 min using a refrigerated centrifuge. The total cell pellet mass was approximately 1–2 g. The cell pellet was gently washed 1–2 times with pre-chilled PBS under sterile conditions to avoid contamination. After washing, the samples were either snap-frozen in liquid nitrogen or stored at −80 °C. The samples were shipped on dry ice to Jinwei BioTech (Nanjing, China) for whole-genome sequencing. Sequencing was performed using Illumina second-generation sequencing technology and the PacBio third-generation high-throughput sequencing platform. The obtained genomic sequence data were analyzed and annotated using databases including COG (COG-NCBI, accessed on 27 November 2024), GO (Gene Ontology Resource, accessed on 27 November 2024), KEGG (KEGG: Kyoto Encyclopedia of Genes and Genomes, accessed on 28 November 2024), CAZY (CAZy-Home, accessed on 27 November 2024), CARD (The Comprehensive Antibiotic Resistance Database, accessed on 28 November 2024), and VFDB (Validated Antibody Database, antibodies, siRNA/shRNA, ELISA, cDNA clones, proteins/peptides, and biochemicals, accessed on 28 November 2024) to predict genes and annotate their functions.

    2.8. Fibrinolytic Enzyme Activity Assay

    The method for determining fibrinolytic enzyme activity was adapted from the literature [20]. Specifically, 20 μL of the sample was spotted onto a solid separation agar plate and incubated at 37 °C for 12 h. The diameter of the clear zone formed around the sample was then measured. Fibrinolytic enzyme activity was calculated by constructing a standard curve using urokinase, and the enzyme activity (IU/mL) of the fibrinolytic enzyme was determined by comparison with the urokinase units.

    2.9. Analytical Method

    The method for detecting MK-7 in the fermentation broth was adapted from the literature with slight modifications [21]. A total of 15 mL of extraction solvent (n-hexane:isopropanol in a 2:1 volume ratio) was added to 30 mL of fermentation broth after 6 days of fermentation. The mixture was then extracted for 12 h under dark conditions at 16 °C and 200 rpm. After extraction, the solution was centrifuged to collect the extract for further analysis. The extract was analyzed using high-performance liquid chromatography (HPLC) equipped with a UV detector. A C18 column (250 mm × 4.6 mm, 5 μm) was used, with methanol:dichloromethane (9:1, v/v) as the mobile phase, a flow rate of 1 mL/min, and a column temperature of 40 °C. Detection was carried out at a wavelength of 248 nm. Under the aforementioned conditions, the MK-7 standards with concentrations of 100 mg/L, 80 mg/L, 60 mg/L, 40 mg/L, and 20 mg/L were individually subjected to detection. Based on the peak areas obtained from the detection, the standard curve for MK-7 was determined to be y = 14.285x + 11.9 (R2 = 0.9998), where x denotes the concentration of MK-7 and y represents the peak area value.

    4. Conclusions

    Menaquinone-7 (MK-7), as a crucial drug for the treatment of osteoporosis and cardiovascular diseases, holds significant market potential. Since the discovery of vitamin K2 in 1929, research on MK-7 has continued for nearly a century. Although previous studies have improved the titer of MK-7, it still falls short of industrial demand.

    In this study, a wild strain of Bacillus subtilis, capable of producing MK-7, was isolated from commercially available natto fermentation agents. Shake-flask experiments confirmed that the MK-7 titer of this strain could reach 75.18 mg/L, which was designated as L-5 for subsequent experiments. This study aimed to enhance the MK-7 production capacity of L-5 by combining traditional mutagenesis and metabolic engineering techniques. Initially, using ARTP mutagenesis technology combined with protoplast technology, resistant fusion strains of HNA and DPA were obtained, and the titer was increased to 196.68 mg/L. This strain, R-8, was used for further research. To explore the genetic factors responsible for the increased MK-7 titer, whole-genome sequencing and resequencing were performed on R-8 and the parental strain L-5. This study focused on the MK-7 biosynthetic pathway and identified several mutations in key enzymes. Four mutants were found in the key enzymes of the MK-7 biosynthetic pathway, namely, MenD (S249L), MenA (S196L), Dxs (N60D, Q185H), and Dxr (T200Q). To verify whether these mutation sites are effective, overexpression plasmids for the mutants menD (S249L), menA (S196L), Dxs (N60D, Q185H), and Dxr (T200Q), as well as the wild-type menD, menA, Dxs, and Dxr from strain L-5, were constructed and introduced into strain R-8 to create recombinant strains. The results of shake-flask fermentation showed that strains overexpressing the four mutants produced higher titers of MK-7 than those overexpressing the wild-type genes from L-5, with increases of 19.65 mg, 20.71 mg, 17.35 mg, and 16.28 mg, respectively. These findings suggest that the mutations in the key enzymes’ genetic loci in the MK-7 pathway are beneficial, as they likely improve enzyme activity or expression, thereby enhancing the metabolic flux of MK-7 synthesis. To further enhance the production of MK-7, the four mutants menD (S249L), menA (S196L), Dxs (N60D, Q185H), and Dxr (T200Q) were expressed in tandem. The recombinant strain R-89, expressing these genes, achieved an MK-7 titer of 239.65 mg/L, which is a significant increase in production compared to the parental strain R-8. In addition, strain R-89 also demonstrated good genetic stability. This study provides guidance and theoretical support for the future engineering of key enzymes in the MK-7 biosynthetic pathway.



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    Meng Li www.mdpi.com

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