Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

Vitamin K supplementation and bone mineral density in dialysis: results of the double-blind, randomized, placebo-controlled RenaKvit trial.

BACKGROUND: Vitamin K deficiency is highly prevalent in patients on dialysis and may contribute to their low bone mineral density (BMD) and increased risk of fracture. This study investigated the effect of menaquinone-7 (MK-7) supplementation on BMD in patients on chronic dialysis. METHODS: In a multicentre, double-blind, placebo-controlled intervention trial, 123 patients on chronic dialysis were randomised to a daily oral supplement of either MK-7 360 µg or placebo for 2 years. BMD of the distal radius (1/3, mid, ultradistal and total), femoral neck, lumbar spine (L1-L4) and whole body was assessed by dual-energy X-ray absorptiometry. Serum levels of vitamin K1 and MK-7 and plasma levels of total osteocalcin, dephosphorylated-uncarboxylated matrix Gla protein and protein induced by vitamin K absence II were measured to assess vitamin K status. RESULTS: After 2 years, an accelerated BMD loss of the 1/3 distal radius was found with MK-7 supplementation {mean difference of changes relative to placebo -0.023 g/cm2 [95% confidence interval (CI) -0.039 to -0.008]}, whereas the decrease in lumbar spine BMD seen in the placebo group was prevented [mean difference of changes between groups 0.050 g/cm2 (95% CI 0.015-0.085)]. No significant effects were observed at the remaining skeletal sites. Vitamin K status strongly improved in MK-7-supplemented participants. CONCLUSION: Compared with placebo, an accelerated BMD loss of the 1/3 distal radius was found after 2 years of MK-7 supplementation, whereas a decline in lumbar spine BMD was prevented. As such, MK-7 supplementation might modify BMD site-specifically in patients on dialysis. In aggregate, our findings do not support MK-7 supplementation to preserve bone in patients on dialysis.

The study of bioavailability and endogenous circadian rhythm of menaquinone-7, a form of vitamin K2, in healthy subjects.

Menaquinone-7 (MK-7), a multipotent vitamin K2, possesses a wide range of biological activities, a precise curative effect and excellent safety. A simple and rapid LC-APCI-MS/MS method for the determination of MK-7 in human plasma with single liquid-liquid extraction (LLE) extraction and 4·5-min analysis time has been developed and validated. Four per cent bovine serum albumin (BSA) was used as surrogate matrix for standard curves and endogenous baseline subtraction. This method was reproducible and reliable and was used to analyse of MK-7 in human plasma. The endogenous circadian rhythm and bioavailability of MK-7 were investigated in two randomised single-dose, open, one-way clinical trials (Study I and Study II). A total of five healthy male subjects were enrolled in Study I and 12 healthy male subjects in Study II. Single-dose (1 mg) of MK-7 was given to each subject under fasting condition, and all eligible subjects were given a restricting VK2 diet for 4 d prior to drug administration and during the trial. The experiment results of Study I demonstrated that endogenous MK-7 has no circadian rhythm in individuals. Both studies showed MK-7 are absorbed with peak plasma concentrations at about 6 h after intake and has a very long half-life time.

Brij-58 supplementation enhances menaquinone-7 biosynthesis and secretion in Bacillus natto.

Menaquinone-7 is a form of vitamin K2 that has been shown to have numerous healthy benefits. In this study, several surfactants were investigated to enhance the production of menaquinone-7 in Bacillus natto. Results showed that Brij-58 supplementation influenced the cell membrane via adsorption, and changed the interfacial tension of fermentation broth, while the changes in the state and the composition of the cell membrane enhanced the secretion and biosynthesis of menaquinone-7. The total production and secretion rate of menaquinone-7 increased by 48.0% and 56.2% respectively. During fermentation, the integrity of the cell membrane decreased by 82.9% while the permeability increased by 158% when the maximum secretory rate was reached. Furthermore, Brij-58 supplementation induced the stress response in bacteria, resulting in hyperpolarization of the membrane, and increased membrane ATPase activity. Finally, changes in fatty acid composition increased membrane fluidity by 30.1%. This study provided an effective strategy to enhance menaquinone-7 yield in Bacillus natto and revealed the mechanism of Brij-58 supplementation in menaquinone-7 production. KEY POINTS: • MK-7 yield in Bacillus natto was significantly increased by Brij-58 supplementation. • Brij-58 could be adsorbed on cell surface and change fermentation environment. • Brij-58 supplementation could affect the state and composition of the cell membrane.

Development, Validation, and Two-Year Application of Rapid and Simple LC-MS/MS-Based Method for the Determination of K2MK-7 in Blood Samples.

Biological properties of menaquinone-7, one of the vitamin K2 vitamers (K2MK-7), both those proven and those that remain to be investigated, arouse extensive interest that goes beyond the strictly scientific framework. The most important of them is the prevention of age-related diseases, considering that we live in the times identified as the era of aging societies and many people are exposed to the vitamin K2MK-7 deficiency. Therefore, an effective analytical protocol that can be adopted as a diagnostic and preventive analytics tool is needed. Herein, a simple sample preparation method followed by the liquid chromatography-tandem mass spectrometry-based method (LC-MS/MS), was used for the selective and sensitive determination of K2MK-7 in serum samples. Under the optimized conditions, using 500 µL of serum and the same amount of n-hexane, the reproducibility and the accuracy were obtained in the ranges of 89-97% and 86-110%, respectively, and the limit of detection value was 0.01 ng/mL. This method was used for the routine analysis. Statistical interpretation of the data from 518 samples obtained during 2 years of practice allowed for obtaining information on the content and distribution of K2MK-7 in the Polish population, broken down by the sex and age groups.

Bottom-up synthetic biology approach for improving the efficiency of menaquinone-7 synthesis in Bacillus subtilis.

BACKGROUND: Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis. Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. RESULTS: We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. CONCLUSIONS: In summary, a "high-efficiency, low-carbon, cofactor-recycling" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

Role of vitamin K2 in bone-vascular crosstalk.

Vitamin K (VK) is a fat-soluble vitamin that is indispensable for the activation of vitamin K-dependent proteins (VKDPs). It has been shown to play an important role in the proper calcium deposit at the bone level, hindering that on the vascular walls. The deficiency of this vitamin in European populations is frequent and unknown. It is related to several factors, poor dietary intake, altered intestinal absorption or altered production by bacteria, indicating possible dysbiosis. For Vitamin K2 (VK2), there is currently no official reference daily intake (RDI). However, the effects of VK2 on the improvement of health in cardiovascular diseases, on bone metabolism, on chronic kidney diseases have been the subject of research in recent decades. The microbiota in the gastrointestinal tract plays an important role: Bacteroides are primarily capable of synthetizing very long chain forms of menaquinones and, in addition to the bacteria present in the intestinal flora, VK2 is also produced by bacteria used in food fermentation processes. This review provides an update on the current literature regarding the origin of VK2 and its implications in what is called the "calcium paradox", namely the lack of calcium in the bone and its storage in the wall of the vessel.

Optimisation of the fermentation media to enhance the production of the bioactive isomer of vitamin menaquinone-7.

Menaquinone-7 (MK-7) offers significant health benefits; however, only the all-trans form is biologically active. MK-7 produced through fermentation can occur as all-trans and cis isomers, and the therapeutic value of the resulting MK-7 is exclusively determined by the quantity of the all-trans isomer. Therefore, this study aimed to investigate the effect of the media composition on the isomer profile obtained from fermentation and determine the optimum media combination to increase the concentration of the all-trans isomer and diminish the production of cis MK-7. For this purpose, design of experiments (DOE) was used to screen the most effective nutrients, and a central composite face-centred design (CCF) was employed to optimise the media components. The optimum media consisted of 1% (w/v) glucose, 2% (w/v) yeast extract, 2% (w/v) soy peptone, 2% (w/v) tryptone, and 0.1% (w/v) CaCl2. This composition resulted in an average all-trans and cis isomer concentration of 36.366 mg/L and 1.225 mg/L, respectively. In addition, the optimised media enabled an all-trans isomer concentration 12.2-fold greater and a cis isomer concentration 2.9-fold less than the unoptimised media. This study was the first to consider the development of an optimised fermentation media to enhance the production of the bioactive isomer of MK-7 and minimise the concentration of the inactive isomer. Furthermore, this media is commercially promising, as it will improve the process productivity and reduce the costs associated with the industrial fermentation of the vitamin.

A study of hydrophobins-modified menaquinone-7 on osteoblastic cells differentiation.

Menaquinone-7 is involved in bone metabolism and can be used to prevent and treat osteoporosis. However, as a fat-soluble vitamin, menaquinone-7 has poor water solubility. As a surfactant, hydrophobins can change the affinity/hydrophobicity of the covered interface. In this study, menaquinone-7 was modified by hydrophobins, and the different addition ratios were explored. Moreover, Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and water contact angle (WCA) measurements indicated that hydrophobins effectively bind to menaquinone-7 and greatly increase the hydrophilicity of the surface of menaquinone-7. Studies on the metabolism of MC3T3-E1 cells showed that compared with native menaquinone-7, HGFI-modified menaquinone-7 can significantly promote osteoblast differentiation but inhibit osteoclast differentiation. Besides, the Mito-Tracker Green experiments show that HGFI-modified menaquinone-7 can significantly promote the activity of mitochondria in cells. These findings indicate that hydrophobins can be used as an effective biomaterial to modify menaquinone-7, promote the formation of osteoblasts, and better to bone balance.

Simultaneous Determination of Vitamins D3 (Calcitriol, Cholecalciferol) and K2 (Menaquinone-4 and Menaquinone-7) in Dietary Supplements by UHPLC.

The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market. Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. In this regard, the simultaneous determination of vitamin D3 (calcitriol-CT and cholecalciferol-CHL) and K2 (menaquinone-4-MK-4 and menaquinone-7-MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04-0.05 µg/mL, respectively. The validation results indicated that the method had reasonable linearity (R2 ≥ 0.9990), good recovery (>82%), satisfactory intra-day precision (≤1.9%) and inter-day precision (≤3.5%), and high selectivity and specificity. The validated UHPLC method was demonstrated to be precise, accurate, and robust for the simultaneous determination of vitamins D3 (CT and CHL) and K2 (MK-4 and MK-7) in dietary supplements.

Effect of Low-Dose Vitamin K2 Supplementation on Bone Mineral Density in Middle-Aged and Elderly Chinese: A Randomized Controlled Study.

Previous studies indicated a positive effect of vitamin K2 (VK2) supplementation on bone turnover biomarkers and bone mineral density (BMD), but the doses varied, and few studies have focused on the difference between VK2 supplementation alone and in combination with calcium and vitamin D3. The aim of this study was to explore a low and effective dose of VK2 for improving BMD, and to examine whether the co-supplementation of VK2, calcium and vitamin D3 would bring greater effects. In this trial, a total of 311 community-dwelling men and postmenopausal women aged 50 and 75 years were randomly assigned to four groups, receiving placebo, 50 µg/day, 90 µg/day or co-supplementation with calcium (500 mg/day) and vitamin D3 (10 µg/day) for 1 year. At the endpoint, the bone loss of femoral neck was significantly lower in postmenopausal women in the two 90 µg groups (treatment × time, p = 0.006) compared with placebo, but no effects in men. Serum biomarkers cOC/ucOC ratio increased in the intervention groups (treatment × time, p < 0.001). VK2 supplementation in dose of 90 µg/day performed a significant effect on reducing bone loss in postmenopausal women, but in combination with calcium and vitamin D3 brought no additional effects.Trial registration This trial was registered at http://www.chictr.org.cn as chiCTR1800019240.

The effect of aeration and mixing in developing a dairy-based functional food rich in menaquinone-7.

Vitamin menaquinone-7 (MK-7) supplementation improves bone health and reduces the incidence of osteoporosis. Despite the recent developments in MK-7 fermentation using Bacillus subtilis natto, low fermentation yields, as well as complicated downstream processing steps, are still the main reasons for the expensive final product. To overcome these issues, developing a fermented dairy-based product rich in MK-7 by avoiding costly downstream steps and optimising the fermentation operating conditions to enhance the MK-7 concentration would be an alternative approach. The present study, therefore, aims to evaluate the role of agitation and aeration as the key operating conditions on MK-7 production by Bacillus subtilis natto using a milk media. The agitation and aeration rates of 525 RPM and 5 VVM were found to be the optimum levels leading to the production of 3.54 mg/L of MK-7. Further, the sensory evaluation was performed to compare the sensory properties of the freeze-dried fermented samples with non-fermented milk samples. The results illustrated that the fermented samples had a significant saltiness with intense aroma resulting in the less acceptability of them by the panellists.

Menaquinone-7 production in engineered Escherichia coli.

Menaquinone-7 (MK-7), a highly valuable member of the vitamin K2 series, is an essential nutrient for humans. In this study, to develop engineered Escherichia coli strains for MK-7 production, heterogeneous heptaprenyl pyrophosphate synthetase (HepPPS) was introduced, and MK-7 production was first achieved in engineered E. coli by overexpression of Bacillus subtilis-derived HepPPS (BsHepPPS). Then, by optimizing the enzyme expression of the heterogenous mevalonic acid (MVA) pathway and the BsHepPPS, the titre of MK-7 increased to 2.3 µM, which was 22-fold higher than that of the original strain. The competitive pathways of MK-7 were further investigated by deletion of ubiCA or ispB. Finally, the scale-up fermentation of the engineered E. coli in a 5-L fermenter was studied under aerobic conditions using glucose, and 13.6 µM (8.8 mg/L) MK-7 was achieved. Additionally, metabolite analysis revealed a new bottleneck in the MK-7 pathway at ubiE, suggesting an avenue for further optimization. This report is the first to describe the metabolic engineering of MK-7 in E. coli, which provides a new perspective for MK-7 production.

Biofilm reactors as a promising method for vitamin K (menaquinone-7) production.

Menaquinone-7 (MK-7) is the most potent subtype of vitamin K with extraordinarily high half-life in the circulatory system. Therefore, MK-7 plays a critical role in promoting human wellbeing today. Studies on MK-7 every year show more and more magnificent benefits of it in preventing cardiovascular diseases and osteoporosis to battling cancer cells, Alzheimer's and Parkinson's diseases. Thus, it needs to be supplemented to daily diet for accumulative and long-term benefits. Chemical synthesis of MK-7 produces a significant cis-isomer form of it, which has no biological activity. Fortunately, due to its key role in electron transfer in bacteria, trans-MK-7 is biosynthesized by especially Gram-positive strains mainly Bacillus genus. Concordantly, MK-7 could be produced via solid or liquid state fermentation strategies. In either regime, when static fermentation is applied in the absence of agitation and aeration, operational issues arise such as heat and mass transfer inefficiencies. Thus, scaling up the process becomes a challenge. On the other hand, studies have indicated that biofilm and pellicle formation that occur in static fermentations are key characteristics for extracellular MK-7 secretion. Therefore, this review covers the most recent discoveries of the therapeutic properties of MK-7 and optimization attempts at increasing its biosynthesis in different media compositions and effective growth parameters as well as the cutting-edge use of biofilm reactors where B. subtilis cells have the infrastructures to form mature biofilm formations on plastic composite supports. Biofilm reactors therefore can provide robust extracellular MK-7 secretion while simultaneously enduring high agitation and aeration rates, which then address the scale-up and operational issues associated with static fermentation strategies.

Intracellular response of Bacillus natto in response to different oxygen supply and its influence on menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7) plays an important role in blood clotting, cardiovascular disease and anti-osteoporosis, and has been wildly used in the food additives and pharmaceutical industries. The aim of this study was to investigate the mechanism of menaquinone-7 biosynthesis in response to different oxygen supplies in Bacillus natto. The differences of fermentation performance, intracellular metabolites, oxidative stress reaction and enzyme activities of Bacillus natto R127 were analyzed under different KLa. Glycerol consumption rate and MK-7 yield at 24.76 min- 1 was 2.1 and 7.02 times of that at 18.23 min- 1. Oxidative stress analysis showed the cell generated more active oxygen and possessed higher antioxidant capacity at high oxygen supply condition. Meanwhile, high pyruvate kinase and high cytochrome c oxidase activities were also observed at 24.76 min- 1. Furthermore, comparative metabolomics analyses concluded series of biomarkers for high MK-7 biosynthesis and cell rapid growth. Besides, several metabolic responses including low glyceraldehyde-3-phosphate accumulation, low flux from pyruvate to lactic acid, high active TCA pathway, were also found to be associated with high MK-7 accumulation at high oxygen supply conditions. These findings provided the information for better understanding of oxygen effect on MK-7 biosynthesis and lay a foundation for further improvement of MK-7 production as well.

Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.

Sphingobacterium terrae sp. nov., isolated from oil-contaminated soil.

A cream-coloured, Gram-stain-negative, non-motile and rod-shaped bacterium, designated strain Brt-FT, was isolated from oil-contaminated soil. Strain Brt-FT was able to grow from 15 to 45 °C, pH 6.5-10.5 and 0-4.5 % (w/v) NaCl concentration. This strain was taxonomically characterized by a polyphasic approach. Based on 16S rRNA gene sequence analysis, strain Brt-FT represented a member of the genus Sphingobacterium and shared highest sequence similarity with Sphingobacterium chuzhouense DH-5T (99.4 %); Sphingobacterium gobiense H7T (95.7 %) and Sphingobacterium arenae H-12T (94.3 %). The only respiratory quinone was menaquinone-7, the major polar lipid was phosphatidylethanolamine and the predominant fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0, and iso-C17 : 0 3-OH. The DNA G+C content was 43.4 mol%. The DNA-DNA relatedness value between strain Brt-FT and S. chuzhouense KCTC 42746T was 35.7 %, which falls below the threshold value of 70 % for the strain to be considered as novel. The morphological, physiological, chemotaxonomic and phylogenetic analyses clearly distinguished this strain from its closest phylogenetic neighbours. Thus, strain Brt-FT represents a novel species of the genus Sphingobacterium, for which the name Sphingobacteriumterrae sp. nov. is proposed. The type strain is Brt-FT (=KEMB 9005-691T=KACC 19392T=JCM 32159T).

Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.

Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (Menaquinone-7) production in biofilm reactors.

Menaquinone-7 (MK-7) is the key form of vitamin K used as a dietary supplement and its production revolves around Bacillus subtilis natto. Current fermentation strategies, which suggest static fermentations without aeration and agitation, can be problematic for large scale MK-7 production due to biofilm formation. The use of biofilm reactors, therefore, is proposed in the present study, which could utilize both agitation and aeration without interrupting MK-7 secretion. In this study, biofilm reactors were constructed using the selected plastic composite support (PCS) and B. subtilis natto strain for MK-7 production. Using response surface methodology (RSM), optimum growth parameters including temperature, pH, and agitation were determined in a glycerol-based medium. Results were presented in a statistical model (R 2 = 0.90), leading to optimum growth conditions of temperature (35 °C), agitation (200 rpm) and pH (6.58). Model-predicted MK-7 concentration was validated and MK-7 concentration of 12.09 mg/L was produced in the biofilm reactor. The obtained concentration was 58% higher as compared to the suspended-cell culture (7.67 mg/L). The results of this study will provide a critical step towards improved industrial scale production of MK-7.