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Menaquinone (MK) in bacterial membrane is an attractive target for the development of novel therapeutic agents. Mining the untapped chemical diversity encoded by Gram-negative bacteria presents an opportunity to identify additional MK-binding antibiotics (MBAs). By MK-binding motif searching of bioinformatically predicted linear non-ribosomal peptides from 14,298 sequenced genomes of 45 underexplored Gram-negative bacterial genera, here we identify a novel MBA structural family, including silvmeb and pseudomeb, using structure prediction-guided chemical synthesis. Both MBAs show rapid bacteriolysis by MK-dependent membrane depolarization to achieve their potent activities against a panel of Gram-positive pathogens. Furthermore, both MBAs are proven to be effective against methicillin-resistant Staphylococcus aureus in a murine peritonitis-sepsis model. Our findings suggest that MBAs are a kind of structurally diverse and still underexplored antibacterial lipodepsipeptide class. The interrogation of underexplored bacterial taxa using synthetic bioinformatic natural product methods is an appealing strategy for discovering novel biomedically relevant agents to confront the crisis of antimicrobial resistance., (© 2024. The Author(s).)

By utilizing the conformational selectivity of biosynthesis and the flexibility of chemical synthesis, researchers have formulated metabolic engineering-based semi-synthetic approaches that initiate with the final product's structure and identify key biosynthesis intermediates. Nonetheless, these tailored semi-synthetic routes focused on end-products, neglecting the possibility of biobased intermediates as a platform for derivatization. To address this challenge, this studyproposed a novel strategy resembling chemosynthesis-style divergent exploration to amplify the significance of biobased intermediates, in the case of geranylgeraniol (GGOH). Using the novel bifunctional terpene synthase PTTC066 and systematic metabolic engineering modifications, the engineered yeast straindemonstrated high GGOH production levels (3.32 g/L, 0.039 g/L/h). This platformenabled the semi-synthesis of various pharmaceuticals, including the anti-ulcer drug teprenone, the osteoporosis treatment drug menaquinone-4, and introduced a novel route for synthesizingα-tocotrienol. This study offers a fresh outlook on semi-synthetic approaches, opening avenues for improvements, substitutions, and innovations in industrial production processes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tiangang, Liu reports financial support was provided by Wuhan Hesheng Technology Co., Ltd. Wuhan University and Wuhan Hesheng Technology Co., Ltd have applied for several patents based on this work. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Intervertebral disc degeneration (IDD) is a progressive and chronic disease, the mechanisms have been studied extensively as a whole, while the cellular heterogeneity of cells in nucleus pulposus (NP) tissues remained controversial for a long time. This study conducted integrated analysis through single-cell sequencing analysis, weighted gene co-expression network analysis (WGCNA), and differential expression analysis, to systematically decipher the longitudinal alterations of distinct NP subtypes, and also analyzed the most essential genes in the development of IDD. Then, this study further conducted structural biology method to discover the potential lead compounds through a suite of advanced approaches like high-throughput screening (HTVS), pharmaceutical characteristics assessment, CDOCKER module as well as molecular dynamics simulation, etc., aiming to ameliorate the progression of IDD. Totally 5 NP subpopulations were identified with distinct biological functions based on their unique gene expression patterns. The predominant dynamics changes mainly involved RegNPs and EffNPs, the RegNPs were mainly aggregated in normal NP tissues and drastically decreased in degenerative NP, while EffNPs, as pathogenic subtype, exhibited opposite phenomenon. Importantly, this study further reported the essential roles of Menaquinone in alleviating degenerative NP cells for the first time, which could provide solid evidence for the application of nutritional therapy in the treatment of IDD. This study combined scRNA-seq, bulk-RNA seq and HTVS techniques to systematically decipher the longitudinal changes of NP subtypes during IDD. EffNPs were considered to be 'chief culprit' in IDD progression, while the novel natural drug Menaquinone could reverse this phenomenon.Communicated by Ramaswamy H. Sarma.

This computational study addresses the question of how large membrane-bound proteins of electron transport chains facilitate fast vector-based charge transport. We study electron transfer reactions following ultrafast initial charge separation induced by absorption of light by P 800 primary pair and leading to the electron localization at the A 0 cofactor. Two subsequent, much slower reactions, electron transfer to the iron-sulfur cluster F x and reduction of the menaquinone (MQ) cofactor, are studied by combining molecular dynamics simulations, electronic structure calculations, and theoretical modeling. The low value of the electronic coupling between A 0 and F x brings this reaction to the microsecond time scale even at the zero activation barrier. In contrast, A 0 -MQ electron transfer occurs on a subnanosecond time scale and might become the preferred route for charge transport. We elucidate mechanistic properties of the protein medium allowing fast, vectorial charge transfer. The electric field is high and inhomogeneous inside the protein and is coupled to high polarizabilities of cofactors to significantly lower the reaction barrier. The A 0 -MQ separation puts this reaction at the edge between the plateau characterizing the reaction dynamical control and exponential falloff due to electronic tunneling. A strong separation in relaxation times of the medium dynamics for the forward and backward reactions promotes vectorial charge transfer.

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., (© 2024 Wiley Periodicals LLC.)

A high cell-surface hydrophobic bacterium, strain A18 T , was isolated from a waste digestion system in Chaozhou, China. Cells of strain A18 T were Gram-stain-positive, aerobic, non-spore-forming, non-motile, and rod-shaped. Phylogenetic analyses based on the 16S rRNA gene showed that strain A18 T shared less than 94.2% sequence similarity to all validated species in the family Chitinophagaceae, and formed a distinct lineage close to genera Niabella and Terrimonas in the neighbor-joining tree, indicating that strain A18 T is a novel species. Genome-based phylogenetic analyses revealed that strain A18 T is affiliated to the genus Niabella. The cellular components, including iso-C 15:0 and iso-C 15:1 G as the major fatty acids, menaquinone-7 as the respiratory quinone and a DNA G + C content of 40.54% supported strain A18 T as a member of the genus Niabella. However, the physiological and biochemical properties, such as enzyme activities, carbon source utilization and C 18:0 3-OH as another major fatty acids, distinguished strain A18 T from its close related species. Therefore, the name Niabella digestorum sp. nov. is proposed for this novel species. The type strain is A18 T (= GDMCC 1.3242  T  = KCTC 92386  T )., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Menaquinone-7 (MK-7) is a form of vitamin K 2 with health-beneficial effects. A novel fermentation strategy based on combining soy protein hydrolysates (SPHs) with biofilm-based fermentation was investigated to enhance menaquinone-7 (MK-7) biosynthesis by Bacillus subtilis natto. Results showed the SPHs increased MK-7 yield by 199.4% in two-stage aeration fermentation as compared to the SP-based medium in submerged fermentation, which was related to the formation of robust biofilm with wrinkles and the enhancement of cell viability. Moreover, there was a significant correlation between key genes related to MK-7 and biofilm synthesis, and the quorum sensing (QS) related genes, Spo0A and SinR, were downregulated by 0.64-fold and 0.39-fold respectively, which promoted biofilm matrix synthesis. Meanwhile, SPHs also enhanced the MK-7 precursor, isoprene side chain, supply, and MK-7 assembly efficiency. Improved fermentation performances of bacterial cells during fermentation were attributed to abundant oligopeptides (Mw < 1 kDa) and moderate amino acids, particularly Arg, Asp, and Phe in SPHs. All these results revealed that SPHs were a potential and superior nitrogen source for MK-7 production by Bacillus subtilis natto., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The K family of vitamins includes a collection of molecules with different pharmacokinetic characteristics. Menaquinone-7 (MK-7) has the finest properties and is the most therapeutically beneficial due to its long plasma half-life and outstanding extrahepatic bioavailability. MK-7 exhibits cis-trans isomerism, and merely the all-trans form is biologically efficacious. Therefore, the remedial value of MK-7 end products is exclusively governed by the quantity of all-trans MK-7. Consumers favour fermentation for the production of MK-7; however, it involves several challenges. The low MK-7 yield and extensive downstream processing requirements increase production costs, resulting in an expensive final product that is not universally available. Bacterial cell immobilisation with iron oxide nanoparticles (IONs) can potentially address the limitations of MK-7 fermentation. Uncoated IONs tend to have low stability and can adversely affect cell viability; thus, amine-functionalised IONs, owing to their increased physicochemical stability and biocompatibility, are a favourable alternative. Nonetheless, employing biocompatible IONs for this purpose is only advantageous if the bioactive MK-7 isomer is obtained in the most significant fraction, exploring which formed the aim of this investigation. Two amine-functionalised IONs, namely 3-aminopropyltriethoxysilane (APTES)-coated IONs (IONs@APTES) and L-Lysine (L-Lys)-coated IONs (L-Lys@IONs), were synthesised and characterised, and their impact on various parameters was evaluated. IONs@APTES were superior, and the optimal concentration (300 μ g/mL) increased all-trans MK-7 production and improved its yield relative to the untreated cells by 2.3- and 3.1-fold, respectively. The outcomes of this study present an opportunity to develop an innovative and effective fermentation method that enhances the production of bioactive MK-7., (© 2023. The Author(s).)

Menaquinone-7 (MK-7), a vital vitamin with numerous health benefits, is synthesized and secreted extracellularly by the formation of biofilm, dominantly in Bacillus strains. Our team developed an innovative biofilm reactor utilizing Bacillus subtilis natto cells to foster biofilm growth on plastic composite supports to produce MK-7. Continuous fermentation in biofilm reactors offers a promising strategy for achieving sustainable and efficient production of Menaquinone-7 (MK-7). Unlike conventional batch fermentation, continuous biofilm reactors maintain a steady state of operation, which reduces resource consumption and waste generation, contributing to sustainability. By optimizing fermentation conditions, MK-7 production was significantly enhanced in this study, demonstrating the potential for sustainable industrial-scale production. To determine the optimal operational parameters, various dilution rates were tested. These rates were selected based on their potential to enhance nutrient supply and biofilm stability, thereby improving MK-7 production. By carefully considering the fermentation conditions and systematically varying the dilution rates, MK-7 production was significantly enhanced during continuous fermentation. The MK-7 productivity was found to increase from 0.12 mg/L/h to 0.33 mg/L/h with a dilution rate increment from 0.007 to 0.042 h -1 ). This range was chosen to explore the impact of various nutrient supply rates on MK-7 production and to identify the optimal conditions for maximizing productivity. However, a further increase in the dilution rate to 0.084 h -1 led to reduced productivity at approximately 0.16 mg/L/h, likely due to insufficient retention time for effective biofilm formation. Consequently, a dilution rate of 0.042 h -1 exhibited the highest productivity of 0.33 mg/L/h, outperforming all investigated dilution rates and demonstrating the critical balance between nutrient supply and retention time in continuous fermentation. These findings validate the feasibility of operating continuous fermentation at a 0.084 h -1 dilution rate, corresponding to a 48 h retention time, to achieve the highest MK-7 productivity compared to conventional batch fermentation. The significant advancements achieved in enhancing Menaquinone-7 (MK-7) productivity through continuous fermentation at optimal dilution rates in the present work indicate promising prospects for even greater efficiency and sustainability in MK-7 production through future developments., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide and there is a huge unmet need to find safer and more effective drugs. Vitamin K has been found to regulate lipid metabolism in the liver. However, the effects of vitamin K2 on NAFLD is unclear. This study aims to evaluate the preventive and therapeutic effects of vitamin K2 in the process of fatty liver formation and to explore molecular mechanisms the associated with lipid metabolism. A non-alcoholic fatty liver model was established by high-fat diet administration for three months. Vitamin K2 significantly reduced the body weight, abdominal circumference and body fat percentage of NAFLD mice. Vitamin K2 also showed histological benefits in reducing hepatic steatosis. NAFLD mice induced by high-fat diet showed increased HMGR while vitamin K2 intervention could reverse the pathological lterations. Adiponectin (APN) is an endogenous bioactive polypeptide or protein secreted by adipocytes. We detected APN, SOD, AlaDH and other indicators that may affect the state of high-fat diet mice, but the experimental results showed that the above indicators did not change significantly. It is worth noting that the effect of vitamin K2 supplementation on the lipid-lowering effect of uc OC in vivo needs to be further explored. This study first reported the protective effect of vitamin K2 on high-fat diet-induced NAFLD in mice. The protective effect of vitamin K2 may be related to the improvement of lipid metabolism disorder in NAFLD., (© 2024. The Author(s).)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia due to insulin deficiency and/or resistance. Vitamin K (VK) is a group of fat-soluble molecules, including naturally occurring vitamin K1 (phylloquinone). vitamin K2 (menaquinone), and synthetic vitamin K3 (menadione). Beyond coagulation, the health benefits of VK have been described to play different roles in both physiological and pathological processes such as inflammation, energy metabolism, neuroprotection, cellular growth, and survival. It was aimed to observe the antioxidant and/or neuroprotective activity of vitamin K1 in our model of chick embryo diabetic neuropathy (DN) induced by streptozotocin (STZ). Ninety White Leghorn, fertile and 0-day-old SPF (specific pathogen-free) eggs (57 ± 4 gr) were used in the study. Chick embryo blood brain tissues were taken for biochemical evaluation. Plasma insulin and glucose levels were measured. In addition, brain tissue total antioxidant level (TAS), total oxidant level (TOS), malondialdehyde (MDA), and vascular endothelial growth factor (VEGF) levels were measured. Plasma glucose levels were higher in the STZ-treated groups and lower in the treatment groups. Plasma insulin levels were observed to be higher in STZ groups in groups treated with high VK. Low TAS, high MDA, TOS, and VEGF levels were recorded in brain tissue STZ groups. Low VEGF, TOS, and MDA levels were recorded in the group treated with the highest VK, while high TAS levels were observed. In our STZ-induced chick embryo diabetic neuropathy model, we observed that VK1 reduced oxidant damage by showing antioxidant properties or by modulating antioxidant enzymes., (© 2023 The Authors. Journal of Biochemical and Molecular Toxicology published by Wiley Periodicals LLC.)

Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the structure of a menaquinol:organohalide oxidoreductase obtained by cryo-EM. The membrane-bound protein was isolated from Desulfitobacterium hafniense strain TCE1 as a PceA 2 B 2 complex catalysing the dechlorination of tetrachloroethene. Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. In this work, the structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria., (© 2023. The Author(s).)

Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m/z = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

A Gram-strain positive, aerobic, endospore-forming bacterial strain (JJ-246 T ) was isolated from the rhizosphere of Zea mays. The 16S rRNA gene sequence similarity comparisons showed a most closely relationship to Paenibacillus oenotherae DT7-4 T (98.4%) and Paenibacillus xanthinolyticus 11N27 T (98.0%). The pairwise average nucleotide identity and digital DNA-DNA hybridisation values of the JJ-246 T genome assembly against publicly available Paenibacillus type strain genomes were below 82% and 33%, respectively. The draft genome of JJ-246 T shared many putative plant-beneficial functions contributing (PBFC) genes, related to plant root colonisation, oxidative stress protection, degradation of aromatic compounds, plant growth-promoting traits, disease resistance, drug and heavy metal resistance, and nutrient acquisition. The quinone system of strain JJ-246 T , the polar lipid profile and the major fatty acids were congruent with those reported for members of the genus Paenibacillus. JJ-246 T was shown to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus plantiphilus sp. nov. is proposed, with JJ-246 T (= LMG 32093 T  = CCM 9089 T  = CIP 111893 T ) as the type strain., (© 2023. The Author(s).)

Menaquinone-7 is a form of vitamin K 2 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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Functional membrane microdomains (FMMs) that are mainly composed of scaffold proteins and polyisoprenoids play important roles in diverse cellular physiological processes in bacteria. The aim of this study was to identify the correlation between MK-7 and FMMs and then regulate the MK-7 biosynthesis through FMMs. Firstly, the relationship between FMMs and MK-7 on the cell membrane was determined by fluorescent labeling. Secondly, we demonstrated that MK-7 is a key polyisoprenoid component of FMMs by analyzing the changes in the content of MK-7 on cell membrane and the changes in the membrane order before and after destroying the integrity of FMMs. Subsequently, the subcellular localization of some key enzymes in MK-7 synthesis was explored by visual analysis, and the intracellular free pathway enzymes Fni, IspA, HepT and YuxO were localized to FMMs through FloA to achieve the compartmentalization of MK-7 synthesis pathway. Finally, a high MK-7 production strain BS3AT was successfully obtained. The production of MK-7 reached 300.3 mg/L in shake flask and 464.2 mg/L in 3 L fermenter.

Intervertebral disc (IVD) degeneration is a common cause of low back pain in diabetes mellitus type 2 (T2DM) patients. Its pathogenesis and the vitamin (vit.) K2 influence on this disease remain unclear. Lumbar motion segments of male Zucker Diabetes Fatty (ZDF) rats (non-diabetic [control] and diabetic; fed without or with vit. K2) were used. Femur lengths and vertebral epiphyseal cross-section areas were measured. IVDs were histopathologically examined. Protein synthesis and gene expression of isolated IVD fibrochondrocytes were analyzed. T2DM rats showed histopathological IVD degeneration. Femur lengths and epiphyseal areas were smaller in T2DM rats regardless of vit. K2 feeding. Fibrochondrocytes synthesized interleukin (IL)-24 and IL-10 with no major differences between groups. Alpha smooth muscle actin (αSMA) was strongly expressed, especially in cells of vit. K2-treated animals. Gene expression of aggrecan was low, and that of collagen type 2 was high in IVD cells of diabetic animals, whether treated with vit. K2 or not. Suppressor of cytokine signaling ( Socs ) 3 and heme oxygenase ( Hmox )1 gene expression was highest in the cells of diabetic animals treated with vit. K2. Vit. K2 influenced the expression of some stress-associated markers in IVD cells of diabetic rats, but not that of IL-10 and IL-24.

Menaquinone-7 (MK-7), a valuable member of the vitamin K2 series, is an essential nutrient for humans. It is used for treating coagulation disorders, and osteoporosis, promoting liver function recovery, and preventing cardiovascular diseases. In this study, to further improve the metabolic synthesis of MK-7 by the mutant strain, the effect of surfactants on the metabolic synthesis of MK-7 by the mutant strain Bacillus subtilis 168 KO-SinR (BS168 KO-SinR) was analyzed. The scanning electron microscopy and flow cytometry results showed that the addition of surfactants changed the permeability of the cell membrane of the mutant strain and the structural components of the biofilm. When 0.7% Tween-80 was added into the medium, the extracellular and intracellular synthesis of MK-7 reached 28.8 mg/L and 59.2 mg/L, respectively, increasing the total synthesis of MK-7 by 80.3%. Quantitative real-time PCR showed that the addition of surfactant significantly increased the expression level of MK-7 synthesis-related genes, and the electron microscopy results showed that the addition of surfactant changed the permeability of the cell membrane. The research results of this paper can serve as a reference for the industrial development of MK-7 prepared by fermentation., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

During aging, bone marrow mesenchymal stromal cells (MSCs)-the precursors of osteoblasts-undergo cellular senescence, losing their osteogenic potential and acquiring a pro-inflammatory secretory phenotype. These dysfunctions cause bone loss and lead to osteoporosis. Prevention and intervention at an early stage of bone loss are important, and naturally active compounds could represent a valid help in addition to diet. Here, we tested the hypothesis that the combination of two pro-osteogenic factors, namely orthosilicic acid (OA) and vitamin K2 (VK2), and three other anti-inflammatory compounds, namely curcumin (CUR), polydatin (PD) and quercetin (QCT)-that mirror the nutraceutical BlastiMin Complex ® (Mivell, Italy)-would be effective in promoting MSC osteogenesis, even of replicative senescent cells (sMSCs), and inhibiting their pro-inflammatory phenotype in vitro. Results showed that when used at non-cytotoxic doses, (i) the association of OA and VK2 promoted MSC differentiation into osteoblasts, even when cultured without other pro-differentiating factors; and (ii) CUR, PD and QCT exerted an anti-inflammatory effect on sMSCs, and also synergized with OA and VK2 in promoting the expression of the pivotal osteogenic marker ALP in these cells. Overall, these data suggest a potential role of using a combination of all of these natural compounds as a supplement to prevent or control the progression of age-related osteoporosis.

Abstract: Myocardial infarction is among the leading causes of mortality. Menaquinone-4 (MK-4), a vitamin K2 analog, might play a role in rescuing cardiac ischemia/reperfusion (I/R) injury. This work aimed to discover the potential cardioprotective role of MK-4 against myocardial I/R injury in rats. Thirty-two rats were categorized into 3 groups: (I/R) control group: subjected to I/R protocol (received vehicle), MK-4 preconditioning group: MK-4 infusion for 20 minutes before the I/R protocol, and MK-4 postconditioning group: MK-4 infusion for 20 minutes at the start of the reperfusion phase. The hearts were placed in the Langendorff apparatus, and the left ventricular developed pressure (LVDP), heart rate (HR), + (LV dP/dt) max, - (LV dP/dt) max, and Tau were calculated. The necrotic mass was determined by staining it with nitro blue tetrazolium. Creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C- reactive protein (CRP), as well as cardiac superoxide dismutase (SOD), nitric oxide (NOx), malondialdehyde (MDA), and glutathione (GSH) levels were all evaluated. MK-4 postconditioning significantly reduced myocardial infarct size; increased LVDP, + (LV dp/dt) max, - (LV dp/dt) max, and HR; reduced Tau, CK-MB, LDH, CRP, IL-6, TNF-α, MDA, and NOx levels; and increased SOD activity, whereas no significant difference in the GSH level was detected. In conclusion, these data imply that MK-4 may protect the heart from the consequences of I/R., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Vitamin K occupies a unique and often obscured place among its fellow fat-soluble vitamins. Evidence is mounting, however, that vitamin K (VK) may play an important role in the visual system apart from the hepatic carboxylation of hemostatic-related proteins. However, to our knowledge, no review covering the topic has appeared in the medical literature. Recent studies have confirmed that matrix Gla protein (MGP), a vitamin K-dependent protein (VKDP), is essential for the regulation of intraocular pressure in mice. The PREDIMED (Prevención con Dieta Mediterránea) study, a randomized trial involving 5860 adults at risk for cardiovascular disease, demonstrated a 29% reduction in the risk of cataract surgery in participants with the highest tertile of dietary vitamin K1 (PK) intake compared with those with the lowest tertile. However, the specific requirements of the eye and visual system (EVS) for VK, and what might constitute an optimized VK status, is currently unknown and largely unexplored. It is, therefore, the intention of this narrative review to provide an introduction concerning VK and the visual system, review ocular VK biology, and provide some historical context for recent discoveries. Potential opportunities and gaps in current research efforts will be touched upon in the hope of raising awareness and encouraging continued VK-related investigations in this important and highly specialized sensory system.

Diverse aerobic bacteria use atmospheric H 2 as an energy source for growth and survival 1 . This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments 2,3 . Atmospheric H 2 oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily 4,5 . However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H 2 amid ambient levels of the catalytic poison O 2 and how the derived electrons are transferred to the respiratory chain 1 . Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H 2 to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H 2 at the expense of O 2 , and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H 2 oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H 2 oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H 2 in ambient air., (© 2023. The Author(s).)

The effect of vitamin K on bovine endometrial epithelial cells has not been thoroughly investigated. The objective of this study was to examine the effect of the biologically active form of vitamin K, menaquinone-4, on gene expression in bovine endometrial epithelial cells. First, we examined the mRNA and protein expression levels of UBIAD1, a menaquinone-4 biosynthetic enzyme. Second, we screened for potential target genes of menaquinone-4 in bovine endometrial epithelial cells using RNA-sequencing. We found 50 differentially expressed genes; 42 were upregulated, and 8 were downregulated. Among them, a dose-dependent response to menaquinone-4 was observed for the top three upregulated (TRIB3, IL6, and TNFAIP3) and downregulated (CDC6, ORC1, and RRM2) genes. It has been suggested that these genes play important roles in reproductive events. In addition, GDF15 and VEGFA, which are important for cellular functions as they are commonly involved in pathways, such as positive regulation of cell communication, cell differentiation, and positive regulation of MAPK cascade, were upregulated in endometrial epithelial cells by menaquinone-4 treatment. To the best of our knowledge, this is the first study showing the expression of UBIAD1 in the bovine uterus. Moreover, the study determined menaquinone-4 target genes in bovine endometrial epithelial cells, which may positively affect pregnancy with alteration of gene expression in cattle uterus., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aminoglycosides (AG) have been used against Gram-negative bacteria for decades. Yet, how bacterial metabolism and environmental conditions modify AG toxicity is poorly understood. Here, we show that the level of AG susceptibility varies depending on the nature of the respiratory chain that Escherichia coli uses for growth, i.e., oxygen, nitrate, or fumarate. We show that all components of the fumarate respiratory chain, namely, hydrogenases 2 and 3, the formate hydrogenlyase complex, menaquinone, and fumarate reductase are required for AG-mediated killing under fumarate respiratory conditions. In addition, we show that the AAA+ ATPase RavA and its Von Wildebrand domain-containing partner, ViaA, are essential for AG to act under fumarate respiratory conditions. This effect was true for all AG that were tested but not for antibiotics from other classes. In addition, we show that the sensitizing effect of RavA-ViaA is due to increased gentamicin uptake in a proton motive force-dependent manner. Interestingly, the sensitizing effect of RavA-ViaA was prominent in poor energy conservation conditions, i.e., with fumarate, but dispensable under high energy conservation conditions, i.e., in the presence of nitrate or oxygen. We propose that RavA-ViaA can facilitate uptake of AG across the membrane in low-energy cellular states. IMPORTANCE Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose. Elucidation of AG-sensitization mechanisms in bacteria would allow reduced effective doses of AG. Here, we have identified the molecular components involved in anaerobic fumarate respiration that are required for AG to kill. In addition to oxidoreductases and menaquinone, this includes new molecular players, RavA, an AAA+ ATPase, and ViaA, its partner that has the VWA motif. Remarkably, the influence of RavA-ViaA on AG susceptibility varies according to the type of bioenergetic metabolism used by E. coli. This is a significant advance because anaerobiosis is well known to reduce the antibacterial activity of AG. This study highlights the critical importance of the relationship between culture conditions, metabolism, and antibiotic susceptibility.

Menaquinones (MKs) are electron carriers in bacterial respiratory chains. In Staphylococcus aureus ( Sau ), MKs are essential for aerobic and anaerobic respiration. As MKs are redox-active, their biosynthesis likely requires tight regulation to prevent disruption of cellular redox balance. We recently found that the Mycobacterium tuberculosis MenD, the first committed enzyme of the MK biosynthesis pathway, is allosterically inhibited by the downstream metabolite 1,4-dihydroxy-2-naphthoic acid (DHNA). To understand if this is a conserved mechanism in phylogenetically distant genera that also use MK, we investigated whether the Sau- MenD is allosterically inhibited by DHNA. Our results show that DHNA binds to and inhibits the SEPHCHC synthase activity of Sau -MenD enzymes. We identified residues in the DHNA binding pocket that are important for catalysis (Arg98, Lys283, Lys309) and inhibition (Arg98, Lys283). Furthermore, we showed that exogenous DHNA inhibits the growth of Sau , an effect that can be rescued by supplementing the growth medium with MK-4. Our results demonstrate that, despite a lack of strict conservation of the DHNA binding pocket between Mtb -MenD and Sau -MenD, feedback inhibition by DHNA is a conserved mechanism in Sau -MenD and hence the Sau MK biosynthesis pathway. These findings may have implications for the development of anti-staphylococcal agents targeting MK biosynthesis. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Type III polyketide synthases (PKSs) found across Streptomyces species are primarily known for synthesis of a vast repertoire of clinically and industrially relevant secondary metabolites. However, our understanding of the functional relevance of these bioactive metabolites in Streptomyces physiology is still limited. Recently, a role of type III PKS harboring gene cluster in producing alternate electron carrier, polyketide quinone (PkQ) was established in a related member of the Actinobacteria , Mycobacteria , highlighting the critical role these secondary metabolites play in primary cellular metabolism of the producer organism. Here, we report the developmental stage-specific transcriptional regulation of homologous type III PKS containing gene cluster in freshwater Streptomyces sp. strain MNU77. Gene expression analysis revealed the type III PKS gene cluster to be stringently regulated, with significant upregulation observed during the dormant sporulation stage of Streptomyces sp. MNU77. In contrast, the expression levels of only known electron carrier, menaquinone biosynthetic genes were interestingly found to be downregulated. Our liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of a metabolite extract from the Streptomyces sp. MNU77 spores also showed 10 times more metabolic abundance of PkQs than menaquinones. Furthermore, through heterologous complementation studies, we demonstrate that Streptomyces sp. MNU77 type III PKS rescues a respiratory defect of the Mycobacterium smegmatis type III PKS deletion mutant. Together, our studies reveal that freshwater Streptomyces sp. MNU77 robustly produces novel PkQs during the sporulation stage, suggesting utilization of PkQs as alternate electron carriers across Actinobacteria during dormant hypoxic conditions. IMPORTANCE The complex developmental life cycle of Streptomyces sp. mandates efficient cellular respiratory reconfiguration for a smooth transition from aerated nutrient-rich vegetative hyphal growth to the hypoxic-dormant sporulation stage. Polyketide quinones (PkQs) have recently been identified as a class of alternate electron carriers from a related member of the Actinobacteria , Mycobacteria , that facilitates maintenance of membrane potential in oxygen-deficient niches. Our studies with the newly identified freshwater Streptomyces sp. strain MNU77 show conditional transcriptional upregulation and metabolic abundance of PkQs in the spore state of the Streptomyces life cycle. In parallel, the levels of menaquinones, the only known Streptomyces electron carrier, were downregulated, suggesting deployment of PkQs as universal electron carriers in low-oxygen, unfavorable conditions across the Actinobacteria family.

Ulcerative colitis (UC) is a chronic recurrent inflammatory illness of the gastrointestinal system. The purpose of this study was to explore the alleviating effect of vitamin K2 (VK2) on UC, as well as its mechanism. C57BL/6J mice were given 3% DSS for seven days to establish UC, and they then received VK2 (15, 30, or 60 mg/kg·bw) and 5-aminosalicylic acid (100 mg/kg·bw) for two weeks. We recorded the clinical signs, body weights, colon lengths, and histological changes during the experiment. We detected the inflammatory factor expressions using enzyme-linked immunosorbent assay (ELISA) kits, and we detected the tight junction proteins using Western blotting. We analyzed the intestinal microbiota alterations and short-chain fatty acids (SCFAs) using 16S rRNA sequencing and targeted metabolomics. According to the results, VK2 restored the colon lengths, improved the colonic histopathology, reduced the levels of proinflammatory cytokines (such as IL-1β, TNF-α, and IL-6), and boosted the level of the immunosuppressive cytokine IL-10 in the colon tissues of the colitis mice. Moreover, VK2 promoted the expression of mucin and tight junction proteins (such as occludin and zonula occludens-1) in order to preserve the intestinal mucosal barrier function and prevent UC in mice. Additionally, after the VK2 intervention, the SCFAs and SCFA-producing genera, such as Eubacterium&#95;ruminantium&#95;group and Faecalibaculum , were elevated in the colon. In conclusion, VK2 alleviated the DSS-induced colitis in the mice, perhaps by boosting the dominant intestinal microflora, such as Faecalibaculum , by reducing intestinal microflora dysbiosis, and by modulating the expression of SCFAs, inflammatory factors, and intestinal barrier proteins.

The membranous quinone/quinol pool is essential for the majority of life forms and its composition has been widely used as a biomarker in microbial taxonomy. The most abundant quinone is menaquinone (MK), which serves as an essential redox mediator in various electron transport chains of aerobic and anaerobic respiration. Several methylated derivatives of MK, designated methylmenaquinones (MMKs), have been reported to be present in members of various microbial phyla possessing either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). Due to their low redox midpoint potentials, MMKs have been proposed to be specifically involved in appropriate electron transport chains of anaerobic respiration. The class C radical SAM methyltransferases MqnK, MenK and MenK2 have recently been shown to catalyse specific MK methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesise 8-MMK, 7-MMK and 7,8-dimethylmenaquinone (DMMK). MqnK, MenK and MenK2 from organisms such as Wolinella succinogenes, Adlercreutzia equolifaciens, Collinsella tanakaei, Ferrimonas marina and Syntrophus aciditrophicus have been functionally produced in Escherichia coli, enabling extensive quinone/quinol pool engineering of the native MK and 2-demethylmenaquinone (DMK). Cluster and phylogenetic analyses of available MK and MMK methyltransferase sequences revealed signature motifs that allowed the discrimination of MenK/MqnK/MenK2 family enzymes from other radical SAM enzymes and the identification of C-7-specific menaquinone methyltransferases of the MenK2 subfamily. It is envisaged that this knowledge will help to predict the methylation status of the menaquinone/menaquinol pool of any microbial species (or even a microbial community) from its (meta)genome., (Copyright © 2023. Published by Elsevier Inc.)

Menaquinone (MK) is an essential component of the electron transport chain (ETC) in the gram-variable Mycobacterium tuberculosis and many Gram-positive pathogens. Three genes in the M. tuberculosis genome were annotated as methyltransferases involved in lipoquinone synthesis in mycobacteria. Heterologous expression of Rv0558 complemented an ubiE (the quinone C -methyltransferase involved in ubiquinone and menaquinone synthesis) deletion in Escherichia coli, and expression in a wild-type E. coli strain increased quinone C -methyltransferase specific activity by threefold. Rv0558 encodes a canonical C -methyltransferase or, more specifically, a S -adenosylmethionine/demethylmenaquinol methyltransferase. Partially purified recombinant protein catalyzed the formation of MK from demethylmenaquinone (DMK), although the activity of the recombinant protein was low and appeared to require a cofactor or intact membrane structure for activity. Membrane preparations from irradiated M. tuberculosis also showed poor activity; however, membrane preparations from wild-type Mycobacterium smegmatis showed robust, substrate-dependent activity. The apparent K m values for demethylmenaquinone and SAM were 14 ± 5.0 and 17 ± 7.0 μM, respectively. Interestingly, addition of dithiothreitol, dithionite, NADH, or other substrates of primary dehydrogenases to reaction mixtures containing membrane preparations stimulated the activity. Thus, these observations strongly suggest that demethylmenaquinol is the actual substrate of MenG. Ro 48-8071, previously reported to inhibit mycobacterial MK synthesis and growth, inhibited Rv0558 activity with an IC 50 value of 5.1 ± 0.5 μM, and DG70 (GSK1733953A), first described as a respiration inhibitor in M. tuberculosis, inhibits MenG activity with an IC 50 value of 2.6 ± 0.6 μM.

Vitamin K is the common name for a group of compounds recognized as essential for blood clotting. The group comprises phylloquinone (K1)-a 2-methyl-3-phytyl-1,4-naphthoquinone; menaquinone (K2, MK)-a group of compounds with an unsaturated side chain in position 3 of a different number of isoprene units and a 1,4-naphthoquinone group and menadione (K3, MD)-a group of synthetic, water-soluble compounds 2-methyl-1,4-naphthoquinone. However, recent epidemiological studies suggest that vitamin K has various benefits that go beyond blood coagulation processes. A dietary intake of K1 is inversely associated with the risk of pancreatic cancer, K2 has the potential to induce a differentiation in leukemia cells or apoptosis of various types of cancer cells, and K3 has a documented anti-cancer effect. A healthy diet rich in fruit and vegetables ensures an optimal supply of K1 and K2, though consumers often prefer supplements. Interestingly, the synthetic form of vitamin K-menadione-appears in the cell during the metabolism of phylloquinone and is a precursor of MK-4, a form of vitamin K2 inaccessible in food. With this in mind, the purpose of this review is to emphasize the importance of vitamin K as a micronutrient, which not only has a beneficial effect on blood clotting and the skeleton, but also reduces the risk of cancer and other pro-inflammatory diseases. A proper diet should be a basic and common preventive procedure, resulting in a healthier society and reduced burden on healthcare systems.

Phylloquinone (vitamin K1) and menaquinones (vitamin K2 family) are essential for post-translational γ-carboxylation of a small number of proteins, including clotting factors. These modified proteins have now been implicated in diverse physiological and pathological processes including cancer. Vitamin K intake has been inversely associated with cancer incidence and mortality in observational studies. Newly discovered functions of vitamin K in cancer cells include activation of the steroid and xenobiotic receptor (SXR) and regulation of oxidative stress, apoptosis, and autophagy. We provide an update of vitamin K biology, non-canonical mechanisms of vitamin K actions, the potential functions of vitamin K-dependent proteins in cancer, and observational trials on vitamin K intake and cancer., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Tuberculosis remains a public health crisis and a health security threat. There is an urgent need to develop new antituberculosis drugs with novel modes of action to cure drug-resistant tuberculosis and shorten the chemotherapy period by sterilizing tissues infected with dormant bacteria. Lysocin E is an antibiotic that showed antibacterial activity against Staphylococcus aureus by binding to its menaquinone (commonly known as vitamin K 2 ). Unlike S. aureus, menaquinone is essential in both growing and dormant Mycobacterium tuberculosis. This study aims to evaluate the antituberculosis activities of lysocin E and decipher its mode of action. We show that lysocin E has high in vitro activity against both drug-susceptible and drug-resistant Mycobacterium tuberculosis var. tuberculosis and dormant mycobacteria. Lysocin E is likely bound to menaquinone, causing M. tuberculosis membrane disruption, inhibition of oxygen consumption, and ATP synthesis. Thus, we have concluded that the high antituberculosis activity of lysocin E is attributable to its synergistic effects of membrane disruption and respiratory inhibition. The efficacy of lysocin E against intracellular M. tuberculosis in macrophages was lower than its potent activity against M. tuberculosis in culture medium, probably due to its low ability to penetrate cells, but its efficacy in mice was still superior to that of streptomycin. Our findings indicate that lysocin E is a promising lead compound for the development of a new tuberculosis drug that cures drug-resistant and latent tuberculosis in a shorter period.

Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ-glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ-carboxyglutamate (Gla) of the vitamin K-dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla-protein by western blot analysis and using a cell-free system to determine the GGCX activity by HPLC. Trans MK7H 2 showed a higher ability to carboxylate the 70 KDa GLA-protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H 2 was demonstrated, comparable with both K1H 2 and oxidized trans MK7 but less than trans MK7H 2 . For the first time, a difference of biological activity between cis and trans configuration of menaquinone-7 has been reported., (© 2022 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

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., (© 2022. The Author(s).)

UbiA prenyltransferase domain-containing protein-1 (UBIAD1) is responsible for the biosynthesis of menaquinone-4 (MK-4), a cofactor for extrahepatic carboxylation of vitamin K-dependent (VKD) proteins. Genetic variations of UBIAD1 are mainly associated with Schnyder corneal dystrophy (SCD), a disease characterized by abnormal accumulation of cholesterol in the cornea. Results from in vitro studies demonstrate that SCD-associated UBIAD1 mutations are defective in MK-4 biosynthesis. However, SCD patients do not exhibit typical phenotypes associated with defects of MK-4 or VKD carboxylation. Here, we coupled UBIAD1's biosynthetic activity of MK-4 with VKD carboxylation in HEK293 cells that stably express a chimeric VKD reporter protein. The endogenous Ubiad1 gene in these cells was knocked out by CRISPR-Cas9-mediated genome editing. The effect of UBIAD1 mutations on MK-4 biosynthesis and VKD carboxylation was evaluated in Ubiad1-deficient reporter cells by determining the production of MK-4 or by measuring the efficiency of reporter-protein carboxylation. Our results show that the hot-spot mutation N102S has a moderate impact on MK-4 biosynthesis (retained ˜ 82% activity) but does not affect VKD carboxylation. However, the G186R mutation significantly affected both MK-4 biosynthesis and VKD carboxylation. Other mutations exhibit varying degrees of effects on MK-4 biosynthesis and VKD carboxylation. These results are consistent with in vivo results obtained from gene knock-in mice and SCD patients. Our findings suggest that UBIAD1's MK-4 biosynthetic activity does not directly correlate with the phenotypes of SCD patients. The established cell-based assays in this study provide a powerful tool for the functional studies of UBIAD1 in a cellular milieu., (© 2021 Federation of European Biochemical Societies.)

A previous study reported that the Mycobacterium smegmatis (Msm) protein MSMEG&#95;2295 is a repressor controlling the expression of several genes, including that for MSMEG&#95;5125, a putative isoprenoid binding protein belonging to the YceI family, and DinB2, a DNA damage repair enzyme. This repressor is encoded by the first gene of the operon that also expresses the gene for DinB2. Targeted inhibition of MSMEG&#95;5125 using CRISPRi technology resulted in a significant loss of Msm's respiratory activity and viability. Since this protein has been predicted to be an isoprenoid binding protein, we suspected a role of menaquinones, which are isoprenoid naphthoquinones, in the observed phenomenon. Accordingly, we tested whether MSMEG&#95;5125's deficiency-induced lethality could be reversed by adding menaquinone. The result was positive, implying cooperation between MSMEG&#95;5125 and menaquinone in bringing about respiration. Inhibition of MSMEG&#95;5125 expression led to the induction of MSMEG&#95;0089 and 2296 , two hallmark genes of the MSMEG&#95;2295 regulon. This result suggests that when MSMEG&#95;5125 becomes limiting, a feedback-loop derepresses the MSMEG&#95;2295 regulon genes, including its own. Interestingly, menaquinone functioned as an inducer of MSMEG&#95;5125 , indicating that it is likely to mediate the feedback mechanism. This result also strengthens our hypothesis that the functions of menaquinone and MSMEG&#95;5125 are interrelated. Menaquinone also induced the MSMEG&#95;2295-controlled operon MSMEG&#95;2295-2294 ( dinB2 ) not induced following the inactivation of MSMEG&#95;5125 . Therefore, the activation mechanism of MSMEG&#95;2295-regulated genes may not be the same for all, although derepression is likely to be a common feature. In vitro , menaquinone abolished MSMEG&#95;2295's DNA binding activity by interacting with it, confirming its role as an inducer. Therefore, a menaquinone-MSMEG&#95;5125-regulated gene expression circuit controls Msm respiration and possibly oxidative stress-induced DNA damage repair.

Background: Vitamin K is a term that comprises a family of structurally related quinones, phylloquinone (PK) and the menaquinones (MKn), that share a common naphthoquinone ring but vary in sidechain length (n) and saturation. Dietary PK is a biosynthetic precursor to tissue menaquinone-4 (MK4), but little is known about the absorption and metabolism of dietary MKn., Objective: To characterize the absorption and metabolism of dietary MKn relative to PK., Methods: In the 4-week diet study, 10-week-old male and female C57BL/6 mice were pair-fed a vitamin K deficient diet (control) or a diet supplemented with 5.0 μmol/kg total PK, MK4, and/or MK9 (separately and in combination). In the 1-week stable isotope study, 12-week-old mice were pair-fed diets containing 2.2 μmol/kg PK (unlabeled control), 2H7PK, 13C11MK4, 2H7MK7, or 2H7MK9. Vitamin K tissue content was quantified by HPLC and/or LC-MS, and concentrations were compared by sex and diet group using 2-factor ANOVA., Results: Regardless of the form(s) of vitamin K provided in the diet, tissue MK4 concentrations did not differ across equimolar supplemented groups in the kidney, adipose, reproductive organ, bone, or pancreas in either males or females in the diet study (all P values > 0.05). Isotopic labeling confirmed the naphthoquinone ring of MK4 in tissues originated from the administered dietary PK or MKn. Despite equimolar supplementation, accumulation of the administered dietary form differed across diet groups in small intestinal segments (all P values < 0.002) and the liver (P < 0.001). Female mice had greater total vitamin K than males in every tissue examined (P < 0.05)., Conclusions: Dietary PK, MK4, MK7, and MK9 all served as precursors to tissue MK4 in mice. This study expands our understanding of vitamin K metabolism and supports a common conversion mechanism of all dietary vitamin K forms to MK4. Further investigation of the metabolism and physiological roles of MK4 that may be independent of classical vitamin K function is warranted., (© The Author(s) 2021. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Background: The study and application of microbial consortia are topics of interest in the fields of metabolic engineering and synthetic biology. In this study, we report the design and optimisation of Elizabethkingia meningoseptica and Escherichia coli co-culture, which bypass certain limitations found during the molecular modification of E. meningoseptica, such as resistance to many antibiotics and fewer available molecular tools., Results: The octaprenyl pyrophosphate synthase from E. meningoseptica sp. F2 (EmOPPS) was expressed, purified, and identified in the present study. Then, owing to the low vitamin K2 production by E. coli or E. meningoseptica sp. F2 monoculture, we introduced the E. meningoseptica and E. coli co-culture strategy to improve vitamin K2 biosynthesis. We achieved production titres of 32 mg/L by introducing vitamin K2 synthesis-related genes from E. meningoseptica sp. F2 into E. coli, which were approximately three-fold more than the titre achieved with E. meningoseptica sp. F2 monoculture. This study establishes a foundation for further engineering of MK-n (n = 4, 5, 6, 7, 8) in a co-cultivation system of E. meningoseptica and E. coli. Finally, we analysed the surface morphology, esterase activity, and membrane permeability of these microbial consortia using scanning electron microscopy, confocal laser scanning microscopy, and flow cytometry, respectively. The results showed that the co-cultured bacteria were closely linked and that lipase activity and membrane permeability improved, which may be conducive to the exchange of substances between bacteria., Conclusions: Our results demonstrated that co-culture engineering can be a useful method in the broad field of metabolic engineering of strains with restricted molecular modifications., (© 2022. The Author(s).)

Corynebacterium glutamicum is a preferentially aerobic gram-positive bacterium belonging to the phylum Actinobacteria, which also includes the pathogen Mycobacterium tuberculosis. In these bacteria, respiratory complexes III and IV form a CIII 2 CIV 2 supercomplex that catalyzes oxidation of menaquinol and reduction of dioxygen to water. We isolated the C. glutamicum supercomplex and used cryo-EM to determine its structure at 2.9 Å resolution. The structure shows a central CIII 2 dimer flanked by a CIV on two sides. A menaquinone is bound in each of the Q N and Q P sites in each CIII and an additional menaquinone is positioned ∼14 Å from heme b L . A di-heme cyt. cc subunit electronically connects each CIII with an adjacent CIV, with the Rieske iron-sulfur protein positioned with the iron near heme b L . Multiple subunits interact to form a convoluted sub-structure at the cytoplasmic side of the supercomplex, which defines a path for proton transfer into CIV., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy., (Copyright © 2021 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Vitamin K (VK) plays many important functions in the body. The most important of them include the contribution in calcium homeostasis and anticoagulation. Vascular calcification (VC) is one of the most important mechanisms of renal pathology. The most potent inhibitor of this process-matrix Gla protein (MGP) is VK-dependent. Chronic kidney disease (CKD) patients, both non-dialysed and hemodialysed, often have VK deficiency. Elevated uncarboxylated matrix Gla protein (ucMGP) levels indirectly reflected VK deficiency and are associated with a higher risk of cardiovascular events in these patients. It has been suggested that VK intake may reduce the VC and related cardiovascular risk. Vitamin K intake has been suggested to reduce VC and the associated cardiovascular risk. The role and possibility of VK supplementation as well as the impact of anticoagulation therapy on VK deficiency in CKD patients is discussed.

Calcium is involved in bone metabolism, regulation of nerve signaling, and release of neurotransmitters. Phosphorus is a structural component of ATP, participates in metabolic energy regulation, and ensures stability to biological membranes and cells. Vitamin D and vitamin K are important for intestinal absorption and renal excretion of calcium and phosphorus. Vitamin D plays a regulatory role in bone formation, carbohydrate metabolism, immune responses, and cardiovascular regulation. Research has linked vitamin D deficiency to the development of diabetes mellitus, hypertension, cancer, and osteoporosis. Vitamin K has been associated with a reduced risk of osteoporosis, cancer, and cardiovascular diseases (due to improved vascular elasticity). This review highlights the importance of vitamins D and K in the metabolism of calcium and phosphorus and explores various molecular mechanisms that help maintain the system's mineral homeostasis. Moreover, the paper reviews the enzyme nattokinase's role in thrombotic prevention due to its fibrinolytic activity.

The effect of dietary vitamin K 3 (VK3) on ruminant animals is not fully investigated. The aim of this study was to examine the effects of dietary VK3 on lactation performance, rumen characteristics, and VK1 and menaquinone (MK, or VK2) dynamics in the rumen, plasma, and milk of dairy cows. Eight Holstein dairy cows in late lactation periods were used in two crossover trials including a control (nontreatment) and a 50 or 200 mg/day (d) VK3 supplementation group. After 14 days, plasma, ruminal fluid, and milk were sampled and their VK1 and MKs contents were measured using fluorescence-high-performance liquid chromatography (HPLC). Milk production was unchanged after feeding 50 mg/day VK3 but marginally decreased after feeding 200 mg/day VK3. The molar ratio of propionate in ruminal fluid was significantly increased on feeding 200 mg/day VK3. Additionally, MK-4 concentrations significantly increased in both plasma and milk after VK3 feeding (50 and 200 mg/day). In ruminal fluid, MK-4 concentrations increased after 200 mg/day VK3 feeding. These results suggest that VK3 may be a good source of MK-4, the biologically active form of VK, in Holstein dairy cows during their late lactation periods. This study provides a basis for understanding the physiological role of VK in dairy cows., (© 2022 Japanese Society of Animal Science.)

To date, there is no functional characterization of EmGGPPS (from Elizabethkingia meningoseptica sp.F2) as enzymes catalyzing GGPP. In this research, maltose-binding protein (MBP), disulfide bond A (DbsA), disulfide bond C (DbsC), and two other small protein tags, GB1 (Protein G B1 domain) and ZZ (Protein A IgG ZZ repeat domain), were used as fusion partners to construct an EmGGPPS fusion expression system. The results indicated that the expression of MBP-EmGGPPS was higher than that of the other four fusion proteins in E. coli BL21 (DE3). Additionally, using EmGGPPS as a catalyst for the production of GGPP was verified using a color complementation assay in Escherichia coli. In parallel with it, the enzyme activity experiment in vitro showed that the EmGGPPS protein could produce GGPP, GPP and FPP. Finally, we successfully demonstrated MK-4 production in engineered E. coli by overexpression of EmGGPPS., (Copyright © 2021. Published by Elsevier Inc.)

The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)