Correction: Tang et al. Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop. Nutrients 2022, 14, 1504.

In the original publication [...].

Preparation of Cellulose-Based Flocculant and Its Application in the Enrichment of Vitamin K2 in Fermentation Supernatant.

Nutritional food supplements and pharmaceutical products produced with vitamin K2 as raw materials a very promising market in the global scope. The main production method of vitamin K2 is microbial fermentation, but approximately 50% of vitamin K2 synthesized by the main production strain Bacillus subtilis natto exists in extracellular form, which is not easy to separate and extract. In order to solve this problem, in this study, we synthesized a novel cellulose flocculant, MCC-g-LMA, by grafting reaction using microcrystalline cellulose (MCC) and lauryl methacrylate (LMA) as monomers, and ammonium persulfate as an initiator to flocculate VK2 from the fermentation supernatant. The flocculant was characterized by Fourier transform infrared spectroscopy (FTIR), elemental analysis, and scanning electron microscopy (SEM), and the grafting reaction was successful. When the flocculant dosage was 48.0 mg/L and pH was 5.0, the flocculation rate of the MCC-g-LMA on the fermentation supernatant reached 85.3%, and the enrichment rate of VK2 reached 90.0%. Furthermore, we explored the flocculation mechanism of VK2 by the MCC-g-LMA and speculated that the flocculation mechanism mainly included adsorption bridging, hydrophobic association and net trapping and sweep effect. In this study, the extraction method for trace high-value biological products in the fermentation supernatant was improved, which provided a method and theoretical basis for the efficient separation and purification of VK2 and other terpenoids.

Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop.

Vitamin K2, a natural fat-soluble vitamin, is a potent neuroprotective molecule, owing to its antioxidant effect, but its mechanism has not been fully elucidated. Therefore, we stimulated SH-SY5Y cells with 6-hydroxydopamine (6-OHDA) in a proper dose-dependent manner, followed by a treatment of vitamin K2. In the presence of 6-OHDA, cell viability was reduced, the mitochondrial membrane potential was decreased, and the accumulation of reactive oxygen species (ROS) was increased. Moreover, the treatment of 6-OHDA promoted mitochondria-mediated apoptosis and abnormal mitochondrial fission and fusion. However, vitamin K2 significantly suppressed 6-OHDA-induced changes. Vitamin K2 played a significant part in apoptosis by upregulating and downregulating Bcl-2 and Bax protein expressions, respectively, which inhibited mitochondrial depolarization, and ROS accumulation to maintain mitochondrial structure and functional stabilities. Additionally, vitamin K2 significantly inhibited the 6-OHDA-induced downregulation of the MFN1/2 level and upregulation of the DRP1 level, respectively, and this enabled cells to maintain the dynamic balance of mitochondrial fusion and fission. Furthermore, vitamin K2 treatments downregulated the expression level of p62 and upregulated the expression level of LC3A in 6-OHDA-treated cells via the PINK1/Parkin signaling pathway, thereby promoting mitophagy. Moreover, it induced mitochondrial biogenesis in 6-OHDA damaged cells by promoting the expression of PGC-1α, NRF1, and TFAM. These indicated that vitamin K2 can release mitochondrial damage, and that this effect is related to the participation of vitamin K2 in the regulation of the mitochondrial quality-control loop, through the maintenance of the mitochondrial quality-control system, and repair mitochondrial dysfunction, thereby alleviating neuronal cell death mediated by mitochondrial damage.

Cloning and functional characterization of the geranylgeranyl diphosphate synthase(GGPPS)from Elizabethkingia meningoseptica sp.F2.

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.

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.

Correction to: Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

The published online version contains some errors.

Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

Synthesis of the carboxymethyl cellulose magnetic nanoparticles for efficient immobilization of prenyltransferase NovQ.

Prenyltransferase NovQ immobilized carboxymethyl cellulose magnetic nanoparticles (NCMNs) were successfully synthesized via a valuable approach integrated from nanocomposite preparation, and applied for the production of vitamin K2 using menadione hydroquinol and dimethylallyl diphosphate (DMAPP) as substrates. To investigate the interaction between nanoparticles and NovQ, we characterized the nanocomposite, and revealed that carboxymethyl cellulose (CMC) and Fe3O4 formed a core-shell structure to absorb NovQ in the reaction systems, resulting from the high affinity of immobilized materials. Meanwhile, NCMNs with excellent pH and temperature tolerance, enhanced prenylated activity, and improved stability were found. Molecular docking analysis was also conducted to justify the contribution of multiple amino acids and effect of nanoparticles on catalytic properties of NovQ. Taken together, our study introduces a promising strategy to prepare magnetic nanoparticles and improve the performance of catalyst, which aims for opening new orientations for synthesis of magnetic nanoparticles used for prenyltransferase immobilization.

Construction of a novel MK-4 biosynthetic pathway in Pichia pastoris through heterologous expression of HsUBIAD1.

BACKGROUND: With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. RESULTS: Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. CONCLUSIONS: In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.

Effect of static magnetic field on morphology and growth metabolism of Flavobacterium sp. m1-14.

Increasing evidence shows that static magnetic fields (SMFs) can affect microbial growth metabolism, but the specific mechanism is still unclear. In this study, we have investigated the effect of moderate-strength SMFs on growth and vitamin K2 biosynthesis of Flavobacterium sp. m1-14. First, we designed a series of different moderate-strength magnetic field intensities (0, 50, 100, 150, 190 mT) and exposure times (0, 24, 48, 72, 120 h). With the optimization of static magnetic field intensity and exposure time, biomass and vitamin K2 production significantly increased compared to control. The maximum vitamin K2 concentration and biomass were achieved when exposed to 100 mT SMF for 48 h; compared with the control group, they increased by 71.3% and 86.8%, respectively. Interestingly, it was found that both the cell viability and morphology changed significantly after SMF treatment. Second, the adenosine triphosphate (ATP) and glucose-6-phosphate dehydrogenase (G6PDH) metabolism is more vigorous after exposed to 100 mT SMF. This change affects the cell energy metabolism and fermentation behavior, and may partially explain the changes in bacterial biomass and vitamin K2 production. The results show that moderate-strength SMFs may be a promising method to promote bacterial growth and secondary metabolite synthesis.

Improvement of menaquinone-7 production by Bacillus subtilis natto in a novel residue-free medium by increasing the redox potential.

Bacillus subtilis natto is a GRAS bacterium. Nattokinase, with fibrinolytic and antithrombotic activities, is one of the major products of this organism. It is being gradually recognized that B. subtilis natto can also be used as a biosynthetic strain for vitamin K2, which has phenomenal benefits, such as effects in the prevention of cardiovascular diseases and osteoporosis along with antitumor effects. Knocking out of the aprN gene by homologous recombination could improve the redox potential and slightly increase the concentration of MK-7. By detecting the change in redox potential during the growth of B. subtilis natto, a good oxygen supply and state of the cell membrane were found to be beneficial to vitamin K2 synthesis. A two-step RSM was used to optimize the operation parameters and substrate concentration in the new residue-free fermentation culture. The optimal conditions for the residue-free medium and control were determined. The optimum concentrations of soybean flour, corn flour, and peptone were 78.9, 72.4, and 24.8 g/L, respectively. The optimum rotational speed and volume of the culture medium using a shaking flask were 117 rpm and 10%, respectively. The state and composition of the cell membranes were more stable when engineered bacteria were cultured in this residue-free fermentation medium. Finally, the concentration of MK-7 increased by 37% to 18.9 mg/L, and the fermentation time was shortened by 24 h.