The biological effects of static magnetic fields (SMF) have long been a research hotspot in academia. While the impact of magnetic fields on microbial morphogenesis is closely linked to microbial fermentation efficiency, the specific mechanism remains incompletely elucidated. In this study, the vitamin K2-producing strain Flavobacterium sp. m1-14 was exposed to a static magnetic field of up to 9 Tesla (T) for 24 h. It was observed that the bacterial cells shrank, showing an overall decreasing trend in size. The length, width, and aspect ratio decreased by approximately 25.27 %, 14.28 %, and 17.95 %, respectively. Furthermore, the physiological and biochemical properties of the bacteria underwent significant changes. Specifically, the cell membrane permeability increased by approximately 6.2 %; the activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase decreased by about 57.5 % and 34.7 %, respectively; and the membrane potential decreased significantly. In addition, intracellular ATP levels decreased by approximately 12 %, a change directly attributed to impaired ATP metabolism. Investigations into the key morphological regulatory genes mreB and ftsZ revealed that their transcription levels were unregulated by 190 % and 38 %, respectively-likely a stress response induced by cellular energy deficiency. Under conditions of high mreB and ftsZ expression, cells reduce their size to minimize metabolic loss, thereby adapting to extreme environments.

Bacillus coagulans has attracted considerable attention because of its notable probiotic properties and high stress tolerance; however, the mechanisms underlying its acid tolerance remain poorly understood. This study employed adaptive laboratory evolution to enhance the acid tolerance of B. coagulans, enabling it to grow stably at pH 3.5. The evolved strains exhibited significant alterations in their metabolic profiles and transcriptional regulation. Among the 279 differentially expressed metabolites, 171 were upregulated and 108 downregulated, including specific amino acid and antioxidant metabolites. Using RNA-seq, 1476 differentially expressed genes (DEGs) were identified in the evolved strains, including 788 significantly upregulated and 688 downregulated genes. Further KEGG enrichment analysis of 1476 DEGs, in terms of energy metabolism, the upregulation of key glycolytic and TCA cycle genes (including pgi, gap, pyk, cs, pdhA, pdhB, pdhC, and mdh) enhanced central carbon metabolic flux, thereby facilitating rapid ATP production to meet the immediate energy demands of cells under acidic conditions. At the nitrogen transport and metabolism level, the downregulation of pyrimidine pathway genes (carA, carB, pyrB, pyrC, pyrD, pyrE, pyrF) and purine pathway genes (purH, purN, purM, purF, purL, purQ, purS, and purC) reduced the production of toxic derivatives that would otherwise exacerbate bacterial vulnerability under acid stress. Additionally, molecular chaperones, DNA damage repair systems, and ribosomal subunits enhanced genome stability. This study presents new insights into the molecular mechanisms underlying the resistance of B. coagulans to acidic environments, which provide a theoretical foundation for its application in probiotics and industrial processes.

To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.

The development of effective and safe insulin analogs remains pivotal in advancing diabetes management. This study addresses the limitations of existing insulin therapies by introducing insulin lisargine, a novel long-acting insulin analog that resolves impurity formation associated with trypsin cleavage in glargine insulin. Insulin lisargine is characterized by glycine substitution at A21 and the addition of lysine and arginine at B31 and B32, respectively. High-performance liquid chromatography (HPLC) and mass spectrometry confirmed its high purity and precise molecular weight. X-ray crystallography at 2.0 Å resolution revealed structural features closely resembling human insulin, crucial for optimizing drug formulations and understanding receptor interactions.In vivo experiments demonstrated that insulin lisargine exhibits superior glucose-lowering effects compared to glargine insulin (Lantus). At a dosage of 1.5 IU/kg, lisargine achieved glucose-lowering effects equivalent to glargine in normal rats. However, at 5 IU/kg, it significantly outperformed glargine in type 1 diabetic rats. Long-term safety assessments revealed a comparable safety profile between lisargine and glargine, with no significant toxicity observed. These findings position insulin lisargine as a promising candidate for diabetes management, offering enhanced blood glucose control, improved production efficiency, and reliable safety. The study’s findings provide a foundation for the development of more effective insulin analogs, addressing critical needs in diabetes therapy.

Menaquinone-7, a form of vitamin K2, plays a critical role in the treatment of hemorrhagic diseases caused by vitamin K deficiency and in the prevention of bone fractures. Microbial fermentation has emerged as a promising method for MK-7 production due to its high product optical purity and significant physiological activity. However, the current production efficiency of MK-7 remains insufficient to meet industrial demands. In this study, we employed a combination of single-factor experiments, Plackett-Burman design, steepest ascent experiments, and Box-Behnken design to optimize the fermentation medium for MK-7 production by Bacillus subtilis BS-ΔackA. Through regression analysis and consideration of practical production constraints, the optimal fermentation medium parameters were determined as follows: 20 g/L sucrose, 20.7 g/L glycerol, 47.3 g/L soy peptone, 4 g/L yeast extract, and 1.9 g/L KH2PO4, 0.1 g/L MgSO4·7 H2O. Under these optimized conditions, the MK-7 yield reached 154.6 ± 1.32 mg/L. The experimental results demonstrated excellent stability and reliability, aligning closely with the model predictions. This study significantly enhances MK-7 production at the shake-flask fermentation level, providing valuable insights for large-scale industrial applications.

Menaquinone-7 (MK-7), a biologically active form of vitamin K2,plays a critical role in preventing osteoporosis and cardiovascular calcification, making it a valuable component in dietary supplements. This study investigated the fermentation process of Bacillus subtilis BS018, which produces MK-7, by integrating fermentation kinetics. Based on this, a feeding strategy was developed and validated at the 5 L bioreactor, and fermentation experiments were conducted in a 1 T bioreactor. The selected fermentation kinetic model effectively described the fermentation process of BS018, with kinetic parameter analysis indicating that MK-7 is a non-growth-associated product. In the 5 L bioreactor, the MK-7 yield of the batch supplemented with 10g/L (grams of added glycerol per volume of fermentation broth) glycerol at 24 hours of fermentation was 1.72 times that of the non-fed batch. At the 1-ton bioreactor, the MK-7 yield reached 72.65 mg/L

The filamentous fungus Rhizopus oryzae is one of the main industrial strains for the production of a series of important chemicals such as ethanol, lactic acid, and fumaric acid. However, the lack of efficient gene editing tools suitable for R. oryzae makes it difficult to apply technical methods such as metabolic engineering regulation and synthetic biology modification. A CRISPR-Cas9 system suitable for efficient genome editing in R. oryzae was developed. Firstly, four endogenous U6 promoters of R. oryzae were identified and screened with the highest transcriptional activity for application to sgRNA transcription. It was then determined that the U6 promoter mediated CRISPR/Cas9 system has the ability to efficiently edit the genome of R. oryzae through NHEJ and HDR-mediated events. Furthermore, the newly constructed CRISPR-Cas9 dual sgRNAs system can simultaneously disrupt or insert different fragments of the R. oryzae genome. Finally, this CRISPR-Cas9 system was applied to the genome editing of R. oryzae by knocking out pyruvate carboxylase gene (PYC) and pyruvate decarboxylase gene (pdcA) and knocking in phosphofructokinase (pfkB) from Escherichia coli and L-lactate dehydrogenase (L-LDH) from Heyndrickxia coagulans, which resulted in a substantial increase in L-LA production. In summary, this study showed that the CRISPR/Cas9-based genome editing tool is efficient for manipulating genes in R. oryzae.

Numerous natural compounds are recognized for their anti-inflammatory properties attributed to antioxidant effects and the modulation of key inflammatory factors. Among them, astaxanthin (AST), a potent carotenoid antioxidant, remains relatively underexplored regarding its anti-inflammatory mechanisms and specific molecular targets. In this study, human monocytic leukemia cell-derived macrophages (THP-1) were selected as experimental cells, and lipopolysaccharides (LPS) served as inflammatory stimuli. Upon LPS treatment, the oxidative stress was significantly increased, accompanied by remarkable cellular damage. Moreover, LPSs escalated the expression of inflammation-related molecules. Our results demonstrate that AST intervention could effectively alleviate LPS-induced oxidative stress, facilitate cellular repair, and significantly attenuate inflammation. Further exploration of the anti-inflammatory mechanism revealed AST could substantially inhibit NF-κB translocation and activation, and mitigate inflammatory factor production by hindering NF-κB through the antioxidant mechanism. We further confirmed that AST exhibited protective effects against cell damage and reduced the injury from inflammatory cytokines by activating p53 and inhibiting STAT3. In addition, utilizing network pharmacology and in silico calculations based on molecular docking, molecular dynamics simulation, we identified interleukin-6 (IL-6) as a prominent core target of AST anti-inflammation, which was further validated by the RNA interference experiment. This IL-6 binding capacity actually enabled AST to curb the positive feedback loop of inflammatory factors, averting the onset of possible inflammatory storms. Therefore, this study offers a new possibility for the application and development of astaxanthin as a popular dietary supplement of anti-inflammatory or immunomodulatory function.

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.

Menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K3 (VK3) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK3 concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76

Protein-polysaccharide systems show promise for probiotic encapsulation and delivery in food applications. This study investigated the formation of nanoparticles from zein and soluble soybean polysaccharide (SSPS) through antisolvent precipitation and their efficacy in encapsulating Bacillus subtilis. At pH 6.0, zein and SSPS formed composite aggregates (ZPS) that effectively encapsulated B. subtilis, as confirmed by scanning electron microscopy (SEM). Fourier-transform infrared spectroscopy (FTIR) analysis revealed the presence of hydrogen bonding and electrostatic interactions within the ZPS. The encapsulated probiotics exhibited significantly enhanced viability compared to free probiotics: 3.13-fold in simulated gastrointestinal digestion, 3.20-fold during pasteurization, and 1.50-fold in storage conditions. In vivo experiments in rats showed that oral administration of encapsulated probiotics increased the abundance of beneficial gut bacteria. These findings underscore the potential of zein-SSPS nanoparticles for probiotic protection and delivery, presenting a promising strategy for enhancing probiotic efficacy in food and nutraceutical applications.

Abstract The structural characterization of insulin analogs is crucial for their clinical applications. In previous study, we developed a novel long-acting insulin analog called insulin lisargine, which exhibits superior sustained-release properties. However, its structure remains unclear and requires thorough investigation. We determined the amino acid sequence of insulin lisargine using mass spectrometry and analyzed its secondary structure using circular dichroism spectroscopy. Furthermore, we employed X-ray crystallography experiments at the Shanghai Synchrotron Radiation Facility to elucidate the crystal structure of the insulin lisargine analog, achieving a resolution of approximately 2.0Å. Comparison with the human insulin structure in the Protein Data Bank revealed structural similarities between the two. These findings suggest promising prospects for the insulin lisargine analog and provide essential foundational information for subsequent drug design and optimization. In summary, we conducted a comprehensive structural study on the insulin lisargine analog, shedding light on its potential applications in the field. The obtained insights pave the way for future drug development and optimization efforts.

In the original publication [...].

Owing to the recognized therapeutic characteristics of G. lucidum, it is one of the most extensively researched mushrooms as a chemopreventive agent and as a functional food. It is a known wood-degrading basidiomycete possessing numerous pharmacological functions and is termed a natural pharmacy store due to its rich number of active compounds which have proved to portray numerous therapeutic properties. This current review highlights studies on the potentialities of G. lucidum extracts as functional ingredients on organoleptic and nutritional properties of food products (e.g., dairy, wine, beverage, bakery, meat, and other products). In addition, the study delved into various aspects of encapsulated G. lucidum extracts, their morphological and rheological characteristics, prebiotic and immunomodulatory importance, the effects on apoptosis, autophagy, cancer therapy, inflammatory responses, oxidative stress, antioxidant activities, and safety concerns. These findings have significant implications for the development of new products in the food and pharmaceutical industries. On the other hand, the various active compounds extracted from G. lucidum exhibited no toxic or adverse effects, and the appeal for it as a dietary food, natural remedy, and health-fortifying food is drastically increasing as well as attracting the interest of both the industrial and scientific communities. Furthermore, the formation of functional foods based on G. lucidum appears to have actual promise and exciting prospects in nutrition, food, and pharmaceutical sciences.

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.