[Enzyme metabolism and functions in vitamin biosynthesis pathways].

Vitamins, as indispensable organic compounds in life activities, demonstrate a complex and refined metabolic network in organisms. This network involves the coordination of multiple enzymes and the integration of various metabolic pathways. Despite the achievements in metabolic engineering and catalytic mechanism research, the lack of studies regarding detailed enzymatic properties for a large number of key enzymes limits the enhancement of vitamin production efficiency and hinders the in-depth understanding and optimization of vitamin synthesis mechanisms. Such limitations not only restrict the industrial application of vitamins but also impede the development of related bio-technologies. This study comprehensively reviews the research progress in the enzymes involved in vitamin biosynthesis and details the current status of research on the enzymes of 13 vitamin synthesis pathways, including their catalytic mechanisms, kinetic properties, and applications in biology. In addition, this study compares the properties of enzymes involved in vitamin metabolic pathways and the glycolysis pathway, and reveals the characteristics of catalytic efficiency and substrate affinity of enzymes in vitamin synthesis pathways. Furthermore, this study discusses the potential and prospects of applying deep learning methods to the research on properties of enzymes associated with vitamin biosynthesis, giving new insights into the production and optimization of vitamins.

A programmable CRISPR/dCas9-based epigenetic editing system enabling loci-targeted histone citrullination and precise transcription regulation.

Histone citrullination, an important post-translational modification mediated by peptidyl arginine deiminases, is essential for many physiological processes and epigenetic regulation. However, the causal relationship between histone citrullination and specific gene regulation remains unresolved. In this study, we develop a programmable epigenetic editor by fusing the peptidyl arginine deiminase PPAD from Porphyromonas gingivalis with dCas9. With the assistance of gRNA, PPAD-dCas9 can recruit peptidyl arginine deiminases to specific genomic loci, enabling direct manipulation of the epigenetic landscape and regulation of gene expression. Our citrullination editor allows for site-specific manipulation of histone H3R2,8,17 and 26 at target human gene loci, resulting in the activation or suppression of different genes in a locus-specific manner. Moreover, the epigenetic effects of the citrullination editor are specific and sustained. This epigenetic editor offers an accurate and efficient tool for exploring gene regulation of histone citrullination.

Increasing Vitamin K2 Synthesis in Bacillus subtilis by Controlling the Expression of MenD and Stabilizing MenA.

As an indispensable member of the family of lipid vitamins, vitamin K2 (MK-7) plays an important role in blood coagulation, cardiovascular health, and kidney health. Microbial fermentation is favored due to its high utilization rate of raw materials, simple operation, and moderate conditions. However, the biosynthesis pathway of vitamin K2 in microorganisms is highly complex, which hinders its industrial production in microbial cell factories. One of the major challenges is the stable expression and deregulation of key enzymes in the vitamin K2 biosynthesis pathway, which remains unclear and has undergone little investigation. In this study, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid synthase (MenD) and 1,4-dihydroxy-2-naphthoate polyprenyltransferase (MenA) were identified as pivotal enzymes in the biosynthesis of vitamin K2. To investigate the catalytic efficiency of MenD in the biosynthesis pathway of vitamin K2, structure-based mutation design and site-directed mutagenesis were performed. Three mutation sites were identified in MenD: A115Y, R96 M, and R323M, which improve the expression level and protein stability. Meanwhile, the MenA mutant T290M, which exhibits improved protein stability, was obtained by modifying its hydrophobic stacking structure. Finally, an engineered strain noted ZQ13 that combinatorially overexpressed MenD (A115Y) and MenA (T290M) mutants was constructed and achieved 338.37 mg/L vitamin K2 production in a 3-L fermenter.