Advancing VB₁₂ Production: Insights intoEnhancing VB₁₂ Titer in Ensifer adhaerens Casida A through ARTP Mutagenesis and Multiomics Analysis

Ensifer adhaerens, a microorganism recognized for its capacity to synthesize vitamin B₁₂ (VB₁₂), has garnered significant attention in recent years. Nonetheless, its practical application has been limited by low production yields. Atmospheric and room-temperature plasma (ARTP) mutagenesis was utilized to improve VB₁₂ production and examine the associated mechanisms. Three high-yielding mutant strainsBCA-24, BCB-14, and BCC-27were isolated through multiple rounds of mutagenesis. The VB₁₂ titer of the highly productive mutant strain, BCA-24, rose significantly from 65.64 mg/L to 104.54 mg/L. Genome resequencing identified 14 mutated genes, of which seven (<i>atpA</i>, <i>gntR</i>, <i>fusA</i>, <i>cobQ</i>, <i>ribD</i>, <i>cirA</i>, and <i>UP</i>) were functionally validated through overexpression in wild-type strains and found to positively influence VB₁₂ biosynthesis. Notably, coexpression of the <i>cobQ</i> and <i>UP</i> mutant genes in strain BCA-24 resulted in a VB₁₂ titer of 163.68 mg/L. Transcriptomic analysis indicated that critical pathways related to energy metabolism, S-adenosylmethionine (SAM), 5-aminolevulinic acid (5-ALA), and riboflavin synthesis were significantly upregulated in BCA-24 relative to the wild type. A multiomics approach clarified the mechanisms through which these mutations increase VB₁₂ production, including enhanced transcription and translation, optimized energy supply, and improved product efflux. The identification of novel candidate genes in Ensifer adhaerens, which have not been previously studied, provides valuable resources for future genetic engineering aimed at enhancing VB₁₂ production efficiency. This study offers practical improvements in microbial VB₁₂ production while also delivering essential insights into the genetic and metabolic regulation of this important biosynthetic pathway.