Advancing VB₁₂ Production: Insights into Enhancing VB₁₂ Titer in <i>Ensifer adhaerens</i> Casida A through ARTP Mutagenesis and Multiomics Analysis

<i>Ensifer adhaerens</i>, 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 <i>Ensifer adhaerens</i>, 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.