Abstract
BACKGROUND: Vitamin B(12) (VB(12)) has a wide range of applications and high economic value. In this study, a new strain with high VB(12) production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB(12) production via fermentation in recent years, the directional modification of the S305 strain to obtain a strain suitable for the industrial production of VB(12) has great potential and commercial value. RESULTS: 16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) analysis showed that the high-yielding VB(12) strain was a E. adhaerens strain and that its VB(12) synthesis pathway genes were highly similar to related genes of strains of this and other species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB(12) yields of the S305 strain were more than double those of the Casida A strain under different medium components. Multiple genes with significantly upregulated and downregulated transcription were identified by comparing the transcription intensity of different genes through transcriptome sequencing. KEGG enrichment analysis of the porphyrin metabolism pathway identified 9 significantly upregulated and downregulated differentially expressed genes (DEGs) in the VB(12) synthesis pathway, including 7 transcriptionally upregulated genes (cobA, cobT, hemA, cobJ, cobN, cobR, and cobP) that were episomally overexpressed in the Casida A strain. The results showed that the VB(12) yield of the overexpressed strain was higher than that of the wild-type strain. Notably, the strains overexpressing the cobA and cobT genes exhibited the most significant increases in VB(12) yield, i.e., 31.4% and 24.8%, respectively. The VB(12) yield of the S305 strain in shake-flask culture was improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the strain. CONCLUSION: Phylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains were quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB(12) synthesis pathway could increase the yield of VB(12), further improving the VB(12) yield of the E. adhaerens S305 strain.