Research on the targeted improvement of the yield of a new VB12-producing strain, Ensifer adhaerens S305, based on genome and transcriptome analysis

Abstract

Abstract Background Vitamin B12 (VB12) has wide application and high economic value. In this study, a new strain with high VB12 production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB12 production via fermentation in recent years, the directional modification of the S305 strain towards a strain suitable for the industrial production of VB12 has great potential and commercial value. Results 16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) value analysis showed that the high-yielding VB12 strain belongs to E. adhaerens and that its VB12 synthesis pathway genes are highly similar to related genes of strains of this species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB12 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 VB12 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 VB12 yield of the overexpressed strain was higher than that of the wild strain. Notably, the strains overexpressing the cobA and cobT genes had the most significant increases in VB12 yield, i.e., 31.4% and 24.8%, respectively. The VB12 yield of the S305 strain in shake-flask culture improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the S305 strain. Conclusion Phylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains are quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB12 synthesis pathway can increase the yield of VB12, further improving the VB12 yield of the E. adhaerens S305 strain.