Linking metagenomic insight to cultivable microbes: isolation of a vitamin B12-producing Sphingomonad from Indonesian tempeh

Tempeh, a famous traditional Indonesian fermented soybean product, reportedly contains vitamin B12. Although Enterobacteriaceae have been previously implicated in vitamin B12 production in tempeh, the function of Sphingomonadaceae, which is abundant in some tempeh samples, remains unknown. This study aimed to identify and characterize vitamin B12-producing bacteria from Empang (EMP) tempeh, with a focus on the understudied Sphingomonad. Metagenomic analysis focusing on vitamin B12 biosynthesis genes revealed that Sphingomonad genes had a complete set gene that required for producing the vitamin. A total of 44 yellow-pigmented isolates, characteristic of Sphingomonas, were screened via a vitamin B12 assay, three of which demonstrated potential for de novo biosynthesis. On the basis of 16S rRNA gene analysis, all three isolates were identified as Sphingomonas paucimobilis. Whole-genome sequencing and annotation of the EMP5-4 isolate revealed a complete gene set for the aerobic vitamin B12 biosynthesis pathway, including hem genes, cob genes and cobalamin riboswitches. Vitamin B12 production was confirmed through fermentation in a TSB medium with cobalt and DMBI supplementation and quantified at 0.949 µg/mL via HPLC. A genome-based safety assessment identified only low confidence antibiotic resistance and virulence genes, and a hemolysis assay revealed no red blood cell lysis, suggesting minimal pathogenicity. These findings demonstrate that S. paucimobilis from tempeh may serve as a novel microbial source of vitamin B12, supporting its potential application in functional foods for individuals who avoid animal-derived products.