LC-MS/MS spectra and genomic annotations for CN-CbI conversion by CCFM1526

The presence of Ado-Cbl in the fermentation product was confirmed by comparing the retention time and characteristic fragment ions (m/z = 147.09, m/z = 359.10, m/z = 486.25, m/z = 665.30) with those of the Ado-Cbl standard.II. Sequence Alignment of CobA Homologous ProteinsTable 1 presents the amino acid sequence alignment of the CobA homologous protein from CCFM1526 compared to that from Lactobacillus paragasseri JCM 5343.

The table contains two entries, with "Variable Sites" indicating positions of amino acid variation, "Identity (%)" representing the percentage of sequence identity, and "Divergence (%)" indicating the percentage of sequence difference. The alignment results show that the CobA protein sequences from the two strains share 98.9% identity, confirming that CCFM1526 possesses the key enzyme gene required for catalyzing the synthesis of Ado-Cbl.III.

Annotation of Genes Related to CN-Cbl MetabolismTable 2, derived from the whole-genome sequencing and annotation system analysis of CCFM1526, lists genes associated with the uptake and transformation of CN-Cbl. These include multiple genes homologous to btuD, which encode vitamin B₁₂ transport ATP-binding proteins, as well as genes involved in electron transfer, such as those belonging to the flavin reductase family and genes encoding flavodoxin proteins (nfr1, nfr2, and yvqK).