Biochemical characterization of the mannojirimycin biosynthetic enzymes reveals metal ion- and pH-dependent catalysis in Bacillus atrophaeus MBLB1156 isolated from red fermented bean curd (Hong fu ru).

1-Deoxynojirimycin (DNJ) and its C2 epimer, 1-deoxymannojirimycin (DMJ), are pharmaceutically relevant iminosugars. Although the gabT1-yktc1-gutB1 gene cluster has been implicated in the biosynthesis of mannojirimycin (MJ), a common precursor of DNJ and DMJ, the coordinated biochemical properties of the pathway enzymes remain poorly understood. Here, we isolated a DMJ-producing strain, Bacillus atrophaeus MBLB1156, from Hong fu ru and identified a gabT1-yktc1-gutB1 cluster by whole-genome sequencing. Comparative genomic analysis of 99 Bacillus genomes further revealed that the cluster of MBLB1156 belongs to a distinct evolutionary lineage. Biochemical analyses showed that rBatGabT1 preferentially aminated fructose-6-phosphate using L-glutamate, while rBatYktc1 exhibited metal ion- and pH-dependent phosphatase activity. rBatGutB1 was characterized as a Zn2+-binding NAD(P)-dependent dehydrogenase with an alkaline pH optimum. The assembled three-enzyme cascade synthesized MJ, which was subsequently converted to DMJ by chemical reduction, providing biochemical evidence for the proposed biosynthetic pathway. Cell extract assays further showed that DMJ production was favored under neutral to mildly alkaline conditions despite the more alkaline pH optima of rBatGabT1 and rBatGutB1, indicating that the distinct biochemical properties of the three enzymes collectively influence pathway activity. These findings advance our biochemical understanding of MJ biosynthesis of B. atrophaeus and provide a foundation for metabolic engineering and biocatalytic production of iminosugars.