Late Stage Mannan Metabolism in Cellvibrio japonicus Requires the Combined Action of a Mannosyl-Glucose Phosphorylase and a Mannobiose Epimerase

Abstract

Manno-oligosaccharides and their metabolism play important roles in gut health, pharmaceutical development, and renewable chemical production. While the degradation of manno-oligosaccharides has been previously studied, interest in bacterial mannobiose epimerases and mannoside phosphorylases is increasing because these enzymes provide a replacement for bacterial phosphotransferase systems as part of synthetic biology applications. In this report, we have physiologically and biochemically characterized the mannobiose epimerase and mannoside phosphorylase from the Gram-negative saprophyte Cellvibrio japonicus, which is a bacterium that does not possess a phosphotransferase system for sugar import. While the initial stages of mannan degradation by C. japonicus have been studied, the physiological importance and biochemical activities of the mannosyl-glucose phosphorylase (Mgp130A) and mannobiose epimerase (EpiA) predicted for latter stages of mannan metabolism were uncharacterized. After functional mutational analysis and biochemical assays of these two enzymes, we observed that both were essential for utilization of linear mannan, mannobiose, and mannotriose; however, only Mgp130A was critical for mannosyl-glucose cleavage. A new plasmid (pJKN5) created during this study allowed for improved complementation analysis and uncovered a surprising toxic effect of galactose-substituted manno-oligosaccharides in strains lacking the epiA gene. Enzyme assay of Mgp130A revealed an enzyme with a high specific activity compared to other bacterial enzymes. Overall, this study advanced our understanding of C. japonicus mannan metabolism and contributed to the growing characterization of bacterial glycoside phosphorylases and epimerases important for biotechnology.