(576d) Comparative Engineering of Escherichia coli for Cellobiose Utilization: Hydrolysis Versus Phosphorolysis

Microbial biocatalysts capable of cellobiose assimilation are of interest in bioconversion of cellulosic materials as they could be directly used without the need for beta-glucosidase.  When these microbes are used with cellulase enzymes, removal of cellobiose accelerates cellulolytic activities since cellobiose is a potent inhibitor for cellulases. Previously, we reported a successful metabolic engineering strategy to establish a novel cellulose assimilation pathway in E. coli by utilizing the native LacY for cellobiose uptake and a Sacchrophagus cellobiose phosphorylase for intracellular phosphorolysis. This presentation provides a careful comparison in the two mechanisms of cellobiose assimilation, hydrolysis vs. phosphorolysis, between two otherwise isogenic E. coli strains. Interestingly, better tolerance to inhibitors, acetate and butanol, by cells assimilating cellobiose phosphorolytically was observed in both anaerobic and aerobic conditions, suggesting that benefits of the more energetically favorable phosphorolysis could be extended to conditions not typically considered to be energy-limited. We also investigated the possibility to direct the favorable energy metabolism to recombinant protein production. We show here that cells undergoing cellobiose phosphorolysis produced three and five fold more recombinant green fluorescent protein and a beta-xylosidase, respectively,  than cells assimilating cellobiose hydrolytically.  Like hydrolysis cells, cells undergoing cellobiose phosphorolysis initially consumed xylose in simultaneous with cellobiose in a mixed sugar fermentation (5% cellobiose +5% xylose).  However, xylose utilization came to a complete halt after only about 60% consumption whereas the hydrolysis cells were able to ferment both sugars to near completion.  These results provide new insights into the new metabolic engineering strategy of cellobiose assimilation. To our best knowledge, this is the first comparison study in E. coli.