Identification of the Required and Sufficient Carbohydrate-Active Enzymes (CAZymes) for Cellulose Deconstruction in Cellvibrio japonicus

Abstract

Cellvibrio japonicus is a Gram-negative saprophytic bacterium proficient at utilizing a broad range of plant and animal polysaccharides. Previous work established that C. japonicus uses a suite of Carbohydrate-Active enZymes (CAZymes) for the efficient deconstruction of recalcitrant substrates, such as cellulose. Current genome annotation suggests that over 20 genes are predicted to encode CAZymes for cellulose degradation, with previous work suggesting that only a subset of these genes are essential. Our current work combined transcriptomic analysis, mutant strain generation coupled with growth phenotyping, and heterologous expression studies to identify the genes that encode the essential cellulose-specific CAZymes for growth using cellulose. Specifically, we found six CAZyme-encoding required for cellulose deconstruction in C. japonicus. These six gens are cel3B, cel5B, cel6A, lpmo10B, cbp2D, and cbp2E, which encode a βglucosidase, an endoglucanase, an exoglucanase, a lytic polysaccharide monooxygenase, and two carbohydrate-binding proteins, respectively. Interestingly, we found that while these CAZyme-encoding genes are necessary for growth using cellulose by C. japonicus, they are not sufficient when heterologously expressed in Escherichia coli. When expressing these genes in E. coli, we observed that E. coli was only able to utilize cellooligosaccharides and soluble forms of cellulose, but not insoluble cellulose. Additionally, we obtained evidence that suggests C. japonicus rapidly uptakes cello-oligosaccharides generated during the deconstruction of cellulose. Using a combination of thin-layer chromatography and linked enzyme assays, we observed that deconstruction of cellulose and uptake of derivative cellodextrins occurs minimally at similar rates. Our revised model of cellulose utilization by C. japonicus can be applied more broadly to ecological studies probing bacterial cellulose degradation in the context of global carbon cycling, as well as provide insights to improve industrial enzymatic conversion of lignocellulosic biomass for renewable fuels and chemicals.