Diversity of potential cellulolytic organisms and cellulases

Abstract

Cellulose, hemicelluloses, pectin and lignin are major constituents of plant cell walls; chitin primarily constitutes fungal cell walls and the exoskeletons of arthropods. These are the most abundant polysaccharides on Earth; hence, enzymes involved in their degradation are vital to the global carbon and nitrogen cycles. In addition to their ecological importance, cellulose- and chitin-degrading enzymes are key to a variety of industrial, medical and agricultural processes. CHAPTER 1 provides a detailed review of the chemistry and biochemistry of cellulose and chitin, the mechanisms of their degradation as well as the enzymes involved and their biotechnological significance. CHAPTER 2 presents a comprehensive genomic analysis of the taxonomic distribution of potential cellulolytic, xylanolytic, pectinolytic, ligninolytic and chitinolytic enzyme families. 50,258 protein sequences drawn from the CAZy database were found unequally distributed across diverse lineages of bacteria, fungi, plants, animals, protists, archaea and viruses. 85% were from four major bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes) and two major fungal phyla (Ascomycota, Basidiomycota), skewed partly by sequencing efforts towards these groups. The genomic capability to decompose complex polymers was shown to be far from universal and more likely associated with the various physiological roles of the enzymes. Moreover, many taxa, some previously reported as plant polysaccharide degraders, could be inferred as useful decomposers. Notably, soil actinomycetes present great potential for biotechnology. In particular, several less well-known species have not yet been tested for cellulase activity and may provide candidates for future microbiological and biochemical studies. CHAPTER 3 presents an evolutionary study of non-homologous cellulases from glycoside hydrolase (GH) families. A comparison of cellulase 3D structures reveals three distinct clusters displaying (β/α)8-barrel, β-jelly roll, and (α/α)6-barrel fold, consistent with convergent evolution. 6,423 sequences from the largest family, GH5, displayed tremendous sequence divergence, partly due to the diverse multi-modular architectures in 28% of these (mostly bacterial) proteins. Phylogenetic analysis of GH5 catalytic domains showed that most clusters were polyspecific in their substrate specificity and that structural diversity in the loop regions of these enzymes produces varied topologies of the substrate-binding cavity. Accurate prediction of enzyme specificity of GH5 family remains a challenge due to the abundance of promiscuous enzymes and inadequate biochemical characterization data. Nonetheless, this study has important implications for understanding the evolution of the GH5 family and serves as a guide for choosing targets for future biochemical, structural and protein engineering studies. Driven by biotechnological applications, much effort has been dedicated to discovering novel cellulases and chitinases. However, not many bacterial decomposers have genome sequences available. CHAPTER 4 addresses this limitation by providing a whole genome sequencing study of 43 bacterial strains isolated from diverse environments. Whole genome-based identification revealed that these isolates belong to two phyla, Firmicutes (Bacillus, Paenibacillus) and Proteobacteria (Klebsiella, Stenotrophomonas, Aeromonas, Plesiomonas). Of note, three isolates represent previously unreported species of the genera Stenotrophomonas, Aeromonas and Paenibacillus. This study demonstrates not only the molecular basis for cellulose/chitin degradation by these isolates but has also elucidated their complete genomes as critical references for future microbiological and taxonomical studies.