Lignocellulose degradation at high temperatures: the use of multi-domain, surface layer associated enzymes by the extremely thermophilic genus Caldicellulosiruptor

Caldicellulosiruptor species are extremely thermophilic, anaerobic bacteria isolated from globally distributed terrestrial hot springs. These microorganisms attach to and hydrolyze a wide range of complex polysaccharides found in lignocellulosic biomass. This process is mediated by novel, multi-domain enzymes containing glycoside hydrolase (GH), polysaccharide lyase (PL), and carbohydrate binding module (CBM) domains. Several of these large proteins are associated with the cell envelope via surface layer homology (SLH) domains.  In total, eight homologous groups of catalytic, GH- or PL-containing SLH proteins can be identified in the twelve Caldicellulosiruptor genomes sequenced to date. Full-length (>150 kDa) and truncated recombinant versions of several of these proteins were produced in E. coli and evaluated on a variety of polysaccharide substrates. To understand the contribution of individual domains to the catalytic and binding functions of these multi-domain proteins, truncation mutants containing various sets of domains from the largest Caldicellulosiruptor GH, Calkro_0111 (2435 amino acids) were examined. To analyze these proteins in vivo, a SLH xylanase (Calkro_0402) was knocked into a genetically tractable species (C. bescii). The resulting strain showed improved ability to attach to and degrade xylan substrates when compared to the parent strain. Immunofluorescence imaging with antibodies raised against Calkro_0402 and Calkro_0111 confirm these proteins are associated with the S-layer and show unique localization patterns on the cell surface.   Fundamental understanding of the role that catalytic multi-domain enzymes, particularly those associated with the cell surface, play in plant biomass recruitment and degradation by Caldicellulosiruptor species will enable optimization of engineered strains for biomass processing.