Parsing in vivo and in vitro contributions to microcrystalline cellulose hydrolysis by multidomain glycoside hydrolases in the Caldicellulosiruptor bescii secretome

Abstract

Six multidomain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation.

Genomic and physiological analyses reveal that extremely thermophilicCaldicellulosiruptor changbaiensisdeploys unique cellulose attachment mechanisms

“…3). Prior in vitro biochemical analyses on the synergy of cellulase mixtures from C. bescii had observed that a mixture of three cellulases, CelA, CelC and CelE (ACE cellulases) worked synergistically to hydrolyze cellulose as well as a mixture of all six C. bescii cellulases [21]. One could hypothesize, then, that members of the genus Caldicellulosiruptor that possess all three of these enzymes would be among the most cellulolytic.…”

“…Three additional species, C. kronotskyensis , C. danielii , and C. naganoensis also share a similar organization of their GDL [36], including the presence of CelA, CelC and CelE. The contributions of CelD and CelF to cellulose hydrolysis or solubilization are low [22,21] and likely not to impact the ability of C. changbaiensis to efficiently hydrolyze cellulose.…”

“…Alternately, sequence divergence of ACE cellulase orthologs may play a larger role in the catalytic capacity of cellulolytic members from the genus Caldicellulosiruptor . Of the ACE cellulases, CelA is a key player, supported by its unique hydrolysis mechanism [8], the severe reduction in cellulose hydrolysis by C. bescii celA gene deletion mutant [65,22], and biochemical analysis of GDL enzyme synergy [21]. Prior comparison of CelA orthologs from C. bescii and C. danielii found Cb CelA to be a superior enzyme [36], indicating that GDL sequences have diverged during speciation, making it likely that the ACE cellulases from C. changbaiensis may not demonstrate the same catalytic efficiency as C. bescii .…”

Novel multidomain, multifunctional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species

“…A distinguishing feature of the genus Caldicellulosiruptor is the variety of glycoside hydrolases (GHs) encoded in its species' genomes, several of which have multiple domains and multiple catalytic sites. These GHs are designed to degrade complex carbohydrates in the plant cell wall at temperatures greater than 70°C and are either tethered to the bacterium's surface (S)‐layer through an S‐layer homology domain (SLH) or released into the secretome . The GHs responsible for the majority of extracellular cellulose and lignocellulose degradation are concentrated in a single genomic region, termed the glucan degradation locus (GDL) .…”

“…The GHs responsible for the majority of extracellular cellulose and lignocellulose degradation are concentrated in a single genomic region, termed the glucan degradation locus (GDL) . Several full‐length enzymes or individual GH domains from the GDL of species, including C. bescii and C. saccharolyticus , have previously been characterized . These GHs in the GDL contain multiple carbohydrate binding module Family 3 (CBM3) domains, interspersed among catalytic domains.…”