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

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.