The unexpected importance of actinobacterial GH12 in hydrolysis of crystalline cellulose

Production of biofuels via enzymatic hydrolysis of complex biomass is a subject of intense global interest. Here, we investigated compost enrichments as sources of novel thermophilic enzymatic mixtures, referred to as cocktails, for cellulose hydrolysis. Bacterial consortia from compost were adapted to microcrystalline cellulose (MCC). Enzyme assays indicated enrichment supernatants had high levels of glycoside hydrolase activities. Isolated supernatants were then used to successfully saccharify MCC and 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc])-pretreated switchgrass. Microbial community compositional analysis revealed that Actinobacteria dominated early passages of two enrichment lineages. One lineage sustained actinobacterial dominance over two-week intervals, while the community profile of a second lineage changed from predominantly Actinobacteria to a mixture of Bacteroidetes and Firmicutes. These data suggest a succession in primary and secondary cellulose degraders in these cultures that may be important in understanding the mechanism of cellulose hydrolysis by thermophilic bacterial populations. The supernatant from the Actinobacterial-dominated lineage was effective at saccharification of crystalline cellulose. Comparative metagenomic and proteomic analyses revealed differential expression of glycoside hydrolases from Actinobacteria were responsible for the changes in observed activities on biomass substrates. The abundance of GH12 correlated with activity of the supernatants on microcrystalline cellulose. This result was unexpected because GH12 is commonly characterized as an endoglucanase with negligible activity on crystalline substrates. Heterologous expression of the Actinobcaterial GH12, along with GH6 and GH48 demonstrated that it had high activity on CMC and pNPC substrates. Heterologous expression has provided purified enzymes to formulate mixtures for high-temperature saccharification of biomass substrates with crystalline cellulose.