Functional Analysis of the Glucan Degradation Locus in Caldicellulosiruptor bescii Reveals Essential Roles of Component Glycoside Hydrolases in Plant Biomass Deconstruction
Authors: Conway Jonathan M. , McKinley Bennett S. , Seals Nathaniel L. , Hernandez Diana , Khatibi Piyum A. , Poudel Suresh , Giannone Richard J. , Hettich Robert L. , Williams-Rhaesa Amanda M. , Lipscomb Gina L. , Adams Michael W. W. , Kelly Robert M. 
Published: October 2017

The ability to hydrolyze microcrystalline cellulose is an uncommon feature in the microbial world, but it can be exploited for conversion of lignocellulosic feedstocks into biobased fuels and chemicals. Understanding the physiological and biochemical mechanisms by which microorganisms deconstruct cellulosic material is key to achieving this objective. The glucan degradation locus (GDL) in the genomes of extremely thermophilic
Caldicellulosiruptor
species encodes polysaccharide lyases (PLs), unique cellulose binding proteins (tāpirins), and putative posttranslational modifying enzymes, in addition to multidomain, multifunctional glycoside hydrolases (GHs), thereby representing an alternative paradigm for plant biomass degradation compared to fungal or cellulosomal systems. To examine the individual and collective
in vivo
roles of the glycolytic enzymes, the six GH genes in the GDL of
Caldicellulosiruptor bescii
were systematically deleted, and the extents to which the resulting mutant strains could solubilize microcrystalline cellulose (Avicel) and plant biomass (switchgrass or poplar) were examined. Three of the GDL enzymes, Athe_1867 (CelA) (GH9-CBM3-CBM3-CBM3-GH48), Athe_1859 (GH5-CBM3-CBM3-GH44), and Athe_1857 (GH10-CBM3-CBM3-GH48), acted synergistically
in vivo
and accounted for 92% of naked microcrystalline cellulose (Avicel) degradation. However, the relative importance of the GDL GHs varied for the plant biomass substrates tested. Furthermore, mixed cultures of mutant strains showed that switchgrass solubilization depended on the secretome-bound enzymes collectively produced by the culture, not on the specific strain from which they came. These results demonstrate that certain GDL GHs are primarily responsible for the degradation of microcrystalline cellulose-containing substrates by
C. bescii
and provide new insights into the workings of a novel microbial mechanism for lignocellulose utilization.

IMPORTANCE
The efficient and extensive degradation of complex polysaccharides in lignocellulosic biomass, particularly microcrystalline cellulose, remains a major barrier to its use as a renewable feedstock for the production of fuels and chemicals. Extremely thermophilic bacteria from the genus
Caldicellulosiruptor
rapidly degrade plant biomass to fermentable sugars at temperatures of 70 to 78°C, although the specific mechanism by which this occurs is not clear. Previous comparative genomic studies identified a genomic locus found only in certain
Caldicellulosiruptor
species that was hypothesized to be mainly responsible for microcrystalline cellulose degradation. By systematically deleting genes in this locus in
Caldicellulosiruptor bescii
, the nuanced, substrate-specific
in vivo
roles of glycolytic enzymes in deconstructing crystalline cellulose and plant biomasses could be discerned. The results here point to synergism of three multidomain cellulases in
C. bescii
, working in conjunction with the aggregate secreted enzyme inventory, as the key to the plant biomass degradation ability of this extreme thermophile.