Caldicellulosiruptor species are anaerobic bacteria that are the most thermophilic (T
opt = 70-78°C) microorganisms currently known to degrade lignocellulosic biomass. Large (~170-230 kDa) multi-domain cellulases and hemicellulases, found in a single genomic locus termed the glucan degradation locus (GDL), are of interest for dissecting the biomass degradation ability of these species. Using recently developed genetic techniques in
C. bescii, a replicating vector was developed to over-express enzymes in
C. bescii for purification and characterization, while maintaining their native glycosylation. All six GDL enzymes from
C. bescii (CelA-CelF), and four additional novel GDL enzymes from
C. danielii and
C. morganii, were expressed and characterized using this system. A simplex centroid mixture experimental design was used to examine enzyme cocktails containing the six
C. bescii GDL enzymes
in vitro, and a model relating enzyme composition to glucan conversion was constructed. In parallel, genetic tools were used to construct knockout strains of
C. bescii with deletion of various combinations of the 6 GDL genes to assess their role
in vivo.
C. bescii strain RKCB123
ÎcelA-celE was not able to degrade microcrystalline cellulose and was extremely impaired in its ability to degrade lignocellulosic substrates switchgrass and poplar. Finally, enzyme mixtures optimized by the
in vitro model were examined alone and when added to cultures containing knockout strain RKCB123 (
ÎcelA-celE). Optimized enzyme cocktails alone performed only 20% the microcrystalline cellulose degradation of wild type
C. bescii, while with strain RKCB123 they restored degradation to wild type levels. This suggests mass action effects caused by sugar fermentation performed by
C. bescii cells helps to drive biomass degradation by these enzymes. The ability of
C. bescii to efficiently degrade lignocellulosic biomass using the enzymes of the GDL, along with our ability to manipulate this species genetically, make
C. bescii of interest as a platform organism for industrial biotechnology applications. Recent work, combining a
C. bescii strain engineered to produce ethanol with transgenic poplar lines showed nearly 90% carbohydrate conversion and fermentation, serving as a proof-of-concept for the conversion of renewable biomass feedstocks by
C. bescii to bio-based fuels and chemicals.
References
[1] Conway JM, Crosby JR, McKinley BS, Seals NL, Adams MWW, Kelly RM. 2018. Parsing in vivo and in vitro contributions to microcrystalline cellulose hydrolysis by multiâdomain glycoside hydrolases in the Caldicellulosiruptor bescii secretome. Biotechnol Bioeng. 115:2426-2440.
[2] Conway JM, McKinley BS, Seals NL, Hernandez D, Khatibi PA, Poudel S, Giannone RJ, Hettich RL, Williams-Rhaesa AM, Lipscomb GL, Adams MWW, Kelly RM. 2017. Functional analysis of the Glucan Degradation Locus (GDL) in Caldicellulosiruptor bescii reveals essential roles of component glycoside hydrolases in plant biomass deconstruction. Appl Environ Microbiol. 83:e01828-01817.
[3] Conway JM, Crosby JR, Hren AP, Southerland RT, Lee LL, Lunin VV, Alahuhta P, Himmel ME, Bomble YJ, Adams MWW, Kelly RM. 2018. Novel multidomain, multifunctional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species. AIChE J. 64:4218-4228.
[4] Straub CT, Khatibi PA, Wang JP, Conway JM, Williams-Rhaesa AM, Peszlen IM, Chiang VL, Adams MWW, Kelly RM. 2019. Quantitative fermentation of unpretreated transgenic poplar by Caldicellulosiruptor bescii. Nat Commun 10:3548.
