Abstract
BackgroundEconomic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.Methodology/Principal FindingsIn this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.Conclusions/SignificanceOverall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.
Highlights
Plant cell walls consist of several intertwined heterogeneous polymers, primarily composed of cellulose, hemicellulose, pectin, and lignin
The anaerobic, thermophilic, Gram-positive bacterium Clostridium thermocellum possesses this diversity in catalytic capability [1], making this organism an attractive candidate for lignocellulosic biomass deconstruction and conversion for cellulosic ethanol production [2]
The catalytic units are non-covalently attached to the scaffoldin via the high affinity Type I interaction between dockerin domains borne by the catalytic units with the cohesins on the scaffoldin [8,9]
Summary
Plant cell walls consist of several intertwined heterogeneous polymers, primarily composed of cellulose, hemicellulose (substituted xylan), pectin, and lignin. The anaerobic, thermophilic, Gram-positive bacterium Clostridium thermocellum possesses this diversity in catalytic capability [1], making this organism an attractive candidate for lignocellulosic biomass deconstruction and conversion for cellulosic ethanol production [2]. High efficiency cellulose hydrolysis is aided by the cell surface attached multienzyme protein complex termed the cellulosome [4,5,6]. The catalytic units are non-covalently attached to the scaffoldin via the high affinity Type I interaction between dockerin domains borne by the catalytic units with the cohesins on the scaffoldin [8,9]. The entire scaffoldin with bound subunits is attached to the cell surface via the high affinity Type II interaction between the dockerin domain of CipA and the cohesin(s) borne by the anchoring proteins (OlpB, SdbA, Orf2p) [10]. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes
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