Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

Abstract

BackgroundClostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.ResultsRelative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.ConclusionsRelative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H2 synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.

The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

“…tengcongensis makes ethanol without an annotated adhE (14,15). Transcriptomic and proteomic studies in T. saccharolyticum and C. thermocellum have shown adhE to be highly expressed, although other putative adh genes were detected as well (16)(17)(18)(19). AdhE also has been shown to play a role in ethanol tolerance (20).…”

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

“…2), and indeed no ethanol production is detected from this strain (18). The phylogenetically related, thermophilic Firmicute C. thermocellum, however, encodes an NADH-dependent AdhE (Cthe0423), which is one of the most highly expressed genes (24) and abundant proteins (25) in C. thermocellum and is the key enzyme in ethanol production in this organism. Based on its known thermostability, coenzyme specificity (NADH-dependent), similarity in codon use, and favorable catalytic stoichiometry (26), this adhE was a promising candidate to generate an ethanol production pathway in C. bescii.…”

Atypical Glycolysis in Clostridium thermocellum

“…Gene clo1313_0489 has been annotated as a glucokinase and shown to be both transcribed (14) and translated (15). There have been several reports showing the absence or very low activity of ATP-dependent glucokinase in C. thermocellum (10,11,16) and one account showing high activity of glucokinase (9).…”

“…Two genes have been annotated as potential phosphofructokinases, clo1313_0997 and clo1313_1876 (12). Both genes have been shown to be transcribed (14) and translated (15). There have been two reports describing ATP-linked phosphofructokinase activity (9,17).…”