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

Thomas Rydzak,Peyman Ezzati,Peter D Mcqueen,John A Wilkins,Vic Spicer,Richard Sparling,David B Levin,Ravi C Dwivedi,Oleg V Krokhin,Dmitry Shamshurin

doi:10.1186/1471-2180-12-214

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.

Highlights

Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass
Metabolic engineering strategies to improve product yields in C. thermocellum [15] and related species [16] have been only moderately successful and at times resulted in unpredicted changes in product yields [12]
Growth and end-product synthesis In this study, we investigated the relative abundance profiles (RAI) of core metabolic proteins in exponential phase cultures, and changes in protein expression in response to growth phase

Summary

Introduction

Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. Clostridium thermocellum ATCC 27405, an anaerobic, Gram-positive thermophilic bacterium, is capable of cellulosome-mediated breakdown of (hemi)cellulose [1,2] and simultaneous fermentation of resulting cellooligosaccharides into hydrogen (H2) and ethanol [3,4,5] This reduces the need for separate cellulase production, cellulose hydrolysis, and fermentation, which could improve economic viability of industrial cellulosic biofuel production [4,6,7]. Its high temperature growth optimum aids in H2 recovery [11], and the availability of annotated genome sequence (GenBank accession number ZP_00312459.1) allows for deduction of metabolic pathways in silico, expression studies by microarray and proteomic analysis, and genetic engineering [12,13,14] It is an attractive model for biofuel production via consolidated bioprocesing. While many of the genes and proteins involved in pyruvate catabolism and product formation have been verified via RT-PCR [22], enzyme activity assays [4], and purification [23,24], a more thorough understanding of metabolic and regulatory networks must be attained

Methods

Results

Conclusion