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.

BackgroundBiofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum can rapidly solubilize and ferment cellulosic biomass, making it a promising candidate microorganism for consolidated bioprocessing for biofuel production, but increases in product yield and titer are still needed.ResultsHere, we started with an engineered C. thermocellum strain where the central metabolic pathways to products other than ethanol had been deleted. After two stages of adaptive evolution, an evolved strain was selected with improved yield and titer. On chemically defined medium with crystalline cellulose as substrate, the evolved strain produced 22.4 ± 1.4 g/L ethanol from 60 g/L cellulose. The resulting yield was about 0.39 gETOH/gGluc eq, which is 75 % of the maximum theoretical yield. Genome resequencing, proteomics, and biochemical analysis were used to examine differences between the original and evolved strains.ConclusionsA two step selection method successfully improved the ethanol yield and the titer. This evolved strain has the highest ethanol yield and titer reported to date for C. thermocellum, and is an important step in the development of this microbe for industrial applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0528-8) contains supplementary material, which is available to authorized users.

SummaryMicrobial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.

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