We apply mass spectrometry-based ReDi proteomics to quantify the Clostridium phytofermentans proteome during fermentation of cellulosic substrates. ReDi proteomics gives accurate, low-cost quantification of an extra and intracellular microbial proteome. When combined with physiological measurements, these methods form a general systems biology strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process.C. phytofermentans expressed more than 100 carbohydrate-active enzymes, of which distinct subsets were upregulated on cellulose and hemicellulose. Numerous extracellular enzymes cleave insoluble plant polysaccharides into oligosaccharides, which are transported into the cell to be further degraded by intracellular carbohydratases. Sugars are catabolized by EMP glycolysis incorporating alternative glycolytic enzymes to maximize the ATP yield of anaerobic metabolism.During cellulosic fermentation, cells adhered to the substrate and altered metabolic processes such as upregulation of tryptophan and nicotinamide synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. These diverse metabolic changes highlight how a systems approach can identify novel ways to optimize cellulosic fermentation.
Cellulose is the world's most abundant renewable, biological energy source (Leschine, 1995). Microbial fermentation of cellulosic biomass could sustainably provide enough ethanol for 65% of US ground transportation fuel at current levels (Somerville, 2006). However, cellulose in plant biomass is packaged into a crystalline matrix, making biomass deconstruction a key roadblock to using it as a feedstock (Houghton et al, 2006). A promising strategy to overcome biomass recalcitrance is consolidated bioprocessing (Lynd et al, 2002), which uses microbes such as Clostridium phytofermentans to both secrete enzymes to depolymerize biomass and then ferment the resulting hexose and pentose sugars to a biofuel such as ethanol. The C. phytofermentans genome encodes 161 carbohydrate-active enzymes (CAZy) including 108 glycoside hydrolases spread across 39 families (Cantarel et al, 2009), highlighting the elaborate set of enzymes needed to breakdown different cellulosic polysaccharides. Faced with the complexity of metabolizing biomass, systems biology strategies are needed to comprehensively identify which cellulolytic and metabolic enzymes are used to ferment different cellulosic substrates.
This study presents a systems-level analysis of how C. phytofermentans ferments different cellulosic substrates that incorporates quantitative mass spectrometry-based proteomics of over 2500 proteins. Protein concentrations within each carbon source treatment were calculated by machine learning-supported spectral counting (Absolute Protein EXpression, APEX) (Lu et al, 2007). Protein levels on hemicellulose and cellulose relative to glucose were determined using reductive methylation (Hsu et al, 2003; Boersema et al, 2009), here called ReDi labeling, to chemically incorporate hydrogen or deuterium isotopes at lysines and N-terminal amines of tryptic peptides. We show that ReDi proteomics gives accurate, low-cost quantification of a microbial proteome and can be used to discern extracellular proteins. Further, we combine these quantitative proteomics with detailed measurements of growth, biomass consumption, fermentation product analyses, and electron microscopy. Together, these methods form a general strategy to evaluate the efficiency of cellulosic bioconversion and to identify enzyme targets to engineer for improving this process (Figure 1).
We found that fermentation of cellulosic substrates by C. phytofermentans involves secretion of numerous CAZy as well as proteins for binding of extracellular solutes, proteolysis, and motility. The most highly expressed protein in the proteome is a secreted protein that appears to compose a surface layer to support the cell and anchor cell surface proteins, including some enzymes for plant degradation. Once the secreted CAZy cleave insoluble plant polysaccharides into oligosaccharides, they are taken into the cell to be further degraded by intracellular CAZy, enabling more efficient sugar transport, conserving energy by phosphorolytic cleavage, and ensuring the sugar monomers were not available to competing microbes. Sugars are catabolized by EMP glycolysis incorporating reversible, PPi-dependent glycolytic enzymes, and pyruvate ferredoxin oxidoreductase. The genome encodes seven alcohol dehydrogenases, among which two iron-dependent enzymes are highly expressed and likely facilitate the high ethanol yields. Growth on cellulose also resulted in indirect changes such as increased tryptophan and nicotinamide synthesis and repression of fatty acid synthesis. We distilled the data into a model showing the highly expressed enzymes enabling efficient cellulosic fermentation by C. phytofermentans (Figure 7). Collectively, these data help understand how bacteria recycle plant biomass works towards enabling the use of plant biomass as a low-cost chemical feedstock.
Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems-level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.