S69: Comprehensive Quantitative Proteomics for Studying Fermentation of Cellulosic biomass by Clostridium phytofermentans

Metabolic engineering of microbes is widely expected to have a great impact on the optimization of producing chemicals and fuels from biomass.  It is anticipated that bioengineering will allow an expanded usage of raw materials such as cellulose showing chemical properties that so far have hindered exploitation for biofuel production.  Analytical techniques, which allow large scale monitoring of biological processes on a molecular level, such as genomics and proteomics, are used as tools to support the process of metabolic engineering.  We have used quantitative proteomics to accurately measure protein expression changes in the cellulose-degrading microbe Clostridium phytofermentans when grown on glucose, hemicelluloses, and cellulose and we were able to quantify more than 2,500 proteins under these conditions.  Absolute protein concentrations were estimated using a machine learning-supported spectral counting approach, and relative protein concentration changes were accurately measured after using reductive dimethylation as an effective differential stable isotope labeling strategy.  Our data provides an accurate picture of how Clostridium phytofermentans adapts to growth on different biomass sources.  The use of proteomics also allowed us to distinguish between relative concentration changes of intra and extracellular proteins, which is of particular interest as secretion of enzymes is needed to degrade cellulose.  The presented study gives a systems-level understanding of how Clostridium phytofermentans ferments biomass and provides an empirical basis to identify targets for industrial cellulosic fermentation.  We believe mass spectrometry-based proteomics can be applied to create data sets that serve as blueprints for metabolic engineering projects in biofuel production.