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Production of biofuels and other value-added products from lignocellulose breakdown requires the coordinated metabolic activity of varied microorganisms. The increasing global demand for biofuels encourages the development and optimization of production strategies. Optimization in turn requires a thorough understanding of the microbial mechanisms and metabolic pathways behind the formation of each product of interest. Hydrolysis of lignocellulosic biomass is a bottleneck in its industrial use and often affects yield efficiency. The accessibility of the biomass to the microorganisms is the key to the release of sugars that are then taken up as substrates and subsequently transformed into the desired products. While the effects of different metabolic intermediates in the overall production of biofuel and other relevant products have been studied, the role of proteins and their activity under anaerobic conditions has not been widely explored. Shifts in enzyme production may inform the state of the microorganisms involved; thus, acquiring insights into the protein production and enzyme activity could be an effective resource to optimize production strategies. The application of proteomic analysis is currently a promising strategy in this area. This review deals on the aspects of enzymes and proteomics of bioprocesses of biofuels production using lignocellulosic biomass as substrate.