(719c) Combinatorial Platform for Evaluation of Protein Complexes Involved in Biomass Deconstruction

In nature, many approaches have evolved for the process of cellulose utilization. Typically, this process will involve many different kinds of enzyme activities, distinct arrangements of enzyme domains, and other binding proteins. These individual parts are then assembled into at least four different types of enzyme complexes. Abundant evidence for this natural diversity has been given by genomic sequencing of known microbes and fungi, and can also be deduced from bioinformatic analysis of the metagenomic sequences isolated from all organisms present in a natural environment. Yet, with this plethora of natural examples, efficient methods to produce individual target polypeptides or to produce the multitude of combinations of these enzymes are not available. Moreover, efficient methods to determine the function of target polypeptides as separate enzymes or, more definitively, as parts of complex mixtures containing other proteins and enzymes are not available. In this presentation, we describe how cell-free translation can be used to address these experimental challenges. Our results demonstrate the ability to evaluate genes in multiple enzyme architectures, allow combinatorial arrangements of genes and proteins, and facilitate quantitative detection and analysis of products without a need to obtain purified preparations of target polypeptides. With this approach, high-throughput analysis of the proteomic complexity of biomass transformation is now a reasonable experimental endeavor. This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494).