(464c) Functional Genomic Analysis of Escherichia Coli Using Sequential Cell-Free Protein Synthesis

With the vast amount of sequence information emerging in the post-genomic era, great efforts are being made to understand the function of every gene product and how each one leads to a specific cellular fate. The current methods used for such characterization, mainly microarray technologies, have several challenges associated with them, such as the need to achieve adequate expression, stability, and biological activity of a large variety of protein targets. Furthermore, many of these techniques fail to define protein function in the context of the dynamic metabolic networks that exist in vivo. In light of these limitations, it is clear that new technologies are needed in order to exploit the abundance of genomic information presently available. We have designed a sequential cell-free protein expression system, which has overcome the aforementioned challenges and has proved to be an efficient system for protein function characterization.

The developed platform was employed for the functional expression and analysis of the complete Escherichia coli (E. coli) K-12 genome, with the aim of identifying proteins that influence the in vitro metabolism and the transcription, translation, and protein folding processes in an E. coli-based cell-free protein synthesis (CFPS) system. After three rounds of screening, we identified over 100 proteins with significantly positive effects on protein expression and folding and ~50 proteins with negative effects. The identified proteins exhibited diverse metabolic activities, such as affecting amino acid and nucleic acid stability, supplying and regenerating energy, and enhancing protein expression, stability, and activation. Encouragingly, ~98% of the effectors influenced the cell-free system in manners that were consistent with their in vivo metabolic functions, while a few of the proteins affected the system in unanticipated, yet very interesting, ways.

These observations can be used to guide alteration of the CFPS system in order to improve productivity. Possible changes include rational metabolic engineering of the CFPS extract source cell genome and/or modification of the CFPS reaction composition and conditions. For instance, non-essential genes that encode negative effects can be deleted from the source cell genome or the deleterious gene products can be selectively removed from the cell extract prior to use. Alternatively, if proteins are identified as positive effectors, duplicate copies of the genes can be incorporated into the chromosome for overexpression of the gene products prior to cell extract preparation; the proteins can be co-expressed during the cell-free reaction; or they can be independently produced and added to the cell-free extract at time of use.

We have demonstrated that the use of the sequential cell-free protein expression system, which enabled us to assess 1000s of protein functions in parallel, allows for the precise and rapid identification of effectors of the in vitro metabolic system and potential targets that may increase the productivity and convenience of CFPS through rational metabolic engineering. Although all observations may not be directly applicable for improving in vivo metabolism, many of them may generate reasonable hypotheses to improve our understanding and utilization of the living organism.