(643d) Cell-Free Metabolic Engineering for Heterologous in vivo Pathway Optimization

Cell-free metabolic engineering methods offer unique methods for rapidly prototyping heterologous pathways. In cases where substrates or intermediates are toxic and where non-native products are desired, optimization of entire heterologous pathways in vivo can be laborious and inefficient. Cell-free prototyping offers an effective alternative. Previous such efforts have focused on the use of lysate-based systems incorporating cell-free protein synthesis, to enable high-throughput testing of large numbers of gene variants and the use of combinatorial methods for optimizing pathway enzyme loadings. This study pursues an alternative approach, where purified enzymes are recombined to create a reconstituted pathway. This approach allows individual and collective characterization, enabling the parameterization of a kinetic model describing the entire pathway. Once validated, the model can be optimized for maximum throughput. In this case, a non-native product pathway in Pseudomonas putida KT 2440 has been optimized using this cell-free method, resulting in a significant increase in the titer of several target aromatic catabolic intermediates. This success bolsters the case for the use of Pseudomonas spp. to convert lignin monomers to high-value products. The method will be compared with a directed evolution approach, to demonstrate the increased efficacy of the rational cell-free approach compared to the stochastic method. Longer-term potential for the use of cell-free systems as conversion platforms will also be discussed.