(169f) Enhancement of PHAscl Specificity of the P. Oleovorans PHAmcl Synthase Via Error-Prone PCR

For biologically produced PHAs (polyhydroxyalkanoates), as with many types of plastics, the synthesis of block copolymers presents an opportunity to engineer novel functionality and improved toughness. Such block copolymers can be synthesized with a PHA polymerase that has a broad substrate specificity. Seeking to further improve the selectivity of the Pseudomonas oleovorans medium chain length (mcl) PHA synthase toward short chain length (scl) monomers, we created a large library of mutants via error-prone PCR. Estimating our library size at ~1x10⁶ clones we chose to develop a selection approach as opposed to the more laborious colony screening procedures employed in similar studies. Using a selection approach requires that those cells producing high levels of PHA_scl grow faster than those with minimal PHA_scl production. To create this growth rate advantage we employed a modified E. coli strain with minimized metabolic functionality designed via elementary mode analysis to couple PHA_scl production to cell growth rate . Testing in minimal media with glucose as the primary carbon source showed a ~100% increase in cell growth rate in cells expressing the class I synthase from Ralstonia eutropha , a native PHA_scl producer, compared to the same cells expressing the wild type P. oleovorans synthase. Based on this significant growth advantage we performed a selection on the population of mutants in a feedback controlled cytostat, shown previously to dramatically enhance mutant selection time over conventional chemostats or cells passaging procedures. [1]

1. Gilbert, A., D.P. Sangurdekar, and F. Srienc, Rapid strain improvement through optimized evolution in the cytostat. Biotechnology and Bioengineering, 2009. 103(3): p. 500-512.