Enzyme Screening and Engineering for Improvement of Novel Biosynthetic Pathways

Biological synthesis is increasingly being pursued as an alternative to traditional organic synthesis for the production of chemical compounds.  As known biochemical pathways are transferred to non-native hosts and new pathways are designed, access to genes encoding desired enzymatic activities with appropriately high reaction rates is often a limiting factor.  In this presentation, I will discuss our efforts to identify enzymes with appropriate activity and selectivity in the context of two novel biosynthetic pathways.  In the first example, we have exploited the promiscuous activity of four different enzymes in order to build a versatile platform pathway for the synthesis of various 3-hydroxyalkanoic acids.  These products can be subsequently dehydrated to yield the unsaturated acids, or hydrogenated to produce the saturated product, but only if enzymes with the appropriate specificity and promiscuity can be found.  Examining the product profiles in vivo has led to greater understanding of the constituent enzymes in the absence of access to the substrates needed for in vitro analysis.  I will discuss our findings to date with this pathway, including an analysis of enzyme variants obtained through bioprospecting and rational design, as well as challenges that we face towards linking in vitro characterization to in vivo performance.  Using a second model pathway for the production of glucaric acid, I will also describe opportunities to use experimental data to guide the selection of additional enzyme variants that may have higher target activities through network analysis of protein sequences.