Enzyme design and screening to facilitate pathway engineering

Microbes are being increasingly used as “microbial chemical factories” to produce compounds that include bulk and specialty chemicals, materials, pharmaceuticals, and biofuels. As new products are desired through biological conversion, new enzyme catalysts are needed to mediate these transformations. 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, 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.