(692e) Bioprospecting to Discover Keto-Aryl Reductases with Enhanced Specificity Towards Longer-Chain, Aliphatic Substrates

Medium-chain length aliphatic compounds (C4-C10) have promising uses as polymeric precursors, biofuels, flavors and fragrances, and pharmaceutical precursors. The reverse beta-oxidation (RBO) cycle, where aliphatic CoA molecules are condensed and then reduced, is commonly used to make these chemicals. Cycles of condensation result in longer molecules, and precursor feeds can be varied to alter terminal R-groups (i.e. adding a branched methyl group). Implementing this method has been successful in making numerous molecules; however, products suffer from low titers and are made as heterogeneous mixtures due to the promiscuity of several pathway enzymes, leading to costly downstream separations. The reduction of keto-aryl-CoA is thought to be an excellent step to add specificity to the RBO pathway due to the equilibrium for the condensation reaction being heavily favored in the reverse direction, which will permit only selective reductases to form the desired reduced product. We generated a sequence similarity network of 5,000 putative reductases based on the often-used keto-aryl reductase PhaB from Cupriavidus necator. From this network, we selected 15 reductases located in various protein clusters and at different phylogenetic distances from PhaB to screen in vivo for higher specificity for 3-oxo-pentanoyl-CoA (C5) over 3-oxo-butanoyl-CoA (C4) substrates. From this initial panning experiment, we identified two reductases that showed at least a two-fold increase in specificity for the C5 to C4 compounds compared to PhaB. Using this knowledge, we decided to test the enzymes that cluster near those with high specificity as well as those that cluster near PhaB to see if these enzymes have similar specificities or if a more catalytically active variant could be found. Enzymes near PhaB in the similarity network all showed lower specificity towards C5 to C4 products than the previously identified high-specificity reductases or PhaB. Searching the clusters of enzymes containing the high-specificity reductases identified ones that had similar or higher specificity to the C5 compounds. In vitro activity of these enzymes is currently being investigated as are potential mechanisms for enhanced specificity. We hope this method of searching through enzyme sequence can be applied to other enzyme classes to enable identification of clusters with desired activities.