(309d) Understanding Substrate Specificity of 2-Ketoacid Decarboxylases to Improve Isobutanol Production in Saccharomyces Cerevisiae

2-ketoacid decarboxylases (KDCs) are thiamine pyrophosphate-dependent enzymes important in catabolism of amino acids and accept a wide range of 2-ketoacid substrates. In microbial production of the biofuel isobutanol, a KDC is required to convert 2-ketoisovalerate (KIV) to isobutyraldehyde. However, KDCs that have been characterized to date possess either weak activity on KIV or low specificity that results in unwanted side reactions. One major side reaction is the decarboxylation of pyruvate. Pyruvate is an upstream metabolite in the isobutanol pathway, and KDC activity toward pyruvate can significantly reduce isobutanol production. Another important side activity is the decarboxylation of phenylpyruvate, which can deplete the cell’s aromatic amino acid pools. To better understand the determinants of KDC substrate specificity and activity, we have characterized a large number of diverse KDC homologs through a combination of in vitro enzyme assays and in vivo growth complementation. Additionally, by expressing these homologs in a KDC-null strain of the yeast Saccharomyces cerevisiae, we can directly characterize the enzymes in an industrially relevant host. Several previously uncharacterized KDCs show interesting substrate specificity profiles, and further analysis of this large substrate specificity dataset using bioinformatic and machine learning tools will further shed light on this enzyme class and guide future efforts to identify KDCs best suited for isobutanol production.