Engineering of 2‐ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae

ABSTRACT Isobutanol is a fusel alcohol that can be produced microbially for use as a biofuel or upgraded into sustainable aviation fuel (SAF). A key enzyme in the isobutanol biosynthetic pathway is 2‐ketoacid decarboxylase (KDC), which irreversibly decarboxylates 2‐ketoisovalerate (KIV) to yield isobutyraldehyde. However, many previously characterized KDC enzymes also act promiscuously on other 2‐ketoacids, (e.g., pyruvate) to produce a related aldehyde (e.g., acetaldehyde). This unwanted side reaction is especially important when isobutanol is produced in Saccharomyces cerevisiae ( S. cerevisiae ) because it leads to pyruvate being diverted to ethanol. In order to make S. cerevisiae a strict isobutanologen, a KDC enzyme that is specific for KIV must be deployed. In this study, we used a combination of cell‐based and in vitro enzyme assays to investigate KDC substrate specificity, characterizing a large set of homologs for KIV, pyruvate, and phenylpyruvate (PPV) activity. A diverse range of substrate specificities was discovered, and some previously uncharacterized KDCs were revealed to have high KIV activity and low pyruvate activity. Multi‐site saturation mutagenesis (SSM) of one of these KDCs identified mutants with increased KIV activity, while maintaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to when using the previously characterized Lactococcus lactis KdcA, though with some ethanol also produced. The KDCs identified here show promise for production of isobutanol and other alcohols derived from 2‐ketoacids, and the dataset of newly characterized KDCs can inform future efforts to understand and engineer substrate specificity in KDCs.