Study identifies enzymes that could improve isobutanol production in Saccharomyces cerevisiae

Background/Objective

Isobutanol is a fusel alcohol with advantages over ethanol as a biofuel or platform for sustainable aviation fuel. Some microbes naturally produce isobutanol at low levels with the enzyme 2-ketoacid decarboxylase (KDC), which converts 2-Ketoisovalerate (KIV) to isobutyraldehyde, but many previously characterized KDC enzymes also act on other 2-ketoacids (e.g. pyruvate). When isobutanol is produced in Saccharomyces cerevisiae, this unwanted side reaction can divert pyruvate to ethanol. A KDC enzyme specific for KIV is required to make S. cerevisiae a strict isobutanologen.

Approach

Researchers used cell-based and in vitro assays to investigate KDC substrate specificity, screening a diverse set of 45 natural KDC homologs and multisite saturation mutagenesis (SSM) libraries of two homologs.

Results

Screening identified previously uncharacterized KDCs with high KIV and low pyruvate activity. SSM of one of these KDCs identified mutants with improved KIV activity relative to the wild-type enzyme while retaining low levels of pyruvate activity. In a KIV bioconversion experiment, bioprospected and engineered KDCs allowed similar KIV consumption to the previously characterized Lactococcus lactis KdcA, which is highly active on KIV but also has significant activity on pyruvate. All KDCs tested also enabled some ethanol production.

Impact

KDCs identified here have the potential to improve production of isobutanol and other fusel alcohols with a reduction in unwanted ethanol. The dataset of newly characterized KDCs can guide future efforts to engineer substrate specificity in KDC enzymes.

Dietrich, J. J., et al. Engineering of 2-ketoacid Decarboxylases for Production of Isobutanol and Other Fusel Alcohols in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 123, 1036–1049. (2026). [DOI:10.1002/bit.70150]