Elimination of biosynthetic pathways for l-valine and l-isoleucine in mitochondria enhances isobutanol production in engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae has a natural ability to produce higher alcohols, making it a promising candidate for production of isobutanol. However, the several pathways competing with isobutanol biosynthesis lead to production of substantial amounts of l-valine and l-isoleucine in mitochondria and isobutyrate, l-leucine, and ethanol in cytosol. To increase flux to isobutanol by removing by-product formation, the genes associated with formation of l-valine (BAT1), l-isoleucine (ILV1), isobutyrate (ALD6), l-leucine (LEU1), and ethanol (ADH1) were disrupted to construct the S. cerevisiae WΔGBIALA1_2vec strain. This strain showed 8.9 and 8.6 folds increases in isobutanol concentration and yield, respectively, relative the corresponding values of the background strain on glucose medium. In a bioreactor fermentation with a gas trapping system, the WΔGBIALA1_2vec strain produced 662 mg/L isobutanol concentration with a yield of 6.71 mgisobutanol/gglucose. With elimination of the competing pathways, the WΔGBIALA1_2vec strain would serve as a platform strain for isobutanol production.