Metabolic Engineering for the Production of Isobutanol and 3- Methyl-1-butanol in Saccharomyces cerevisiae

Abstract

Higher alcohols including isobutanol and 3-methyl-1-butanol have received much attention as biofuels because of their higher energy density and lower moisture absorption property compared with ethanol. Saccharomyces cerevisiae naturally generates small amounts of isobutanol and 3-methyl-1-butanol via amino acid biosynthesis pathway and Ehrlich pathway involved in amino acid degradation. 2-ketoisovalerate and 2-ketoisocaproate, the intermediates of Val and Leu biosynthetic pathways are first converted into corresponding aldehydes by decarboxylation, and then converted into isobutanol and 3-methyl-1-butanol, respectively, by alcohol dehydrogenases. In this study, production levels of isobutanol and 3-methyl-1-butanol were increased by deleting ALD6 gene encoding aldehyde dehydrogenase and BAT1 involved in Val and Leu production from 2-ketoisovalerate and 2-ketoisocaproate. In addition, we overexpressed LEU3Δ601, a LEU3 mutant which is a transcriptional activator of genes in the Val and Leu biosynthesis pathway, but lacking the feedback inhibition by Leu, as well as ILV2, ILV5, ILV6, ARO10, and ADH2 genes. To increase 3-methyl-1-butanol production, LEU2 gene in the Leu biosynthetic pathway was also overexpressed. The engineered yeast strain produced 377 mg/L isobutanol and 250 mg/L 3-methyl-1-butanol from 10% glucose, resulting in 20- and 17-fold increases in production titers compared with wild type.