(191be) Engineering a Novel 3-Methyl-1-Butanol Biosynthetic Pathway in Escherichia coli

Production of branched-chain higher alcohols as gasoline substitutes via a coupled branched-chain amino acid biosynthesis pathway and Ehrlich degradation pathway has been well established. In this work, we constructed a novel pathway for production of isobutanol and 3-methyl-1-butanol (3MB) in E.coli. In addition, we explored its potential for specific 3MB biosynthesis using metabolic engineering, bioprospecting, and protein engineering approaches. Preferential production of 3MB over the one-carbon shorter byproduct, isobutanol, involves selective termination of carbon chain elongation at the desired chain length, which has been shown to represent a challenge in various engineered pathways. To overcome this challenge, we selected the long-chain favoring keto acid dehydrogenase complex (KDHC) as the termination enzyme. Subsequently, downstream modification enzymes aldehyde dehydrogenase (AldH) and alcohol dehydrogenases (ADH) with enhanced product selectivity were obtained using structure-guided protein engineered and bioprospecting, respectively. The flux distribution between the desired chain elongation step and the pre-mature termination step was also improved through titrating the expression levels of the two competing enzymes. Taken together, the optimized pathway yielded a ratio of 3:1 3MB to isobutanol, a 400% increase over the non-engineered pathway.