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

Authors: 
Black, W., University of California, Irvine
Seki, K., University of California, Irvine
Jenic, A., University of California, Irvine
Wang, Y., University of California, Irvine
Li, H., University of California, Irvine
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.