We propose to metabolically engineer P. pastoris, an industrial powerhouse in enzyme production, to produce higher (>C2) and branched-chain alcohols and their corresponding acetate esters using glucose or methanol as the sole carbon source.
Within the next 2 years, the following Specific Aims will be pursued:
Aim 1. Construction of a set of yeast expression cassettes that can be used to enhance chemical production in P. pastoris and other methylotrophs grown on glucose or methanol;
Aim 2. Overexpression of the 2-keto acid degradation pathway genes (2-keto acid decarboxylase and alcohol dehydrogenase) to enhance alcohol production from 2-keto acids;
Aim 3. Overexpression of L-valine biosynthetic pathway genes (acetolactate synthase, acetohy- droxyacid reductoisomerase and dihydroxyacid dehydratase) to further increase the pool of 2-ketoisovalerate and subsequently the production titer of isobutanol.
Aim 4: Overexpression of a broad-substrate-range alcohol-O-acyltransferase to produce acetate esters of isobutanol and other higher branched-chain alcohols.
