Engineering the Valine Assimilation Pathway to Produce Biochemicals and Fuels in S. Cerevisiae

Growing energy demand and sustainability concerns have stimulated a renewed interest in the engineering of branched-chain amino acid assimilation pathways.  The Ehrlich pathway, in particular, has been the subject of recent efforts for its ability to produce branched-chain alcohols such as isobutanol.  In this study, we explore alternative valine assimilation routes as a production platform for renewable biochemicals and fuels in the yeast Saccharomyces cerevisiae.  We were able to successfully reconstitute a heterologous valine assimilation pathway in yeast and target expression to the cytoplasm or mitochondria through the use of an N-terminal tag from a mitochondrial ATPase (Su9) found in N. crassa.  While pathway in vitro activities were comparable after 48h, product titers were observed to be much larger when expression was targeted to the mitochondria as compared to the cytoplasm.  In this poster, I further discuss the potential and performance of this platform and examine the effects of pathway localization.