초록
<P><B>Abstract</B></P> <P>Medium-chain esters such as isobutyl acetate (IBAc) and isoamyl acetate (IAAc) are high-volume solvents, flavors and fragrances. In this work, we engineered synthetic metabolic pathways in <I>Escherichia</I> co<I>li</I> for the total biosynthesis of IBAc and IAAc directly from glucose. Our pathways harnessed the power of natural amino acid biosynthesis. In particular, the native valine and leucine pathways in <I>E. coli</I> were utilized to supply the precursors. Then alcohol acyltransferases from various organisms were investigated on their capability to catalyze esterification reactions. It was discovered that ATF1 from <I>Saccharomyces cerevisiae</I> was the best enzyme for the formation of both IBAc and IAAc in <I>E. coli. In vitro</I> biochemical characterization of ATF1 confirmed the fermentation results and provided rational guidance for future enzyme engineering. We also performed strain improvement by removing byproduct pathways (Δ<I>ldh</I>, Δ<I>poxB</I>, Δ<I>pta</I>) and increased the production of both target chemicals. Then the best IBAc producing strain was used for scale-up fermentation in a 1.3-L benchtop bioreactor. 36g/L of IBAc was produced after 72h fermentation. This work demonstrates the feasibility of total biosynthesis of medium-chain esters as renewable chemicals.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We constructed synthetic metabolic pathways to produce isobutyl acetate and isoamyl acetate directly from glucose. </LI> <LI> Optimal alcohol acyltransferases were identified for the esterification step by bioprospecting. </LI> <LI> Enzyme kinetics and protein expression level explained the <I>in vivo</I> productivity of alcohol acyltransferases. </LI> <LI> Production of esters was improved by blocking competing pathways. </LI> <LI> Scale-up fermentation in a 1.3L fermentor achieved a titer of 36g/L of isobutyl acetate. </LI> </UL> </P>