초록
<P><B>Abstract</B></P> <P>Branched-chain fatty acids (BCFAs) are key precursors of branched-chain fuels, which have cold-flow properties superior to straight chain fuels. BCFA production in Gram-negative bacterial hosts is inherently challenging because it competes directly with essential and efficient straight-chain fatty acid (SCFA) biosynthesis. Previously, <I>Escherichia coli</I> strains engineered for BCFA production also co-produced a large percentage of SCFA, complicating efficient isolation of BCFA. Here, we identified a key bottleneck in BCFA production: incomplete lipoylation of 2-oxoacid dehydrogenases. We engineered two protein lipoylation pathways that not only restored 2-oxoacid dehydrogenase lipoylation, but also increased BCFA production dramatically. <I>E. coli</I> expressing an optimized lipoylation pathway produced 276mg/L BCFA, comprising 85% of the total free fatty acids (FFAs). Furthermore, we fine-tuned BCFA branch positions, yielding strains specifically producing ante-iso or odd-chain iso BCFA as 77% of total FFA, separately. When coupled with an engineered branched-chain amino acid pathway to enrich the branched-chain α-ketoacid pool, BCFA can be produced from glucose at 181mg/L and 72% of total FFA. While <I>E. coli</I> can metabolize BCFAs, we demonstrated that they are not incorporated into the cell membrane, allowing our system to produce a high percentage of BCFA without affecting membrane fluidity. Overall, this work establishes a platform for high percentage BCFA production, providing the basis for efficient and specific production of a variety of branched-chain hydrocarbons in engineered bacterial hosts.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Discovered that BKD overexpression depletes native enzyme lipoylation. </LI> <LI> Lipoylation can be complemented by engineered protein lipoylation pathways. </LI> <LI> Branched-chain fatty acids are produced in high percentages. </LI> <LI> <I>E. coli</I> cannot incorporate BCFA to its cell membrane, but can metabolize BCFA. </LI> </UL> </P>