초록
<P><B>Abstract</B></P> <P>In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse β-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the <I>trans</I>-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and β-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from <I>Vitreoscilla</I> hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse β-oxidation pathway produced n-alcohols at titers of 1.8 g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).</P> <P><B>Highlights</B></P> <P> <UL> <LI> Anaerobic production of medium chain n-alcohols by a β-reduction pathway. </LI> <LI> Improved variants of TER, thiolase, and β-ketoreductases were identified. </LI> <LI> Inclusion of an anaerobic-responsive promoter increased production. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>