초록
<P><B>Abstract</B></P> <P>High titer 1-butanol production in <I>Escherichia coli</I> has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a <I>pta</I> deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in <I>E. coli</I> by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Improvement of 1-butanol production in <I>E. coli</I> was done by resolving CoA imbalance. </LI> <LI> Fine-tuning AdhE2 expression with cysteine addition achieved 18.3g/L 1-butanol titer. </LI> <LI> Metabolomics-driven approach can be used to optimize the 1-butanol producing <I>E. coli</I>. </LI> </UL> </P>