초록
<P><I>n</I>-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for <I>n</I>-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled <I>in vitro</I> from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Recombinant versions of eight thermophilic enzymes (β-ketothiolase [Thl], 3-hydroxybutyryl-CoA dehydrogenase [Hbd], and 3-hydroxybutyryl-CoA dehydratase [Crt] from <I>Caldanaerobacter subterraneus</I> subsp. <I>tengcongensis</I>; <I>trans</I>-2-enoyl-CoA reductase [Ter] from <I>Spirochaeta thermophila</I>; bifunctional acetaldehyde dehydrogenase/alcohol dehydrogenase [AdhE] from <I>Clostridium thermocellum</I>; and AdhE, aldehyde dehydrogenase [Bad], and butanol dehydrogenase [Bdh] from <I>Thermoanaerobacter</I> sp. strain X514) were utilized to examine three possible pathways for <I>n</I>-butanol. These pathways differed in the two steps required to convert butyryl-CoA to <I>n</I>-butanol: Thl-Hbd-Crt-Ter-AdhE (<I>C. thermocellum</I>), Thl-Hbd-Crt-Ter-AdhE (<I>Thermoanaerobacter</I> X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. <I>n</I>-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to <I>in vitro</I> experiments, was used to determine relative enzyme ratios needed to maximize <I>n</I>-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to <I>n</I>-butanol was observed <I>in vitro</I>, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to <I>n</I>-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic <I>n</I>-butanol production at elevated temperatures.</P>