초록
<P><B>Abstract</B></P> <P>Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the <I>Klebsiella oxytoca</I> diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratase's interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An <I>in silico</I> approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by <I>in vitro</I> assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered <I>Escherichia coli</I>'s xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve <I>de novo</I> biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Enabling and enhancing dehydratase-based non-native biocatalysis. </LI> <LI> Docking studies and <I>in silico</I> analysis identify candidate mutants. </LI> <LI> Rationally engineered dehydratase improves activity towards BTO by 5 folds. </LI> <LI> Achieving <I>de novo</I> production of 1,4-butanediol at 209mg/L from xylose. </LI> </UL> </P>