State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China.
College of Engineering, The University of Georgia, Athens, GA 30602, USA.
Metab Eng. 2017 Mar;40:148-156. doi: 10.1016/j.ymben.2017.02.003. Epub 2017 Feb 13.
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 Klebsiella oxytoca 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 in silico 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 in vitro 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 Escherichia coli'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 de novo 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.
建立微生物生产化学品的新合成途径通常需要通过改造酶来克服途径瓶颈，以增强生物催化作用，甚至实现非天然催化作用。二醇脱水酶因其与 C2 和 C3 二醇的相互作用而得到了广泛的研究。然而，利用这些见解来实现对具有两个以上羟基的非天然底物的催化作用的尝试一直受到低效率的困扰。在这里，我们通过理性设计，使产氧克雷伯氏菌二醇脱水酶能够并增强对非天然 C4 三醇 1,2,4-丁三醇的催化活性。我们分析了脱水酶与 1,2-丙二醇和甘油的相互作用，这使我们提出了合理构想的假说。然后，我们开发了一种基于计算的方法来识别和筛选具有所需活性的候选突变体。这导致了一种工程化的二醇脱水酶，其对 1,2,4-丁三醇的催化活性比野生型高近 5 倍，这是通过体外测定确定的。基于这一结果，我们进一步扩展了 1,2,4-丁三醇途径，建立了一种新型的 1,4-丁二醇生产平台。我们对大肠杆菌的木糖代谢途径进行了工程改造，以增强从 224mg/L 到 1506mg/L 的 1,2,4-丁三醇的生物合成。通过在工程菌株中引入完整的途径，我们实现了从木糖出发以 209mg/L 的浓度从头合成 1,4-丁二醇。这项工作扩展了二醇脱水酶催化的底物谱，并为建立涉及脱水酶基生物催化的新型生物合成途径提供了阐明。
