Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA.
Department of Chemical and Biomolecular Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, USA
Appl Environ Microbiol. 2018 Mar 19;84(7). doi: 10.1128/AEM.02307-17. Print 2018 Apr 1.
Engineering the Wood-Ljungdahl pathway (WLP) in the established industrial organism would allow for the conversion of carbohydrates into butanol, acetone, and other metabolites at higher yields than are currently possible, while minimizing CO and H release. To this effect, we expressed 11 core genes coding for enzymes and accessory proteins of the WLP in The engineered WLP in showed functionality of the eastern branch of the pathway based on the formation of labeled 5,10-methylenetetrahydrofolate from C-labeled formate, as well as functionality of the western branch as evidenced by the formation of CO from CO However, the lack of labeling in acetate and butyrate pools indicated that the connection between the two branches is not functional. The focus of our investigation then centered on the functional expression of the acetyl-coenzyme A (CoA) synthase (ACS), which forms a complex with the CO dehydrogenase (CODH) and serves to link the two branches of the WLP. The CODH/ACS complex catalyzes the reduction of CO to CO and the condensation of CO with a methyl group to form acetyl-CoA, respectively. Here, we show the simultaneous activities of the two recombinant enzymes. We demonstrate the classical ACS carbonyl carbon exchange assay, whereby the carbonyl carbon of acetyl-CoA is exchanged with the CO carbon. Our data suggest that the low heterologous expression of ACS may limit the functionality of the heterologous WLP in The bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from was heterologously expressed in the obligate heterotroph The functional activity of the CODH was confirmed through both the oxidation and reduction of CO, as had previously been shown for the heterologous CODH from Significantly, a novel assay for ACS exchange activity using C-tracers was developed and used to confirm functional ACS expression.
在已建立的工业生物体内工程化 Wood-Ljungdahl 途径（WLP）将允许将碳水化合物转化为丁醇、丙酮和其他代谢物，产率高于目前可能的水平，同时最大限度地减少 CO 和 H 的释放。为此，我们在 中表达了 11 个核心基因，这些基因编码 WLP 的酶和辅助蛋白。 在 中，基于 C 标记的甲酸盐形成标记的 5,10-亚甲基四氢叶酸，以及基于 CO 形成 CO 的事实，证明了该途径的东部分支具有功能；然而，由于缺乏在乙酸盐和丁酸盐池中的标记，表明两个分支之间的连接没有功能。然后，我们的研究重点集中在乙酰辅酶 A（CoA）合酶（ACS）的功能表达上，该酶与 CO 脱氢酶（CODH）形成复合物，用于连接 WLP 的两个分支。CODH/ACS 复合物分别催化 CO 还原为 CO 和 CO 与甲基形成乙酰-CoA。在这里，我们展示了两个重组酶的同时活性。我们展示了经典的 ACS 羰基碳交换测定法，其中乙酰-CoA 的羰基碳与 CO 的碳交换。我们的数据表明，ACS 的低异源表达可能限制了 中的异源 WLP 的功能。 来自 的双功能一氧化碳脱氢酶/乙酰辅酶 A 合酶（CODH/ACS）在需氧异养菌 中异源表达。CODH 的功能活性通过 CO 的氧化和还原得到证实，这与先前从 中异源 CODH 的情况相同。值得注意的是，开发了一种使用 C 示踪剂的新型 ACS 交换活性测定法，并用于确认功能性 ACS 表达。
