Department of Chemistry, University of California, Berkeley , Berkeley, California 94720-1460, United States.
Department of Molecular and Cell Biology, University of California, Berkeley , Berkeley, California 94720-1460, United States.
J Am Chem Soc. 2017 Oct 18;139(41):14526-14532. doi: 10.1021/jacs.7b07400. Epub 2017 Oct 9.
Cell-based synthesis offers many opportunities for preparing small molecules from simple renewable carbon sources by telescoping multiple reactions into a single fermentation step. One challenge in this area is the development of enzymatic carbon-carbon bond forming cycles that enable a modular disconnection of a target structure into cellular building blocks. In this regard, synthetic pathways based on thiolase enzymes to catalyze the initial carbon-carbon bond forming step between acyl coenzyme A (CoA) substrates offer a versatile route for biological synthesis, but the substrate diversity of such pathways is currently limited. In this report, we describe the identification and biochemical characterization of a thiolase-ketoreductase pair involved in production of branched acids in the roundworm, Ascaris suum, that demonstrates selectivity for forming products with an α-methyl branch using a propionyl-CoA extender unit. Engineering synthetic pathways for production of α-methyl acids in Escherichia coli using these enzymes allows the construction of microbial strains that produce either chiral 2-methyl-3-hydroxy acids (1.1 ± 0.2 g L) or branched enoic acids (1.12 ± 0.06 g L) in the presence of a dehydratase at 44% and 87% yield of fed propionate, respectively. In vitro characterization along with in vivo analysis indicates that the ketoreductase is the key driver for selectivity, forming predominantly α-branched products even when paired with a thiolase that highly prefers unbranched linear products. Our results expand the utility of thiolase-based pathways and provide biosynthetic access to α-branched compounds as precursors for polymers and other chemicals.
基于细胞的合成方法提供了许多机会，可以通过将多个反应缩合到单个发酵步骤中，从简单的可再生碳源中制备小分子。该领域的一个挑战是开发酶促碳-碳键形成循环，使目标结构能够模块化地断开为细胞构建块。在这方面，基于硫解酶的合成途径来催化酰基辅酶 A (CoA) 底物之间的初始碳-碳键形成步骤，为生物合成提供了一种多功能途径，但此类途径的底物多样性目前受到限制。在本报告中，我们描述了一种在蛔虫 Ascaris suum 中参与支链酸生产的硫解酶-酮还原酶对的鉴定和生化特征，该酶对使用丙酰 CoA 延伸单元形成具有α-甲基支链的产物具有选择性。使用这些酶在大肠杆菌中构建生产α-甲基酸的合成途径，可以构建出能够在脱水酶存在下生产手性 2-甲基-3-羟基酸（1.1 ± 0.2 g/L）或支链烯酸（1.12 ± 0.06 g/L）的微生物菌株，分别以 44%和 87%的进料丙酸盐产率。体外表征和体内分析表明，酮还原酶是选择性的关键驱动因素，即使与高度偏爱无支链线性产物的硫解酶配对，也主要形成α-支化产物。我们的研究结果扩展了基于硫解酶的途径的实用性，并为α-支化化合物作为聚合物和其他化学品的前体提供了生物合成途径。
