Discovery and Engineering of Pathways for Production of α-Branched Organic Acids

被引:17
作者
Blaisse, Michael R. [1 ]
Dong, Hongjun [1 ]
Fu, Beverly [1 ]
Chang, Michelle C. Y. [1 ,2 ]
机构
[1] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA
[2] Univ Calif Berkeley, Dept Mol & Cell Biol, 229 Stanley Hall, Berkeley, CA 94720 USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
ENOYL-COA HYDRATASE; SUBSTRATE-SPECIFICITY; THIOLASE SUPERFAMILY; RAT-LIVER; ENZYME; DEHYDROGENASE; BIOSYNTHESIS; MECHANISM; IDENTIFICATION; STRAIN;
D O I
10.1021/jacs.7b07400
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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 alpha-methyl branch using a propionyl-CoA extender unit. Engineering synthetic pathways for production of alpha-methyl acids in Escherichia soli using these enzymes allows the construction of microbial strains that produce either chiral 2-methyl-3-hydroxy acids (1.1 +/- 0.2 g L-1) or branched enoic acids (1.12 +/- 0.06 g L-1) 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 alpha-branched products even when:paired with a thiolase that highly prefers unbrandied linear products. Our results expand the utility of thiolase-based pathways and provide biosynthetic access to alpha-branched compounds as precursors for polymers and other chemical's.
引用
收藏
页码:14526 / 14532
页数:7
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