Ketosynthase mutants enable short-chain fatty acid biosynthesis in E. coli

被引:3
作者
Mains, Kathryn [1 ]
Fox, Jerome M. [1 ]
机构
[1] Univ Colorado, Dept Chem & Biol Engn, 3415 Colorado Ave, Boulder, CO 80303 USA
关键词
Fatty acid synthesis; Biocatalytic networks; Enzyme cascades; Kinetic models; Ketosynthases; Acyl-ACPs; Free fatty acids; Oleochemicals; ACYL CARRIER PROTEIN; ESCHERICHIA-COLI; SYNTHASE; GENE; ORGANIZATION; METABOLISM; EXPRESSION; PATHWAY; BIOLOGY; CLONING;
D O I
10.1016/j.ymben.2023.03.008
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Cells build fatty acids in tightly regulated assembly lines, or fatty acid synthases (FASs), in which beta-ketoacyl-acyl carrier protein (ACP) synthases (KSs) catalyze sequential carbon-carbon bond forming reactions that generate acyl-ACPs of varying lengths-precursors for a diverse set of lipids and oleochemicals. To date, most efforts to control fatty acid synthesis in engineered microbes have focused on modifying termination enzymes such as acyl-ACP thioesterases, which release free fatty acids from acyl-ACPs. Changes to the substrate specificity of KSs provide an alternative-and, perhaps, more generalizable-approach that focuses on controlling the acyl-ACPs available for downstream products. This study combines mutants of FabF and FabB, the two elongating KSs of the E. coli FAS, with in vitro and in vivo analyses to explore the use of KS mutants to control fatty acid synthesis. In vitro, single amino acid substitutions in the gating loop and acyl binding pocket of FabF shifted the product profiles of reconstituted FASs toward short chains and showed that KS mutants, alone, can cause large shifts in average length (i.e., 6.5-13.5). FabB, which is essential for unsaturated fatty acid synthesis, blunted this effect in vivo, but exogenously added cis-vaccenic acid (C18:1) enabled sufficient transcriptional repression of FabB to restore it. Strikingly, a single mutant of FabB afforded titers of octanoic acid as high as those generated by an engineered thioesterase. Findings indicate that fatty acid synthesis must be decoupled from microbial growth to resolve the influence of KS mutants on fatty acid profiles but show that these mutants offer a versatile approach for tuning FAS outputs.
引用
收藏
页码:118 / 127
页数:10
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