Highly Active C 8 -Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy

Néstor J. Hernández Lozada, Rung Yi Lai, Trevor R. Simmons, Kelsey A. Thomas, Ratul Chowdhury, Costas D. Maranas, Brian F. Pfleger

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

Abstract

Microbial metabolism is an attractive route for producing medium chain length fatty acids, e.g., octanoic acid, used in the oleochemical industry. One challenge to this strategy is the lack of enzymes that are both highly active in a microbial host and selective toward substrates with desired chain length. Of the many steps in fatty acid biosynthesis, the thioesterase is the most widely used enzyme for controlling chain length. Thioesterases hydrolyze the thioester bond between fatty acids and the acyl-carrier protein (ACP) or coenzyme A (CoA) cofactor. The functional role of thioesterases varies between organisms (i.e., bacteria vs plant) and therefore so do the substrate specificities. As a result, microbial biocatalysts that utilize a heterologous thioesterase either produce high titers of fatty acids with mixed chain lengths or low titers of products with a narrow chain length distribution. To search for highly active enzymes that selectively hydrolyze octanoyl-ACP, we developed a genetic selection based on the lipoic acid requirement of Escherichia coli. We used the selection to identify variants in a randomly mutagenized library of the C 8 -specific Cuphea palustris FatB1 thioesterase. After optimizing expression of the thioesterase, E. coli cultures produced 1.7 g/L of octanoic acid with >90% specificity from a single chromosomal copy of this thioesterase. In vitro studies confirmed the mutant thioesterase possessed a 15-fold increase in k cat compared to its native sequence. The high level of specific activity allowed for low levels of expression while maintaining fatty acid titer. The low expression requirement will allow metabolic engineers to use more cellular resources to address other limitations in the pathway and maximize overall productivity.

Original languageEnglish (US)
Pages (from-to)2205-2215
Number of pages11
JournalACS Synthetic Biology
Volume7
Issue number9
DOIs
StatePublished - Sep 21 2018

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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