Highly Active C₈-Acyl-ACP Thioesterase Variant Isolated by a Synthetic Selection Strategy

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-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 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.

Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase

“…The new thioesterases were based on Cuphea palustris (CpFatB1) (29) and Cuphea avigera pulcherrima (CaFatB3) (30) with varying mutations and truncations (SI Appendix, Fig. S1), two of which were designed according to recent work of Lozada et al (31). Surprisingly, all new thioesterases showed an improvement in 1octanol production compared with Tes3 (15), but the performance varied widely in response to changes in the inducer concentration ( Fig.…”

“…With these improvements, the internally produced 1-octanol now exceeded the titer (0.75 mM) at which the underivatized product affected the growth of the strain in the absence of solvent overlay. Lozada et al (31) reported that 'CpFatB1-4 was most effective when the expression level was low, as the strain expressing it showed a growth defect at higher expression levels. This was also observed in our study (SI Appendix, Fig.…”

The Phylogeny and Functional Characterization of Peanut Acyl-ACP Thioesterases

“…The substrate specificity of FATs often determines the composition of oils. Thus, many plant FATs have been isolated and remolded to produce oleochemicals with desired chain lengths (Lozada et al 2018). Peanut is an important oil crop (with seed oil content up to 50%), and its genome possesses 21 FATs, which are far more than any other species ( Supplemental Tables S1, S3).…”

From directed evolution to computational enzyme engineering—A review

“…The best design exhibited a 27‐fold improvement in NADH‐dependent activity. Other successes from the same group include, OptGraft for grafting a binding site from one protein into another protein scaffold, rational design to obtain 200‐fold higher D‐hydantoinase activity in Bacillus stearothermophilus using just two amino acid changes, OptZyme for redesigning enzymes by improving binding to a transition state analogue instead of the substrate as it correlates with greater turnover, IPRO Suite of programs for fully‐automated protein redesign, and altering substrate specificity of thioesterase enzyme from long‐chain fatty acyl ACP to medium‐chain ones …”

“…Recent trends in directed evolution has seen attempts and successes at improving proteins with the biological proviso of still being relevant to the metabolic pathways they belong to, thus creating novel whole cell chemical factories for synthesis of value‐added chemicals . More recently, biochemists have aimed at dialing in novel functionalities in enzymatic proteins which are not seen in nature .…”