Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. KEY POINTS: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.

• PMID: 36976306
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Journal Article

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Valle-Rodríguez JO, Siewers V, Nielsen J, Shi S

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