Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

Paul Santner, László Krisztián Szabó, Santiago Nahuel Chanquia, Aske Høj Merrild, Frank Hollmann, Selin Kara, Bekir Engin Eser*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity.

Original languageEnglish
Pages (from-to)4038-4046
Number of pages9
JournalChemCatChem
Volume13
Issue number18
DOIs
Publication statusPublished - 2021

Bibliographical note

Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.

Keywords

  • Biocatalysis
  • Drop-in Biofuels
  • Fatty Acid Photodecarboxylase
  • Photoenzyme
  • Protein Engineering

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