TY - JOUR

T1 - Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

AU - Santner, Paul

AU - Szabó, László Krisztián

AU - Chanquia, Santiago Nahuel

AU - Merrild, Aske Høj

AU - Hollmann, Frank

AU - Kara, Selin

AU - Eser, Bekir Engin

N1 - Funding Information: The authors thank the Novo Nordisk Foundation, Light‐BioFuels project, grant No. NNF19OC0057522. Publisher Copyright: © 2021 Wiley-VCH GmbH

PY - 2021/9/17

Y1 - 2021/9/17

N2 - 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.

AB - 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.

KW - Biocatalysis

KW - Drop-in Biofuels

KW - Fatty Acid Photodecarboxylase

KW - Photoenzyme

KW - Protein Engineering

UR - http://www.scopus.com/inward/record.url?scp=85111354890&partnerID=8YFLogxK

U2 - 10.1002/cctc.202100840

DO - 10.1002/cctc.202100840

M3 - Article

AN - SCOPUS:85111354890

VL - 13

SP - 4038

EP - 4046

JO - CHEMCATCHEM

JF - CHEMCATCHEM

SN - 1867-3880

IS - 18

ER -