Optimization and Engineering of Fatty Acid Photodecarboxylase for Substrate Specificity

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.

Immobilization and Application of Fatty Acid Photodecarboxylase in Deep Eutectic Solvents

“…Depending on the case thioredoxin was present or not (Table S1 and Figure S2). The protein expression protocol was adapted from previous literature [32] . BL21‐Gold (DE3) E. coli cells transformed with the plasmid containing Cv FAP were grown in TB media under continuous shaking (150 rpm), with a temperature of 37 °C until an OD 600 of 0.6 was reached.…”

Photobiocatalysis in Continuous Flow

“…Unfortunately, the first three mentioned enzymes still do not have wide applications in biocatalysis (Scrutton, 2017). On the other hand, the recently discovered photodecarboxylase shows promising results for its application in the production of drop-in fuels via decarboxylation of fatty acids, which are present in natural fats and oils, using blue light (Huijbers et al, 2018;Santner et al, 2021). Despite being an interesting field; it should be taken into account that, in comparison to the application of other fields of photobiocatalysis, the use of photoenzymes is still in its infancy (Björn, 2018).…”

Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

“…[7][8][9][10] Protein engineering approaches have been extensively studied using rational, semi-rational, or random methods. [11][12][13] CHMO Acineto , a cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, is the first reported and most extensively studied BVMO. A saturation mutagenesis library involving mutation of 35 residues was designed to increase the activity of CHMO Acineto on 2-butanone.…”

“…Protein engineering approaches have been extensively studied using rational, semi‐rational, or random methods [11–13] . CHMO Acineto , a cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871, is the first reported and most extensively studied BVMO.…”