Utilizing photosystem I to drive CYP153A monooxygenase activity by light

Nowadays, the chemical industry has a great interest in the sustainable production of chemicals such as ω-hydroxylated hydrocarbons or epoxides as chemical feedstocks. As an alternative to the chemical synthesis, the enzymatic approach is considered promising. CYP153A oxidoreductases, a family of the P450 monooxygenase superfamily, are known to catalyze diverse oxidation reactions, e.g. the terminal hydroxylation of various carbon hydrates or the epoxidation of carbon double bounds in a highly regio- and stereoselective manner. In situ, CYP153A enzymes are part of a NAD(P)H dependent three component system consisting of a reductase and a ferredoxin as an electron carrier.

The accessibility of NAD(P)H as the electron donor is one of the most relevant limitations for the enzymatic biocatalysis. Various NAD(P)H regeneration systems are available but are also harboring unfavorable downsides. A strategy to overcome the dependence of NAD(P)H is the functional coupling of the CYP153A enzymes to the photosynthetic electron transport chain to enable a light dependent biocatalysis. As a proof of concept, a photosystem I (PSI) driven cascade was established, utilizing combinations of two ferredoxins from Thermosynechococcus vestitus and Acinetobacter sp. OC4 as an electron mediator to enable the monooxygenase activity of two CYP153A candidates from Polaromonas sp. JS666 or Gordonia rubripertincta CWB2.

Active PSI trimers were isolated from the thermophilic cyanobacterium T. vestitus via hydrophobic interaction chromatography, whereas the ferredoxins and monooxygenases were expressed as recombinant proteins in E. coli and purified via Ni‑NTA affinity chromatography. For light driven biocatalysis, the conversion of different alkanes to their respective alkanols, the formation of perillyl alcohol from limonene and the epoxidation of aromatic and aliphatic substrates were tested, analyzed and quantified via GC-FID. In addition, P700+ reduction kinetics were established as a screening tool for electron acceptors for PSI.

In conclusion, the formation of 1-octanol, 1- hexanol, perillyl alcohol and 1,2-epoxyhexane using the in vitro cascade serves as a proof of concept for PSI driven monooxygenase activity and paves the way for the implementation of CYP153A enzymes in cyanobacteria for in vivo models. Additionally, P700+ reduction kinetics could be utilized to determine the electron transport efficiency between PSI and various potential electron acceptors.