Fungal cytochrome P450 monooxygenases of Fusarium oxysporum for the synthesis of ω-hydroxy fatty acids in engineered Saccharomyces cerevisiae

Abstract

BackgroundOmega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom.ResultsTo gain a deep insight on the biochemical role of fungal P450s towards the production of omega hydroxy fatty acids, two cytochrome P450 monooxygenases from Fusarium oxysporum (FoCYP), FoCYP539A7 and FoCYP655C2; were identified, cloned, and heterologously expressed in Saccharomyces cerevisiae. For the efficient production of ω-OHFAs, the S. cerevisiae was engineered to disrupt the acyl-CoA oxidase enzyme and the β-oxidation pathway inactivated (ΔPox1) S. cerevisiae mutant was generated. To elucidate the significance of the interaction of redox mechanism, FoCYPs were reconstituted with the heterologous and homologous reductase systems - S. cerevisiae CPR (ScCPR) and F. oxysporum CPR (FoCPR). To further improve the yield, the effect of pH was analyzed and the homologous FoCYP-FoCPR system efficiently hydroxylated caprylic acid, capric acid and lauric acid into their respective ω-hydroxy fatty acids with 56%, 79% and 67% conversion. Furthermore, based on computational simulations, we identified the key residues (Asn106 of FoCYP539A7 and Arg235 of FoCYP655C2) responsible for the recognition of fatty acids and demonstrated the structural insights of the active site of FoCYPs.ConclusionFungal CYP monooxygenases, FoCYP539A7 and FoCYP655C2 with its homologous redox partner, FoCPR constitutes a promising catalyst due to its high regio- and stereo-selectivity in the hydroxylation of fatty acids and in the substantial production of industrially valuable ω-hydroxy fatty acids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0228-2) contains supplementary material, which is available to authorized users.

Diverse Metabolic Capacities of Fungi for Bioremediation

“…CYP63A2 P450 monooxygenase from white-rot fungus P. chrysosporium oxidized crude oil aliphatic hydrocarbon n-alkanes, endocrine-disrupting long-chain alkylphenols (APs), mutagenic/carcinogenic fused-ring high molecular weight PAHs (HMW-PAHs) [98]. F. oxysporum CYP monooxygenases were promising catalysts in significant production of xhydroxy fatty acids [13]. Pre-induction of the P450 monooxygenase before application in degradation studies could result in enhanced PAH removal [125].…”

“…The broad substrate specificity of laccase for attacking multiple compounds makes application of recombinant enzyme expression techniques for production of high activity, environmental friendly enzyme desirable. Another such example can be that of the expression of cytochrome P450 monooxygenase from F. oxysporum in Saccharomyces cerevisae [13]. For exploiting the tremendous potential of fungi for azo dye degradation, there is a need for understanding cellular mechanisms for azo dye degradation.…”

“…Mycoremediation is a form of bioremediation in which fungi are used to decontaminate contaminated areas. There has been growing interest in the unique capacity of fungi to degrade such pollutants by employing a variety of extracellular and intracellular enzyme systems including peroxidases and cytochrome P450 respectively for detoxification and biodegradation [13][14][15]. Figure 1 shows the different mechanisms adopted by fungi for bioremediation of toxic, recalcitrant compounds and this review assesses the multifaceted role of fungi in the bioremediation of xenobiotic compounds with reference to features employed by the fungi for detoxification and subsequent bioremediation of toxic waste.…”

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…Furthermore, no diterminal oxidation of nalkanes by AlkB has been reported. Ω-oxidation of medium-chain fatty acids was possible but only to a limited extent 19,66,67,69 .…”

“…Application of Candida tropicalis for direct ω-oxidation of MCFAs is thus not a feasible option. Other CYP450 enzymes are able to ω-oxidize MCFAs, such as CYP153A, FoCYP and CYP450-BM3 monooxygenases [66][67][68][69] . However, those monooxygenases are not 100% selective and/or display low activity on MCFAs.…”