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

Abstract

Background: Omega 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.

Results: To 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.

Conclusion: Fungal 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.

Figure 1

Reaction scheme of omega hydroxylation of fatty acids by Fusarium oxysporum cytochrome P450 monooxygenases ( FoCYP ) with the heterologous ( ScCPR ) and homologous ( FoCPR ) reductases. FoCYP539A7 can hydroxylate caprylic acid (C8), capric acid (C10) and lauric acid (C12) into their respective ω-hydroxy fatty acids, whereas FoCYP655C2 can hydroxylate only capric acid and lauric acid.

Figure 2

CO Binding analysis of microsomes of FoCYP539A7 and FoCYP655C2 expressed in S. cerevisiae. The solid line represents FoCYP539A7 and the dotted line represents FoCYP655C2. Yeast expression was carried out in S. cerevisiae cells using 4% galactose, 2 mM 5-ALA at 30°C.

Figure 3

Schematic representation of the strategy used to disrupt pox1 gene of S. cerevisiae INV S c1 by PCR-mediated short-region homologous recombination. The HisMX cassette was used to replace the pox1gene. The double alleles of pox1 are replaced by the HisMX auxotrophic marker through homologous recombination

Figure 4

Reaction profiles of hydroxylation of fatty acids by FoCYP539A7 and FoCYP655C2 with the heterologous ( ScCPR ) reductase. ΔPox1 mutant S. cerevisiae cells harboring the CYP39A7-ScCPR and CYP655C2-ScCPR reconstituted systems were induced with 4% galactose, 2 mM 5-ALA and 500 μM of substrates: caprylic acid (C8), capric acid (C10) and lauric acid (C12) were added and cultured at pH 7.0. Samples collected at 10 hr intervals were extracted, trimethylsilyl derivatized and analyzed by GC.

Figure 5

5 Significance of homologous FoCYP-FoCPR reconstituted system in the hydroxylation of fatty acids. (A) Reaction profile of hydroxylation of fatty acids by FoCYP539A7 and FoCYP655C2 with the homologous (FoCPR) reductase. (B) Comparative analysis on the catalytic conversion of fatty acids by FoCYP539A7 and FoCYP655C2 with the heterologous (ScCPR) and homologous (FoCPR) reductases. Data were plotted from the 50 hr biotransformation reaction samples. ΔPox1 mutant S. cerevisiae cells harboring the CYP539A7-FoCPR, CYP655C2-FoCPR, CYP39A7-ScCPR and CYP655C2-ScCPR reconstituted systems were induced with 4% galactose, 2 mM 5-ALA and 500 μM of substrates: caprylic acid (C8), capric acid (C10) and lauric acid (C12) were added and cultured at pH 7.0. Samples collected at 10 hr intervals were extracted, trimethylsilyl derivatized and analyzed by GC.

Figure 6

Final yield (mg/L) of ω-hydroxy fatty acids by FoCYP539A7 and FoCYP655C2 with the homologous reductase ( FoCPR ) in the biotransformation carried out at pH 5.5 and pH 7.0. Data were plotted from the 50 hr biotransformation reaction samples. ΔPox1 mutant S. cerevisiae cells harboring the CYP539A7-FoCPR and CYP655C2-FoCPR reconstituted systems were induced with 4% galactose, 2 mM 5-ALA and 500 μM of substrates: caprylic acid (C8), capric acid (C10) and lauric acid (C12) were added and cultured at pH 5.5 and pH 7.0. Samples collected at 10 hr intervals were extracted, trimethylsilyl derivatized and analyzed by GC.

Figure 7

Superimposition of docked complexes of fatty acids in the active site of FoCYP s. (A) Superimposition of docked complexes of caprylic acid (cyan stick), capric acid (blue stick), and lauric acid (green stick) in the active site of FoCYP539A7 (pink ribbons). Fatty acids show hydrogen bond interaction with Asn106 of FoCYP539A7 and the ω carbon faces towards the ferric atom of heme. (B) Superimposition of docked complexes of capric acid (blue stick), lauric acid (green stick) and myristic acid (orange sticks) in the active site of FoCYP655C2 (cyan ribbons). Fatty acids show hydrogen bond interaction with Arg235 of FoCYP539A7 and the ω carbon faces towards the ferric atom of heme except myristic acid. The oxygen and nitrogen are represented in red and blue, and heme is represented as red sticks.