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

Microbial Cell Factories

Research

Saravanan Prabhu Nadarajan¹  Hyungdon Yun¹  Hyun Ho Park²  Pradeepraj Durairaj²  Pyung-Gang Lee³  Eunok Jung³  Byung-Gee Kim³  Sailesh Malla⁴ 

[1] Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea;School of Biotechnology, Yeungnam University, Gyeongsan, South Korea;School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea;School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea;Current position: Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Copenhagen, Denmark;

关键词: Cytochrome P450;    Cytochrome P450 reductase;    Omega fatty acid hydroxylase;    cDNA gene cloning;    Heterologous expression;    Saccharomyces cerevisiae;   

DOI  :  10.1186/s12934-015-0228-2

 received in 2014-12-06, accepted in 2015-03-12,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Durairaj et al.; licensee BioMed Central. 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.