Exploring cryptic Baeyer-Villiger monooxygenases from fungi

FERRONI F; TOLMIE C; SMIT M; OPPERMAN DJ

Manchester

Congreso; Biotrans 2013; 2013

Baeyer-Villiger monooxygenases (BVMOs) are flavoenzymes that catalyze the oxidation of linear and cyclic ketones to esters or lactones, respectively, using NAD(P)H and molecular oxygen[1]. Although Baeyer-Villiger oxidation reactions have been observed in whole cells of Fusarium, Penicillium and Aspergillus species, with the exception of a BVMO from Cylindrocarpon radicicola, all of the BVMOs cloned and heterologously expressed in E. coli to date have been from bacteria[2]. Aspergillus flavus is a filamentous fungus ubiquitous in the environment that produces mycotoxins called aflatoxins, as secondary metabolites. Within the aflatoxin biosynthesis pathway, the BVMO moxY is responsible for conversion of versicolorone to versiconol acetate, or hydroxyversicolorone to versiconal hemiacetal acetate[3]. In addition to moxY, A. flavus genome mining has revealed another 25 putative BVMOs. cDNA sequencing of A. flavus grown under standard laboratory culture conditions, shows that none of the BVMO genes are expressed. Similarly, whole-cell biocatalysis of various ketones using resting-cells showed only alcohol production from ketone reduction by native KRED/ADHs. In an attempt to induce these silent/cryptic BVMOs, A. flavus were grown using different growth media, in the presence of various substrates as well as euchromatin promotors (small molecule epigenetic modifiers). BVMO activity towards 2-methylcyclohexanone could be induced using these methods. Multiple alignment and phylogenetic relationships of the 26 A. flavus BVMOs with well characterized BVMOs, grouped the sequences into 6 distinct clusters. Cluster V contains 4 A. flavus BVMOs related to ACMO from Comamonas sp. Cloning and heterologous expression in E. coli yielded soluble active enzymes. Biotransformations of various cyclic, bicyclic, aromatic and linear ketones with recombinant E. coli whole-cells expressing the various BVMOs revealed functional divergence between these closely related/clustered BVMOs. Similarly, differences between heteroatom oxidation were observed during sulfoxidation, where either the sulfoxide or sulfone could be produced. [1] H. Leisch, K. Morley, P. C. K. Lau, Chemical reviews 2011, 111, 4165?222. [2] F. Leipold, R. Wardenga, U. T. Bornscheuer, Applied microbiology and biotechnology 2012, 94, 705?17. [3] Y. Wen, H. Hatabayashi, H. Arai, H. K. Kitamoto, K. Yabe, Applied and environmental microbiology 2005, 71, 3192?8.