Two-component systems for ketone oxidation: type II Baeyer-Villiger monooxygenases.

DIZANZO, MARIA P.; BIANCHI, DARIO A.; RIAL, DANIELA V.; CECCOLI, ROMINA D.; DIZANZO, MARIA P.; BIANCHI, DARIO A.; RIAL, DANIELA V.; CECCOLI, ROMINA D.

San Miguel de Tucuman

Congreso; XII Congreso Argentino de Microbiología General; 2017

Asociación Civil de Microbiología General (SAMIGE)

The Baeyer-Villiger biooxidation of ketones is a valuable reaction for the preparation of esters or lactones in both academic and industrial fields. Baeyer-Villiger monooxygenases (BVMOs) are enzymes that catalyze the insertion of one atom from molecular oxygen into the substrate, whereas the other atom is reduced to water at the expense of NAD(P)H. In particular, type II BVMOs are multicomponent enzymes formed by a flavin reductase that reduces FMN in a NADH-dependent manner and a monooxygenase subunit that oxidizes the substrate at the expense of reduced FMN. These enzymes are interesting systems for biocatalytic applications due to their ability to accept bicyclic ketones as substrates and to use NADH instead of the more expensive NADPH. In this work, we identified one gene coding for a putative monooxygenase component in the genomes of Bradyrhizobium diazoefficiens and Mycobacterium tuberculosis by bioinformatic analysis. The retrieved proteins shared 31-35 % sequence identity with other monooxygenase components of type II BVMOs from Pseudomonas putida previously reported [1]. A similar analysis was performed for the putative flavin reductase genes using the sequences of known reductases as queries for blast searches. Several genes were retrieved and we selected those that located near to the identified monooxygenase genes in these genomes and encoded proteins with sequence homology to the flavin reductase Fred from P. putida [1]. We cloned each pair of selected genes coding for the monooxygenase and reductase subunits and expressed them in Escherichia coli. The flavin reductase components were soluble whereas both monooxygenase components were obtained in the insoluble fractions. In order to overcome this drawback, different expression conditions were assayed. We tested diverse induction temperatures, inducer concentrations, culture media, host E. coli strains and the co-expression of molecular chaperones. Then, we evaluated the biocatalytic activity of these BVMOs by biotransformations in recombinant whole-cell systems (growing and/or resting cells) with the ketone (±)‐cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one as a model substrate. Our results indicated that the rhizobial type II BVMO was not active under the assayed conditions and the type II BVMO from M. tuberculosis was able to oxidize this fused ketone. Funded by ANPCyT, CONICET, UNR.[1] Iwaki,H, Grosse, S, Bergeron, H, Leisch, H, Morley, K, Hasegawa, Y, Lau, PCK (2013).Appl Environ Microbiol, 79, 3282‐3293.