TWO-COMPONENT SYSTEMS FOR KETONE OXIDATION: TYPE II BAEYER-VILLIGER MONOOXYGENASES

ROMINA D CECCOLI, MARÍA P DIZANZO, DARIO A BIANCHI, DANIELA V RIAL

San Miguel de Tucuman

Congreso; Congreso Argentino de Microbiología General SAMIGE; 2017

The Baeyer-Villiger biooxidation of ketones is a valuable reaction for the preparation of esters orlactones in both academic and industrial fields. Baeyer-Villiger monooxygenases (BVMOs) areenzymes that catalyze the insertion of one atom from molecular oxygen into the substrate, whereasthe other atom is reduced to water at the expense of NAD(P)H. In particular, type II BVMOs aremulticomponent enzymes formed by a flavin reductase that reduces FMN in a NADH-dependentmanner 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 acceptbicyclic 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 ofBradyrhizobium diazoefficiens and Mycobacterium tuberculosis by bioinformatic analysis. Theretrieved proteins shared 31-35 % sequence identity with other monooxygenase components of type IIBVMOs from Pseudomonas putida previously reported [1]. A similar analysis was performed for theputative flavin reductase genes using the sequences of known reductases as queries for blastsearches. Several genes were retrieved and we selected those that located near to the identifiedmonooxygenase genes in these genomes and encoded proteins with sequence homology to the flavinreductase Fred from P. putida [1]. We cloned each pair of selected genes coding for themonooxygenase and reductase subunits and expressed them in Escherichia coli. The flavin reductasecomponents were soluble whereas both monooxygenase components were obtained in the insolublefractions. In order to overcome this drawback, different expression conditions were assayed. Wetested diverse induction temperatures, inducer concentrations, culture media, host E. coli strains andthe co-expression of molecular chaperones. Then, we evaluated the biocatalytic activity of theseBVMOs by biotransformations in recombinant whole-cell systems (growing and/or resting cells) withthe ketone (±)-cis-bicyclo[3.2.0]hept-2-en-6-one as a model substrate. Our results indicated that therhizobial 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.