Functional assembly of camphor converting two-component Baeyer–Villiger monooxygenases with a flavin reductase from E. coli

• Published in: Applied microbiology and biotechnology 2014-05, Vol.98 (9), p.3975-3986
• Main Authors: Kadow, Maria, Balke, Kathleen, Willetts, Andrew, Bornscheuer, Uwe T, Bäckvall, J.-E
• Format: Article
• Language: English
• Subjects: Analysis; Baeyer-Villiger monooxygenases; Biocatalysts; Biochemistry; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Camphor; Camphor - metabolism; Catalysis; Coenzymes - metabolism; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - metabolism; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Flavin Mononucleotide - metabolism; Flavin reductase; FMN Reductase - genetics; FMN Reductase - metabolism; Fre; Fusion protein; Gene Expression; Genes; ketones; Life Sciences; Microbial Genetics and Genomics; Microbiology; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Multi-component flavin-dependent monooxygenases; open reading frames; Organic chemistry; Oxidases; Physiological aspects; Polypeptides; Proteins; Pseudomonas putida; Pseudomonas putida - enzymology; Pseudomonas putida - metabolism; Pseudomonas putida NCIMB10007; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Studies
• ISSN: 0175-7598; 1432-0614; 1432-0614
• DOI: 10.1007/s00253-013-5338-3

The major limitation in the synthetic application of two-component Baeyer–Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.