Abstract
Flavonoids are a large and important group of phytochemicals with a great variety of bioactivities. The addition of methyl groups during biosynthesis of flavonoids and other polyphenols enhances their bioactivities and increases their stability. In a previous study of our research group, we detected a novel flavonoid O-methyltransferase activity in Streptomyces albidoflavus J1074, which led to the heterologous biosynthesis of homohesperetin from hesperetin in feeding cultures. In this study, we identify the O-methyltransferase responsible for the generation of this methylated flavonoid through the construction of a knockout mutant of the gene XNR_0417, which was selected after a blast analysis using the sequence of a caffeic acid 3'-O-methyltransferase from Zea mays against the genome of S. albidoflavus J1074. This mutant strain, S. albidoflavus ∆XNR_0417, was no longer able to produce homohesperetin after hesperetin feeding. Subsequently, we carried out a genetic complementation of the mutant strain in order to confirm that the enzyme encoded by XNR_0417 is responsible for the observed O-methyltransferase activity. This new strain, S. albidoflavus SP43-XNR_0417, was able to produce not only homohesperetin from hesperetin, but also different mono-, di-, tri- and tetra-methylated derivatives on other flavanones, flavones and stilbenes, revealing a broad substrate flexibility. Additionally, in vitro experiments were conducted using the purified enzyme on the substrates previously tested in vivo, demonstrating doubtless the capability of XNR_0417 to generate various methylated derivatives.