Research Group BIONUC (Biotechnology of Nutraceuticals and Bioactive Compounds), Departamento de Biología Funcional, Área de Microbiología, Universidad de Oviedo, Oviedo, Principality of Asturias, Spain.
IUOPA (Instituto Universitario de Oncología del Principado de Asturias), Oviedo, Principality of Asturias, Spain.
Microb Cell Fact. 2024 Oct 5;23(1):265. doi: 10.1186/s12934-024-02541-8.
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.
类黄酮是一类具有多种生物活性的重要植物化学物质。在类黄酮和其他多酚的生物合成过程中添加甲基可增强其生物活性并提高其稳定性。在我们研究小组的先前研究中，我们在白色链霉菌 J1074 中检测到一种新型的类黄酮 O-甲基转移酶活性，该酶可导致橙皮素在喂食培养物中异源生物合成同系物橙皮素。在这项研究中，我们通过构建基因 XNR_0417 的敲除突变体来确定负责生成这种甲基化类黄酮的 O-甲基转移酶，该基因是使用玉米咖啡酸 3'-O-甲基转移酶的序列对白色链霉菌 J1074 的基因组进行 Blast 分析后选择的。该突变株，白色链霉菌 ∆XNR_0417，在橙皮素喂食后不再能够产生橙皮素。随后，我们对突变株进行了遗传互补，以确认 XNR_0417 编码的酶负责观察到的 O-甲基转移酶活性。该新菌株，白色链霉菌 SP43-XNR_0417，不仅能够从橙皮素生产橙皮素，而且还能够在其他黄酮、黄酮醇和芪类化合物上生产不同的单、二、三、四甲基化衍生物，显示出广泛的底物灵活性。此外，使用先前在体内测试过的底物进行了体外实验，证明了 XNR_0417 无疑有能力生成各种甲基化衍生物。
