Department of Food Chemistry and Biocatalysis, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland.
Department of Genetics, Plant Breeding and Seed Production, Wroclaw University of Environmental and Life Sciences, Wrocław, Poland.
Microb Cell Fact. 2022 Aug 29;21(1):175. doi: 10.1186/s12934-022-01899-x.
The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast.
Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity.
This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.
酚类化合物（尤其是类黄酮）的区域选择性羟化仍然是经典有机化学的瓶颈，可使用具有高活性和特异性的酶来解决。已知酵母红冬孢酵母 KCh735 可催化黄酮和黄烷酮的 C-8 羟化。该反应涉及酶 F8H（类黄酮 C8-羟化酶），但尚未鉴定出其特定基因。在这项工作中，我们介绍了酵母中第一个 F8H 邻位羟化酶的鉴定、异源表达和特性。
差异转录组分析和与细菌单加氧酶的同源性，包括黄素依赖单加氧酶的 FAD 依赖性基序和 GD 基序，提供了一组编码序列，其中鉴定出 RgF8H。系统发育分析表明，RgF8H 是一种对黄酮类底物具有活性的黄素单加氧酶组的成员。重组蛋白分析表明，该酶催化柚皮素、橙皮素、圣草酚、杨梅素、芹菜素、木犀草素、白杨素、染料木素和 7,4'-二羟基黄酮的 C8-羟化。酶活性需要 C7-OH 基团的存在，表明其为邻位羟化机制。该酶需要 NADPH 辅酶来再生辅基，具有非常低的羟过黄素解偶联率，并且添加 FAD 可显著提高其活性。
本研究鉴定了第一个负责黄酮类化合物（F8H）区域选择性 C8-羟化的酵母羟化酶。对该酶进行了生化表征，并与巨大芽孢杆菌葡萄糖脱氢酶反应进行了体外级联应用。在大肠杆菌中的高体内活性使其能够进一步应用于合成生物学，以生产罕见的高抗氧化化合物。
