操纵茶树类黄酮3'-羟基化酶生物合成B环-3',4'-二羟基黄酮类化合物

Engineering of a flavonoid 3'-hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3',4'-dihydroxylated flavones

摘要:

[目的]操纵茶树类黄酮3'-羟基化酶，生物合成B环-3'，4'-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。[方法]构建了4个茶树类黄酮3'-羟基化酶基因（CsF3'H）和拟南芥的P450还原酶基因（ATR）融合表达质粒：SUMO-CsF3'H[7-517]：：ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]：：ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]：：ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]：：ATR2[75-711]3 AA，分别转化大肠杆菌菌株TOP10、DH5α和BL21，获得12个转化菌株S1-S12；构建了茶树类黄酮3'-羟基化酶基因CsF3'H表达质粒pYES-Dest52-CsF3'H，转化酵母菌株WAT11，得到转化菌株S13；构建了茶树类黄酮3'-羟基化酶基因CsF3'H表达质粒pES-URA-CsF3'H，及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因AtFLS的融合表达质粒pES-HIS-CsF3H：：AtFLS 9AA，二者共转化酵母菌株WAT11，获得转化菌株S14。[结果]转化SUMO-CsF3'H[28-517]：：ATR1[49-688]3 AA质粒的TOP10菌株S6在25℃条件下发酵，转化效率最高，能将1000 μmol/L柚皮素、二氢山奈酚和山奈酚，分别转化生成287.93 μmol/L圣草酚、131.76 μmol/L二氢槲皮素和188.62 μmol/L槲皮素。发酵菌株S13能分别将1000 μmol/L柚皮素、二氢山奈酚和山奈酚，最多能转化生成734.32 μmol/L圣草酚、446.07 μmol/L二氢槲皮素和594.64 μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素，在发酵36-48 h中，最多能生成1412.16 μmol/L圣草酚、490.25 μmol/L山奈酚、445.75 μmol/L槲皮素、66.75 μmol/L二氢槲皮素和73.50 μmol/L二氢山奈酚。[结论]本研究首次将茶树类黄酮3'-羟基化酶基因应用于B环-3'，4'-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。

Abstract:

[Objective] A flavonoid 3'-hydroxylase from tea plant was engineered to synthesize B-3',4'-dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin. [Methods] Four articifical P450 constructs harboring both flavonoid 3'-hydroxylase gene from Camellia sinensis (CsF3'H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pYES-Dest52-CsF3'H harboring CsF3'H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pES-HIS-CsF3H::AtFLS 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pES-URA-CsF3'H and pES-HIS-CsF3H::AtFLS 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14. [Results] Strain S6 generated highest bioconversion efficiency at 25℃ among all E. coli strains during 24 h fernentation. Supplemented with 1000 μmol/L naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were 734.32 μmol/L, 446.07 μmol/L and 594.64 μmol/L respectively. Supplemented with 5 mmol/L naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were 1412.16 μmol/L, 490.25 μmol/L, 445.75 μmol/L, 66.75 μmol/L and 73.50 μmol/L during 36-48 h fermentaion respectively. [Conclusion] CsF3'H was engineered for biosynthesis of B-3',4'-dihydroxylated flavone.