[从茶树(Camellia sinensis)中提取用于生物合成B-3′,4′-二羟基黄酮的类黄酮3′-羟化酶工程]。

微生物学报 Pub Date : 2017-03-04
Tianshan Zhou, Youben Yu, Bin Xiao, Lu Bao, Yuefang Gao
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引用次数: 0

摘要

目的:利用茶树类黄酮3′-羟化酶合成B-3′,4′-二羟化黄酮,如戊二醇、二氢槲皮素和槲皮素。方法:将含有茶树类黄酮3′-羟化酶基因(csf3′h)和拟南芥类P450还原酶基因(ATR1或ATR2)的4个人工P450构建体导入大肠杆菌菌株TOP10、DH5α和BL21,得到工程菌株S1 ~ S12。将携带CsF3'H基因的质粒pYES-Dest52-CsF3'H引入酵母(Saccharomyces cerevisiae WAT11,菌株S13)。将山茶黄酮3-羟化酶基因(CsF3H)与拟南芥黄酮醇合成酶基因(AtFLS)融合构建质粒ps - his -CsF3H::AtFLS 9aa。然后将质粒pES-URA-CsF3'H和pES-HIS-CsF3H::AtFLS 9aa共同导入酵母S. cerevisiae WAT11,命名为菌株S14。结果:菌株S6在25℃发酵24 h时生物转化效率最高。在柚皮素、二氢山奈酚和山奈酚添加量为1000 μmol/L的条件下,菌株S13产生的碘二醇、二氢槲皮素和槲皮素的最大产量分别为734.32 μmol/L、446.07 μmol/L和594.64 μmol/L。在柚皮素添加量为5 mmol/L的条件下,菌株S14发酵36 ~ 48 h时,产生的最大蒽醌、山奈酚、槲皮素、二氢槲皮素和二氢山奈酚的量分别为1412.16 μmol/L、490.25 μmol/L、445.75 μmol/L、66.75 μmol/L和73.50 μmol/L。结论:CsF3'H用于B-3',4'-二羟基黄酮的生物合成。
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[Engineering of a flavonoid 3'-hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3',4'-dihydroxylated flavones].

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.

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期刊介绍: Acta Microbiologica Sinica(AMS) is a peer-reviewed monthly (one volume per year)international journal,founded in 1953.It covers a wide range of topics in the areas of general and applied microbiology.The journal publishes original papers,reviews in microbiological science,and short communications describing unusual observations. Acta Microbiologica Sinica has been indexed in Index Copernicus (IC),Chemical Abstract (CA),Excerpt Medica Database (EMBASE),AJ of Viniti (Russia),Biological Abstracts (BA),Chinese Science Citation Database (CSCD),China National Knowledge Infrastructure(CNKI),Institute of Scientific and Technical Information of China(ISTIC),Chinese Journal Citation Report(CJCR),Chinese Biological Abstracts,Chinese Pharmaceutical Abstracts,Chinese Medical Abstracts and Chinese Science Abstracts.
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