Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A
Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A
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DOI：
10.1104/pp.19.00254
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发表时间：
2019-07-01
期刊：
影响因子：
7.4
通讯作者：
Bohlmann, Joerg
中科院分区：
文献类型：
--
作者：
Irmisch, Sandra;Ruebsam, Henriette;Bohlmann, Joerg
植物代谢产物Montbretin A(MBA)及其前体mini-MBA是治疗2型糖尿病的潜在新药。这些复杂的酰化黄酮醇糖苷仅少量存在于观赏植物蒙特布里亚(Crocosmia X CroCosmiiflora)的球茎中。我们的目标是利用蒙特伯里亚基因通过代谢工程来改造本氏烟草，以增加迷你MBA和MBA的产量。两个蒙特布莱亚UDP依赖的糖基转移酶(UGT)CcUGT1和CcUGT2催化MBA生物合成的前两个途径特异性中间体的形成，杨梅素3-O-鼠李糖苷和杨梅素3-O-葡萄糖鼠李糖苷。在以前的工作中，这些UGT在N.benthamiana中的表达导致了少量的山奈酚糖苷，而不是杨梅素糖苷，这表明杨梅素是有限的。在这里，我们研究了蒙脱氏菌基因和黄酮醇生物合成的酶，以促进杨梅素的形成。我们鉴定了两种黄酮羟基酶，一种是黄酮醇合成酶，一种是类黄酮3‘-羟基酶(F3’H)，另一种是类黄酮3‘5’-羟基酶(F3‘5’H)。蒙特布里亚黄酮醇合成酶将二氢杨梅素转化为杨梅素。出乎意料的是，在细胞色素P450的细胞色素P45B亚家族中，蒙脱氏菌F3‘5’H与F3‘HS的序列相关性比与已知F3’5‘H活性的序列更高。在M.benthamiana中瞬时表达蒙氏黄酮醇生物合成基因和蒙脱酵母MYB转录因子的组合导致杨梅素可用于MBA的生物合成。在N.benthamiana中瞬时共表达蒙氏黄酮醇生物合成基因与CcUGT1和CcUGT2相结合，产生了2 mg g(-1)的MBA途径特异性化合物杨梅素3-O-葡萄糖鼠李糖苷。另外，Montbretia酰基转移酶CcAT1的表达导致了在N.benthamiana中可检测到的mini-MBA水平。
The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana. We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g(-1) fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.