基于尿苷二磷酸葡萄糖原位再生构建酵母合成红景天苷体系的研究

Salidroside (8-O-β-D-glucoside of tyrosol) is an important plant secondary metabolite in Rhodiola and has been demonstrated to have adaptogenic capacity including anti-cancer, anti-hypoxia, anti-diabetic and so on. In order to overcome the drawbacks of obtaining salidroside from plant resources and chemical synthesis, microbial synthesis of salidroside is gaining considerable interesting. Researchers have mainly focused on improving of the tyrosol biosynthesis and its corresponding UDP-glycosyltransferases (UGTs) expression in recombinant microorganism, but ignored the regeneration of uridine-diphosphate-glucose (UDPG). In this project, we will choose yeast, which have the ability of tyrosol biosynthesis, as host. Rhodiola cDNA library will be constructed using SMART (Switching Mechanism At 5' end of the RNA Transcript) technology, followed by high throughput screening for candidate genes of UGT and SuSy（sucrose synthase）. A series of fusion protein will be constructed with the codon-optimized candidate genes. Recombinant yeast with the fusion proteins will be screened to establish an in situ regeneration of UDP-glucose pathway for salidroside biosynthesis. Theoretical principles and key technologies are expected to be provided for microbial salidroside production with the established system of salidroside biosynthesis in yeast and its recommend regulation proposals based on the analysis of transcription data and metabolic flux.

红景天苷（酪醇8-O-β-D-葡萄糖苷）是红景天属植物特征次生代谢产物，具有抗肿瘤、耐缺氧、降血糖等重要功效。为克服依靠植物资源和化学合成技术获取红景天苷的不足，人们开始致力于微生物合成法的研究，但主要集中于苷元酪醇合成途径和UDP-葡萄糖基转移酶（UGTs）的表达，却忽略了尿苷二磷酸葡萄糖（UDPG）的再生。本项目拟通过UGT和SuSy（蔗糖合酶）的融合表达以构建UDPG再生途径：选择已具有酪醇合成能力的酵母为宿主，利用SMART技术建库和高通量筛选法获取红景天UGT和SuSy候选基因；采用候选UGT和SuSy的优化序列构建其融合基因的酵母表达体系；通过全细胞催化验证和筛选鉴定，建立基于UDPG原位再生的酵母合成红景天苷体系。基于重组菌转录组和代谢流分析，初步探索体系的调控机理，为实现微生物法高效合成红景天苷提供理论基础和关键技术。

红景天苷（酪醇8-O-β-D-葡萄糖苷）是红景天属植物特征次生代谢产物，具有抗肿瘤、耐缺氧、降血糖等重要功效。人们在研究微生物法红景天苷时主要集中于苷元酪醇合成途径和UDP-葡萄糖基转移酶（UGTs）的表达，却忽略了尿苷二磷酸葡萄糖（UDPG）的再生。本项目进行了基于尿苷二磷酸葡萄糖原位再生构建酵母合成红景天苷体系的研究。构建了2套酵母UDPG再生合成红景天苷体系，分别为基于蔗糖合酶AtSUS1和糖基转移酶RsUGT72B14的UDPG原位再生实现重组酿酒酵母BY4742合成红景天苷和基于焦磷酸化酶ScGalU和RsUGT72B14的UDPG原位再生实现重组酿酒酵母W303合成红景天苷。研究采用密码子优化方法实现了重组基因的高效表达、应用底物流加调控策略进一步提高了重组菌红景天苷产量。结果表明，构建UDPG原位再生体系可以有效提高重组酿酒酵母红景天苷合成效率，最大产量可达29.26mg/L，为揭示红景天苷合成调控机理和实现微生物法高效合成技术的转化应用提供了基础。