【作者】 马斌；

【导师】 王风清；

【作者基本信息】 华东理工大学 ， 生物化工， 2019， 硕士

【摘要】 随着代谢工程和合成生物学的迅猛发展,利用微生物异源合成天然产物引起了极大关注。广藿香醇是一种自广藿香提取的天然倍半萜化合物,具有重要的经济价值,广泛应用于香水和化妆品的定香剂,也具有神经保护、抗炎和抗癌等医药作用,有旺盛的市场需求。酿酒酵母是合成天然产物的重要宿主,本课题利用酿酒酵母自身的甲羟戊酸代谢途径构建了广藿香醇的合成路径,获得了高效生产广藿香醇的酿酒酵母细胞。首先,在酿酒酵母中构建了广藿香醇的合成途径。通过pESC-URA高拷贝质粒引入广藿香来源经密码子优化的广藿香醇合成酶基因,该基因编码一种焦磷酸金合欢酯(FPP)环化酶,催化FPP生产广藿香醇,在酿酒酵母甲羟戊酸途径的基础上成功构建了广藿香醇合成途径,产量为1.02mg/L。随后,为了提高广藿香醇的产量,对广藿香醇合成路线进行了优化改造。1.为了增加广藿香醇合成酶对前体FPP的利用,采取了基因融合策略,把FPP合成酶和广藿香醇合成酶基因进行了融合表达,使得广藿香醇产量提升了一倍,达到了 2.01mg/L。2.在酵母基因组上整合了tHMGR、IDI1、upc2-1基因,对酿酒酵母中甲羟戊酸途径进行了强化,随后,使用葡萄糖浓度相关型启动子PHXTI替换原ERG9基因的启动子,弱化了酵母原有FPP的主要代谢路径—麦角甾醇合成路线提升了广藿香醇的生产能力,产量达到了 59.16 mg/L。3.为了进一步减少FPP的其它代谢消耗路线,删除与法尼醇合成有关的基因DPP1、LPP1,降低了 FPP向法尼醇的转化,但是,广藿香醇的产量未能得到提高。随后,对构建成功的工程菌REL003-FPTS菌株进行了发酵放大,使广藿香醇的产量达到了 466.81 mg/L,远高于已知文献所报道的最高产量。此外,为了增加广藿香醇的水溶性,降低广藿香醇的挥发性,对广藿香醇糖基化反应进行了探索。更多还原

【Abstract】 With the rapid development of metabolic engineering and synthetic biology,heterologous biosynthesis of natural products in microorganism caused great attention.Patchoulol,extracted from patchouli oil,is a natural sesquiterpene compound,has important economic value and is widely used in perfumes and cosmetics.It also has neuroprotective,anti-inflammatory,anti-cancer activities.Saccharomyces cerevisiae is an important platform organism for the synthesis of natural products.This study constructed the synthesis of patchoulol in S.cerevisiae,and constructed recombinant strain which produced high-yield patchoulol.Firstly,the synthetic pathway of patchouli alcohol was constructed.Patchoulol synthase is a kind of famesyl pyrophosphate(FPP)cyclases which catalyzes the formation of patchoulol by FPP,was codon-optimized and introduced into Saccharomyces cerevisiae by high-copy plamid pESC-URA,resulting in the synthesis of patchoulol,approximately 1.02mg/L.In order to increase the yield of patchouli alcohol,metabolic engineering strategy was adopted to optimize the synthetic pathway of patchoulol.1.In order to increase the utilization of precursor FPP,a gene fusion strategy was adopted.The fusion of farnesyl diphosphate synthase and patchoulol synthase increased the production of patchoulol up to to 2.01mg/L.2.The gene tHMGR,IDI1,and upc2-1 were integrated into the yeast genome to enhance the rate-limiting step of the mevalonate pathway in Saccharomyces cerevisiae,and ERG9 was weaked by glucose concentration-related promoter PHXT1 to reduce metabolic flux from FPP to ergosterol,which increased the yield of patchoulol to 59.16 mg/L.3.The genes DPP1,LPP1,related to the synthesis of farnesol,was knocked out,resulting in the decrease of farnseol,but the yield of patchoulol has no increase.The well-characterized strain REL003-FPTS was amplified in a 5-liter bioreactor,two-phase fermentation and medium-feeding strategy were used to achieve a high production of patchoulol to 466.81 mg/L,which was much higher than the reported up to date.Morever,in order to increase the water-solubility and reduce the volatility of patchoulol,the glycosylation reaction of patchoulol was also explored.更多还原