甘草酸生物合成途径中糖基转移酶的挖掘及产甘草酸的酵母细胞工厂的构建

Identification of genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to understanding of the biosynthetic and medicinal chemistry of this compound, which provides possibility of high level production of glycyrrhizin by biotechnological method or by synthetic biology. All genes but not glycosyltransferase which catalyzes the formation of glycyrrhizin from glycyrrhetic acid involved in the glycyrrhizin biosynthetic pathway were found and verified in the previous studies. Based on this background, the project plans to identify the unknown glycosyltransferase which is essential for revealing the complete glycyrrhizin biosynthesis pathway. The project also plans to metabolically engineer a glycyrrhizin-producing Saccharomyces cerevisiae which aims to balance the contradiction between the huge demand and the shortage supply of glycyrrhizin. Based on our previous constructed high-quality cDNA library of Glycyrrhiza uralensis and the EST analysis, novel candidate genes encoding glycosyltransferases could be provided. The target gene can be identified and verified by in vitro and in vivo enzyme activity assay. Efficient glycyrrhizin pathway was constructed using the modular pathway engineering (MOPE) approach. By adding promoters and terminators into genes which encode β-amyrin synthase(β-AS), CYP88D6,CYP72A154, CPR1 and the identified glycosyltransferase, glycyrrhizin pathway could be constructed. Heterologous production of glycyrrhizin can further be realized by assembling glycyrrhizin pathway into Saccharomyces cerevisiae. Further, the tHMG1 module for expressing the catalytic domain of hydroxy-3-methylglutaryl coenzyme A (tHMG1) will up-regulated to enhance the amount of 2,3-oxidosqualene which is the precursor of β-amyrin. Lanosterol synthase gene(ERG7) will be deleted by PCR-mediated seamless gene deletion technology to divert the metabolic flux from 2,3-oxidosqualene to β-amyrin and then to glycyrrhizin. These strategies thus could improve the fermentative concentration of glycyrrhizin. If this project shall be supported fortunately, the last unknown gene which catalyzes the formation of glycyrrhizin from glycyrrhetic acid will be clear and the efficient heterologous production of glycyrrhizin will be achieved by the engineered S. cerevisiae in this project.

当前随着甘草应用范围的扩大，其供需矛盾十分突出。从基因水平上阐明甘草酸形成的分子机制，利用生物技术改造次生代谢途径提高甘草酸含量或通过代谢工程大规模产甘草酸为解决这一问题提供新策略。甘草中参与甘草酸合成的糖基转移酶基因是其合成途径中最后一个未知关键基因。本研究提出挖掘甘草酸合成途径中的糖基转移酶基因，利用其和已知其它调控基因元件，构建产甘草酸酵母菌株，实现微生物发酵产甘草酸。以实验室前期甘草转录组数据为基础，通过生物信息学分析筛选糖基转移酶候选基因，利用酵母表达体系进行体外催化鉴定和体内酶活验证。构建β-香树脂醇(β-AS),CYP88D6,CYP72A154及所挖掘糖基转移酶基因表达簇，转入酵母细胞构建产甘草酸酵母菌株。通过基因删除、提高限速基因表达等代谢调控增加到甘草酸的代谢流，实现酵母发酵大规模生产甘草酸。该研究对甘草酸分子合成机制解析和甘草酸异源合成有着重要的科学意义和工程价值。

甘草酸前体物质甘草次酸，一种齐墩果烷型五环三萜类皂苷，是甘草最主要的活性成分之一。甘草次酸是一种甜味剂，在皮肤炎症方面有着很好的药效活性，同时广泛用于化妆品、日化用品等生产。由于甘草次酸良好的药用价值和广阔的应用领域，国内外对甘草的需求急剧增长。然而，作为多年生草本植物，甘草生长周期较长，野生甘草资源日益匮乏，已被国家明令禁止采挖。而人工栽培甘草品质退化、质量层次不齐、甘草酸含量低。目前甘草酸主要是从甘草中分离提取得到，而甘草中甘草酸含量低，以甘草为资源的提取分离造成了甘草资源的减少和生态破坏。化学法合成甘草次酸还无法实现。因此本项目通过工业微生物发酵大规模生产甘草次酸为甘草次酸的原料供应提供新的技术方法。.本项目建立了酵母crispr-cas9的基因编辑技术，通过该技术实现酵母基因组的删除和启动子替换等技术操作，建立了多片段酵母体内重组技术，基于该技术实现多个片段在酵母染色体上的整合；2. 构建了甘草酸合成途径的三萜骨架β-Amyrin的酿酒酵母细胞，浓度达到158 mg/L； 3. 构建了产甘草次酸的直接前体11-OXO-β-Amyrin的酿酒酵母细胞，摇瓶发酵水平达到15 mg/L; 4 构建了产甘草次酸的酿酒酵母细胞，通过删除竞争性途径基因ERG27和BTS1等基因，同时过表达甘草次酸合成MVA途径的多个关键基因HMGR等基因，使得酵母产甘草次酸的浓度提高600多倍，达到8mg/L。 .依托本项目实现了通过酵母细胞发酵合成甘草次酸，尽管浓度较低，但因为甘草次酸价格较贵，且目前来源主要基于提取甘草酸之后酸水解催化获得，因此通过本项目取得的进展具备较大的应用前景，但目前存在主要问题CYP88D6和CYP72A154两个基因在酵母细胞表达较低，造成酵母产甘草次酸浓度很低，因此需要进一步优化提高产量，达到工业化前景。