葡萄内生菌合成白藜芦醇的代谢途径构建与功能基因解析

Resveratrol is a natural plant polyphenol having multiple functions. Microbial fermentation has great potential in resveratrol production. Alternaria sp. MG1 is an endophytic fungus previously isolated from grape fruit. It has stable capability in metabolizing glucose into resveratrol in high speed, and thus has good potential in constructing genetic modified resveratrol producing strains. However, the information on the resveratrol biosyntheis pathway and key genes in very limited. In out study, gene functaion annotation of the whole genomes of Alternaria sp. MG1 will be carried out to obtain the outline of resveratrol biosyntheis pathway. Then, the pathway will be confirmed by consulting to the substrate flows in the pathway obtained by using C isotope labelling method, and all genes involved in the pathway will be identified. After that, influence of precursor and intermediates addition on substrate flow, gene expression, and enzyme activies was investigated, and the key step and related genes was identified accordingly. Finally, RNAi technique will be used to identify the function of the obtained key genes in resveratrol pathway by referring to the change of substrate flows and resveratrol production. At the end, the revseratrol biosynthesis pathway will be identified and illustrated in the levels of substate flows and gene expression, and the key genes and their functions will also be illustrated.It is expected to provide new gene sources for construction of resveratrol producing strain. It is special in using grape endophytic fungi to produce resveratrol and has novel value in illustrating microbial resveratrol biosynthesis pathway and related genes.

白藜芦醇是有多种功能活性的植物多酚和膳食补充剂。微生物发酵法生产白藜芦醇有着重要的产业地位。葡萄内生菌链格孢属MG1能够稳定、快速地将葡萄糖转化为白藜芦醇，在生产菌株的基因改造方面有很好的应用前景，但有关该菌中白藜芦醇合成途径和关键基因并不清楚。项目在基因组和转录组测序的基础上，通过基因组注释和基因组规模代谢网络模型构建，获得大致的合成途径；再结合C同位素标记前体的代谢物跟踪分析，准确解析出途径中代谢流与相关基因；然后根据前体和中间产物添加对途径中代谢流和基因表达的影响，确定关键限速步骤与基因；最后用RNAi技术抑制关键基因表达，根据其对白藜芦醇产量和途径中代谢流的影响，阐明这些基因的地位与功能，最终在代谢流和基因水平上阐明整个白藜芦醇合成途径与功能基因。研究可为白藜芦醇生产菌构建提供新理论与基因资源，在利用葡萄内生菌合成白藜芦醇方面有特色，在揭示微生物中白藜芦醇合成途径与基因方面有创新。

葡萄内生菌链格孢属MG1能够稳定地以葡萄糖为底物合成白藜芦醇，表现出工程菌所不具有优势，但是产物得率很低。为揭示其内在的合成途径与关键基因，从而为进一步调控其代谢途径与提高产物得率奠定基础，本项目进行了以下研究：①通过基因组分析，成功构建了基因组规模代谢网络模型，命名为iYL1539；鉴定了iYL1539中生长必需基因和反应；成功注释到苯丙烷及其下游次生代谢物合成途径，并对白藜芦醇合成途径进行了试运行与湿法实验验证，并将其成功应用于环境对白藜芦醇合成途径扰动的预测与验证，以及过表达4CL和CHS基因对白藜芦醇合成途径的影响。通过转录组数据分析，验证了MG1中白藜芦醇合成途径的关键基因，以及将白藜芦醇转化为白皮杉醇的相关基因。提示我们，该菌在生产白皮杉醇方面会有更好的应用。②通过代谢组、前体添加、13C同位素标记葡萄糖示踪等方法，确定了MG1中白藜芦醇合成途径中代谢流是葡萄糖→苯丙氨酸→肉桂酸→对香豆酸→白藜芦醇，并检测到白皮杉醇的积累。③通过关键酶活的跟踪检测、白藜芦醇合成酶类的细胞定位分析、关键酶类的分离纯化与催化活性鉴定、细胞内酶催化反应体系的构建，确定了MG1合成白藜芦醇的关键酶类。并结合转录组分析中关键酶类的基因表达量变化分析，发现：肉桂酸-4-羟基化酶（C4H）是途径中的限速酶；由肉桂酸转化为对香豆酸，或者由肉桂酰辅酶A转化成对香豆酰辅酶A的步骤为白藜芦醇合成的限速步骤。④对MG1中4CL和CHS在酵母中进行了外源克隆表达，验证了CHS具有合成柚皮素和白藜芦醇的双重活性，4CL能够催化合成柚皮素，是芪类化合物和黄酮合成途径的关键酶。.项目研究在首次揭示微生物中苯丙烷-白藜芦醇合成途径及其关键基因方面有重要的科学意义，在以葡萄糖为起始物，通过微生物发酵法从头合成白藜芦醇方面有很好的应用前景。.项目执行期发表学术论文26篇，其中SCI论文25篇，影响因子大于3.0的16篇，授权国家发明专利4项，培养硕士3名，博士3名，博士后2名，超额完成预期目标。