抗癌化合物灵芝酸单体T和Me的高效生物合成研究

Ganoderic acids (GAs) are unique secondary metabolites of medicinal mushroom Ganoderma lucidum, of which GA-T and GA-Me were proved to have significant anti-tumor and anti-metastasis activities. However, the lack of substantial and efficient supply of these individual GAs has hindered their widespread application and large-scale pre-clinical studies. A couple of key enzymes, responsible for the biosynthesis of these GAs, were identified in our recent research. Based on the accumulation of our previous long-term related work as well as literature information, in this proposal, an engineered strain of G. lucidum will be constructed as the first step, which could produce more GA-T and GA-Me compared to the wild type. Then, detailed omics analyses (including transcriptomics, proteomics and metabolomics) in fermentation of the engineered strain vs. the wild type will be conducted, so as to clarify the affecting law and acting mechanism of GA biosynthesis in the engineered strain and to find out new potential targets for further metabolic engineering. After the engineered cell factory is re-improved by metabolic engineering, its adaptability to the bioreactor cultivation environment will be investigated. In short, by effectively combining metabolic engineering, omics and bioreactor engineering research, new ideas and new methodologies for reaching hyper-production of these individual GAs will be proposed, and a large-scale highly-efficient production of GA-T and GA-Me is to be achieved. The work will provide a typical example and insights to studies on secondary metabolite production by other microorganisms (especially higher fungi) from strain improvement to fermentation technology innovation.

灵芝酸是药用高等真菌灵芝特有的次生代谢产物。其中，灵芝酸单体T和Me具有良好的抗肿瘤及抑制肿瘤转移的活性。然而，缺乏大量、有效的供给是阻碍其广泛应用以及临床前的深入研究的瓶颈。我们的前期工作鉴定获得了数个参与合成灵芝酸单体的关键酶基因。本研究基于以往的工作积累及文献报道的信息，首先构建高产灵芝酸单体T和Me的灵芝基因工程菌株，再就工程菌和野生菌的发酵进行组学（比较转录谱、蛋白质谱和代谢物谱）分析，揭示工程菌株中灵芝酸合成的规律和受调控机制，挖掘菌株代谢改造的新靶点，进而开展工程菌的再改造。最后，进行优化的工程菌（细胞工厂）在反应器培养环境的适配性研究。通过有机结合代谢工程、组学及生物反应器技术，提出灵芝酸单体高效生物合成的新思路新方法，也为其他微生物（特别是高等真菌）次生代谢产物的发酵高产研发提供借鉴和范例。

灵芝是我国传统的名贵药用真菌，它对于辅助肿瘤治疗、提高人体免疫力、延缓衰老等具良好功效。这些重要生理活性与灵芝细胞内丰富的灵芝酸类三萜类物质密切相关。随着合成生物学技术的迅猛发展，利用异源宿主合成灵芝酸被认为是一个富有潜力的高效生产方式。. 本研究首先建立了以酿酒酵母为底盘、筛选参与灵芝酸生物合成催化元件的平台。从已报道的219个灵芝CYP基因中优先选择了82个可能参与灵芝酸生物合成的候选元件。通过构建各个CYP的表达载体、并分别和辅助CYP功能发挥的CPR基因GL19526（GlCPR）共同导入高产羊毛甾醇的酿酒酵母底盘细胞来系统筛选。率先报道了CYP5150L8基因参与抗癌活性物质3-羟基-羊毛甾-8，24-二烯-26-酸（HLDOA）的异源生物合成。在此基础上拓展灵芝酸生物合成步骤，发现了CYP5139G1可催化HLDOA羟基化反应，合成一种新的灵芝酸，经UPLC-MS、NMR分析确定其为3，28-二羟基-羊毛甾-8，24-二烯-26-酸（DHLDOA）。实验表明DHLDOA具有抗非小细胞肺癌细胞活性。结合发酵工程研究，实现了新颖灵芝酸单体的高效发酵。以上工作不仅为通过合成生物学手段高效制造灵芝酸提供了基因元件，也为解析完整的灵芝酸生物合成途径、实现灵芝酸类物质高效生产奠定了重要基础。. 本项目同时开展了灵芝真菌培养及基因编辑技术的开发，率先报道了基于CRISPR基因编辑的高等真菌功能基因改造技术平台。结合创建的基因敲除和回补，证实了cyp5150l8基因在灵芝酸生物合成中发挥必需作用，gl26097基因 (编码钙信号下游的Cys2-His2锌指转录因子)在调控灵芝酸生物合成中发挥重要作用。所创建的基于CRISPR-Cas9的灵芝基因中断和回补技术有望用于其他担子菌的基因中断。本研究为解析高等真菌的基因的功能和目标代谢物的生物合成途径奠定重要基础，也为促进高等真菌的分子育种和生物技术应用提供了重要技术平台。. 已发表论文10篇，包括在生物工程一流期刊Biotechnol Bioeng, Appl Microbiol Biotechnol，相关研究成果受到国内外同行的引用和采用，产生了良好学术影响力。已获授权国家发明专利2项、公开1项，培养博硕研究生5名。