仿动物甾体激素代谢模式创建甾体药物合成微生物细胞工厂的研究

The synthesis of steroids in the industry is a complicated process ,which is lengthy, low yield and seriously polluting. Green processes, especially the replacement of hard and dangerous chemical reactions by microbial transformations, are the most pressing needs of the steroid pharmaceutical industry, which are also national great demands for industry transformation and upgrading in our country as a huge producer of steroids. The primary synthesis pathways of steroids in the industry are highly similar to the steroidogenic pathway in animals, and there are many common processes in the synthesis of four kinds of steroid hormones, on the basis of which we propose a research plan to develope artificial cascade transformation pathways for the synthesis of steroids in microorganisms by deconstructing, extracting, and mimicking the steroidogenic pathway of steroids in animals. According to the feasibility analyses on steroids tolerance, steroid transport capacity, P450s heterologous expression and genome editing, a Mycobactial model is designed to carry out the research plan, which involves five cytochrome P450 monooxygenases and two hydroxysteroid dehydrogenases. In this work, systematic strategies will be employed, including the selection and optimization of biological components and parts, the adaptive modification of microbial chassises, the construction of cascade conversion pathways of steroids, and the high-throughput evolution of enzymes, pathways, and engineered strains by developing steroid-detecting biosensors based on steroid receptors from animals. Through the above researches, we expect to provide a guidance for the development of microbial cell factories in the synthesis of steroids and create a kind of innovative industrial system for the green synthesis of steroids.

甾体药物的生产，工艺复杂、路线长、收率低、污染重。我国是甾体制药大国，开发绿色生产技术，推动微生物转化对众多高难化学反应的替代是甾体制药工业技术变革的大方向，也是推动我国甾体制药产业转型升级的重大需求。基于甾体制药工业主体生产路线与动物甾体激素生成途径高度相似这一事实，申请者针对两者在四大类甾体激素合成过程中的共性，通过解构抽提动物甾体激素代谢途径，提出在微生物中“模仿动物甾体激素代谢模式”组建甾体级联转化途径，实现甾体药物微生物代谢合成的研究思路。基于甾体毒性耐受、甾体转运能力、P450酶异源表达和基因组连续编辑等的可行性，申请者提出了以分枝杆菌为底盘的工作模型，内容涉及5个P450酶系和2个羟基甾体脱氢酶的元器件筛选优化、底盘适配性改造、级联转化途径构建以及基于甾体激素受体传感的高通量进化研究等。期望通过以上研究为甾体制药微生物细胞工厂的研发提供理论指导，为甾体制药工业开创绿色新体系。

甾体药物立体结构复杂，其工业制造过程十分繁琐、路线长、收率低、污染重、生产成本高。我国是甾体制药大国，开发绿色生产技术，推动微生物转化对众多高难化学反应的替代，从而简化甾体制药路线，是甾体制药工业技术变革的大方向，也是推动我国甾体制药产业转型升级的重大需求。本项目基于甾体制药工业主体生产路线与动物甾体激素生成途径高度相似这一事实，通过解构抽提动物甾体激素代谢途径，以甾体制药工业中植物甾醇生物转化路线的分枝杆菌为底盘，基于“模仿动物甾体激素代谢模式”，通过元器件筛选优化、底盘适配性改造、细胞壁结构改造、代谢毒性耐受性的提高以及基于甾体激素受体传感的高通量进化研究等工作，设计构建了多条甾体级联转化途径，实现了黄体酮、C21-羟基孕甾、17-烯孕酮、孕甾-4,16-双烯、孕甾-1,4,16-三烯和谷内酯等细胞工厂的构建。其中由植物甾醇一步转化生产黄体酮、C21-羟基孕甾和17-烯孕酮这三种产物，为国内外首创，十分便于C21孕激素和肾上腺皮质激素的工业化生产，拓展了植物甾醇生物转化路线在甾体制药工业中的应用范围、提高了应用优势，使植物甾醇生物转化体系完全覆盖了甾体制工业中传统的薯蓣皂素工业体系，为完全取代薯蓣皂素路线奠定了基础，其中C21-羟基孕甾的生产技术已转让给甾体制药企业用于黄体酮的生产，实现了应用转化。此外，为了能有效表达膜结合甾体脱氢酶，我们以链霉菌为底盘通过表达系统的设计和优化，成功开发了以孕甾-16-烯为底物转化生产孕甾-4,16-双烯和孕甾-1,4,16-三烯的生产技术，可把剑麻工业的废弃物——剑麻皂素变废为宝转化为了高附加值的肾上腺皮质激素前体，是植物甾醇生物转化路线的有益补充。本项目已发表学术论文11篇，获省部级技术发明一等奖1项，培养博士生9人和博士后2人，圆满完成了既定工作目标。