甾体化合物绿色生物制造：从生物转化到微生物从头合成

doi:10.12211/2096-8280.2021-061

合成生物学 ›› 2021, Vol. 2 ›› Issue (6): 942-963.DOI: 10.12211/2096-8280.2021-061

甾体化合物绿色生物制造：从生物转化到微生物从头合成

熊亮斌¹^,², 宋璐¹, 赵云秋¹, 刘坤¹, 刘勇军¹, 王风清¹, 魏东芝¹

^1.华东理工大学，鲁华生物技术研究所，生物反应器国家重点实验室，上海 200237
^2.上海健康医学院，协同科研中心，上海 201318

收稿日期:2021-06-02 修回日期:2021-07-20 出版日期:2021-12-31 发布日期:2022-01-21
通讯作者: 王风清,魏东芝
作者简介:熊亮斌（1986—），男，助理研究员。研究方向为代谢工程及合成生物学。E-mail：lbxiong2010@163.com
宋璐（1995—），女，博士研究生。研究方向为生物催化，代谢工程及合成生物学。E-mail：s13127579253@163.com
王风清（1977—），男，副教授，博士生导师。研究方向为生物催化，萜类、甾体类等天然活性化合物的微生物合成生物学研究。E-mail：fqwang@ecust.edu.cn
魏东芝（1963—），男，教授，博士生导师。研究方向为生物催化，萜类、甾体类等天然活性化合物的微生物合成生物学研究。E-mail：dzhwei@ecust.edu.cn
基金资助:
国家自然科学基金(21776075);中国博士后基金面上项目(2020M671028)

Green biomanufacturing of steroids: from biotransformation to de novo synthesis by microorganisms

Liangbin XIONG¹^,², Lu SONG¹, Yunqiu ZHAO¹, Kun LIU¹, Yongjun LIU¹, Fengqing WANG¹, Dongzhi WEI¹

^1.State Key Laboratory of Bioreactor Engineering，Newworld Institute of Biotechnology，East China University of Science and Technology，Shanghai 200237，China
^2.Collaborative Innovation Center for Biomedicine，Shanghai University of Medicine and Health Sciences，Shanghai 201318，China

Received:2021-06-02 Revised:2021-07-20 Online:2021-12-31 Published:2022-01-21
Contact: Fengqing WANG, Dongzhi WEI

摘要/Abstract

摘要：

甾体化合物（简称甾体）分布广泛、功能卓越，在机体生长、物种繁育以及代谢调控等方面，发挥着难以取代的生理功能。因此，天然甾体及其衍生物被广泛用于生殖健康、内分泌调控等领域，是器官移植、重症感染等许多危重疾病的“刚需药”和“救命药”。甾体结构复杂、构型精巧，很难通过化学全合成来生产，当前主要以天然甾体皂素或甾醇为原料，通过化学与生物转化相结合的半合成法获得。然而，甾体药物的生产路线长、工艺复杂、收率低，涉及大量有毒有害试剂和重金属催化剂的使用，污水废渣排放量大、处理难度高，总体成本居高不下。为改变此局面，推动产业的转型升级，大力开发绿色生物制造技术是行业健康发展的大势所趋。当前，甾体制药工业正处于以生物催化转化取代化学合成的产业升级阶段，随着高效酶和细胞转化的成功应用，传统的甾体生产模式正发生着深刻变化。在此基础上，若能进一步利用合成生物学技术，创建可高效从头合成甾体的微生物细胞工厂，则将彻底改变甾体制药的工业模式，切实实现甾体药物的绿色制造。近年来，已有利用微生物从头合成部分甾体化合物的报道，然而由于甾体的天然合成机制异常复杂，如何实现细胞工厂的高效生产，仍是目前面临的主要挑战。本文从甾体药物生产方式的演变出发，系统综述了甾体生物催化转化和从头合成的最新进展，重点阐述了甾体生物催化转化酶的挖掘及改造、微生物代谢转化甾醇机制的解析及转化细胞工厂的开发、微生物从头合成甾体人工路线的创建三部分内容，以期对甾体药物绿色生物制造的现状和趋势做出合理的总结与展望。

关键词: 甾体化合物, 绿色生物制造, 生物催化转化, 从头生物合成, 微生物细胞工厂

Abstract:

Steroids are widely distributed in natural organisms and play essential roles in growth, breeding, metabolic regulation, and endocrine homeostasis. To date, diverse steroids have been proved to have numerous therapeutic effects on reproductive health, endocrine regulation, inflammation, etc. and can be used as life-saving drugs for some serious diseases, such as cancer, organ transplantation, and serious infection. Therefore, the industrial synthesis of steroid drugs has developed rapidly and steroid drugs have become the second largest drug category just ranked after antibiotics. Due to the complex structure and delicate configuration, steroids are difficult to be produced at an industrial scale by chemical total synthesis. At present, steroids are mainly produced by semi-synthesis ways with natural steroidal sapogenins or sterols as raw materials via the combination of chemical and biological transformations. However, the above routes are long and complex resulting in the low yield and the overuse of toxic reagents and heavy metal catalysts, thus resulting in a large quantity of wastewater and residue as well as high production costs. Therefore, it is necessary to develop green biomanufacturing technologies, such as biocatalysis, biotransformation, and biosynthesis, for the industrial production of steroids. Currently, the production mode of steroids has been profoundly changed due to the successful application of enzyme-catalyzed reactions and microbial transformations in the industrial production of steroids. It is expected that the production of steroids will be changed into a biomanufacturing mode if the robust microbial cell factories for the de novo biosynthesis of steroids can be developed via the biosynthetic way. The de novo synthesis of steroids by microorganisms has been materialized. However, due to the extraordinarily complex metabolic mechanism of steroids in the nature, the efficient production of steroids in engineered hosts still remains a challenge. Here, starting from the evolution of steroidal pharmaceutical industry, we systematically review recent advances in biomanufacturing technologies of steroids, including the identification and application of steroidal biocatalysis and biotransformation, the characterization and modification of the metabolic mechanisms of steroids in certain microorganisms, and the development of de novo biosynthesis pathways of steroids in engineered cell factories. From the above three aspects, this review provides a reasonable summary and prospect for the current status and future development trend of green bio-manufacturing technologies in the steroidal pharmaceutical industry.

Key words: steroids, green biomanufacturing, biocatalysis and biotransformation, de novo biosynthesis, microbial cell factories

中图分类号:

Q819

熊亮斌, 宋璐, 赵云秋, 刘坤, 刘勇军, 王风清, 魏东芝. 甾体化合物绿色生物制造：从生物转化到微生物从头合成[J]. 合成生物学, 2021, 2(6): 942-963.

Liangbin XIONG, Lu SONG, Yunqiu ZHAO, Kun LIU, Yongjun LIU, Fengqing WANG, Dongzhi WEI. Green biomanufacturing of steroids: from biotransformation to de novo synthesis by microorganisms[J]. Synthetic Biology Journal, 2021, 2(6): 942-963.

图/表 6

图1 基于Marker降解的甾体半合成工艺体系［24］

Fig. 1 The semi-synthesis of steroids based on the Marker degradation technology

图2 基于甾醇微生物转化的甾体半合成工艺体系［24］

Fig. 2 The semi-synthesis of steroids based on the microbial transformation of sterols

表1 甾体化合物羟基化反应的类型

Tab. 1 Types of steroidal hydroxylation

反应类型	羟基化酶	微生物来源
11β-OH^[33-34]	CYP103168	新月弯孢霉
11β-OH^[33-34]	CYP5311B2	犁头霉菌
11α-OH^[36-37]	CYP5311B1 CYP68L8、CYP68J5	蓝色犁头霉AS3.65 赭曲霉TCCC41060
9α-OH^[35]	KshA/B、TDO	普提达假单胞菌F117
7β-OH^[38-39]	P450-BM3	巨大芽孢杆菌
7β-OH^[38-39]	CYP107D1	链霉菌
25-OH^[40-41]	CYP105A1	灰色链霉菌
25-OH^[40-41]	CYP107-Vdh	Pseudonocardia autotrophica
19-OH^[42-43]	STH10	Thanatephorus cucumeris NBRC 6298

图3 甾醇的微生物分解途径

Fig. 3 The catabolic pathway of sterols in microorganisms

图4 不同生物体内的甾醇合成路线

Fig. 4 The biosynthetic pathway of sterols in organisms

图5 甾体激素生物合成途径

Fig. 5 The biosynthesis pathway of steroidal hormones

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甾体化合物绿色生物制造：从生物转化到微生物从头合成

Green biomanufacturing of steroids: from biotransformation to de novo synthesis by microorganisms

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