梭菌正丁醇代谢工程研究进展

doi:10.12211/2096-8280.2020-080

合成生物学 ›› 2021, Vol. 2 ›› Issue (2): 194-221.DOI: 10.12211/2096-8280.2020-080

梭菌正丁醇代谢工程研究进展

闻志强¹, 孙小曼¹, 汪庆卓¹, 李亚楠¹, 刘文正¹, 蒋宇², 杨晟^2,³

^1.南京师范大学食品与制药工程学院，江苏南京　210046
^2.中国科学院上海生命科学研究院湖州工业生物技术研发中心，浙江湖州 313000
^3.中国科学院分子植物科学卓越创新中心，中科院合成生物学重点实验室，上海 200032

收稿日期:2020-10-22 修回日期:2021-02-09 出版日期:2021-04-30 发布日期:2021-04-30
通讯作者: 杨晟
作者简介:闻志强（1985—），男，博士，讲师，研究方向为合成梭菌菌群代谢工程。E-mail：zqwen@njnu.edu.cn|杨晟（1973—），男，博士，研究员，研究方向为合成生物催化剂工程与基因编辑。E-mail：syang@sibs.ac.cn
基金资助:
国家自然科学基金杰出青年科学基金(21825804);国家自然科学基金创新研究群体科学基金(31921006);国家自然科学基金青年项目(21706133)

Recent advances in metabolic engineering of clostridia for n-butanol production

Zhiqiang WEN¹, Xiaoman SUN¹, Qingzhuo WANG¹, Yanan LI¹, Wenzheng LIU¹, Yu JIANG², Sheng YANG^2,³

^1.School of Food Science and Pharmaceutical Engineering，Nanjing Normal University，Nanjing 210046，Jiangsu，China
^2.Huzhou Center of Industrial Biotechnology，Shanghai Institutes of Biological Sciences，Chinese Academy of Sciences，Huzhou 313000，Zhejiang，China
^3.Key Laboratory of Synthetic Biology，CAS Center for Excellence in Molecular Plant Sciences，Shanghai Institute of Plant Physiology and Ecology，Chinese Academy of Sciences，Shanghai 200032，China

Received:2020-10-22 Revised:2021-02-09 Online:2021-04-30 Published:2021-04-30
Contact: Sheng YANG

摘要/Abstract

摘要：

正丁醇是大宗化学品和可再生、替代性车用燃料，可由微生物发酵生产，以替代现有的高污染/不可持续的石油炼制方法。本文首先回顾和比较了各种正丁醇合成途径和底盘细胞，指出梭菌是天然的正丁醇细胞工厂，且在丁醇产量和生产强度上有明显优势，但仍受制于菌株性能不足，具体表现在菌株遗传改造困难，正丁醇产量不高，副产物较多，正丁醇合成途径刚性强，以及底物利用效率低等方面。幸运的是，合成生物学的发展加速了产正丁醇梭菌的遗传操作工具开发。很多遗传操作工具如TargeTron、CRISPR/Cas系统介导的基因和碱基编辑工具已经被开发出来。梭菌内已经可以高效实现靶标基因插入、删除、替换、点突变以及表达水平调控等各种操作，这为梭菌正丁醇代谢工程奠定了良好的基础。正丁醇合成途径的增强及副产物如丙酮、乙酸、丁酸等竞争途径的弱化或者删除，提升了丁醇的产量、比例；同时，一些非常规梭菌被代谢工程改造用于同型丁醇发酵，实现丁醇与丙酮生产的解耦；另外，遗传操作工具还为梭菌的戊糖转运/代谢途径以及碳源代谢抑制效应的调控机制的解析和重构提供了便利，极大改善了梭菌戊糖利用效率。相信在合成生物技术的驱动下，梭菌生产正丁醇的成本将大幅降低，最终走向市场。

关键词: 遗传操作工具, 正丁醇产量, 正丁醇比例, 途径重构, 戊糖代谢

Abstract:

n-Butanol is a bulk chemical and renewable and alternative vehicle fuel. It can be produced from biomass by some microorganisms, which has potential to replace the unsustainable and environment-hazardous petroleum refining methods. In this work, we review and compare various metabolic pathways and chassis cells for n-butanol production, and highlight that clostridia are natural cell factories for n-butanol production with obvious advantages in butanol titer and productivity. However, strains from this species are still unsatisfactory, which includes inefficient strain genetic modification, low n-butanol yield/ratio, rigid n-butanol synthesis pathway, and low substrate utilization spectrum.Fortunately, synthetic biotechnology has significantly accelerated the development of genetic manipulation tools, and many of them including TargeTron, allelic-exchange, CRISPR/Cas system mediated gene and base editing tools have been developed and applied in clostridia. Various genetic operations such as insertion, deletion, substitution, site mutation, and regulation of target gene expression can be efficiently implemented in clostridia, which laid a foundation for its metabolic engineering. As a result, significant progress has been made in increasing n-butanol titer/yield/ratio, reconstructing and refining the n-butanol synthesis pathway in clostridial chassis, as well as enhancing pentose utilization. The enhancement of the n-butanol pathway and the weakening or deletion of pathways for producing by-products such as acetone, acetic acid, butyric acid have increased butanol titer and ratio.In addition, unconventional clostridia including cellulolytic and gas-fermenting strains have been metabolically engineered for homo-butanol fermentation through decoupling with acetone production. Moreover, genetic manipulation tools also facilitate the reconstruction of the clostridial pentose (xylose and arabinose) transport/metabolism pathway and analysis of carbon catabolite repression (CCR) mechanism, which greatly improved the utilization of pentose. In this article, we review the above metabolic engineering strategies and important milestones of n-butanol production, and address the current bottlenecks and future trends. With the driving of synthetic biotechnology, the cost of n-butanol production by clostridia will be reduced, making it eventually competitive in the market.

Key words: genetic tools, butanol titer, butanol ratio, pathway reconstruction, pentose metabolism

中图分类号:

Q815

闻志强, 孙小曼, 汪庆卓, 李亚楠, 刘文正, 蒋宇, 杨晟. 梭菌正丁醇代谢工程研究进展[J]. 合成生物学, 2021, 2(2): 194-221.

Zhiqiang WEN, Xiaoman SUN, Qingzhuo WANG, Yanan LI, Wenzheng LIU, Yu JIANG, Sheng YANG. Recent advances in metabolic engineering of clostridia for n-butanol production[J]. Synthetic Biology Journal, 2021, 2(2): 194-221.

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Recent advances in metabolic engineering of clostridia for n-butanol production

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