液滴微流控技术在微生物工程菌株选育中的应用进展  

Advances and applications of droplet-based microfluidics in evolution and screening of engineered microbial strains

作　　者：涂然 李世新[3] 李昊霓 王猛 TU Ran;LI Shixin;LI Haoni;WANG Meng(College of Environmental and Resources,Chongqing Technology and Business University,Chongqing 400067,China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing,Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]重庆工商大学环境与资源学院,重庆400067 [2]中国科学院天津工业生物技术研究所,中国科学院低碳合成工程生物学重点实验室,天津300308 [3]天津科技大学生物工程学院,天津300457

出　　处：《合成生物学》2023年第1期165-184,共20页Synthetic Biology Journal

基　　金：国家重点研发计划“绿色生物制造”重点专项(2021YFC2100201,2021YFC2103302);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-003,TSBICIP-KJGG-006)。

摘　　要：微生物工程菌株是生物制造的重要基础,但大多数的工程菌株需要进化改造才能适用于生物制造。在菌种选育过程中,如何高效地筛选获得具有目标性状的微生物工程菌株是进行生物制造应用的关键影响因素之一。液滴微流控技术作为近年来发展起来的一种基于微芯片的高通量检测筛选技术,可以生成大小均一、相互独立的微体积液滴小室,并应用于单细胞的培养、检测和分离,在微生物菌株改造尤其是分泌型菌株的改造中得到广泛应用。本文首先概述液滴微流控技术的组成部分,对关键性的技术进行简要介绍;其次根据液滴检测信号的来源、液滴筛选流程的难易程度和液滴分选仪器的适用范围,对液滴微流控技术在工程菌株选育中的应用进行总结分析;最后对液滴微流控技术在应用中存在的问题和研究方向进行展望,为深化其在微生物合成生物学中的应用提供指导。Microbial strains are perquisites for biomanufacting through microbial culture and fermentation. However,most strains usually need to be engineered to improve their performances for industrial applications. Therefore how to efficiently screen and isolate robust strains is a critical step of strain engineering. As an advanced high-throughput screening technology, droplet-based microfluidics developed with micro-chips can generate highly independent and uniform micro-or nano-liter droplets, in which single cells can be encapsulated, inoculated, detected, and analyzed for strain engineering. It is especially useful in the evolution of microbial strains for producing extracellular products. In this review, we first introduce the basic components of the droplet-based microfluidic system and the main steps involved in the strain screening. We then summarize key factors for the application of the droplet-based microfluidic technology in strain engineering, such as the signal sources of droplet detection, the difficulties of handling droplet screening, and the scopes of droplet sorting instruments. Based on the instruments used for the droplet sorting, we group the application cases into two types either via fluorescence-activated droplet sorting(FADS) using microfluidic equipment or via fluorescence-activated cell sorting(FACS) using flow cytometry instrument. While FADS using single-layer water-in-oil droplet can be further classified into cellular signature, fluorescent reporter protein, and substrate-based reaction according to the signal sources, FACS can be divided into double-layers water-in-oil-in-water(W/O/W) droplet or microgel droplet according to the droplet property. Finally, we outline challenges and prospects for the droplet microfluidic technology, and provide some guidelines for its applications in synthetic biology. Compared with traditional screening methods such as shaking flask or microplate with a throughput of hundreds to thousands of samples per day in milli-or micro-liter volume, the droplet-base

关 键 词：液滴微流控技术 单细胞分析 细胞工厂 高通量筛选 进化改造 

分 类 号：Q939.97[生物学—微生物学]