液滴微流控技术在微生物工程菌株选育中的应用进展

doi:10.12211/2096-8280.2021-105

合成生物学 ›› 2023, Vol. 4 ›› Issue (1): 165-184.DOI: 10.12211/2096-8280.2021-105

液滴微流控技术在微生物工程菌株选育中的应用进展

涂然¹^,², 李世新³, 李昊霓³, 王猛²

^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

收稿日期:2021-12-05 修回日期:2022-01-18 出版日期:2023-02-28 发布日期:2023-03-07
通讯作者: 王猛
作者简介:涂然（1979—），女，博士，正高级工程师，教授，硕士生导师。研究方向为合成生物学、高通量检测和筛选技术等。E-mail： tu_r@ctbu.edu.cn
王猛（1982—），男，博士，研究员，博士生导师。研究方向为合成生物学、高通量自动化技术等。 E-mail： wangmeng@tib.cas.cn
第一联系人：涂然（1979—），女，博士，正高级工程师，教授，硕士生导师。研究方向为合成生物学、高通量检测和筛选技术等。
基金资助:
国家重点研发计划“绿色生物制造”重点专项(2021YFC2100201);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-003)

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

Ran TU¹^,², Shixin LI³, Haoni LI³, Meng WANG²

^1.College of Environmental and Resources，Chongqing Technology and Business University，Chongqing 400067，China
^2.Key Laboratory of Engineering Biology for Low-Carbon Manufacturing，Tianjin Institute of Industrial Biotechnology，Chinese Academy of Sciences，Tianjin 300308，China
^3.College of Biotechnology，Tianjin University of Science & Technology，Tianjin 300457，China

Received:2021-12-05 Revised:2022-01-18 Online:2023-02-28 Published:2023-03-07
Contact: Meng WANG

摘要/Abstract

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。

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

Abstract:

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-based microfluidic technology can achieve millions of samples per day in pico- or nano-liter volume, resulting in an increase of thousand-folds in screening speed and cost-saving for million-folds. By integrating with an automated station, the droplet-based microfluidic technology can be further improved for its screening efficiencies and application potentials in microbial synthetic biology. {L-End}

Key words: droplet-based microfluidics, single-cell analysis, cell factory, high-throughput screening, direct evolution

中图分类号:

Q939.97

涂然, 李世新, 李昊霓, 王猛. 液滴微流控技术在微生物工程菌株选育中的应用进展[J]. 合成生物学, 2023, 4(1): 165-184.

Ran TU, Shixin LI, Haoni LI, Meng WANG. Advances and applications of droplet-based microfluidics in evolution and screening of engineered microbial strains[J]. Synthetic Biology Journal, 2023, 4(1): 165-184.

图/表 9

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液滴类型	液滴信号来源	优缺点	通量	代表性应用案例
单层液滴	细胞自身信号（荧光、拉曼、浊度、细胞图像）	优点：操作简单，细胞直接包埋在液滴中缺点：需要特殊软件，例如拉曼和图像识别软件	荧光300 Hz；拉曼1～5 Hz；图像识别5～10 Hz；浊度240 Hz	色素：核黄素 ^{[ 103]}；拉曼：虾青素 ^{[ 78]}；图形识别：人成红细胞白血病细胞 ^{[ 106]}；散射光：细胞浊度 ^{[ 82]}
	细胞荧光蛋白（目标蛋白和外源荧光蛋白融合、生产菌自身含生物传感器、生产菌和感应菌共包埋培养）	优点：操作简单，细胞直接包埋在液滴中缺点：需要构建特定生物传感器	150～300 Hz	生产菌自身含生物传感器：3-脱氢莽草酸 ^{[ 107]}；生产菌和感应菌共培养：香豆酸酵母菌和大肠杆菌感应报告菌 ^{[ 109]}
	细胞和底物（同时共包埋在液滴）	优点：操作简单，细胞和底物直接包埋在液滴中缺点：不适合酶促反应快的样品	300～2000 Hz	大肠杆菌：半乳糖苷酶 ^{[ 92]}、硫酯酶 ^{[ 47, 56]}等；枯草芽孢杆菌：淀粉酶 ^{[ 114]}；丝状真菌：纤维素酶 ^{[ 119]}
	细胞和底物（细胞先包埋在液滴，底物后加入）	优点：可以通过液滴融合添加试剂，用于酶促反应快的样品缺点：液滴融合操作复杂	300～1000 Hz	漆酶 ^{[ 112]}、脂肪酶 ^{[ 122]}、乙醇 ^{[ 123]}等

液滴类型	液滴信号来源	优缺点	通量	代表性应用案例
单层液滴	细胞自身信号（荧光、拉曼、浊度、细胞图像）	优点：操作简单，细胞直接包埋在液滴中缺点：需要特殊软件，例如拉曼和图像识别软件	荧光300 Hz；拉曼1～5 Hz；图像识别5～10 Hz；浊度240 Hz	色素：核黄素 ^{[ 103]}；拉曼：虾青素 ^{[ 78]}；图形识别：人成红细胞白血病细胞 ^{[ 106]}；散射光：细胞浊度 ^{[ 82]}
	细胞荧光蛋白（目标蛋白和外源荧光蛋白融合、生产菌自身含生物传感器、生产菌和感应菌共包埋培养）	优点：操作简单，细胞直接包埋在液滴中缺点：需要构建特定生物传感器	150～300 Hz	生产菌自身含生物传感器：3-脱氢莽草酸 ^{[ 107]}；生产菌和感应菌共培养：香豆酸酵母菌和大肠杆菌感应报告菌 ^{[ 109]}
	细胞和底物（同时共包埋在液滴）	优点：操作简单，细胞和底物直接包埋在液滴中缺点：不适合酶促反应快的样品	300～2000 Hz	大肠杆菌：半乳糖苷酶 ^{[ 92]}、硫酯酶 ^{[ 47, 56]}等；枯草芽孢杆菌：淀粉酶 ^{[ 114]}；丝状真菌：纤维素酶 ^{[ 119]}
	细胞和底物（细胞先包埋在液滴，底物后加入）	优点：可以通过液滴融合添加试剂，用于酶促反应快的样品缺点：液滴融合操作复杂	300～1000 Hz	漆酶 ^{[ 112]}、脂肪酶 ^{[ 122]}、乙醇 ^{[ 123]}等

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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

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