合成生物学表型测试生物反应器及其装备化研究进展

doi:10.12211/2096-8280.2023-067

合成生物学 ›› 2024, Vol. 5 ›› Issue (1): 16-37.DOI: 10.12211/2096-8280.2023-067

合成生物学表型测试生物反应器及其装备化研究进展

郭肖杰¹^,², 剪兴金¹^,², 王立言³, 张翀¹^,²^,⁴, 邢新会¹^,²^,⁴^,⁵

^1.清华大学化学工程系，生物化工研究所，北京 100084
^2.工业生物催化教育部重点实验室，北京 100084
^3.清华大学无锡应用技术研究院生物育种中心，无锡 214000
^4.清华大学合成与系统生物学中心，北京 100084
^5.清华大学深圳国际研究生院生物医药与健康工程研究院，深圳 518055

收稿日期:2023-09-19 修回日期:2023-11-02 出版日期:2024-02-29 发布日期:2024-03-20
通讯作者: 邢新会
作者简介:郭肖杰（1990—），男，博士研究生。研究方向为液滴微流控及其在微生物领域的装备化应用。 E-mail：gxj20@mails.tsinghua.edu.cn
邢新会（1965—），男，教授。研究方向为生物化工、生物育种技术及装备等。 E-mail：xhxing@tsinghua.edu.cn
基金资助:
国家重点研发计划(2018YFA0901500);国家自然科学基金重大仪器专项(21627812);深圳市可持续发展专项(KCXFZ20201221173207022)

Progress in bioreactors and instruments for phenotype testing with synthetic biology research

Xiaojie GUO¹^,², Xingjin JIAN¹^,², Liyan WANG³, Chong ZHANG¹^,²^,⁴, Xinhui XING¹^,²^,⁴^,⁵

^1.Institute of Biochemical Engineering，Department of Chemical Engineering，Tsinghua University，Beijing 100084，China
^2.Key Laboratory for Industrial Biocatalysis of the Ministry of Education，Beijing 100084，China
^3.Biobreeding Center，Wuxi Research Institute of Applied Technologies，Tsinghua University，Wuxi 214072，Jiangsu，China
^4.Center for Synthetic and Systems Biology，Tsinghua University，Beijing 100084，China
^5.Institute of Biopharmaceutical and Health Engineering，Tsinghua Shenzhen International Graduate School，Shenzhen 518055，Guangdong，China

Received:2023-09-19 Revised:2023-11-02 Online:2024-02-29 Published:2024-03-20
Contact: Xinhui XING

摘要/Abstract

摘要：

合成生物学经过多年的发展，形成了典型的细胞工厂创制“设计-构建-测试-学习”（design-build-test-learn， DBTL）循环，成为支撑面向加速生物制造发展的智慧生物育种的重要方法。其中，测试环节是对前期所设计与构建的生物体系进行表型测试，以提供大量数据用于后续的学习和迭代升级。测试阶段的通量主要依赖于细胞自身或其培养测试所使用的生物反应器及其装备，是整个DBTL流程的限速步骤。本文系统综述了合成生物学表型测试所开发的面向不同通量的生物反应器及装备，从单细胞检测和筛选及不同尺度生物反应器包括皮纳升级生物反应器、微升级生物反应器、毫升级生物反应器和升级生物反应器等，系统地介绍了各自的特点和应用场景。同时，指出了现有生物反应器及其装备的应用潜力、面临的挑战与发展趋势，为合成生物学表型测试技术研究提供重要参考。

关键词: 合成生物学, 高通量筛选, 液滴微流控, 生物反应器, 微孔板, 微型管式生物反应器

Abstract:

Over the past years, synthetic biology has seen significant development, establishing a typical "Design-Build-Test-Learn (DBTL)" cycle for engineering cell factories. This cycle has been becoming an enabling methodology for smart breeding to accelerate the development of biomanufacturing. In the DBTL cycle, the testing step primarily aims to evaluate the phenotypes of constructed cell factories, which can provide a large amount of data for further learning and iterative optimization. Due to the complexity of cellular metabolic networks and regulatory mechanisms, as well as the complicated associations between genotypes and phenotypes, the design and construction of cell factories have traditionally involved long-term and labor-intensive iterative experiments. In synthetic biology, the construction of cell factories with designed synthetic pathways is often combined with random mutation and evolution to build up a large screening library, which always requires a high throughput and efficient technology and equipment in the testing step. The testing step is the rate-limiting process in the entire DBTL cycle, and its efficiency is largely dependent on chassis cells themselves, as well as the throughput of bioreactors and instruments needed for their phenotype testing. Here, this review article focuses on an overview of bioreactors and instruments with different throughput scales used for the phenotype testing in synthetic biology. We introduce their characteristics and application scenarios, including single-cell detecting and screening technology as well as pico-, nano-, micro-, milli-, and liter-scale bioreactors. Moreover, this article also points out the application potential of existing phenotype testing bioreactors and instruments, and illustrates how they can be selected for specific research purposes. Finally, the challenges and perspectives for phenotype testing bioreactors and instruments are summarized, which hopefully provides a reference for a wide range of synthetic biology researchers to properly select and use phenotype testing instruments.

Key words: synthetic biology, high throughput screening, droplet microfluidics, bioreactor, micro-well plate, micro-tube bioreactor

中图分类号:

Q816

郭肖杰, 剪兴金, 王立言, 张翀, 邢新会. 合成生物学表型测试生物反应器及其装备化研究进展[J]. 合成生物学, 2024, 5(1): 16-37.

Xiaojie GUO, Xingjin JIAN, Liyan WANG, Chong ZHANG, Xinhui XING. Progress in bioreactors and instruments for phenotype testing with synthetic biology research[J]. Synthetic Biology Journal, 2024, 5(1): 16-37.

图/表 8

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类型		典型装备	特点	主要不足	相关重要文献
单细胞	细胞自体	FACS、RACS、Namo cell、Single-cell printer	通量高，装备成熟且种类多	无法对细胞进行培养，细胞裂解物或外泌物无法作为检测指标	Sun et al.^[5] Gross et al.^[14] Zhang et al.^[39]
皮纳升级	微孔阵列	单细胞光导系统	通量高，装备类型多，可检测细胞裂解物和外泌物	芯片工艺复杂，操作烦琐	Lindstrom et al.^[48] Weibel et al.^[58]
	微液滴	FADS、RADS、IADS	通量高，装备类型多，可检测细胞裂解物和外泌物	存在液滴融合、内容物泄漏等风险	Jiang et al.^[178] Sanchez er al.^[63] Fu et al.^[102]
微升级	微液滴	MMC、MISS cell、Millidrop	细胞培养性能好，相关装备自动化和微型化	液滴稳定性易受影响，脂溶性物质易逃逸出液滴	Baraban et al.^[132] Jian et al.^[35] Jakiela et al.^[120]
微升级	96孔板	酶标仪、Bioscreen生长曲线测定仪	使用方便，适用性广	蒸发问题、边缘效应、孔距离小易交叉污染、溶氧受限	Mansoury et al.^[131] Duetz et al.^[144]
毫升级	深孔板	Biolector、酶标仪	使用方便，适用性广	孔间距较小易交叉污染	Klockner et al.^[150] Betts et al.^[142] Running et al.^[179]
	试管、摇瓶	Micro-24 bioreactor	使用方便，适用性广	相关自动化装备缺乏
	搅拌釜生物反应器	Ambr@15	使用范围广，过程参数易精确控制，利于后期放大	操作烦琐	Karimi et al.^[159] 丁宁，等^[161] Warr et al.^[164]
	微型管式生物反应器	EVOL cell	无气泡渗透供氧，内部流体剪切力低，易集成装备化开发	黏度大或含有颗粒的培养基以及易形成生物膜的菌株不适用	Yang et al.^[73] Li et al.^[75] Jian et al.^[135]
升级	搅拌釜生物反应器	多联罐、平行反应器以及其配套线检测装备（如BODS）	使用范围广，过程参数易精确控制，利于后期工艺放大	体积增大、搅拌釜反应器内部的物质混合和传递难度增加	Garcia-ochoa et al.^[143] Marques et al.^[147] Betts et al.^[157]

类型		典型装备	特点	主要不足	相关重要文献
单细胞	细胞自体	FACS、RACS、Namo cell、Single-cell printer	通量高，装备成熟且种类多	无法对细胞进行培养，细胞裂解物或外泌物无法作为检测指标	Sun et al.^[5] Gross et al.^[14] Zhang et al.^[39]
皮纳升级	微孔阵列	单细胞光导系统	通量高，装备类型多，可检测细胞裂解物和外泌物	芯片工艺复杂，操作烦琐	Lindstrom et al.^[48] Weibel et al.^[58]
	微液滴	FADS、RADS、IADS	通量高，装备类型多，可检测细胞裂解物和外泌物	存在液滴融合、内容物泄漏等风险	Jiang et al.^[178] Sanchez er al.^[63] Fu et al.^[102]
微升级	微液滴	MMC、MISS cell、Millidrop	细胞培养性能好，相关装备自动化和微型化	液滴稳定性易受影响，脂溶性物质易逃逸出液滴	Baraban et al.^[132] Jian et al.^[35] Jakiela et al.^[120]
微升级	96孔板	酶标仪、Bioscreen生长曲线测定仪	使用方便，适用性广	蒸发问题、边缘效应、孔距离小易交叉污染、溶氧受限	Mansoury et al.^[131] Duetz et al.^[144]
毫升级	深孔板	Biolector、酶标仪	使用方便，适用性广	孔间距较小易交叉污染	Klockner et al.^[150] Betts et al.^[142] Running et al.^[179]
	试管、摇瓶	Micro-24 bioreactor	使用方便，适用性广	相关自动化装备缺乏
	搅拌釜生物反应器	Ambr@15	使用范围广，过程参数易精确控制，利于后期放大	操作烦琐	Karimi et al.^[159] 丁宁，等^[161] Warr et al.^[164]
	微型管式生物反应器	EVOL cell	无气泡渗透供氧，内部流体剪切力低，易集成装备化开发	黏度大或含有颗粒的培养基以及易形成生物膜的菌株不适用	Yang et al.^[73] Li et al.^[75] Jian et al.^[135]
升级	搅拌釜生物反应器	多联罐、平行反应器以及其配套线检测装备（如BODS）	使用范围广，过程参数易精确控制，利于后期工艺放大	体积增大、搅拌釜反应器内部的物质混合和传递难度增加	Garcia-ochoa et al.^[143] Marques et al.^[147] Betts et al.^[157]

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合成生物学表型测试生物反应器及其装备化研究进展

Progress in bioreactors and instruments for phenotype testing with synthetic biology research

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