Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction

SUN Mengchu; LU Liangyu; SHEN Xiaolin; SUN Xinxiao; WANG Jia; YUAN Qipeng

doi:10.12211/2096-8280.2023-017

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Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction

Invited Review | 更新时间：2025-03-20

- Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction
- Synthetic Biology Journal Vol. 4, Issue 5, Pages: 947-965(2023)
- 作者机构：
  
  北京化工大学化工资源有效利用国家重点实验室，北京软物质科学与工程高精尖创新中心，北京 100029
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2023-017
  CLC： Q812
- Received：28 February 2023，
  
  Revised：2023-04-17，
  
  Published：31 October 2023
- 稿件说明：
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孙梦楚，陆亮宇，申晓林，孙新晓，王佳，袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建［J］. 合成生物学， 2023， 4（5）： 947-965

SUN Mengchu， LU Liangyu， SHEN Xiaolin， SUN Xinxiao， WANG Jia， YUAN Qipeng. Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction［J］. Synthetic Biology Journal， 2023， 4（5）： 947-965
孙梦楚，陆亮宇，申晓林，孙新晓，王佳，袁其朋. 基于荧光检测的高通量筛选技术和装备助力细胞工厂构建［J］. 合成生物学， 2023， 4（5）： 947-965 DOI： 10.12211/2096-8280.2023-017.

SUN Mengchu， LU Liangyu， SHEN Xiaolin， SUN Xinxiao， WANG Jia， YUAN Qipeng. Fluorescence detection-based high-throughput screening systems and devices facilitate cell factories construction［J］. Synthetic Biology Journal， 2023， 4（5）： 947-965 DOI： 10.12211/2096-8280.2023-017.

摘要

微生物工业制造是以微生物细胞工厂为核心，利用低成本、可再生资源为原料，实现高附加值化合物的绿色生产。依赖于“设计-构建-测试-学习”（DBTL）循环的微生物细胞工厂开发过程中“测试”阶段已成为制约合成生物学和代谢工程发展的瓶颈之一。基于微量滴定板（MTP）高通量自动化筛选平台极大降低了高通量筛选过程的劳动强度，流式细胞术和液滴微流控技术的发展大幅度提高了筛选通量。尤其是荧光激活液滴分选（FADS）高通量筛选技术的开发为自动化、高通量和低消耗筛选工作提供了新的解决方案。本文综述了不同高通量筛选技术在合成生物学和代谢工程领域应用的主要进展，重点介绍了近几年荧光激活细胞分选技术（FACS）和FADS在微生物细胞工厂和酶定向进化方面的应用实例，关注了待测分子与荧光信号偶联的常用策略，并简单介绍目前国内外基于液滴微流控技术高通量筛选装备的研发情况。

Abstract

Microbial industrial manufacturing focuses on c

onstruction of microbial cell factories using low-cost

renewable resources as materials to achieve sustainable production of value-added compounds. The "test" stage in the development of microbial factories that relies on the "Design-Built-Test-Learn" cycle has quickly become one of the bottlenecks restricting the development of synthetic biology and metabolic engineering. To accelerate DBTL cycling

high-throughput screening techniques need to match the size of the library during the testing phase. Microtiter plates (MTP)

as a traditional screening method

uses the optical changes of metabolites in microliters of culture medium for detection and analysis

which can meet the repeated detection and accurate determination of mutants in the library

and also have the ability to screen high-yield strains of extracellular metabolites. However

this screening method is time-consuming and has low throughput. The automated platform is a good solution to the limitations of low screening throughput of microplates. However

the high cost of automation equipment and equipment maintenance makes this method not universal. At present

the main method of high-throughput screening is fluorescence-activated cell sorting (FACS)

which can reach a screening throughput of about 100 000 cells per second. However

FACS is limited to detecting intracellular fluorescence signals associated with target products or metabolite fluorescence signals bound to membranes. This problem is well solved by droplet microfluidic technology

which embeds and cultures single cells in monodisperse and picoliter droplets; each droplet acts as a separate microreactor to achieve genotype and phenotype coupling. In the process of screening of huge mutant libraries by droplet microfluidic technology

the screening throughput can reach 10

per day

which effectively improves the work efficiency

and also shows great advantages in experimental cost

realizing the development of microbial cell factories with high screening throughput and low cost an

d the screening of highly active enzyme variants. In conclusion

based on the high-throughput automated screening platform using microtiter plates

the human labor investment of the high-throughput screening process is greatly reduced

and the development of FACS and droplet microfluidic technology further improves the throughput. In particular

the development of fluorescence-activated droplet sorting (FADS) high-throughput screening technology opens up the possibility for automated

high-throughput

and low-consumption screening. This paper reviews the main progress of the application of different high-throughput screening techniques in the field of synthetic biology. Emphasis will be put on the application of fluorescence-activated cell sorting and FADS in microbial cell factories and enzyme directed evolution in recent years

especially the common strategies of coupling the molecules to be tested with fluorescence signals. We also briefly introduce the current research and development of high-throughput screening equipment based on droplet microfluidic technology.

关键词

Keywords

references

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State Key Laboratory of Chemical Resource Engineering， Beijing Advanced Innovation Center for Soft Matter Science and EngineeringBeijing University of Chemical Technology

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