Abstract:

Microbial industrial manufacturing focuses on construction 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 10⁷ 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 and 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.

Key words: flow cytometry, fluorescence-activated droplet sorting, microbial factories, enzyme directed evolution

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。

关键词: 流式细胞术, 荧光激活液滴分选, 微生物细胞工厂, 酶定向进化