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

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