Advanced strategies in high-throughput droplet screening for enzyme engineering

Yan, Wenxin; Li, Xiang; Zhao, Danshan; Xie, Meng; Li, Ting; Qian, Lu; Ye, Chao; Shi, Tianqiong; Wu, Lina; Wang, Yuetong

doi:10.1016/j.bios.2023.115972

Cited by 2 publications

(1 citation statement)

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“…Moreover, it would be preferable to incubate the droplets using a continuous flow device instead of a batch container to ensure homogeneous oxygen availability [ 46 ]. For detecting and separating single yeast cells, our approach can be combined with fluorescent image-based sorting technologies that use image processing and dielectrophoretic or microvalve sorting [ 47 – 50 ]. This will enable us to screen yeast cells that secrete active agonistic peptides in the future.…”

Section: Resultsmentioning

confidence: 99%

Droplet-based microfluidic platform for detecting agonistic peptides that are self-secreted by yeast expressing a G-protein-coupled receptor

Asama,

Liu,

Tominaga

et al. 2024

Microb Cell Fact

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Background Single-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides. Results The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists. Conclusions We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.

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Section: Resultsmentioning

confidence: 99%