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Label-free high-throughput live-cell sorting of genome-wide random mutagenesis libraries for metabolic traits by
Xixian Wang1,2,3,4, Sen Wang1,2,3,4,5, Zhidian Diao1,2,3,4
1State Key Laboratory of Photoelectric Conversion and Utilization of Solar Energy, Key Laboratory of Shandong Energy Biological Genetic Resources, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China.
We developed a novel Raman-activated cell sorting (RACS) method using positive dielectrophoresis-induced deterministic lateral displacement (pDEP-DLD). This technique enables label-free, high-throughput sorting of rare cells based on their metabolic profiles, significantly advancing synthetic biology applications.
Area of Science:
- Biotechnology
- Cell Sorting
- Metabolic Engineering
Background:
- Single-cell Raman spectroscopy offers label-free metabolic phenotyping but lacks efficient sorting methods for rare cells.
- Raman-activated cell sorting (RACS) has been limited in throughput and applicability to heterogeneous samples.
Purpose of the Study:
- To develop and validate a novel RACS technique for high-throughput, label-free isolation of rare cells based on metabolic traits.
- To demonstrate the platform's capability for rapid screening of mutagenesis libraries for enhanced metabolite production.
Main Methods:
- Development of a positive dielectrophoresis-induced deterministic lateral displacement (pDEP-DLD)-based RACS (pDEP-DLD-RACS) system.
- Application of pDEP-DLD-RACS for sorting pigment- and oil-producing yeasts and screening a large mutagenesis library of *Aurantiochytrium* sp.
Main Results:
- pDEP-DLD-RACS achieved high sorting accuracy (>90%), throughput (~600 cells/min), yield (>85%), and stability (~10 hours).
- The method successfully sorted rare cells while maintaining vitality and enabled label-free screening of >10^5 mutants.
- Two days of pDEP-DLD-RACS screening yielded *Aurantiochytrium* sp. mutants with 58% higher docosahexaenoic acid (DHA) productivity.
Conclusions:
- pDEP-DLD-RACS is a powerful, efficient platform for label-free, single-cell metabolic trait screening from large mutagenesis libraries.
- This technology significantly accelerates strain development in synthetic biology by improving time- and cost-efficiency.
- The platform facilitates direct selection of mutants with enhanced metabolic functions, such as increased triacylglycerol (TAG) synthesis.
