Label-free high-throughput live-cell sorting of genome-wide random mutagenesis libraries for metabolic traits by Raman flow cytometry
Abstract
A full spontaneous single-cell Raman spectrum captures the metabolic phenome in a label-free and noninvasive manner. However, Raman-activated cell sorting (RACS) of rare target cells from highly heterogeneous systems has remained largely conceptual. Here, we present a positive dielectrophoresis-induced deterministic lateral displacement (pDEP-DLD)-based RACS (pDEP-DLD-RACS), in which a modulated pDEP-DLD force is applied to focus, trap, and functionally sort fast-moving single cells in a wide channel. For pigment- and oil-producing yeasts, pDEP-DLD-RACS shows high sorting accuracy (>90%), high throughput (~600 events min −1 ), high yield (>85%), and long stable running time (~10 h), and can sort rare cells while preserving full cellular vitality. Moreover, label-free sorting directly from a genome-wide random mutagenesis library with >10 5 Aurantiochytrium sp. Mutants, based on intracellular docosahexaenoic acid (DHA) content, produces mutant cells with 58% higher DHA productivity in just two RACS runs over two days, representing two-orders-of-magnitude higher time- and cost-efficiency than conventional approaches. This superior trait arises from global remolding of transcriptomes, including enhanced carbon metabolism, reduced intracellular NADPH synthesis rates, and increased triacylglycerol (TAG) synthesis. By enabling direct screening of metabolic traits from genome-wide mutagenesis libraries, pDEP-DLD-RACS is a powerful platform for synthetic biology.
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (18)
Xixian Wang
State 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
Sen Wang
State Key Laboratory of Coal Conversion
Zhidian Diao
State 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
Xibao Hou
Qingdao Single-Cell Biotechnology Co., Ltd
Yanhai Gong
State 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
Qing Sun
Jiaping Zhang
State 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
Lihui Ren
State 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
Yuandong Li
State 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
Yuetong Ji
Qingdao Single-Cell Biotechnology Co., Ltd
Wei Shen
Yifeng Yin
eCyte, Inc
Shi Huang
Faculty of Dentistry, University of Hong Kong
Xiaojin Song
State 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
Qiu Cui
State 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
Yingang Feng
State 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
Jian Xu
Bo Ma