Phylogeny-metabolism dual-directed single-cell genomics for dissecting and mining ecosystem function by FISH-scRACS-seq

Xiaoyan Jing; Yanhai Gong; Zhidian Diao; Yan Ma; Yu Meng; Jie Chen; Yishang Ren; Yuting Liang; Yinchao Li; Weihan Sun; Jia Zhang; Yuetong Ji; Zhiqi Cong; Shengying Li; Bo Ma; Zhisong Cui; Li Ma; Jian Xu

doi:10.1016/j.xinn.2024.100759

Phylogeny-metabolism dual-directed single-cell genomics for dissecting and mining ecosystem function by FISH-scRACS-seq

Innovation (Camb). 2025 Jan 16;6(3):100759. doi: 10.1016/j.xinn.2024.100759. eCollection 2025 Mar 3.

Authors

Xiaoyan Jing^{1

2

3

4}, Yanhai Gong^{1

2

3

4}, Zhidian Diao^{1

2

3

4}, Yan Ma^{1

5}, Yu Meng^{1

2

3

4}, Jie Chen^{1

2

3

4}, Yishang Ren^{1

2

3

4}, Yuting Liang⁶, Yinchao Li⁷, Weihan Sun⁸, Jia Zhang^{1

2

3

4}, Yuetong Ji⁹, Zhiqi Cong^{1

2

3

4}, Shengying Li⁸, Bo Ma^{1

2

3

4}, Zhisong Cui⁷, Li Ma⁸, Jian Xu^{1

2

3

4}

Affiliations

¹ Single-Cell Center, CAS Key Laboratory of Biofuels, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266000, China.
² University of Chinese Academy of Sciences, Beijing 100000, China.
³ Shandong Energy Institute, Qingdao 266000, China.
⁴ Qingdao New Energy Shandong Laboratory, Qingdao 266000, China.
⁵ Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430000, China.
⁶ State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Science, Nanjing 211300, China.
⁷ Marine Bioresource and Environment Research Center, Key Laboratory of Marine Eco-Environmental Science and Technology, First Institute of Oceanography, Ministry of Natural Resources of China, Qingdao 266000, China.
⁸ State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266000, China.
⁹ Qingdao Single-Cell Biotechnology, Co., Ltd., Qingdao 266000, China.

Abstract

Microbiome-wide association studies (MWASs) have uncovered microbial markers linked to ecosystem traits, but the mechanisms underlying their functions can remain elusive. This is largely due to challenges in validating their in situ metabolic activities and tracing such activities to individual genomes. Here, we introduced a phylogeny-metabolism dual-directed single-cell genomics approach called fluorescence-in situ-hybridization-guided single-cell Raman-activated sorting and sequencing (FISH-scRACS-seq). It directly localizes individual cells from target taxon via an FISH probe for marker organism, profiles their in situ metabolic functions via single-cell Raman spectra, sorts cells of target taxonomy and target metabolism, and produces indexed, high-coverage, and precisely-one-cell genomes. From cyclohexane-contaminated seawater, cells representing the MWAS-derived marker taxon of γ-Proteobacteria and that are actively degrading cyclohexane in situ were directly identified via FISH and Raman, respectively, then sorted and sequenced for one-cell full genomes. In such a Pseudoalteromonas fuliginea cell, we discovered a three-component cytochrome P450 system that can convert cyclohexane to cyclohexanol in vitro, representing a previously unknown group of cyclohexane-degrading enzymes and organisms. Therefore, by unveiling enzymes, pathways, genomes, and their in situ cellular functions specifically for those organisms with ecological relevance at one-cell resolution, FISH-scRACS-seq is a rational and generally applicable approach to dissecting and mining microbiota functions.

Keywords: FISH; Fluorescence in situ hybridization; RACS; Raman-activated cell sorting; Single-cell Raman microspectroscopy; microbiota; pollutant degradation.