Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions

Lee, Kang Soo; Pereira, Fátima C.; Palatinszky, Márton; Behrendt, Lars; Alcolombri, Uria; Berry, David; Wagner, Michael; Stocker, Roman

doi:10.1038/s41596-020-00427-8

Cited by 49 publications

(40 citation statements)

References 76 publications

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“…The newly identified sequences are then used to update the initial model and the procedure repeated until no additional homologs are identified. This strategy has been applied to identify new β-defensin members in humans and mice 279 and to discover a cysteine-rich gene family in corals. 287 …”

Section: Discussionmentioning

confidence: 99%

“…Another recent study used optical tweezers and microfluidics to sort complex microbial communities based on the Raman spectra of individual cells. 279 This has numerous applications for downstream single-cell sequencing or cultivation efforts including sorting microbes based on stable isotope labels or natural Raman signals from storage compounds or carotenoids. By processing sorted cells for downstream single-cell sequencing, chemical phenotypes of live individual cells can be directly linked to their genotypes.…”

Section: Gearing Up For the Future: New Frontiers In Enzyme Discoverymentioning

confidence: 99%

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A roadmap for metagenomic enzyme discovery

2021

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Section: Discussionmentioning

confidence: 99%

Section: Gearing Up For the Future: New Frontiers In Enzyme Discoverymentioning

confidence: 99%

A roadmap for metagenomic enzyme discovery

2021

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“…Raman microscopy is one of the few single-cell techniques that provides a direct link between metabolites and source organism in a non-destructive way and at the single-cell level and can be applied to diverse questions in microbiological research using commercially available instrumentation. Raman microscopy was previously applied to single-bacterial natural product imaging 37 and to bacterial sorting based on deuterium incorporation 38 , but to our knowledge MERMAID is the first single-cell pipeline that also provides genomic information. This pipeline can be a one-stop, high-throughput system based on microfluidics.…”

Section: Discussionmentioning

confidence: 99%

Single-cell metabolite detection and genomics reveals uncultivated talented producer

Kogawa

Miyaoka

Hemmerling

et al. 2021

Preprint

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The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), one of the oldest metazoans. However, as most complex microbiomes remain largely uncultivated and lack reference genomes, unequivocally linking metabolic functions to a cellular source is a challenge. Here we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge Theonella swinhoei, previously shown to contain ′Entotheonella′ symbionts providing most of its bioactive substances except for the antifungal aurantosides that lacked biosynthetic gene candidates in the metagenome. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ′Candidatus Poriflexus aureus′ from a new Chloroflexi lineage. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

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“…Unlike MS, RADS is non-invasive but also label-free. RADS could sort cells at a rate of hundreds of cells per minute [up to 500 cells/h achieved (Lee et al, 2020a )]. Wang et al ( 2017 ) ( Figure 3C ) developed RADS platform by placing single-cell Raman spectrum (SCRS) prior to droplet generation, since Raman background of oil medium might adversely affect the system's accuracy.…”

Section: Label-free Sortingmentioning

confidence: 99%

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

Zhang

et al. 2021

Front. Chem.

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Droplet-based microfluidics has been widely applied in enzyme directed evolution (DE), in either cell or cell-free system, due to its low cost and high throughput. As the isolation principles are based on the labeled or label-free characteristics in the droplets, sorting method contributes mostly to the efficiency of the whole system. Fluorescence-activated droplet sorting (FADS) is the mostly applied labeled method but faces challenges of target enzyme scope. Label-free sorting methods show potential to greatly broaden the microfluidic application range. Here, we review the developments of droplet sorting methods through a comprehensive literature survey, including labeled detections [FADS and absorbance-activated droplet sorting (AADS)] and label-free detections [electrochemical-based droplet sorting (ECDS), mass-activated droplet sorting (MADS), Raman-activated droplet sorting (RADS), and nuclear magnetic resonance-based droplet sorting (NMR-DS)]. We highlight recent cases in the last 5 years in which novel enzymes or highly efficient variants are generated by microfluidic DE. In addition, the advantages and challenges of different sorting methods are briefly discussed to provide an outlook for future applications in enzyme DE.

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Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions

Cited by 49 publications

References 76 publications

A roadmap for metagenomic enzyme discovery

A roadmap for metagenomic enzyme discovery

Single-cell metabolite detection and genomics reveals uncultivated talented producer

Recent Advances on Sorting Methods of High-Throughput Droplet-Based Microfluidics in Enzyme Directed Evolution

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