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Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics
Woojoo E Kim1, Katherine Charov1, Mária Džunková2
1Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States.
ACS Chemical Biology
|May 6, 2021
Summary
Researchers used enzyme assays and single-cell genomics to find active microbes in tunicates. This led to the discovery of novel biosynthetic gene clusters, including a new nonribosomal peptide synthetase (NRPS) in marine bacteria.
Area of Science:
- Microbiology and Genomics
- Natural Product Discovery
- Biotechnology
Background:
- Computational methods have identified numerous biosynthetic gene clusters, but lack experimental validation of their in vivo activity.
- Environmental samples, like tunicate microbiomes, are rich sources of microbial diversity with potential for novel natural product discovery.
- Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) are key enzymes in secondary metabolite biosynthesis.
Purpose of the Study:
- To develop and apply an activity-guided approach to identify microbes with active secondary metabolic capabilities.
- To uncover the genetic basis of novel biosynthetic diversity within enriched microbial populations.
- To discover and characterize new biosynthetic gene clusters, specifically NRPS, from marine environments.
Main Methods:
- Fluorescent in situ enzyme assays targeting PKS and NRPS carrier proteins were employed.
- Fluorescence-activated cell sorting (FACS) was used to enrich for enzyme-active microbial cells from tunicate microbiomes.
- Single-cell genomics was performed on sorted cells to determine the genetic makeup of active microbes.
Main Results:
- The study successfully enriched for microbes exhibiting active secondary metabolic functions.
- Single-cell genomics revealed significant biosynthetic diversity within the enzyme-active cell population.
- A novel NRPS gene cluster was identified in a marine Oceanospirillales bacterium, showing similarity to distant bacterial clusters and associated with siderophore biosynthesis.
Conclusions:
- Activity-guided single-cell genomics is an effective strategy for discovering novel bioactive natural products.
- The identified NRPS cluster represents a new avenue for exploring marine-derived secondary metabolites.
- This methodology bridges the gap between computational prediction and experimental validation of biosynthetic gene clusters.
