Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics

Abstract

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using a fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and non-ribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

Synthase-selected sorting approach identifies a beta-lactone synthase in a nudibranch symbiotic bacterium

“…D. californiensis as a putative PKS/NRPS active cell. To date, our studies have focused on the use of the KC-12 probe based on its ability to label carrier proteins (CP) associated with fatty acid (ACPs), polyketide (ACPs), and NRPS (PCPs) synthesis [ 47 ]. As shown schematically in Fig.…”

“…We have previously shown that the probe KC-12 hijacks coenzyme A (CoA) biosynthesis in cells to produce a fluorescently-labeled analog of coenzyme A (Fig. 1 a) [ 46 , 47 ]. This fluorescently labeled CoA can be post-translationally added to the active site serine residue on acyl-carrier proteins (ACP) and peptidyl-carrier proteins (PCPs) associated with PKS and NRPS, respectively [ 46 , 47 ].…”

“…1 a) [ 46 , 47 ]. This fluorescently labeled CoA can be post-translationally added to the active site serine residue on acyl-carrier proteins (ACP) and peptidyl-carrier proteins (PCPs) associated with PKS and NRPS, respectively [ 46 , 47 ]. The uptake of the KC-12 probe in bacterial cells undergoes a two-step process beginning with conversion to its corresponding CoA and then covalent 4’-phosphopantetheinylation of the active site serine residue on ACP and PCP-containing proteins with the modified CoA.…”

Single-cell metabolite detection and genomics reveals uncultivated talented producer

“…This pipeline can be a one-stop, high-throughput system based on microfluidics. Recently, a single-cell analysis method linking metabolic and genomic features has been developed based on labeling of synth(et)ase enzymes ( 39 ). The potential advantage of MERMAID is the detection of various types of intracellular biomolecules with specific molecular features.…”

The natural product biosynthesis potential of the microbiomes of Earth – Bioprospecting for novel anti-microbial agents in the meta-omics era

“…This strategy was applied to extract an enriched fraction of the tunicate microbiome actively expressing bioactive compounds at the time of sampling. Through fluorescent labelling of the carrier proteins involved in NRP and PK biosynthesis and subsequent fluorescence activated cell sorting (FACS), it was possible to enrich the BGC content from 0.77 to 1.17 BGC per Mbp [97] .…”