Marine sponges are prolific sources of bioactive natural products, several of which are produced by bacteria symbiotically associated with the sponge host. Bacteria-derived natural products, and the specialized bacterial symbionts that synthesize them, are not shared among phylogenetically distant sponge hosts. This is in contrast to nonsymbiotic culturable bacteria in which the conservation of natural products and natural product biosynthetic gene clusters (BGCs) is well established. Here, we demonstrate the widespread conservation of a BGC encoding a cryptic ribosomally synthesized and post-translationally modified peptide (RiPP) in microbiomes of phylogenetically and geographically dispersed sponges from the Pacific and Atlantic oceans. Detection of this BGC was enabled by mining for halogenating enzymes in sponge metagenomes, which, in turn, allowed for the description of a broad-spectrum regiospecific peptidyl tryptophan-6-brominase which possessed no chlorination activity. In addition, we demonstrate the cyclodehydrative installation of azoline heterocycles in proteusin RiPPs. This is the first demonstration of halogenation and cyclodehydration for proteusin RiPPs and the enzymes catalyzing these transformations were found to competently interact with other previously described proteusin substrate peptides. Within a sponge microbiome, many different generalized bacterial taxa harbored this BGC with often more than 50 copies of the BGC detected in individual sponge metagenomes. Moreover, the BGC was found in all sponges queried that possess high diversity microbiomes but it was not detected in other marine invertebrate microbiomes. These data shed light on conservation of cryptic natural product biosynthetic potential in marine sponges that was not detected by traditional natural product-to-BGC (meta)genome mining.

Natural product-encoding biosynthetic gene clusters (BGCs) within microbial genomes far outnumber the known natural products; chemical products from such BGCs remain cryptic. These silent BGCs hold promise not only for the elaboration of new natural products but also for the discovery of useful biosynthetic enzymes. Here, we describe a genome mining strategy targeted toward the discovery of substrate promiscuous natural product biosynthetic enzymes. In the genome of the methanotrophic bacterium Methylovulum psychrotolerans Sph1T, we discover a transcriptionally silent natural product BGC that encoded numerous ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. These cryptic RiPP natural products were accessed using heterologous expression of the substrate peptide and biosynthetic enzyme-encoded genes. In line with our genome mining strategy, the RiPP biosynthetic enzymes in this BGC were found to be substrate promiscuous, which allowed us to use them in a combinatorial fashion with a similarly substrate-tolerant cyanobactin biosynthetic enzyme to introduce head-to-tail macrocyclization in the proteusin family of RiPP natural products.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Bioorthogonal derivatization of peptides and proteins enables investigations into their biological function and allows for exploitation of their therapeutic potential, among other varied deliverables. Herein, we describe a marine halogenating enzyme-assisted bioconjugation strategy in which an N-terminal leader peptide guides bromination of a C-terminal Trp residue in genetically encoded peptides and proteins, setting up further Trp arylation by Suzuki–Miyaura reactions. The bromination and subsequent cross-coupling reactions are residue-specific and regiospecific for the indole-6 position, occur under mild aqueous conditions, and do not require any modification of other Trp residues in the substrate peptide and/or protein. Workflows described herein demonstrate the applicability of halogenating enzymes in bioorthogonal conjugation chemistry.

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