Scytodecamide from the Cultured Scytonema sp. UIC†10036 Expands the Chemical and Genetic Diversity of Cyanobactins.

Cyanobactins are a large family of cyanobacterial ribosomally synthesized and post-translationally modified peptides (RiPPs) often associated with biological activities, such as cytotoxicity, antiviral, and antimalarial activities. They are traditionally described as cyclic molecules containing heterocyclized amino acids. However, this definition has been recently challenged by the discovery of short, linear cyanobactins containing three to five amino acids as well as cyanobactins containing no heterocyclized residues. Herein we report the discovery of scytodecamide (1) from the freshwater cyanobacterium Scytonema sp. UIC 10036. Structural elucidation based on mass spectrometry, 1D and 2D NMR spectroscopy, and Marfey's method revealed 1 to be a linear decapeptide with an N-terminal N-methylation and a C-terminal amidation. The genome of Scytonema sp. UIC 10036 was sequenced, and bioinformatic analysis revealed a cyanobactin-like biosynthetic gene cluster consistent with the structure of 1. The discovery of 1 as a novel linear peptide containing an N-terminal N-methylation and a C-terminal amidation expands the chemical and genetic diversity of the cyanobactin family of compounds.

Diazaquinomycin Biosynthetic Gene Clusters from Marine and Freshwater Actinomycetes.

Tuberculosis is an infectious disease of global concern. Members of the diazaquinomycin (DAQ) class of natural products have shown potent and selective activity against drug-resistant Mycobacterium tuberculosis. However, poor solubility has prevented further development of this compound class. Understanding DAQ biosynthesis may provide a viable route for the generation of derivatives with improved properties. We have sequenced the genomes of two actinomycete bacteria that produce distinct DAQ derivatives. While software tools for automated biosynthetic gene cluster (BGC) prediction failed to detect DAQ BGCs, comparative genomics using MAUVE alignment led to the identification of putative BGCs in the marine Streptomyces sp. F001 and in the freshwater Micromonospora sp. B006. Deletion of the identified daq BGC in strain B006 using CRISPR-Cas9 genome editing abolished DAQ production, providing experimental evidence for BGC assignment. A complete model for DAQ biosynthesis is proposed based on the genes identified. Insufficient knowledge of natural product biosynthesis is one of the major challenges of productive genome mining approaches. The results reported here fill a gap in knowledge regarding the genetic basis for the biosynthesis of DAQ antibiotics. Moreover, identification of the daq BGC shall enable future generations of improved derivatives using biosynthetic methods.

Heterologous expression of the diazaquinomycin biosynthetic gene cluster.

Members of the diazaquinomycin class of natural products have shown potent and selective activity against Mycobacterium tuberculosis. However, poor aqueous solubility has prevented extensive studies in animal models thus far. Our long-term goal is to harness knowledge regarding diazaquinomycin biosynthesis towards the generation of derivatives for structure-activity relationship studies. We have previously sequenced the genomes of two diazaquinomycin-producing, actinomycete bacteria and identified putative daq biosynthetic gene clusters. Here, we report the heterologous expression of the daq gene cluster from the marine Streptomyces sp. F001 in S. coelicolor M1152. In addition to serving as functional proof for gene cluster assignment, the heterologous expression system reported here is expected to facilitate investigations aimed at elucidating diazaquinomycin biosynthesis.

Heterologous expression of a putative ClpC chaperone gene leads to induction of a host metabolite.

Genome mining provides exciting opportunities for the discovery of natural products. However, in contrast to traditional bioassay-guided approaches, challenges of genome mining include poor or no expression of biosynthetic gene clusters (BGCs). Additionally, given that thousands of BGCs are now available through extensive genome sequencing, how does one select BGCs for discovery? Synthetic biology techniques can be used for BGC refactoring and activation, whereas resistance-gene-directed genome mining is a promising approach to discover bioactive natural products. Here we report the selection of a BGC by applying a resistance-gene-directed approach, cloning of the silent BGC from Micromonospora sp. B006, promoter exchange, and heterologous expression in Streptomyces coelicolor M1152. While we have yet to identify the encoded compound, we unexpectedly observed induction of a host metabolite, which we hypothesize is due to the presence of a ClpC chaperone gene in the BGC, suggesting that ClpC chaperones may be used for BGC activation.

Complete Genome of Micromonospora sp. Strain B006 Reveals Biosynthetic Potential of a Lake Michigan Actinomycete.

Actinomycete bacteria isolated from freshwater environments are an unexplored source of natural products. Here we report the complete genome of the Great Lakes-derived Micromonospora sp. strain B006, revealing its potential for natural product biosynthesis. The 7-megabase pair chromosome of strain B006 was sequenced using Illumina and Oxford Nanopore technologies followed by Sanger sequencing to close remaining gaps. All identified biosynthetic gene clusters (BGCs) were manually curated. Five known BGCs were identified encoding desferrioxamine, alkyl- O-dihydrogeranylmethoxyhydroquinone, a spore pigment, sioxanthin, and diazepinomicin, which is currently in phase II clinical trials to treat Phelan-McDermid syndrome and co-morbid epilepsy. We report here that strain B006 is indeed a producer of diazepinomicin and at yields higher than previously reported. Moreover, 11 of the 16 identified BGCs are orphan, eight of which were transcriptionally active under the culture condition tested. Orphan BGCs include an enediyne polyketide synthase and an uncharacteristically large, 36-module polyketide synthase-nonribosomal peptide synthetase BGC. We developed a genetics system for Micromonospora sp. B006 that will contribute to deorphaning BGCs in the future. This study is one of the few attempts to report the biosynthetic capacity of a freshwater-derived actinomycete and highlights this resource as a potential reservoir for new natural products.

Biochemical and genetic basis of orsellinic acid biosynthesis and prenylation in a stereaceous basidiomycete.

The prenylphenols are a class of natural products that have been frequently isolated from basidiomycetes, e.g., from the genus Stereum (false turkey tail fungi) and other Russulales as well as from ascomycetes. Biosynthetically, these compounds are considered hybrids, as the orsellinic acid moiety is a polyketide and the prenyl side chain originates from the terpene metabolism, although no literature on the genetic and biochemical background of the biosynthesis is available. In a stereaceous basidiomycete, referred to as BY1, a new prenylphenol, now termed cloquetin, was identified and its structure elucidated by mass spectrometry and nuclear magnetic resonance spectroscopy. Genes for two non-reducing polyketide synthases (PKS1 and PKS2) were identified in the BY1 genome, and heterologously expressed in Aspergillus niger. Product formation identified both PKSs as orsellinic acid synthases. A putative prenyltransferase gene (BYPB) found in the BY1 genome was expressed in Escherichia coli. In vitro characterization showed that BYPB activity depends on bivalent cations and that it uses orsellinic acid as acceptor substrate for the transfer of a prenyl group. The two orsellinic acid synthases support the emerging notion that fungi secure individual metabolic steps or entire pathways by redundant enzymes.

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors.

Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Genome mining reveals the evolutionary origin and biosynthetic potential of basidiomycete polyketide synthases.

Numerous polyketides are known from bacteria, plants, and fungi. However, only a few have been isolated from basidiomycetes. Large scale genome sequencing projects now help anticipate the capacity of basidiomycetes to synthesize polyketides. In this study, we identified and annotated 111 type I and three type III polyketide synthase (PKS) genes from 35 sequenced basidiomycete genomes. Phylogenetic analysis of PKS genes suggests that all main types of fungal iterative PKS had already evolved before the Ascomycota and Basidiomycota diverged. A comparison of genomic and metabolomic data shows that the number of polyketide genes exceeds the number of known polyketide structures by far. Exploiting these results to design degenerate PCR primers, we amplified and cloned the complete sequence of armB, a PKS gene from the melleolide producer Armillaria mellea. We expect this study will serve as a guide for future genomic mining projects to discover structurally diverse mushroom-derived polyketides.

Genome Sequence of Marine-Derived Streptomyces sp. Strain F001, a Producer of Akashin A and Diazaquinomycins.

We report the 9.7-Mb genome sequence of Streptomyces sp. strain F001, isolated from a marine sediment sample from Raja Ampat, Indonesia. F001 produces diazaquinomycins, which exhibit potent and selective antituberculosis activity. In addition, it is also known to produce akashin A, a blue pigment that has shown cytotoxic activity.

Three Redundant Synthetases Secure Redox-Active Pigment Production in the Basidiomycete Paxillus involutus.

The symbiotic fungus Paxillus involutus serves a critical role in maintaining forest ecosystems, which are carbon sinks of global importance. P. involutus produces involutin and other 2,5-diarylcyclopentenone pigments that presumably assist in the oxidative degradation of lignocellulose via Fenton chemistry. Their precise biosynthetic pathways, however, remain obscure. Using a combination of biochemical, genetic, and transcriptomic analyses, in addition to stable-isotope labeling with synthetic precursors, we show that atromentin is the key intermediate. Atromentin is made by tridomain synthetases of high similarity: InvA1, InvA2, and InvA5. An inactive atromentin synthetase, InvA3, gained activity after a domain swap that replaced its native thioesterase domain with that of InvA5. The found degree of multiplex biosynthetic capacity is unprecedented with fungi, and highlights the great importance of the metabolite for the producer.

Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.

The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing.