Nature's Combinatorial Biosynthesis Produces Vatiamides A-F.

Hybrid type I PKS/NRPS biosynthetic pathways typically proceed in a collinear manner wherein one molecular building block is enzymatically incorporated in a sequence that corresponds to gene arrangement. In this work, genome mining combined with the use of a fluorogenic azide-based click probe led to the discovery and characterization of vatiamides A-F, three structurally diverse alkynylated lipopeptides, and their brominated analogues, from the cyanobacterium Moorea producens ASI16Jul14-2. These derive from a unique combinatorial non-collinear PKS/NRPS system encoded by a 90 kb gene cluster in which an upstream PKS cassette interacts with three separate cognate NRPS partners. This is facilitated by a series of promiscuous intermodule PKS-NRPS docking motifs possessing identical amino acid sequences. This interaction confers a new type of combinatorial capacity for creating molecular diversity in microbial systems.

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium.

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa. The structure of 1 was established by detailed 1D and 2D NMR experiments, HRESIMS data, and chemical degradation/chromatographic (e.g., Marfey's analysis) studies. Pure compound 1 was shown to have in vitro cytotoxic activity against several human cancer cell lines in both traditional cell culture and zone inhibition bioassays. Although there was no particular selectivity between the cell lines tested for samoamide A, the most potent activity was observed against H460 human non-small-cell lung cancer cells (IC50 = 1.1 µM). Molecular modeling studies suggested that one possible mechanism of action for 1 is the inhibition of the enzyme dipeptidyl peptidase (CD26, DPP4) at a reported allosteric binding site, which could lead to many downstream pharmacological effects. However, this interaction was moderate when tested in vitro at up to 10 µM and only resulted in about 16% peptidase inhibition. Combining bioassay screening with the cheminformatics strategy of LC-MS/MS molecular networking as a discovery tool expedited the targeted isolation of a natural product possessing both a novel chemical structure and a desired biological activity.

Integrating mass spectrometry and genomics for cyanobacterial metabolite discovery.

Filamentous marine cyanobacteria produce bioactive natural products with both potential therapeutic value and capacity to be harmful to human health. Genome sequencing has revealed that cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The biosynthetic pathways that encode cyanobacterial natural products are mostly uncharacterized, and lack of cyanobacterial genetic tools has largely prevented their heterologous expression. Hence, a combination of cutting edge and traditional techniques has been required to elucidate their secondary metabolite biosynthetic pathways. Here, we review the discovery and refined biochemical understanding of the olefin synthase and fatty acid ACP reductase/aldehyde deformylating oxygenase pathways to hydrocarbons, and the curacin A, jamaicamide A, lyngbyabellin, columbamide, and a trans-acyltransferase macrolactone pathway encoding phormidolide. We integrate into this discussion the use of genomics, mass spectrometric networking, biochemical characterization, and isolation and structure elucidation techniques.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

Collection, Culturing, and Genome Analyses of Tropical Marine Filamentous Benthic Cyanobacteria.

Decreasing sequencing costs has sparked widespread investigation of the use of microbial genomics to accelerate the discovery and development of natural products for therapeutic uses. Tropical marine filamentous cyanobacteria have historically produced many structurally novel natural products, and therefore present an excellent opportunity for the systematic discovery of new metabolites via the information derived from genomics and molecular genetics. Adequate knowledge transfer and institutional know-how are important to maintain the capability for studying filamentous cyanobacteria due to their unusual microbial morphology and characteristics. Here, we describe workflows, procedures, and commentary on sample collection, cultivation, genomic DNA generation, bioinformatics tools, and biosynthetic pathway analysis concerning filamentous cyanobacteria.

Ketoreductase Domain Dysfunction Expands Chemodiversity: Malyngamide Biosynthesis in the Cyanobacterium Okeania hirsuta.

Dozens of type A malyngamides, principally identified by a decorated six-membered cyclohexanone headgroup and methoxylated lyngbic acid tail, have been isolated over several decades. Their environmental sources include macro- and microbiotic organisms, including sea hares, red alga, and cyanobacterial assemblages, but the true producing organism has remained enigmatic. Many type A analogues display potent bioactivity in human-health related assays, spurring an interest in this molecular class and its biosynthetic pathway. Here, we present the discovery of the type A malyngamide biosynthetic pathway in the first sequenced genome of the cyanobacterial genus Okeania. Bioinformatic analysis of two cultured Okeania genome assemblies identified 62 and 68 kb polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) pathways with unusual loading and termination genes. NMR data of malyngamide C acetate derived from 13C-substrate-fed cultures provided evidence that an intact octanoate moiety is transferred to the first KS module via a LipM homologue originally associated with lipoic acid metabolism and implicated an inactive ketoreductase (KR0) as critical for six-membered ring formation, a hallmark of the malyngamide family. Phylogenetic analysis and homology modeling of the penultimate KR0 domain inferred structural cofactor binding and active site alterations as contributory to domain dysfunction, which was confirmed by recombinant protein expression and NADPH binding assay. The carbonyl retained from this KR0 ultimately enables an intramolecular Knoevenagel condensation to form the characteristic cyclohexanone ring. Understanding this critical step allows assignment of a biosynthetic model for all type A malyngamides, whereby well-characterized tailoring modifications explain the surprising proliferation and diversity of analogues.

Biosynthesis of t-Butyl in Apratoxin A: Functional Analysis and Architecture of a PKS Loading Module.

The unusual feature of a t-butyl group is found in several marine-derived natural products including apratoxin A, a Sec61 inhibitor produced by the cyanobacterium Moorea bouillonii PNG 5-198. Here, we determine that the apratoxin A t-butyl group is formed as a pivaloyl acyl carrier protein (ACP) by AprA, the polyketide synthase (PKS) loading module of the apratoxin A biosynthetic pathway. AprA contains an inactive "pseudo" GCN5-related N-acetyltransferase domain (ΨGNAT) flanked by two methyltransferase domains (MT1 and MT2) that differ distinctly in sequence. Structural, biochemical, and precursor incorporation studies reveal that MT2 catalyzes unusually coupled decarboxylation and methylation reactions to transform dimethylmalonyl-ACP, the product of MT1, to pivaloyl-ACP. Further, pivaloyl-ACP synthesis is primed by the fatty acid synthase malonyl acyltransferase (FabD), which compensates for the ΨGNAT and provides the initial acyl-transfer step to form AprA malonyl-ACP. Additionally, images of AprA from negative stain electron microscopy reveal multiple conformations that may facilitate the individual catalytic steps of the multienzyme module.

A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit.

Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here, we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+, and Ni2+ support only a single methyl transfer. MT1 homologues exist within the "GNAT" (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl, or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA, the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT di-domain with bound Mn2+, malonate, and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologues. These results afford an expanded understanding of MT1-GNAT structure and activity and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

Kalkipyrone B, a marine cyanobacterial γ-pyrone possessing cytotoxic and anti-fungal activities.

Bioassay-guided fractionation of two marine cyanobacterial extracts using the H-460 human lung cancer cell line and the OVC-5 human ovarian cancer cell line led to the isolation of three related α-methoxy-ß, ß'-dimethyl-γ-pyrones each containing a modified alkyl chain, one of which was identified as the previously reported kalkipyrone and designated kalkipyrone A. The second compound was an analog designated kalkipyrone B. The third was identified as the recently reported yoshinone A, also isolated from a marine cyanobacterium. Kalkipyrone A and B were obtained from a field-collection of the cyanobacterium Leptolyngbya sp. from Fagasa Bay, American Samoa, while yoshinone A was isolated from a field-collection of cyanobacteria (cf. Schizothrix sp.) from Panama. One-dimensional and two-dimensional NMR experiments were used to determine the overall structures and relative configurations of the kalkipyrones, and the absolute configuration of kalkipyrone B was determined by (1)H NMR analysis of diastereomeric Mosher's esters. Kalkipyrone A showed good cytotoxicity to H-460 human lung cancer cells (EC50=0.9µM), while kalkipyrone B and yoshinone A were less active (EC50=9.0µM and >10µM, respectively). Both kalkipyrone A and B showed moderate toxicity to Saccharomyces cerevisiae ABC16-Monster strain (IC50=14.6 and 13.4µM, respectively), whereas yoshinone A was of low toxicity to this yeast strain (IC50=63.8µM).