A Pseudoalteromonas Clade with Remarkable Biosynthetic Potential.

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we report the biochemical and genomic analysis of Pseudoalteromonas sp. strain HM-SA03, isolated from the blue-ringed octopus, Hapalochlaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides, and four novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through the phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of 10 biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the underexplored potential of Pseudoalteromonas as a source of new natural products.IMPORTANCE This study demonstrates that the Pseudoalteromonas strain HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Genome mining of a fungal endophyte of Taxus yunnanensis (Chinese yew) leads to the discovery of a novel azaphilone polyketide, lijiquinone.

Genome mining of Ascomycete sp. F53 (F53), a fungal endophyte of the traditional Chinese medicinal plant Taxus yunnanensis (Chinese yew), revealed 35 putative specialized metabolite biosynthesis gene clusters, one of which encodes a rarely seen tandem polyketide synthase pathway with close homology to azaphilone biosynthesis pathways. A novel compound, lijiquinone 1, was subsequently isolated from F53 and structurally and functionally characterized. The m/z 385 [M + H+ ]+ compound, comprised of a cyclohexenone side group attached to a core bicyclic ring, displayed cytotoxicity against human myeloma cells (IC50  = 129 µM), as well as antifungal activity against Candida albicans (IC50  = 79 µM) and Cryptococcus albidus (IC50  = 141 µM). Our results suggest that enzymes encoded on the lij gene cluster are responsible for the synthesis of 1 and that the medicinal properties of T. yunnanensis could be partially mediated by this novel azaphilone. This study highlights the utility of combining traditional knowledge with contemporary genomic approaches for the discovery of new bioactive compounds.

Directing the Heterologous Production of Specific Cyanobacterial Toxin Variants.

Microcystins are globally the most commonly occurring freshwater cyanotoxins, causing acute poisoning and chronically inducing hepatocellular carcinoma. However, the detection and toxicological study of microcystins is hampered by the limited availability and high cost of pure toxin standards. Biosynthesis of microcystin variants in a fast-growing heterologous host offers a promising method of achieving reliable and economically viable alternative to isolating toxin from slow-growing cyanobacterial cultures. Here, we report the heterologous expression of recombinant microcystin synthetases in Escherichia coli to produce [d-Asp3]microcystin-LR and microcystin-LR. We assembled a 55 kb hybrid polyketide synthase/nonribosomal peptide synthetase gene cluster from Microcystis aeruginosa PCC 7806 using Red/ET recombineering and replaced the native promoters with an inducible PtetO promoter to yield microcystin titers superior to M. aeruginosa. The expression platform described herein can be tailored to heterologously produce a wide variety of microcystin variants, and potentially other cyanobacterial natural products of commercial relevance.

Heterologous Production of Cyanobacterial Mycosporine-Like Amino Acids Mycosporine-Ornithine and Mycosporine-Lysine in Escherichia coli.

Mycosporine-like amino acids (MAAs) are an important class of secondary metabolites known for their protection against UV radiation and other stress factors. Cyanobacteria produce a variety of MAAs, including shinorine, the active ingredient in many sunscreen creams. Bioinformatic analysis of the genome of the soil-dwelling cyanobacterium Cylindrospermum stagnale PCC 7417 revealed a new gene cluster with homology to MAA synthase from Nostoc punctiforme This newly identified gene cluster is unusual because it has five biosynthesis genes (mylA to mylE), compared to the four found in other MAA gene clusters. Heterologous expression of mylA to mylE in Escherichia coli resulted in the production of mycosporine-lysine and the novel compound mycosporine-ornithine. To our knowledge, this is the first time these compounds have been heterologously produced in E. coli and structurally characterized via direct spectral guidance. This study offers insight into the diversity, biosynthesis, and structure of cyanobacterial MAAs and highlights their amenability to heterologous production methods. IMPORTANCE: Mycosporine-like amino acids (MAAs) are significant from an environmental microbiological perspective as they offer microbes protection against a variety of stress factors, including UV radiation. The heterologous expression of MAAs in E. coli is also significant from a biotechnological perspective as MAAs are the active ingredient in next-generation sunscreens.

Temporal variations in microcystin-producing cells and microcystin concentrations in two fresh water ponds.

The relationship between microcystin production, microcystin-producing cyanobacteria, including Microcystis spp., and various biological and physicochemical parameters in Sankuldhara and Lakshmikund, situated in the same geographical area was studied over a period of 1.5 years. Seasonal variation in cyanobacterial 16S rRNA, Microcystis spp. 16S rRNA, mcyA and mcyB genes were quantitatively determined by real-time PCR. Microcystis was the dominant microcystin producer in both study sites constituting 67% and 97% of the total microcystin-producing cyanobacteria at Sankuldhara and Lakshmikund, respectively. Microcystin concentrations were 2.19-39.60 µg/L and 15.22-128.14 µg/L at Sankuldhara and Lakshmikund, respectively, as determined by LC-MS. Principal component analysis revealed a strong positive correlation between microcystin concentration and the copy number of mcyA and mcyB, chlorophyll a and cyanobacterial biomass at both sites. The higher microcystin concentrations in Lakshmikund pond were attributed to the high copy number of mcy genes present coupled with the pond's eutrophication status, as indicated by high total algal biomass, high chlorophyll a content, high nutrient load and low DO. Therefore, a significant difference in microcystin concentrations, correlating with these various biological and physicochemical parameters, confirms the importance of local environmental variables in the overall regulation of microcystins production.

Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals.

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.

High-titer heterologous production in E. coli of lyngbyatoxin, a protein kinase C activator from an uncultured marine cyanobacterium.

Many chemically complex cyanobacterial polyketides and nonribosomal peptides are of great pharmaceutical interest, but the levels required for exploitation are difficult to achieve from native sources. Here we develop a framework for the expression of these multifunctional cyanobacterial assembly lines in Escherichia coli using the lyngbyatoxin biosynthetic pathway, derived from a marine microbial assemblage dominated by the cyanobacterium Moorea producens. Heterologous expression of this pathway afforded high titers of both lyngbyatoxin A (25.6 mg L(-1)) and its precursor indolactam-V (150 mg L(-1)). Production, isolation, and identification of all expected chemical intermediates of lyngbyatoxin biosynthesis in E. coli also confirmed the previously proposed biosynthetic route, setting a solid chemical foundation for future pathway engineering. The successful production of the nonribosomal peptide lyngbyatoxin A in E. coli also opens the possibility for future heterologous expression, characterization, and exploitation of other cyanobacterial natural product pathways.

On the origins and biosynthesis of tetrodotoxin.

The potent neurotoxin tetrodotoxin (TTX) has been identified from taxonomically diverse marine organisms. TTX possesses a unique cage-like structure, however, its biosynthesis has yet to be elucidated. Biosynthetic studies in the TTX-producing newt Taricha torosa, and in bacterial genera, including Vibrio, have proven inconclusive. Indeed, very few studies have been performed that address the cellular production of TTX. Here we review the sources of TTX described to date and provide evidence for the biosynthesis of TTX by symbiotic microorganisms in higher taxa. Chemical and genetic based biosynthesis studies of TTX undertaken thus far are discussed and we outline approaches which may be useful for expanding upon the current body of knowledge. The complex biosynthesis of structurally similar toxins, that reveal clues into the biosynthetic pathway of TTX, is also presented.

Biosynthesis of toxic naturally-occurring seafood contaminants.

Outbreaks of human illness caused by the consumption of contaminated seafood, continues to be a major problem particularly for the shellfish industry. Toxins from marine, brackish and freshwater environments, which are often produced as a result of harmful algal blooms, have been implicated as the causative agents of these poisonings. Commonly, poisoning events have been grouped into one of six classes, Paralytic Shellfish Poisoning (PSP), Diarrhetic Shellfish Poisoning (DSP), Neurotoxic Shellfish Poisoning (NSP), Ciguatera Fish Poisoning (CFP), Azaspiracid Shellfish Poisoning (AZP), and Amnesiac Shellfish Poisoning (ASP). The causative agents of these specific poisonings along with their biosyntheses are discussed in this review. The highly unusual and complex structures of most common seafood toxins have made them interesting targets for biosynthetic studies. Many of the toxins presented are biosynthesized via complex pathways that have been elucidated either through isotope labelled precursor feeding studies and/or characterization of the genes encoding the producing organism's biosynthetic machinery. Feeding studies key to our understanding of a particular toxin's biosynthesis, such as the incorporation of unusual precursors, as well as unique biosynthetic pathways and rare chemical mechanisms involved in the assembly process are highlighted. More recently, however, modern genomics-based techniques have been used for the elucidation of biosynthetic pathways and these are presented in the context of polyketide, non-ribosomal peptide, and hybrid pathway derived, toxin assembly.