William H. Gerwick

The structure of scytonemin, an ultraviolet sunscreen pigment from the sheaths of cyanobacteria.

Despite knowledge of the existence of the pigment called scytonemin for over 100 years, its structure has remained unsolved until now. This pigment, the first shown to be an effective, photo-stable ultraviolet shield in prokaryotes, is a novel dimeric molecule (molec. wt. 544) of indolic and phenolic subunits and is known only from the sheaths enclosing the cells of cyanobacteria. It is probable that scytonemin is formed from a condensation of tryptophan-and phenylpropanoid-derived subunits. The linkage between these units is unique among natural products and this novel ring structure is here termed the ‘scytoneman skeleton’. Scytonemin absorbs strongly and broadly in the spectral region 325–425 nm (UV-A-violet-blue, with an in vivo maximum at 370 nm). However, there is also major absorption in the UV-C (λ max=250nm) and UV-B (280–320 nm). The pigment has been recently shown to provide significant protection to cyanobacteria against damage by ultraviolet radiation. The pigment occurs in all phylogenetic lines of sheathed cyanobacteria and possibly represents a UV screening strategy far more ancient than that of plant flavonoids and animal melanins. How diverse organisms deal with UV radiation is considered of vital importance to global ecology.

Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth

Significance As global CO2 levels rise and fossil fuel abundance decreases, the development of alternative fuels becomes increasingly imperative. Biologically derived fuels, and specifically those from microalgae, are promising sources, but improvements throughout the production process are required to reduce cost. Increasing lipid yields in microalgae without compromising growth has great potential to improve economic feasibility. We report that disrupting lipid catabolism is a practical approach to increase lipid yields in microalgae without affecting growth or biomass. We developed transgenic strains through targeted metabolic engineering that show increased lipid accumulation, biomass, and lipid yields. The target enzyme’s ubiquity suggests that this approach can be applied broadly to improve the economic feasibility of algal biofuels in other groups of microalgae. Biologically derived fuels are viable alternatives to traditional fossil fuels, and microalgae are a particularly promising source, but improvements are required throughout the production process to increase productivity and reduce cost. Metabolic engineering to increase yields of biofuel-relevant lipids in these organisms without compromising growth is an important aspect of advancing economic feasibility. We report that the targeted knockdown of a multifunctional lipase/phospholipase/acyltransferase increased lipid yields without affecting growth in the diatom Thalassiosira pseudonana. Antisense-expressing knockdown strains 1A6 and 1B1 exhibited wild-type–like growth and increased lipid content under both continuous light and alternating light/dark conditions. Strains 1A6 and 1B1, respectively, contained 2.4- and 3.3-fold higher lipid content than wild-type during exponential growth, and 4.1- and 3.2-fold higher lipid content than wild-type after 40 h of silicon starvation. Analyses of fatty acids, lipid classes, and membrane stability in the transgenic strains suggest a role for this enzyme in membrane lipid turnover and lipid homeostasis. These results demonstrate that targeted metabolic manipulations can be used to increase lipid accumulation in eukaryotic microalgae without compromising growth.

The barbamide biosynthetic gene cluster: a novel marine cyanobacterial system of mixed polyketide synthase (PKS)-non-ribosomal peptide synthetase (NRPS) origin involving an unusual trichloroleucyl starter unit

Barbamide was extracted from the marine cyanobacterium Lyngbya majuscula strain 19L as a chlorinated lipopeptide for its potent molluscicidal activity. Precursor incorporation studies indicated that it is derived from acetate, L-phenylalanine, L-leucine and L-cysteine. The gene cluster responsible for biosynthesis of barbamide (bar) was cloned and characterized in this study. DNA sequence analysis of cosmid pLM49 revealed a cluster of 12 open reading frames (barA-barK) extending 26 kb including the expected polyketide synthase and non-ribosomal peptide synthetase modules and tailoring genes. The genetic architecture and domain organization of the bar cluster supports the assignment based on the apparent co-linearity of the systems. The activity assay of adenylation domains of barD (A(D)), barE (A(E)) and barG (A(G2) for module 2) in an amino acid-dependent ATP-pyrophosphate exchange experiment supports the conclusion that barbamide is synthesized from acetate, L-phenylalanine, L-cysteine and L-leucine with trichloroleucine as a direct precursor by a mixed polyketide synthase/non-ribosomal polypeptide synthetase. Assembly of barbamide includes unique biochemical mechanisms for chlorination, one-carbon truncation during chain elongation, E-double bond formation and thiazole ring formation.

Molecular Networking as a Dereplication Strategy

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogues, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogues.

Biologically active secondary metabolites from marine cyanobacteria

Marine cyanobacteria are a rich source of complex bioactive secondary metabolites which derive from mixed biosynthetic pathways. Recently, several marine cyanobacterial natural products have garnered much attention due to their intriguing structures and exciting anti-proliferative or cancer cell toxic activities. Several other recently discovered secondary metabolites exhibit insightful neurotoxic activities whereas others are showing pronounced anti-inflammatory activity. A number of anti-infective compounds displaying activity against neglected diseases have also been identified, which include viridamides A and B, gallinamide A, dragonamide E, and the almiramides.

Moorea producens gen. nov., sp. nov. and Moorea bouillonii comb. nov., tropical marine cyanobacteria rich in bioactive secondary metabolites.

The filamentous cyanobacterial genus Moorea gen. nov., described here under the provisions of the International Code of Botanical Nomenclature, is a cosmopolitan pan-tropical group abundant in the marine benthos. Members of the genus Moorea are photosynthetic (containing phycocyanin, phycoerythrin, allophycocyanin and chlorophyll a), but non-diazotrophic (lack heterocysts and nitrogenase reductase genes). The cells (discoid and 25-80 µm wide) are arranged in long filaments (<10 cm in length) and often form extensive mats or blooms in shallow water. The cells are surrounded by thick polysaccharide sheaths covered by a rich diversity of heterotrophic micro-organisms. A distinctive character of this genus is its extraordinarily rich production of bioactive secondary metabolites. This is matched by genomes rich in polyketide synthase and non-ribosomal peptide synthetase biosynthetic genes which are dedicated to secondary metabolism. The encoded natural products are sometimes responsible for harmful algae blooms and, due to morphological resemblance to the genus Lyngbya, this group has often been incorrectly cited in the literature. We here describe two species of the genus Moorea: Moorea producens sp. nov. (type species of the genus) with 3L(T) as the nomenclature type, and Moorea bouillonii comb. nov. with PNG5-198(R) as the nomenclature type.

Metamorphic enzyme assembly in polyketide diversification

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a β-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide β-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH1s), decarboxylases (ECH2s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), α-ketoglutarate-dependent enzyme. In a divergent scheme, Cur ECH2 was found to catalyse formation of the α,β enoyl thioester, whereas Jam ECH2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH2 instead of the canonical α,β C = C saturation reaction. Thus, the combination of chlorination and polyketide β-branching, coupled with mechanistic diversification of ECH2 and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

Oxylipin Profiling of the Hypersensitive Response in Arabidopsis thaliana FORMATION OF A NOVEL OXO-PHYTODIENOIC ACID-CONTAINING GALACTOLIPID, ARABIDOPSIDE E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.

Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA) and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

Symplocamide A, a potent cytotoxin and chymotrypsin inhibitor from the marine Cyanobacterium Symploca sp.

Investigation of a Symploca sp. from Papua New Guinea has led to the isolation of symplocamide A (1), a potent cancer cell cytotoxin, which also inhibits serine proteases with a 200-fold greater inhibition of chymotrypsin over trypsin. The complete stereostructure of symplocamide A was determined by detailed NMR and MS analysis as well as chiral HPLC analysis of the component amino acid residues. The presence of several unusual structural features in symplocamide A provides new insights into the pharmacophore model for protease selectivity in this drug class and may underlie the potent cytotoxicity of this compound to H-460 lung cancer cells (IC50=40 nM) as well as neuro-2a neuroblastoma cells (IC50=29 nM).