Consequences of Mixotrophy on Cell Energetic Metabolism in Microchloropsis gaditana Revealed by Genetic Engineering and Metabolic Approaches.

Algae belonging to the Microchloropsis genus are promising organisms for biotech purposes, being able to accumulate large amounts of lipid reserves. These organisms adapt to different trophic conditions, thriving in strict photoautotrophic conditions, as well as in the concomitant presence of light plus reduced external carbon as energy sources (mixotrophy). In this work, we investigated the mixotrophic responses of Microchloropsis gaditana (formerly Nannochloropsis gaditana). Using the Biolog growth test, in which cells are loaded into multiwell plates coated with different organic compounds, we could not find a suitable substrate for Microchloropsis mixotrophy. By contrast, addition of the Lysogeny broth (LB) to the inorganic growth medium had a benefit on growth, enhancing respiratory activity at the expense of photosynthetic performances. To further dissect the role of respiration in Microchloropsis mixotrophy, we focused on the mitochondrial alternative oxidase (AOX), a protein involved in energy management in other algae prospering in mixotrophy. Knocking-out the AOX1 gene by transcription activator-like effector nuclease (TALE-N) led to the loss of capacity to implement growth upon addition of LB supporting the hypothesis that the effect of this medium was related to a provision of reduced carbon. We conclude that mixotrophic growth in Microchloropsis is dominated by respiratory rather than by photosynthetic energetic metabolism and discuss the possible reasons for this behavior in relationship with fatty acid breakdown via ß-oxidation in this oleaginous alga.

Screening for Biologically Annotated Drugs That Trigger Triacylglycerol Accumulation in the Diatom Phaeodactylum.

Microalgae are a promising feedstock for the production of triacylglycerol (TAG) for a variety of potential applications, ranging from food and human health to biofuels and green chemistry. However, obtaining high TAG yields is challenging. A phenotypic assay for the accumulation of oil droplets was developed to screen a library of 1,200 drugs, annotated with pharmacology information, to select compounds that trigger TAG accumulation in the diatom Phaeodactylum tricornutum Using this screen, we identified 34 molecules acting in a dose-dependent manner. Previously characterized targets of these compounds include cell division and cell signaling effectors, membrane receptors and transporters, and sterol metabolism. Among the five compounds possibly acting on sterol metabolism, we focused our study on ethynylestradiol, a synthetic form of estrogen that is used in contraceptive pills and known for its ecological impact as an endocrine disruptor. Ethynylestradiol impaired the production of very-long-chain polyunsaturated fatty acids, destabilized the galactolipid versus phospholipid balance, and triggered the recycling of fatty acids from membrane lipids to TAG. The P. tricornutum transcriptomic response to treatment with ethynylestradiol was consistent with the reallocation of carbon from sterols to acetyl-coenzyme A and TAG. The mode of action and catabolism of ethynylestradiol are unknown but might involve several up-regulated cytochrome P450 proteins. A fatty acid elongase, Δ6-ELO-B1, might be involved in the impairment of very-long-chain polyunsaturated fatty acids and fatty acid turnover. This phenotypic screen opens new perspectives for the exploration of novel bioactive molecules, potential target genes, and pathways controlling TAG biosynthesis. It also unraveled the sensitivity of diatoms to endocrine disruptors, highlighting an impact of anthropogenic pollution on phytoplankton.

Nitric Oxide Mediates Nitrite-Sensing and Acclimation and Triggers a Remodeling of Lipids.

Nitric oxide (NO) is an intermediate of the nitrogen cycle, an industrial pollutant, and a marker of climate change. NO also acts as a gaseous transmitter in a variety of biological processes. The impact of environmental NO needs to be addressed. In diatoms, a dominant phylum in phytoplankton, NO was reported to mediate programmed cell death in response to diatom-derived polyunsaturated aldehydes. Here, using the Phaeodactylum Pt1 strain, 2E,4E-decadienal supplied in the micromolar concentration range led to a nonspecific cell toxicity. We reexamined NO biosynthesis and response in Phaeodactylum NO inhibits cell growth and triggers triacylglycerol (TAG) accumulation. Feeding experiments indicate that NO is not produced from Arg but via conversion of nitrite by the nitrate reductase. Genome-wide transcriptional analysis shows that NO up-regulates the expression of the plastid nitrite reductase and genes involved in the subsequent incorporation of ammonium into amino acids, via both Gln synthesis and Orn-urea pathway. The phosphoenolpyruvate dehydrogenase complex is also up-regulated, leading to the production of acetyl-CoA, which can feed TAG accumulation upon exposure to NO. Transcriptional reprogramming leading to higher TAG content is balanced with a decrease of monogalactosyldiacylglycerol (MGDG) in the plastid via posttranslational inhibition of MGDG synthase enzymatic activity by NO. Intracellular and transient NO emission acts therefore at the basis of a nitrite-sensing and acclimating system, whereas a long exposure to NO can additionally induce a redirection of carbon to neutral lipids and a stress response.

Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum.

Diatoms are prominent marine microalgae, interesting not only from an ecological point of view, but also for their possible use in biotechnology applications. They can be cultivated in phototrophic conditions, using sunlight as the sole energy source. Some diatoms, however, can also grow in a mixotrophic mode, wherein both light and external reduced carbon contribute to biomass accumulation. In this study, we investigated the consequences of mixotrophy on the growth and metabolism of the pennate diatom Phaeodactylum tricornutum, using glycerol as the source of reduced carbon. Transcriptomics, metabolomics, metabolic modelling and physiological data combine to indicate that glycerol affects the central-carbon, carbon-storage and lipid metabolism of the diatom. In particular, provision of glycerol mimics typical responses of nitrogen limitation on lipid metabolism at the level of triacylglycerol accumulation and fatty acid composition. The presence of glycerol, despite provoking features reminiscent of nutrient limitation, neither diminishes photosynthetic activity nor cell growth, revealing essential aspects of the metabolic flexibility of these microalgae and suggesting possible biotechnological applications of mixotrophy.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Membrane glycerolipid remodeling triggered by nitrogen and phosphorus starvation in Phaeodactylum tricornutum.

Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids have not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.

Spiralisones A-D: acylphloroglucinol hemiketals from an Australian marine brown alga, Zonaria spiralis.

An intertidal sample of the Australian marine brown alga, Zonaria spiralis, exhibited promising kinase inhibitory and antibacterial activity. Chemical analysis returned six phloroglucinol-derived lipids, the new hemiketal spiralisones A-D (1-4) and the known chromones 5-6, and the known norsesquiterpenoid apo-9'-fucoxanthinone (7). Structures 1-7 were assigned on the basis of detailed spectroscopic analysis, biosynthetic considerations and total synthesis. Spiralisones undergo facile acid-mediated dehydration to yield the corresponding chromones, revealing for the first time that brown algal chromones may be handling artifacts rather than natural products. Hemiketals 1 and 2, and chromone 6, displayed inhibitory activity against the neurodegenerative disease kinase targets CDK5/p25, CK1δ and GSK3ß, while hemiketals 1, 3 and 4, and chromone 6, displayed growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis (ATCC 6051 and 6633). The promising kinase inhibitory and antibacterial properties of the Z. spiralis extract were attributed to the cumulative effect of many moderately potent phloroglucinol-derived lipid co-metabolites.

Kinase inhibitor scaffolds against neurodegenerative diseases from a Southern Australian ascidian, Didemnum sp.

Screening a library of Southern Australian and Antarctic marine invertebrates and algae for inhibitors of neurodegenerative disease kinase targets casein kinase 1 (CK1δ), cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3ß (GSK3ß) identified a Western Australian Didemnum species (CMB-02127) as a high-priority specimen. Chemical fractionation returned the known aromatic alkaloids ningalins B-D as the major metabolites, together with six minor metabolites, the new ningalins E-G and the known hexacyclic pyrrole alkaloids lamellarins Z, G and A6. All structures were assigned by detailed spectroscopic analysis and literature comparisons, and the structural assignments were supported by biosynthetic considerations. The ningalins showed potent and broad inhibition across the three kinases, while the lamellarins were generally more selective for CDK5. Docking studies using published X-ray crystal structures of CDK5 revealed both scaffolds target the ATP binding pocket.

A search for BACE inhibitors reveals new biosynthetically related pyrrolidones, furanones and pyrroles from a southern Australian marine sponge, Ianthella sp.

Fractionation of a southern Australian marine sponge, Ianthella sp., yielded sixteen metabolites including a new class of pyrrolidone, ianthellidones A-F (1-6), a new class of furanone, ianthellidones G-H (7-8), new and known lamellarins, lamellarins O1 (9), O2 (10), O (11) and Q (12), plus the known 4-hydroxybenzaldehyde (13), 4-hydroxybenzoic acid (14), 4-methoxybenzoic acid (15) and ethyl 4-hydroxybenzoate (16). Structures for all Ianthella metabolites were determined by detailed spectroscopic analysis, supported by a plausible biosynthetic relationship. The ianthellidones were non-cytotoxic towards two human colon cancer cell lines (SW620 and SW620 Ad300), as well as Gram +ve and Gram -ve bacteria, and a fungus. Ianthellidone F (6) and lamellarins O2 (10) and O (11) displayed modest BACE inhibitory properties (IC(50) > 10 µM), while lamellarin O1 (9) was more potent (IC(50) < 10 µM). Lamellarin O (11) exhibited modest cytotoxicity towards SW620 and SW620 Ad300 cell lines (IC(50) > 22 µM), was an inhibitor of the multi-drug resistance efflux pump P-glycoprotein, and displayed selective growth inhibitory activity against the Gram +ve bacterium Bacillus subtilis (ATCC 6633) (IC(50) 2.5 µM).

Heronamides A-C, new polyketide macrolactams from an Australian marine-derived Streptomyces sp. A biosynthetic case for synchronized tandem electrocyclization.

A Streptomyces sp. isolated from a shallow water sediment sample collected off Heron Island, Australia, afforded three new polyketide macrolactams, heronamides A-C (1-3). Structures were assigned to the heronamides on the basis of detailed spectroscopic analysis, chemical derivatization and biosynthetic considerations. A plausible biosynthetic pathway is proposed in which key carbocyclic ring transformations proceed via an unprecedented synchronized tandem electrocyclization. This biosynthesis provides a framework for the assignment of complete relative configurations across all heronamides, and inspires an attractive biomimetic strategy for future total syntheses. Heronamide C elicits a dramatic and reversible non-cytotoxic effect on mammalian cell morphology.

Heterofibrins: inhibitors of lipid droplet formation from a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp.

A bioassay-guided search for inhibitors of lipid droplet formation in a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp., yielded six new compounds, fatty acids heterofibrins A1 (1) and B1 (4), along with related monolactyl and dilactyl esters, heterofibrins A2 (2), B2 (5), A3 (3) and B3 (6). Heterofibrin structures were assigned on the basis of detailed spectroscopic analysis, with comparison to chiral synthetic model compounds. All heterofibrins possess a diyne-ene moiety, while the monolactyl and dilactyl moiety featured in selected heterofibrins is unprecedented in the natural products literature. SAR by co-metabolite studies on the heterofibrins confirmed them to be non-cytotoxic, with the carboxylic acids 1 and 4 inhibiting lipid droplet formation in A431 fibroblast cell lines. Such inhibitors have potential application in the management of obesity, diabetes and atherosclerosis

Nocardiopsins: new FKBP12-binding macrolide polyketides from an Australian marine-derived actinomycete, Nocardiopsis sp.

A marine-derived actinomycete, Nocardiopsis sp. (CMB-M0232), obtained from a sediment sample collected at a depth of 55 m off the coast of Brisbane, Australia, yielded two new macrolide polyketides. Structures for nocardiopsins A and B were assigned by detailed spectroscopic analysis, degradation and chemical derivatization. A Marfey's analysis revealed an unexpected acid-mediated partial racemization of the L-pipecolic acid incorporated within the nocardiopsins. The scope of this racemization was assessed against a selection of natural and synthetic N-acyl pipecolic acids. While the nocardiopsins are not antibacterial, antifungal or cytotoxic, they do exhibit low-micromolar binding to the immunophilin FKBP12, consistent with their structural and biosynthetic relationship to the immunosuppressive agents FK506 and rapamycin. The nocardiopsins represent a new point of entry into what has been a valuable, exclusive and reclusive region of bioactive chemical space--that surrounding the FK506/rapamycin pharmacophore.

Mirabilins revisited: polyketide alkaloids from a southern Australian marine sponge, Clathria sp.

Chemical investigation of a southern Australian marine sponge, Clathria sp., yielded the known mirabilins C, F and G, together with three new analogues, mirabilins H-J. For the first time mirabilins C and F are documented as the underivatized natural products, and a complete absolute stereochemistry is assigned to mirabilin F. Mirabilin I represents the first member of this structure class to incorporate a trans-fused ring junction. Structures for all mirabilins are assigned on the basis of detailed spectroscopic analysis. A plausible polyketide origin is proposed, linking all mirabilins and related sponge alkaloids. Mirabilin cytotoxicity against several human cancer cell lines is discussed.

Naseseazines A and B: a new dimeric diketopiperazine framework from a marine-derived actinomycete, Streptomyces sp.

Chemical analysis of a Streptomyces sp. (CMB-MQ030) isolated from a Fijian marine sediment yielded two new diketopiperazines, naseseazines A and B (1, 2), featuring a new dimeric framework. Structures were determined by detailed spectroscopic analysis and C(3) Marfey's analysis.

The role of PKCdelta and PKCepsilon in the neonatal rat colon in response to hypoxia challenge.

Previous studies have determined that, in response to bacterial endotoxin, the colonic mucosa of the 10-d-old neonatal rat was more susceptible to injury than was the colon of the 25-d-old mature animal. Furthermore, it is known that certain isoforms of protein kinase C (PKC), specifically PKCdelta and PKCepsilon, mediate intestinal inflammatory responses to specific challenges. Therefore, in the present study, we have examined the association between the activation of these PKC isoforms and the enhanced susceptibility to hypoxia-induced challenge. In response to exposure to a hypoxic environment (14% O2/86% N2, 30 min), the degree of inflammation and tissue damage was significantly greater in 10- than in 25-d-old rats. The injury in 10-d-old rats was associated with activation of PKCdelta and PKCepsilon as estimated by translocation of the isoform from cytosolic to membrane fraction of the tissue lysate. There was no activation of either isoform in colons from 25-d-old rats after hypoxia. Pretreatment of 10-d-old rats with epidermal growth factor (EGF) (10 microg/kg) but not 16,16 dimethyl prostaglandin E2 (2 microg/kg) significantly reduced the extent of colonic injury, whereas neither agent was able to exert significant protection of the colonic mucosa of 25-d-old rats. PKC activation associated with hypoxia was not evident after EGF treatment in 10-d-old rats. In 25-d-old rats, prostaglandin E2 treatment was linked with an activation of PKCepsilon only. In conclusion, these data suggest that activation of PKCdelta and PKCepsilon is associated with the enhanced susceptibility to injury evident in suckling neonatal rat colon. EGF-mediated protection of the colon in these animals results in a removal of this PKC activation.