Marine endophytic fungi associated with Halopteris scoparia (Linnaeus) Sauvageau as producers of bioactive secondary metabolites with potential dermocosmetic application.

Marine fungi and, particularly, endophytic species have been recognised as one of the most prolific sources of structurally new and diverse bioactive secondary metabolites with multiple biotechnological applications. Despite the increasing number of bioprospecting studies, very few have already evaluated the cosmeceutical potential of marine fungal compounds. Thus, this study focused on a frequent seaweed in the Portuguese coast, Halopteris scoparia, to identify the endophytic marine fungi associated with this host, and assess their ability to biosynthesise secondary metabolites with antioxidative, enzymatic inhibitory (hyaluronidase, collagenase, elastase and tyrosinase), anti-inflammatory, photoprotective, and antimicrobial (Cutibacterium acnes, Staphylococcus epidermidis and Malassezia furfur) activities. The results revealed eight fungal taxa included in the Ascomycota, and in the most representative taxonomic classes in marine ecosystems (Eurotiomycetes, Sordariomycetes and Dothideomycetes). These fungi were reported for the first time in Portugal and in association with H. scoparia, as far as it is known. The screening analyses showed that most of these endophytic fungi were producers of compounds with relevant biological activities, though those biosynthesised by Penicillium sect. Exilicaulis and Aspergillus chevalieri proved to be the most promising ones for being further exploited by dermocosmetic industry. The chemical analysis of the crude extract from an isolate of A. chevalieri revealed the presence of two bioactive compounds, echinulin and neoechinulin A, which might explain the high antioxidant and UV photoprotective capacities exhibited by the extract. These noteworthy results emphasised the importance of screening the secondary metabolites produced by these marine endophytic fungal strains for other potential bioactivities, and the relevance of investing more efforts in understanding the ecology of halo/osmotolerant fungi.

Simultaneous enrichment of grape pomace with γ-linolenic acid and carotenoids by solid-state fermentation with Zygomycetes fungi and antioxidant potential of the bioprocessed substrates.

Two filamentous fungi (Actinomucor elegans and Umbelopsis isabellina), were tested for their ability to enrich white grape pomace simultaneously with both γ-linolenic acid (GLA) and carotenoids through solid-state fermentation (SSF) processes. U. isabellina presented higher ability to produce GLA-rich lipids (composed mainly of neutral fractions) than A. elegans (the 6-th day of SSF: 378.85 mg/100 g of pomace -U. isabellina and 193.36 mg/100 g of pomace- A. elegans). The amounts of ß-carotene and lutein for both SSFs gradually increased until the end of the fermentation processes. The effect of fermentation time on the phenolic content and antioxidant activity of grape pomace was also studied. The SSF with A. elegans increased significantly total phenolic and flavonoid contents and DPPH scavenging activity of grape popmace. These bioprocessed grape pomaces with significant amounts of carotenoids and GLA-rich lipids (>94% nutritionally-valuable polyunsaturated fatty acids at the sn-2 position) could be very attractive for food industry.

Host metabolite producing endophytic fungi isolated from Hypericum perforatum.

In the present study, endophytic fungi have been isolated from various parts of the medicinal herb Hypericum perforatum (St. John's Wort), which is known as a source of medically important metabolites. The isolated strains were cultured in liquid media and their ability to synthesize hypericin, the secondary metabolite of the host and its suspected precursor, emodin was tested analyzing the extracts of the fermentation broth and the mycelia. The HPLC-UV analysis of the chloroform/methanol extracts of the mycelia revealed that three isolates were able to produce emodin (SZMC 23771, 19.9 ng/mg; SZMC 23772, 20.8 ng/mg; SZMC 23769, 427.9 ng/mg) and one of them also could synthesize hypericin (SZMC 23769, 320.4 ng/mg). These results were also confirmed via UHPLC-HRMS technique both in full scan and MS/MS mode. The strains producing only emodin belong to the section Alternata of the genus Alternaria, while the isolate producing both metabolites was identified as Epicoccum nigrum. The mycelial extracts of E. nigrum and the Alternaria sp. SZMC 23772 showed higher inhibitory activities in the antimicrobial tests against the six selected bacteria compared to the hypericin and emodin standards in the applied concentration (100 µg/mL), while in case of the Alternaria sp. SZMC 23771 lower inhibition activities were observed on Staphylococcus aureus and Streptomyces albus than the pure compounds.

Acidic cultivation of Haematococcus pluvialis for improved astaxanthin production in the presence of a lethal fungus.

An acidic cultivation strategy was developed to prevent contamination of a lethal fungus Paraphysoderma sedebokerensis in Haematococcus pluvialis culture for astaxanthin production. Instead of generally used neutral pH, an acidic condition (pH 4) was applied to the cultivation, resulting in a significant inhibition of the fungal contamination. This could be ascribed to the acidity-associated denaturation of a surface protein of P. sedebokerensis, which plays an important role in recognition of H. pluvialis. Stress relief strategies including stepwise light irradiation and naturally occurring nitrogen deficiency were employed in the induction stage to minimize the reduction of astaxanthin production caused by acidic pH. Accordingly, an astaxanthin titer of 84.8 mg L-1 was obtained, which is 141-fold of that from the completely contaminated culture and double of that without the stress relief methods. This strategy provides a persistent contamination control method that can be used for practical astaxanthin production by H. pluvialis.

2,4-dichlorophenoxyacetic acid-induced oxidative stress: Metabolome and membrane modifications in Umbelopsis isabellina, a herbicide degrader.

The study reports the response to herbicide of the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading fungal strain Umbelopsis isabellina. A comparative analysis covered 41 free amino acids as well as 140 lipid species of fatty acids, phospholipids, acylglycerols, sphingolipids, and sterols. 2,4-D presence led to a decrease in fungal catalase activity, associated with a higher amount of thiobarbituric acid-reactive substances (TBARS). Damage to cells treated with the herbicide resulted in increased membrane permeability and decreased membrane fluidity. Detailed lipidomic profiling showed changes in the fatty acids composition such as an increase in the level of linoleic acid (C18:2). Moreover, an increase in the phosphatidylethanolamine/phosphatidylcholine ratio was observed. Analysis of fungal lipid profiles revealed that the presence of 2,4-D was accompanied by the accumulation of triacylglycerols, a decrease in ergosterol content, and a considerable rise in the level of sphingolipid ceramides. In the exponential phase of growth, increased levels of leucine, glycine, serine, asparagine, and hydroxyproline were found. The results obtained in our study confirmed that in the cultures of U. isabellina oxidative stress was caused by 2,4-D. The herbicide itself forced changes not only to membrane lipids but also to neutral lipids and amino acids, as the difference of tested compounds profiles between 2,4-D-containing and control samples was consequently lower as the pesticide degradation progressed. The presented findings may have a significant impact on the basic understanding of 2,4-D biodegradation and may be applied for process optimization on metabolomic and lipidomic levels.

Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B, morphogenetic factors of Zygomycetes fungi.

(9Z)-Methyl 4-dihydrotrisporate B and (9Z)-methyl trisporate B, pheromones of Zygomycetes fungi, have been synthesized using Stille cross-coupling from previously described cyclohexenone precursors. Conducting the coupling without protection groups allowed for a short and stereospecific synthesis route of the late trisporoids. Stability studies for both the compounds revealed (9Z)-methyl trisporate B to be very unstable against UV irradiation.

Reference gene selection for quantitative real-time PCR in Solanum lycopersicum L. inoculated with the mycorrhizal fungus Rhizophagus irregularis.

The gene expression stability of candidate reference genes in the roots and leaves of Solanum lycopersicum inoculated with arbuscular mycorrhizal fungi was investigated. Eight candidate reference genes including elongation factor 1 α (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), protein phosphatase 2A (PP2Acs), ribosomal protein L2 (RPL2), ß-tubulin (TUB), ubiquitin (UBI) and actin (ACT) were selected, and their expression stability was assessed to determine the most stable internal reference for quantitative PCR normalization in S. lycopersicum inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. The stability of each gene was analysed in leaves and roots together and separated using the geNorm and NormFinder algorithms. Differences were detected between leaves and roots, varying among the best-ranked genes depending on the algorithm used and the tissue analysed. PGK, TUB and EF1 genes showed higher stability in roots, while EF1 and UBI had higher stability in leaves. Statistical algorithms indicated that the GAPDH gene was the least stable under the experimental conditions assayed. Then, we analysed the expression levels of the LePT4 gene, a phosphate transporter whose expression is induced by fungal colonization in host plant roots. No differences were observed when the most stable genes were used as reference genes. However, when GAPDH was used as the reference gene, we observed an overestimation of LePT4 expression. In summary, our results revealed that candidate reference genes present variable stability in S. lycopersicum arbuscular mycorrhizal symbiosis depending on the algorithm and tissue analysed. Thus, reference gene selection is an important issue for obtaining reliable results in gene expression quantification.

Deoxycholic acid transformations catalyzed by selected filamentous fungi.

More than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different phyla, were screened for the ability to convert deoxycholic acid (DCA) to valuable bile acid derivatives. Along with 11 molds which fully degraded DCA, several strains were revealed capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-LCA), 3-keto-DCA, 15ß-hydroxy-DCA and 15ß-hydroxy-12-oxo-LCA as major products from DCA. The last metabolite was found to be a new compound. The ability to catalyze the introduction of a hydroxyl group at the 7(α/ß)-positions of the DCA molecule was shown for 32 strains with the highest 7ß-hydroxylase activity level for Fusarium merismoides VKM F-2310. Curvularia lunata VKM F-644 exhibited 12α-hydroxysteroid dehydrogenase activity and formed 12-keto-LCA from DCA. Acremonium rutilum VKM F-2853 and Neurospora crassa VKM F-875 produced 15ß-hydroxy-DCA and 15ß-hydroxy-12-oxo-LCA, respectively, as major products from DCA, as confirmed by MS and NMR analyses. For most of the positive strains, the described DCA-transforming activity was unreported to date. The presented results expand the knowledge on bile acid metabolism by filamentous fungi, and might be suitable for preparative-scale exploitation aimed at the production of marketed bile acids.