Genome and Metabolome MS-Based Mining of a Marine Strain of Aspergillus affinis

Aspergillus section Circumdati encompasses several species that express both beneficial (e.g., biochemical transformation of steroids and alkaloids, enzymes and metabolites) and harmful compounds (e.g., production of ochratoxin A (OTA)). Given their relevance, it is important to analyze the genetic and metabolic diversity of the species of this section. We sequenced the genome of Aspergillus affinis CMG 70, isolated from sea water, and compared it with the genomes of species from section Circumdati, including A. affinis’s strain type. The A. affinis genome was characterized considering secondary metabolites biosynthetic gene clusters (BGCs), carbohydrate-active enzymes (CAZymes), and transporters. To uncover the biosynthetic potential of A. affinis CMG 70, an untargeted metabolomics (LC-MS/MS) approach was used. Cultivating the fungus in the presence and absence of sea salt showed that A. affinis CMG 70 metabolite profiles are salt dependent. Analyses of the methanolic crude extract revealed the presence of both unknown and well-known Aspergillus compounds, such as ochratoxin A, anti-viral (e.g., 3,5-Di-tert-butyl-4-hydroxybenzoic acid and epigallocatechin), anti-bacterial (e.g., 3-Hydroxybenzyl alcohol, L-pyroglutamic acid, lecanoric acid), antifungal (e.g., L-pyroglutamic acid, 9,12,13-Trihydroxyoctadec-10-enoic acid, hydroxyferulic acid), and chemotherapeutic (e.g., daunomycinone, mitoxantrone) related metabolites. Comparative analysis of 17 genomes from 16 Aspergillus species revealed abundant CAZymes (568 per species), secondary metabolite BGCs (73 per species), and transporters (1359 per species). Some BGCs are highly conserved in this section (e.g., pyranonigrin E and UNII-YC2Q1O94PT (ACR toxin I)), while others are incomplete or completely lost among species (e.g., bikaverin and chaetoglobosins were found exclusively in series Sclerotiorum, while asperlactone seemed completely lost). The results of this study, including genome analysis and metabolome characterization, emphasize the molecular diversity of A. affinis CMG 70, as well as of other species in the section Circumdati.

Bioprocesses with Reduced Ecological Footprint by Marine Debaryomyces hansenii Strain for Potential Applications in Circular Economy

The possibility to perform bioprocesses with reduced ecological footprint to produce natural compounds and catalyzers of industrial interest is pushing the research for salt tolerant microorganisms able to grow on seawater-based media and able to use a wide range of nutrients coming from waste. In this study we focused our attention on a Debaryomyces hansenii marine strain (Mo40). We optimized cultivation in a bioreactor at low pH on seawater-based media containing a mixture of sugars (glucose and xylose) and urea. Under these conditions the strain exhibited high growth rate and biomass yield. In addition, we characterized potential applications of this yeast biomass in food/feed industry. We show that Mo40 can produce a biomass containing 45% proteins and 20% lipids. This strain is also able to degrade phytic acid by a cell-bound phytase activity. These features represent an appealing starting point for obtaining D. hansenii biomass in a cheap and environmentally friendly way, and for potential use as an additive or to replace unsustainable ingredients in the feed or food industries, as this species is included in the QPS EFSA list (Quality Presumption as Safe—European Food Safety Authority).

Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase—Alys1—with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0–8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

Production and identification of two antifungal terpenoids from the Posidonia oceanica epiphytic Ascomycota Mariannaea humicola IG100

Background Marine fungi are an important repository of bioactive molecules with great potential in different technological fields, the annual number of new compounds isolated from marine fungi is impressive and the general trend indicates that it is still on the rise. In this context, the antifungal and antimicrobial activity of the marine strain Mariannaea humicola IG100 was evaluated and two active terpenoids were isolated and characterized. Methods Preliminary screening of activity of marine strain IG100 was carried out by agar plug diffusion methods against fungal (Penicillium griseofulvum TSF04) and bacterial (Bacillus pumilus KB66 and Escherichia coli JM109) strains. Subsequently, inhibition tests were done by using the cultural broth and the organic extract (ethyl acetate, EtOAc) by the agar well diffusion methods. The main active fractions were identified and tested for their antifungal activity against P. griseofulvum TSF04 in a 24 wells microplate at different concentrations (1000, 100, 10 and 1.0 µg/mL). Two active compounds were characterized and their relative MIC measured by the broth micro-dilution methods in a 96-well microplate against Aspergillus flavus IG133, P. griseofulvum TSF04, and Trichoderma pleuroticola IG137. Results Marine strain IG100 presented significant antifungal activity associated with two active compounds, the terpenoids terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. Their MIC values were measured for A. flavus (MIC of 7.9 µg/mL and 31.3 µg/mL for 1 and 2, respectively), P. griseofulvum (MIC of 25 µg/mL and 100 µg/mL for 1 and 2, respectively) and T. pleuroticola (MIC > 500 µg/mL and 125 µg/mL for 1 and 2, respectively). They showed a rather good fungistatic effect. Conclusions In this study, the first marine strain of M. humicola (IG100) was investigated for the production of bioactive molecules. Strain IG100 produced significant amounts of two bioactive terpenoids, terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. The two compounds showed significant antifungal activities against A. flavus IG133, T. pleuroticola IG137 and P. griseofulvum TSF04. Compound 2 was identified for the first time in fungi.

Production and identification of two antifungal terpenoids from the Posidonia oceanica epiphytic Ascomycota Mariannaea humicola IG100.

Background: Marine fungi are an important repository of bioactive molecules with great potential in different technological fields, the annual number of new compounds isolated from marine fungi is impressive and the general trend indicates that it is still on the rise. In this context, the antifungal and antimicrobial activity of the marine strain Mariannaea humicola IG100 was evaluated and two active terpenoids were isolated and characterized.Methods: Preliminary screening of activity of marine strain IG100 was carried out by agar plug diffusion methods against fungal (P. griseofulvum TSF04) and bacterial (Bacillus pumilus KB66 and Escherichia coli JM109) strains. Subsequently, inhibition tests were done by using the cultural broth and the organic extract (ethyl acetate, EtOAc) by the agar well diffusion methods. The main active fractions were identified and tested for their antifungal activity against P. griseofulvum TSF04 in a 24 wells microplate at different concentrations (1000, 100, 10 and 1.0 µg/mL). Two active compounds were characterized and their relative MIC measured by the broth micro-dilution methods in a 96-well microplate against A. flavus IG133, P. griseofulvum TSF04, and T. pleuroticola IG137. Results: Marine strain IG100 presented significant antifungal activity associated with two active compounds, the terpenoids terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. Their MIC values were measured for Aspergillus flavus (MIC of 7.9 µg/mL and 31.3 µg/mL for 1 and 2, respectively), P. griseofulvum (MIC of 25 µg/mL and 100 µg/mL for 1 and 2, respectively) and T. pleuroticola (MIC >500 µg/mL and 125 µg/mL for 1 and 2, respectively). They showed a rather good fungistatic effect. Conclusions: In this study, the first marine strain of M. humicola (IG100) was investigated for the production of bioactive molecules. Strain IG100 produced significant amounts of two bioactive terpenoids, terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. The two compounds showed significant antifungal activities against A. flavus IG133, T. pleuroticola IG137 and P. griseofulvum TSF04. Compound 2 was identified for the first time in fungi.

Production and identification of two antifungal terpenoids from the Posidonia oceanica epiphytic Ascomycota Mariannaea humicola IG100

Background Marine fungi are an important repository of bioactive molecules with great potential in different technological fields, the annual number of new compounds isolated from marine fungi is impressive and the general trend indicates that it is still on the rise. In this context, the antifungal and antimicrobial activity of the marine strain Mariannaea humicola IG100 was evaluated and two active terpenoids were isolated and characterized. Methods Preliminary screening of activity of marine strain IG100 was carried out by agar plug diffusion methods against fungal (P. griseofulvum TSF04) and bacterial (Bacillus pumilus KB66 and Escherichia coli JM109) strains. Subsequently, inhibition tests were done by using the cultural broth and the organic extract (ethyl acetate, EtOAc) by the agar well diffusion methods. The main active fractions were identified and tested for their antifungal activity against P. griseofulvum TSF04 in a 24 wells microplate at different concentrations (1000, 100, 10 and 1.0 µg/mL). Two active compounds were characterized and their relative MIC measured by the broth micro-dilution methods in a 96-well microplate against A. flavus IG133, P. griseofulvum TSF04, and T. pleuroticola IG137. Results Marine strain IG100 presented significant antifungal activity associated to two active compounds, the terpenoids terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. Their MIC values were measured for Aspergillus flavus (MIC of 7.9 µg/mL and 31.3 µg/mL for 1 and 2, respectively), P. griseofulvum (MIC of 25 µg/mL and 100 µg/mL for 1 and 2, respectively) and T. pleuroticola (MIC > 500 µg/mL and 125 µg/mL for 1 and 2, respectively). They showed a rather good fungistatic effect. Conclusions In this study, the first marine strain of M. humicola (IG100) was investigated for the production of bioactive molecules. Strain IG100 produced significant amounts of two bioactive terpenoids, terperstacin 1 and 19-acetyl-4-hydroxydictyodiol 2. The two compounds showed significant antifungal activities against A. flavus IG133, T. pleuroticola IG137 and P. griseofulvum TSF04. Compound 2 was identified for the first time in fungi.

Extracellular polysaccharides (EPS) secreted by a model marine bacterium (Alteromonas spp.)

<p><em>Alteromonas</em> are model copiotrophic marine bacteria that are able to produce highly hydrated extracellular biopolymers mainly composed of polysaccharides (i.e., extracellular polysaccharides, EPS), which have a role in biofilm formation in oceans. Some of the functions of EPS are related to protection against environmental stressors, adhesion to particles, carbon storage and nutrient acquisition. Microbial EPS are largely heterogeneous in composition and structure, and some strains produce different types of EPS in response to different conditions. This study aimed at characterizing the synthesis of polysaccharides secreted from an <em>Alteromonas</em> spp. marine strain isolated from the Biscay Bay, targeting the genes involved in its synthesis. First, the genome of this strain was sequenced and different gene clusters related to the synthesis of EPS were identified. Then, a transcriptomic study was carried out to analyse the expression of EPS synthesis related genes in response to glucose and the EPS composition was preliminary characterized.  The long-term objective is to increase our understanding of the patterns of EPS secretion in <em>Alteromonas</em>, which may have a key role in their association with phytoplankton blooms and adaptation to different environmental conditions.</p>