Bisdechlorogeodin from antarctic Pseudogymnoascus sp. LAMAI 2784 for citrus canker control.

AIMS: Citrus canker caused by Xanthomonas citri subsp. citri (X. citri) is a disease of economic importance. Control of this disease includes the use of metallic copper, which is harmful to the environment and human health. Previous studies showed that the crude extract from the fungus Pseudogymnoascus sp. LAMAI 2784 isolated from Antarctic soil had in vitro antibacterial action against X. citri. The aim of the present study was to expand the applications of this extract. METHODS AND RESULTS: In greenhouse assays, the crude extract was able to reduce bacterial infection on citrus leaves from 1.55 lesions/cm2 (untreated plants) to 0.04 lesions/cm2. Bisdechlorogeodin was identified as the main compound of the bioactive fraction produced by Pseudogymnoascus sp. LAMAI 2784, which inhibited bacterial growth in vitro (IC90 ≈ 156 µg ml-1) and permeated 80% of X. citri cells, indicating that the membrane is the primary target. CONCLUSION: The present results showed that the bioactive fraction of the extract is mainly composed of the compound bisdechlorogeodin, which is likely responsible for the biological activity against X. citri, and the main mechanism of action is the targeting of the cell membrane. This study indicates that bisdechlorogeodin has valuable potential for the control of X. citri.

Biobank of fungi from marine and terrestrial Antarctic environments.

Harsh and extreme environments, such as Antarctica, offer unique opportunities to explore new microbial taxa and biomolecules. Given the limited knowledge on microbial diversity, this study aimed to compile, analyze and compare a subset of the biobank of Antarctic fungi maintained at the UNESP's Central of Microbial Resources (CRM-UNESP). A total of 711 isolates (240 yeasts and 471 filamentous fungi) from marine and terrestrial samples collected at King George Island (South Shetland Islands, Antarctica) were used with the primary objective of investigating their presence in both marine and terrestrial environments. Among the yeasts, 13 genera were found, predominantly belonging to the phylum Basidiomycota. Among the filamentous fungi, 34 genera were represented, predominantly from the phylum Ascomycota. The most abundant genera in the marine samples were Metschnikowia, Mrakia, and Pseudogymnoascus, while in the terrestrial samples, they were Pseudogymnoascus, Leucosporidium, and Mortierella. Most of the genera and species of the CRM-UNESP biobank of Antarctic fungi are being reported as an important target for biotechnological applications. This study showed the relevance of the CRM-UNESP biobank, highlighting the importance of applying standard methods for the preservation of the biological material and associated data (BMaD), as recommended in national and international standards.

Antarctic-derived yeasts: taxonomic identification and resistance to adverse conditions.

Antarctic harsh conditions favor the development of microbial adaptations. In this study, a molecular approach was applied to identify/refine the taxonomy of five yeasts isolated from different Antarctic samples, which were tested against ranges of temperature, UV radiations, salinity, and pH. Based on sequencing and phylogenetic analysis, strain CRM 1839 was confirmed as Naganishia sp., and strains CRM 1874, CRM 1565, CRM 2571, and CRM 2576 were identified as Goffeauzyma gilvescens, Goffeauzyma gastrica, Candida atlantica, and Camptobasidium sp., respectively, being this last one possibly a new species. Growth at different temperatures indicates that these yeasts are psychrotolerant, with the exception of Camptobasidium sp., which presents psychrophilic characteristics. G. gastrica recovered from marine sediment showed the best results of resistance to UV radiation, being able to grow even after the exposure to UVB dose of 9144 J/m² and UVC dose of 6102 J/m². C. atlantica isolated from glacier soil showed high cellular growth from 3 to 10% NaCl. The majority of the strains produced higher biomass at pH 7; nevertheless, G. gilvescens showed higher biomass production at pH 9. The studied Antarctic-derived yeasts have adaptations to extreme conditions, which makes them useful for biotechnological applications and studies of extremophiles.

Antarctic fungi applied to textile dye bioremediation.

Antarctica has one of the most hostile conditions on the planet. The environmental characteristics found in this region favor the development of extremophile microorganisms, which are poorly explored biotechnologically. In this context, this study aimed at selectively isolating fungi with potential for the bioremediation of a textile dye. A total of 11 filamentous fungi were isolated from Antarctic samples after incubation in Minimal Mineral medium with the addition of Sulphur Indigo Blue dye. The Antarctic-derived fungi were submitted to textile dye decolorization analysis and biomass production. Isolates LAMAI 2400 and LAMAI 2402 showed more than 90% of decolorization at 15 °C, whereas at 28 °C these isolates showed 81.86 and 98.89%, respectively. In general, the toxicity of the bioassays, evaluated using Cucumis sativus, was higher than in the control. Both isolates, LAMAI 2400 and LAMAI 2402, were identified as Penicillium cf. oxalicum and classified as mesophilic-psychrotolerant. This fungal species has rarely been reported in the Antarctic environments. The results presented herein indicate the potential of the fungi recovered from Antarctic marine sediments for bioremediation of textile dyes at low and moderate temperatures, broadening the perspectives in the field of Antarctic mycology.

Antarctic environments as a source of bacterial and fungal therapeutic enzymes.

Microbial therapeutic enzymes are the protagonists in the pharmacological treatment of different human diseases. The intrinsic enzymatic characteristics, such as high affinity and specificity to the corresponding substrate, enable effective therapies, with minimal adverse effects and complete remission. However, immunogenicity, short half-life, low enzymatic yield, and low selectivity regarding available enzyme drugs are currently the main obstacles to their development and the broad adherence to therapeutic protocols. By harboring adapted and still unexplored microbial life, environments of extreme conditions, such as Antarctica, become especially important in the prospecting and development of new enzymatic compounds that present higher yields and the possibility of genetic improvement. Antarctic microorganisms have adaptation mechanisms, such as more fluid cell membranes, production of antifreeze proteins and enzymes with more malleable structures, more robust, stable, selective catalytic sites for their respective substrates, and high antioxidant capacity. In this context, this review aims to explore enzymes synthesized by bacteria and fungi from Antarctica as potential drug producers, capable of providing therapeutic efficacy, less adverse effects, and lower production costs with highlight to L-Asparaginase, collagenase, superoxide dismutase and ribonucleases. In addition, this review highlights the unique biotechnological profile of these Antarctic extremophile microorganisms.

Isolation and agricultural potential of penicillic acid against citrus canker.

AIMS: The control of Xanthomonas citri subsp. citri (X. citri), causal agent of citrus canker, relies heavily on integrated agricultural practices involving the use of copper-based chemicals. Considering the need for alternatives to control this disease and the potential of fungi from extreme environments as producers of bioactive metabolites, we isolated and identified a bioactive compound from Penicillium sp. CRM 1540 isolated from Antarctica marine sediment. METHODS AND RESULTS: The potential of compound as an antibacterial agent against X. citri was assessed through in vitro and greenhouse experiments. Molecular taxonomy indicates that this fungus is a possible new species of Penicillium. Results revealed 90% bacterial inhibition in vitro at 25 µg ml- 1 and a decrease in 75.37% of citrus canker symptoms emergency in vivo in treated leaves of Citrus sinensis (L.) Osbeck considering the number of lesions per cm2 (p < 0.05) in comparison with the control. The structure of the active agent was identified as penicillic acid based on a detailed spectroscopic analysis. CONCLUSION: Penicillic acid can be an alternative against citrus canker. SIGNIFICANCE AND IMPACT OF STUDY: Research into extremophile micro-organisms can identify molecules with biotechnological potential and alternatives to current agricultural practices.

Water-Soluble Glutamic Acid Derivatives Produced in Culture by Penicillium solitum IS1-A from King George Island, Maritime Antarctica.

A new method of screening was developed to generate 770 organic and water-soluble fractions from extracts of nine species of marine sponges, from the growth media of 18 species of marine-derived fungi, and from the growth media of 13 species of endophytic fungi. The screening results indicated that water-soluble fractions displayed significant bioactivity in cytotoxic, antibiotic, anti-Leishmania, anti-Trypanosoma cruzi, and inhibition of proteasome assays. Purification of water-soluble fractions from the growth medium of Penicillium solitum IS1-A provided the new glutamic acid derivatives solitumine A (1), solitumine B (2), and solitumidines A-D (3-6). The structures of compounds 1-6 have been established by analysis of spectroscopic data, chemical derivatizations, and vibrational circular dichroism calculations. Although no biological activity could be observed for compounds 1-6, the new structures reported for 1-6 indicate that the investigation of water-soluble natural products represents a relevant strategy in finding new secondary metabolites.

Metal and organic pollutants bioremediation by extremophile microorganisms.

Extremophiles comprise microorganisms that are able to grow and thrive in extreme environments, including in an acidic or alkaline pH, high or low temperatures, high concentrations of pollutants, and salts, among others. These organisms are promising for environmental biotechnology due to their unique physiological and enzymatic characteristics, which allow them to survive in harsh environments. Due to the stability and persistence of these microorganisms under adverse environmental conditions, they can be used for the bioremediation of environments contaminated with extremely recalcitrant pollutants. Here, we provide an overview of extremophiles and the role of "omics" in the field of bioremediation of environmental pollutants, including hydrocarbons, textile dyes and metals.

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp. CBMAI 1063, a laccase hyper-producer strain.

The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.

Pyrene degradation by marine-derived ascomycete: process optimization, toxicity, and metabolic analyses.

Marine-derived fungi are relevant genetic resources for bioremediation of saline environments/processes. Among the five fungi recovered from marine sponges able to degrade pyrene (Py) and benzo[a]pyrene (BaP), Tolypocladium sp. strain CBMAI 1346 and Xylaria sp. CBMAI 1464 presented the best removal rates of Py and BaP, respectively. Since the decrease in BaP was related to mycelial adsorption, a combined strategy was applied for the investigation of Py degradation by the fungus Tolypocladium sp. CBMAI 1346. The selected fungus was able to degrade about 95% of Py after 7 days of incubation (optimized conditions), generating metabolites different from the ones found before optimization. Metabolites and transcriptomic data revealed that the degradation occurred mainly by the cytochrome P450 pathway. Putative monooxygenases and dioxygenases found in the transcriptome may play an important role. After 21 days of degradation, no toxicity was found in the optimized culture conditions. The findings from the present study highlight the potential of marine-derived fungi to degrade environmental pollutants and convey innovative information related to the metabolism of pyrene.

Sequencing and phylogenetic analyses of talaromyces amestolkiae from amazon: A producer of natural colorants.

The population interest in health products is increasing day-by-day. Thus, the demand for natural products to be added in food and pharmaceutical commodity is also rising. Among these additives, colorants, which provides color to products, can be produced by microorganism through bioprocess. Looking for new source of natural colorants, fungi have been employed to this purpose producing novel and safer natural colorants. So, the main goal of this study was to describe a Talaromyces species able to produce natural colorants and investigate nutritional parameters of colorants production using statistical tool. The taxonomy classified the microorganism as Talaromyces amestolkiae. The statistical design evaluated pH and glucose, meat extract and meat peptone concentration as independent variables, and red colorants production as main response. Under the best condition (g/L: glucose 30, meat extract 1, meat peptone 10, and initial pH of 7.0) an increase of 229% in the red colorant production was achieved as compared with the initial media used. The dried fermented broth containing red colorants showed low cytotoxicity against fibroblasts cells (IC50 > 187.5 g/L) and effective antimicrobial activity against S. aureus (MIC of 2.5 g/L). Thus, T. amestolkiae colorants can be attractive to food and pharmaceutical applications as it does not produce toxic compounds and can promote protection against microorganism contaminants. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2684, 2019.

Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process

ABSTRACT Pyrene and benzo[a]pyrene (BaP) are high molecular weight polycyclic aromatic hydrocarbons (PAHs) recalcitrant to microbial attack. Although studies related to the microbial degradation of PAHs have been carried out in the last decades, little is known about degradation of these environmental pollutants by fungi from marine origin. Therefore, this study aimed to select one PAHs degrader among three marine-derived basidiomycete fungi and to study its pyrene detoxification/degradation. Marasmiellus sp. CBMAI 1062 showed higher levels of pyrene and BaP degradation and was subjected to studies related to pyrene degradation optimization using experimental design, acute toxicity, organic carbon removal (TOC), and metabolite evaluation. The experimental design resulted in an efficient pyrene degradation, reducing the experiment time while the PAH concentration applied in the assays was increased. The selected fungus was able to degrade almost 100% of pyrene (0.08 mg mL-1) after 48 h of incubation under saline condition, without generating toxic compounds and with a TOC reduction of 17%. Intermediate metabolites of pyrene degradation were identified, suggesting that the fungus degraded the compound via the cytochrome P450 system and epoxide hydrolases. These results highlight the relevance of marine-derived fungi in the field of PAH bioremediation, adding value to the blue biotechnology.

Isolation, synthesis and bioactivity studies of phomactin terpenoids.

Studies of secondary metabolites (natural products) that cover their isolation, chemical synthesis and bioactivity investigation present myriad opportunities for discovery. For example, the isolation of novel secondary metabolites can inspire advances in chemical synthesis strategies to achieve their practical preparation for biological evaluation. In the process, chemical synthesis can also provide unambiguous structural characterization of the natural products. Although the isolation, chemical synthesis and bioactivity studies of natural products are mutually beneficial, they are often conducted independently. Here, we demonstrate the benefits of a collaborative study of the phomactins, diterpenoid fungal metabolites that serve as antagonists of the platelet activating factor receptor. Our isolation of novel phomactins has spurred the development of a bioinspired, unified approach that achieves the total syntheses of six congeners. We also demonstrate in vitro the beneficial effects of several phomactins in suppressing the rate of repopulation of tumour cells following gamma radiation therapy.

Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process.

Pyrene and benzo[a]pyrene (BaP) are high molecular weight polycyclic aromatic hydrocarbons (PAHs) recalcitrant to microbial attack. Although studies related to the microbial degradation of PAHs have been carried out in the last decades, little is known about degradation of these environmental pollutants by fungi from marine origin. Therefore, this study aimed to select one PAHs degrader among three marine-derived basidiomycete fungi and to study its pyrene detoxification/degradation. Marasmiellus sp. CBMAI 1062 showed higher levels of pyrene and BaP degradation and was subjected to studies related to pyrene degradation optimization using experimental design, acute toxicity, organic carbon removal (TOC), and metabolite evaluation. The experimental design resulted in an efficient pyrene degradation, reducing the experiment time while the PAH concentration applied in the assays was increased. The selected fungus was able to degrade almost 100% of pyrene (0.08mgmL-1) after 48h of incubation under saline condition, without generating toxic compounds and with a TOC reduction of 17%. Intermediate metabolites of pyrene degradation were identified, suggesting that the fungus degraded the compound via the cytochrome P450 system and epoxide hydrolases. These results highlight the relevance of marine-derived fungi in the field of PAH bioremediation, adding value to the blue biotechnology.

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063.

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high ß-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.

Improvement in extracellular protease production by the marine antarctic yeast Rhodotorula mucilaginosa L7.

Microorganisms from extreme and restrictive eco systems, such as the Antarctic continent, are of great interest due to their ability to synthesize products of commercial value. Among these, enzymes from psychrotolerant and psychrophilic microorganisms offer potential economical benefits due to their high activity at low and moderate temperatures. The cold adapted yeast Rhodotorula mucilaginosa L7 was selected out of 97 yeasts isolated from Antarctica as having the highest extracellular proteolytic activity in preliminary tests. The present study was aimed at evaluating the effects of nutrient composition (peptone, rice bran extract, ammonium sulfate, sodium chloride) and physicochemical parameters (temperature and pH) on its proteolytic activity. A 26-2 fractional factorial design experiment followed by a central composite design (CCD 23) was performed to optimize the culture conditions and improve the extracellular proteolytic activity. The results indicated that the presence of peptone in the medium was the most influential factor in protease production. Enzymatic activity was enhanced by the interaction between low glucose and peptone concentrations. The optimization of culture conditions with the aid of mathematical modeling enabled a c. 45% increase in proteolytic activity and at the same time reduced the amount of glucose and peptone required for the culture. Thus culture conditions established in this work may be employed in the biotechnological production of this protease.

Condensation of Macrocyclic Polyketides Produced by Penicillium sp. DRF2 with Mercaptopyruvate Represents a New Fungal Detoxification Pathway.

Application of a refined procedure of experimental design and chemometric analysis to improve the production of curvularin-related polyketides by a marine-derived Penicillium sp. DRF2 resulted in the isolation and identification of cyclothiocurvularins 6-8 and cyclosulfoxicurvularins 10 and 11, novel curvularins condensed with a mercaptolactate residue. Two additional new curvularins, 3 and 4, are also reported. The structures of the sulfur-bearing curvularins were unambiguously established by analysis of spectroscopic data and by X-ray diffraction analysis. Analysis of stable isotope feeding experiments with [U-(13)C3(15)N]-l-cysteine confirmed the presence of the 2-hydroxy-3-mercaptopropanoic acid residue in 6-8 and the oxidized sulfoxide in 10 and 11. Cyclothiocurvularins A (6) and B (7) are formed by spontaneous reaction between 10,11-dehydrocurvularin (2) and mercaptopyruvate (12) obtained by transamination of cysteine. High ratios of [U-(13)C3(15)N]-l-cysteine incorporation into cyclothiocurvularin B (7), the isolation of two diastereomers of cyclothiocurvularins, the lack of cytotoxicity of cyclothiocurvularin B (7) and its methyl ester (8), and the spontaneous formation of cyclothiocurvularins from 10,11-dehydrocurvularin and mercaptopyruvate provide evidence that the formation of cyclothiocurvularins may well correspond to a 10,11-dehydrocurvularin detoxification process by Penicillium sp. DRF2.

Marine-derived fungus Aspergillus cf. tubingensis LAMAI 31: a new genetic resource for xylanase production.

Marine-derived fungi have been reported as relevant producers of enzymes, which can have different properties in comparison with their terrestrial counterparts. The aim of the present study was to select from a collection of 493 marine-derived fungi the best producer of xylanase in order to evaluate the enzymatic production under different conditions. A total of 112 isolates produced xylanase in solid medium containing xylan as the carbon source, with 31 of them able to produce at least 10 U/mL of the enzyme. The best production (49.41 U/mL) was achieved by the strain LAMAI 31, identified as Aspergillus cf. tubingensis. After confirming the lack of pathogenicity (absence of ochratoxin A and fumonisin B2 production) this fungus was submitted to the experimental design in order to evaluate the effect of different variables on the enzymatic production, with the aim of optimizing culture conditions. Three experimental designs (two Plackett-Burman and one factorial fractional) were applied. The best condition for the enzymatic production was defined, resulting in an increase of 12.7 times in comparison with the initial production during the screening experiments. In the validation assay, the peak of xylanase production (561.59 U/mL) was obtained after 96 h of incubation, being the best specific activity achieved after 72 h of incubation. Xylanase from A. cf. tubingensis LAMAI 31 had optimum pH and temperature at 5.0 and 55 °C, respectively, and was shown to be stable at a range of 40-50 °C, and in pH from 3.6 to 7.0. Results from the present work indicate that A. cf. tubingensis LAMAI 31 can be considered as a new genetic resource for xylanase production.

Enhanced textile dye decolorization by marine-derived basidiomycete Peniophora sp. CBMAI 1063 using integrated statistical design.

In the present study, the biotechnological potential of the marine-derived fungus Peniophora sp. CBMAI 1063 was investigated in relation to Reactive Black 5 (RB5) dye decolorization and degradation using an integrated statistical design composed of Plackett-Burman design (P&B), central composite design (CCD), and response surface methodology (RSM). RB5 dye was effectively decolorized (94 %) in saline conditions, without any detection of mutagenic compounds, and simultaneously, 57 % of total organic carbon (TOC) was removed in 7 days. The activity of lignin peroxidase (LiP) was not detected during the process. The gene expression of laccase (Lac) and manganese peroxidase (MnP) enzymes produced during the process was evaluated, and results from this experiment coupled with LC-MS analyses revealed that in the early stage of dye decolorization, a higher MnP gene expression and significant enzymatic activity was detected in Peniophora sp. CBMAI 1063 with the formation of p-Base and TAHNDS compounds. This paper reports innovative data related to the textile dye decolorization by the marine-derived basidiomycete Peniophora sp. CBMAI 1063, showing the metabolites formed and enzymatic action throughout the process in saline condition. The strategy used showed to be an efficient statistical approach that provides an attractive solution for the screening and simultaneous optimization of the degradation process.

Structure and Biogenesis of Roussoellatide, a Dichlorinated Polyketide from the Marine-Derived Fungus Roussoella sp. DLM33.

The structure of the fungal metabolite roussoellatide (1) has been established by spectroscopic and X-ray diffraction analyses. Results from feeding experiments with [1-(13)C]acetate, [2-(13)C]acetate, and [1,2-(13)C]acetate were consistent with a biosynthetic pathway to the unprecedented skeleton of 1 involving Favorskii rearrangements in separate pentaketides, subsequently joined via an intermolecular Diels-Alder reaction.