Isolation and Characterization of an Antioxidant Aryl Polyene Pigment from Antarctic Bacterium Lysobacter sp. A03.

Lysobacter is known as a bacterial genus with biotechnological potential, producing an array of enzymes, antimicrobial metabolites, and bioactive antioxidant compounds, including aryl polyene (APE) pigments that have been described as protecting substances against photooxidative damage and lipid peroxidation. In this study, the pigment extracted from keratinolytic Lysobacter sp. A03 isolated from Antarctic environment was characterized. The results of KOH test, UV-vis spectroscopy, CIELAB color system, 1H-NMR, and FTIR-ATR spectroscopy suggest the pigment is a yellow xanthomonadin-like pigment. The in vitro antioxidant activity of the pigment was confirmed by the scavenging of ABTS and DPPH radicals. In silico analysis of the genome through antiSMASH software was also performed and the secondary metabolite gene clusters for APE and resorcinol synthesis were identified, suggesting that proteins responsible for the pigment biosynthesis are encoded in Lysobacter A03 genome.

Genomic characterization and production of antimicrobial lipopeptides by Bacillus velezensis P45 growing on feather by-products.

AIMS: To investigate the potential of novel Bacillus velezensis P45 as an eco-friendly alternative for bioprocessing poultry by-products into valuable antimicrobial products. METHODS AND RESULTS: The complete genome of B. velezensis P45 was sequenced using the Illumina MiSeq platform, showing 4455 protein and 98 RNA coding sequences according to the annotation on the RAST server. Moreover, the genome contains eight gene clusters for the production of antimicrobial secondary metabolites and 25 putative protease-related genes, which can be related to feather-degrading activity. Then, in vitro tests were performed to determine the production of antimicrobial compounds using feather, feather meal and brain-heart infusion (BHI) cultures. Antimicrobial activity was observed in feather meal and BHI media, reaching 800 and 3200 AU ml-1 against Listeria monocytogenes respectively. Mass spectrometry analysis indicates the production of antimicrobial lipopeptides surfactin, fengycin and iturin. CONCLUSIONS: The biotechnological potential of B. velezensis P45 was deciphered through genome analysis and in vitro studies. This strain produced antimicrobial lipopeptides growing on feather meal, a low-cost substrate. SIGNIFICANCE AND IMPACT OF STUDY: The production of antimicrobial peptides by this keratinolytic strain may represent a sustainable alternative for recycling by-products from poultry industry. Furthermore, whole B. velezensis P45 genome sequence was obtained and deposited.

Diversity of cyclic antimicrobial lipopeptides from Bacillus P34 revealed by functional annotation and comparative genome analysis.

Cyclic lipopeptides (CLPs) from Bacillus strains have demonstrated a wide range of bioactivities making them interesting candidates for different applications in the pharmaceutical, food and biotechnological industries. Genome sequencing, together with phylogenetic analysis of the Bacillus sp. P34, isolated from a freshwater fish gut, showed that the bacterial strain belongs to the Bacillus velezensis group. In silico investigation of metabolic gene clusters of nonribosomal peptide synthetases (NRPS) revealed the genetic elements associated with the synthesis of surfactin, fengycin and iturin family component bacillomycin. Further, an assay was conducted to investigate the production of CLPs in the presence of heat inactivated bacterial cultures or fungal spores. Maximum fengycin concentration was observed at 24 h (2300-2700 mg/mL), while maximum iturin amounts were detected at 48 h (250 mg/mL) in the presence of heat-inactivated spores of Aspergillus niger. Heat-inactivated cells of Listeria monocytogenes caused a reduction of both fengycin and iturin amounts. The production of fengycins A and B and the iturin family component bacillomycin L was confirmed by mass spectrometry analyses. This study reinforces the potential of B. velezensis P34 as a valuable strain for biotechnological production of CLPs recognized as important antimicrobial substances.

Functional genome annotation depicts probiotic properties of Bacillus velezensis FTC01.

An in silico genome analysis of the probiotic Bacillus strain FTC01 was performed. The draft genome comprises 3.9 Mb, with a G + C content of 46.6% and a total of 3941 coding sequences. The species of strain FTC01 was defined as B. velezensis during GenBank genome annotation, following the current nomenclature. Eight gene clusters involved in the synthesis of non-ribosomal lipopeptides, polyketides and bacilysin were found, as well as part of the gene cluster involved in the synthesis of cyclic lipopeptide locillomycin. The production of lipopeptides surfactin and iturin by strain FTC01 was confirmed. In addition, a gene encoding a peptidylprolyl isomerase, involved in bacterial adhesion to the host tissue, beyond twelve genes responsible for acid tolerance and several hydrolase genes were found. These characteristics may help in host colonization and maintenance and may account for the probiotic properties observed for strain FTC01.

A new cold-adapted serine peptidase from Antarctic Lysobacter sp. A03: Insights about enzyme activity at low temperatures.

Currently, there is a great interest for customized biocatalysts that can supply the ongoing demand of industrial processes, but also deal with the growing concern about the environment. In this scenario, cold-adapted enzymes have features that make them very attractive for industrial and biotechnological purposes. Here, we describe A03Pep1, a new cold-adapted serine peptidase isolated from Lysobacter sp. A03 by screening a genomic library. The enzyme is synthesized as a large inactive prepropeptidase that, after intramolecular processing, gives rise to the active form, of 35kDa. The heterologous expression of A03Pep1 was carried out in E. coli cells harboring the vector pGEX-4T-2-a0301. Its activity was optimal at pH 9.0 and 40°C, in the presence of 25mM Ca2+, which may contribute to the thermal stability of the enzyme. The 3D structure modelling predicted a less deep and more open binding pocket in A03Pep1 than that observed in the crystal structure of its mesophilic homologous AprV2, presumably as a way to enhance the probability of substrate binding at low temperatures. These results provide possible approaches in developing new biotechnologically relevant peptidases active at low to moderate temperatures.

Whole-Genome Shotgun Sequence of the Keratinolytic Bacterium Lysobacter sp. A03, Isolated from the Antarctic Environment.

Lysobacter sp. strain A03 is a protease-producing bacterium isolated from decomposing-penguin feathers collected in the Antarctic environment. This strain has the ability to degrade keratin at low temperatures. The A03 genome sequence provides the possibility of finding new genes with biotechnological potential to better understand its cold-adaptation mechanism and survival in cold environments.

Production of selenium-enriched biomass by Enterococcus durans.

Selenium (Se) is an essential micronutrient for several organisms, and there is an increased interest about adequate sources for dietary selenium supplementation. The aim of this study was to evaluate the selenium bioaccumulation capacity of an Enterococcus strain. The isolate LAB18s was identified as Enterococcus durans by the VITEK® 2 system and analysis of both 16S rDNA gene sequence (JX503528) and the 16S-23S rDNA intergenic spacer (ITS). After 24-h incubation, E. durans LAB18s bioaccumulated elevated Se(IV) concentrations, reaching 2.60 and 176.97 mg/g in media containing initial amounts of 15 and 240 mg/l sodium selenite, respectively. The isolate grew optimally and had high selenium bioaccumulation at initial pH of 7.0 and 30 °C. Time course studies showed that E. durans LAB18s displayed the highest bioaccumulation of Se(IV) after 6 h of incubation. Analyses from scanning electron microscopy (SEM) demonstrated the presence of filaments connecting the cells of E. durans LAB18s cultivated in the presence of sodium selenite. It was demonstrated that a considerable amount of Se(IV) was absorbed by E. durans LAB18s. Therefore, this strain may represent an alternative source of organic dietary selenium.

Pigment production by filamentous fungi on agro-industrial byproducts: an eco-friendly alternative.

The search for new sources of natural pigments has increased, mainly because of the toxic effects caused by synthetic dyes used in food, pharmaceutical, textile, and cosmetic industries. Fungi provide a readily available alternative source of natural pigments. In this context, the fungi Penicillium chrysogenum IFL1 and IFL2, Fusarium graminearum IFL3, Monascus purpureus NRRL 1992, and Penicillium vasconiae IFL4 were selected as pigments producers. The fungal identification was performed using ITS and part of the ß-tubulin gene sequencing. Almost all fungi were able to grow and produce water-soluble pigments on agro-industrial residues, with the exception of P. vasconiae that produced pigments only on potato dextrose broth. The production of yellow pigments was predominant and the two strains of P. chrysogenum were the largest producers. In addition, the production of pigments and mycotoxins were evaluated in potato dextrose agar using TOF-MS and TOF-MS/MS. Metabolites as roquefortine C, chrysogine were found in both extracts of P. chrysogenum, as well fusarenone X, diacetoxyscirpenol, and neosolaniol in F. graminearum extract. In the M. purpureus extract, the pigments monascorubrin, rubropunctatin, and the mycotoxin citrinin were found. The crude filtrates have potential to be used in the textile industry; nevertheless, additional pigment purification is required for food and pharmaceutical applications.

Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger.

A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.