A basidiomycetous yeast, strain E2A-C3-II, was isolated from a marine sponge (Hymeniacidon sp.) collected at a depth of 6 m in Fildes Bay, King George Island, Antarctica. The phylogenetic analysis revealed that the yeast isolated is related to Leucosporidium drummii, Leucosporidiella muscorum and to the Leucosporidium scottii group, including Leucosporidiella creatinivora and Leucosporidiella yakutica. The analysis of the nucleotide differences and the genetic distances of the D1/D2 domain of the LSU rDNA gene and 5.8S ITS regions support that strain E2A-C3-II represents a new species. The novel species can be distinguished from L. drummii by its ability to assimilate L-sorbose, L-rhamnose, lactose and ribitol. The maximum temperature for growth was 25 °C. On the basis of morphological, biochemical and physiological characterization, and phylogenetic and nucleotide analysis, a novel basidiomycetous yeast species, Leucosporidium escuderoi f.a., sp. nov., is proposed. The type strain is E2A-C3-II(T) (=CBS 12734(T) =CECT 13080(T)). The Mycobank ( http://www.mycobank.org ) accession number is MB 804654. The nucleotide sequences of D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions obtained in this work have been deposited in Genbank under the Accession numbers JN181009 and JN197600, respectively.
During the characterization of the mycobiota associated with shallow-water marine environments from Antarctic sea, a novel pink yeast species was isolated. Sequence analysis of the D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions revealed that the isolated yeast was closely related to Rhodotorula pallida CBS 320(T) and Rhodotorula benthica CBS 9124(T). On the basis of morphological, biochemical and physiological characterization and phylogenetic analyses, a novel basidiomycetous yeast species, Rhodotorula portillonensis sp. nov., is proposed. The type strain is Pi2(T) (?=?CBS 12733(T) ?=?CECT 13081(T)) which was isolated from shallow-water marine sediment in Fildes Bay, King George Island, Antarctica.
Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.
The diversity of sponge-associated fungi has been poorly investigated in remote geographical areas like Antarctica. In this study, 101 phenotypically different fungal isolates were obtained from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong to the genera Geomyces, Penicillium, Epicoccum, Pseudeurotium, Thelebolus, Cladosporium, Aspergillus, Aureobasidium, Phoma, and Trichocladium but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bioactivities, antimicrobial, antitumoral and antioxidant activities of fungal culture extracts were assayed. Around 51 % of the extracts, mainly from the genus Geomyces and non identified relatives, showed antimicrobial activity against some of the bacteria tested. On the other hand, around 42 % of the extracts showed potent antitumoral activity, Geomyces sp. having the best performance. Finally, the potential of the isolated fungi as producers of antioxidant activity seems to be moderate. Our results suggest that fungi associated with Antarctic sponges, particularly Geomyces, would be valuable sources of antimicrobial and antitumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of fungi associated with Antarctic marine sponges.
The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of L-?-aminoadipic acid, L-cysteine and L-valine into the tripeptide ?-(L-?-aminoadipyl)-L-cysteinyl-D-valine (ACV). This reaction is catalysed by the multienzyme ACV synthetase (ACVS), which is encoded in the filamentous fungus Penicillium chrysogenum by the pcbAB gene. This enzyme contains at least ten catalytic domains. The precise role of the C-terminal domain of this multidomain NRPS still remains obscure. The C-terminal region of ACVS bears the epimerase and the thioesterase domains and may be involved in the epimerization of LLL-ACV to LLD-ACV and in the hydrolysis of the thioester bond. In this work, the conserved motifs (3371)EGHGRE(3376) (located in the putative epimerase domain) and (3629)GWSFG(3633) (located in the thioesterase domain) were changed by site-directed-mutagenesis to LGFGLL and GWAFG, respectively. In addition, the whole thioesterase domain (230 amino acids) and the different parts of this domain were deleted. The activity of these mutant enzymes was assessed in vivo by two different procedures: i) through the quantification of bisACV produced by the fungus and ii) by quantifying the benzylpenicillin production using tailored strains of P. chrysogenum, which lack the pcbAB gene, as host strains. All indicated mutant enzymes showed lower or null activity than the control strain confirming that E3371, H3373, R3375 and E3376 belong to the epimerase active centre. Different fragments included in the C-terminal region of ACVS control thioester hydrolysis. Overexpression of the sequence encoding the ACVS integrated thioesterase domain as a separate (stand-alone) transcriptional unit complemented mutants lacking the integrated thioesterase domain, although with low ACV releasing activity, suggesting that the stand-alone thioesterease interacts with the other ACVS domains.
A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and -75?° C with a half-life of 770 days.
The mechanisms of compartmentalization of intermediates and secretion of penicillins and cephalosporins in ?-lactam antibiotic-producing fungi are of great interest. In Acremonium chrysogenum, there is a compartmentalization of the central steps of the CPC (cephalosporin C) biosynthetic pathway. In the present study, we found in the early CPC cluster a new gene named cefP encoding a putative transmembrane protein containing 11 transmembrane spanner. Targeted inactivation of cefP by gene replacement showed that it is essential for CPC biosynthesis. The disrupted mutant is unable to synthesize cephalosporins and secretes a significant amount of IPN (isopenicillin N), indicating that the mutant is blocked in the conversion of IPN into PenN (penicillin N). The production of cephalosporin in the disrupted mutant was restored by transformation with both cefP and cefR (a regulatory gene located upstream of cefP), but not with cefP alone. Fluorescence microscopy studies with an EGFP (enhanced green fluorescent protein)-SKL (Ser-Lys-Leu) protein (a peroxisomal-targeted marker) as a control showed that the red-fluorescence-labelled CefP protein co-localized in the peroxisomes with the control peroxisomal protein. In summary, CefP is a peroxisomal membrane protein probably involved in the import of IPN into the peroxisomes where it is converted into PenN by the two-component CefD1/CefD2 protein system.
Two new diterpenoids, mulin-12-en-16-al-20-oic acid and 13-?-hydroxy-mulin-11-en-14-one-20-oic acid, were isolated from Azorella madreporica. Their structures were identified on the basis of one-dimensional and two-dimensional NMR experiments. Their antibacterial activity was also tested.
Penicillium chrysogenum converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the penDE gene. In silico analysis of the P. chrysogenum genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding penDE gene. We have termed this gene ial because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the ial gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.
Genetic manipulation of the filamentous fungus Penicillium camemberti has been limited by a lack of suitable genetics tools for this fungus. In particular, there is no available homologous transformation system. In this study, the nitrate reductase (niaD) and orotidine-5-monophosphate decarboxylase (pyrG) genes from Penicillium camemberti were characterized, and their suitability as metabolic molecular markers for transformation was evaluated. The genes were amplified using PCR-related techniques, and sequenced. The niaD gene is flanked by the nitrite reductase (niiA) gene in a divergent arrangement, being part of the putative nitrate assimilation cluster in P. camemberti. pyrG presents several polymorphisms compared with a previously sequenced pyrG gene from another P. camemberti strain, but almost all are silent mutations. Southern blot assays indicate that one copy of each gene is present in P. camemberti. Northern blot assays showed that the pyrG gene is expressed in minimal and rich media, and the niaD gene is expressed in nitrate, but not in reduced nitrogen sources. The functionality of the two genes as transformation markers was established by transforming A. nidulans pyrG- and niaD-deficient strains. Higher transformation efficiencies were obtained with a pyrG-containing plasmid. This is the first study yielding a molecular and functional characterization of P. camemberti genes that would be useful as molecular markers for transformation, opening the way for the future development of a non-antibiotic genetic transformation system for this fungus.
The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM-GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1-CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.
Unlike filamentous fungi and bacteria, very little is known about cultivable yeasts associated with marine sponges, especially those from Antarctic seas. During an expedition to King George Island, in the Antarctica, samples of 11 marine sponges were collected by scuba-diving. From these sponges, 20 psychrotolerant yeast isolates were obtained. Phylogenetic analyses of D1/D2 and ITS rRNA gene sequences revealed that the marine ascomycetous yeast Metschnikowia australis is the predominant organism associated with these invertebrates. Other species found belonged to the Basidiomycota phylum: Cystofilobasidium infirmominiatum, Rhodotorula pinicola, Leucosporidiella creatinivora and a new yeast from the Leucosporidiella genus. None of these yeasts have been previously associated with marine sponges. A screening to estimate the ability of these yeasts as producers of extracellular enzymatic activities at several pH and temperature conditions was performed. Several yeast isolates demonstrated amylolytic, proteolytic, lipolytic or cellulolytic activity, but none of them showed xylanolytic activity under the conditions assayed. To our knowledge, this work is the first description of cultivable yeasts associated with marine sponges from the Antarctic sea.
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