Motifs in the C-terminal region of the Penicillium chrysogenum ACV synthetase are essential for valine epimerization and processivity of tripeptide formation

The first step in the penicillin biosynthetic pathway is the non-ribosomal condensation of l-α-aminoadipic acid, l-cysteine and l-valine into the tripeptide δ-(l-α-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 3371EGHGRE 3376 (located in the putative epimerase domain) and 3629GWSFG 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 par

Cold-active pectinolytic activity produced by filamentous fungi associated with Antarctic marine sponges

BACKGROUND: Pectinase enzymes catalyze the breakdown of pectin, a key component of the plant cell wall. At industrial level, pectinases are used in diverse applications, especially in food-processing industry. Currently, most of the industrial pectinases have optimal activity at mesophilic temperatures. On the contrary, very little is known about the pectinolytic activities from organisms from cold climates such as Antarctica. In this work, 27 filamentous fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new source of cold-active pectinases. RESULTS: In semi-quantitative plate assays, 8 out 27 of these isolates showed pectinolytic activities at 15 °C and one of them, Geomyces sp. strain F09-T3-2, showed the highest production of pectinases in liquid medium containing pectin as sole carbon source. More interesting, Geomyces sp. F09-T3-2 showed optimal pectinolytic activity at 30 °C, 10 °C under the temperature of currently available commer

Heterologous expression, purification and characterization of a highly thermolabile endoxylanase from the Antarctic fungus Cladosporium sp.

© 2018 British Mycological SocietyNumerous endoxylanases from mesophilic fungi have been purified and characterized. However, endoxylanases from cold-adapted fungi, especially those from Antarctica, have been less studied. In this work, a cDNA from the Antarctic fungus Cladosporium sp. with similarity to endoxylanases from glycosyl hydrolase family 10, was cloned and expressed in Pichia pastoris. The pure recombinant enzyme (named XynA) showed optimal activity on xylan at 50 °C and pH 6–7. The enzyme releases xylooligosaccharides but not xylose, indicating that XynA is a classical endoxylanase. The enzyme was most active on xylans with high content of arabinose (rye arabinoylan and wheat arabinoxylan) than on xylans with low content of arabinose (oat spelts xylan, birchwood xylan and beechwood xylan). Finally, XynA showed a very low thermostability. After 20–30 min of incubation at 40 °C, the enzyme was completely inactivated, suggesting that XynA would be the most thermolabile endoxy

Characterization of a novel peroxisome membrane protein essential for conversion of isopenicillin N into cephalosporin C

The mechanisms of compartmentalization of intermediates and secretion of penicillins and cephalosporins in β-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 (enhanc

The developmental regulator Pcz1 affects the production of secondary metabolites in the filamentous fungus Penicillium roqueforti

Penicillium roqueforti is used in the production of several kinds of ripened blue-veined cheeses. In addition, this fungus produces interesting secondary metabolites such as roquefortine C, andrastin A and mycophenolic acid. To date, there is scarce information concerning the regulation of the production of these secondary metabolites. Recently, the gene named pcz1 (Penicillium C6 zinc domain protein 1) was described in P. roqueforti, which encodes for a Zn(II)(2)Cys(6) protein that controls growth and developmental processes in this fungus. However, its effect on secondary metabolism is currently unknown. In this work, we have analyzed how the overexpression and down-regulation of pcz1 affect the production of roquefortine C, andrastin A and mycophenolic acid in P. roqueforti. The three metabolites were drastically reduced in the pcz1 down-regulated strains. However, when pcz1 was overexpressed, only mycophenolic acid was overproduced while, on the contrary, levels of roquefortine C and andrastin A were diminished. Importantly, these results match the expression pattern of key genes involved in the biosynthesis of these metabolites. Taken together, our results suggest that Pcz1 plays a key role in regulating secondary metabolism in the fungus Penicillium roqueforti.This work was supported by project Fondecyt 1120833 and "Proyecto DICYT, Codigo 021743CR, Vicerrectoria de Investigation, Desarrollo e Innovation, Universidad de Santiago de Chile", and MIISSB Iniciativa Cientifica Milenio-MINECON. JFR-A and CG-D have received doctoral fellowships CONICYT-PFCHA/Doctorado National/2013-21130251 and CONICYT-PFCHA/Doctorado National/2014-63140056, respectively

Cold-active xylanase produced by fungi associated with Antarctic marine sponges

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

Cultivable psychrotolerant yeasts associated with Antarctic marine sponges

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.This work was supported by Instituto Anta´rtico Chileno (INACH) grant G_06-10, FONDECYT grant 11090192, ‘‘Programa Bicentenario de Ciencia y Tecnologı´a’’ (Chile) project PDA13, and DICYT-USACH

Biotransformation of Stypotriol triacetate by Aspergillus niger

Biological transformation of the meroditerpenoid, stypotriol triacetate (1) by the fungi Aspergillus niger, Cunninghamella elegans, Gibberella fujikuroi and Mucor plumbeus was studied. The incubation of 1 with A. niger yielded the new compound 6′,14-diacetoxy-stypol-4,5-dione (2) whose structure was established by 1H, 13C and 2D NMR and supported by DFT/GIAO. © 2011 Elsevier B.V. All rights reserved

Production of a heterologous recombinant protein using fragments of the glyceraldehyde-3-phosphate dehydrogenase promoter from Penicillium Camemberti

The biotechnological applications of cheese-ripening fungi have been limited by a lack of genetics tools, in particular the identification and characterization of suitable promoters for protein expression. In this study, the suitability of the glyceraldehyde-3-phosphate dehydrogenase (gpdP) promoter from Penicillium camemberti to drive the production of a recombinant protein was evaluated. The gpdP gene and its promoter were isolated using PCR and Genome Walker. The promoter of gpdP has two regions with high identity to the regulatory elements gpd-box and ct-box previously described in Aspergillusnidulans. Two fragments of the promoter containing the gpd- and ct-box or the ct-box alone were used to drive the in vivo production of recombinant β-galactosidase using A. nidulans as host. Our results indicate that larger fragment containing gpd-box enhances the production of β-galactosidase activity levels respect to ct-box alone, and that both boxes are necessary to obtain maximal enzymatic activity production. The smaller fragment (187 nt) containing the ct-box alone was able to trigger up to 27% of b-galactosidase activity, and to our knowledge this is the smallest fragment from a gpd gene used to produce a recombinant protein. Differences were not observed when glycerol, galactose or glucose were used as carbon sources, suggesting that the promoter activity is carbohydrate-independent. This is the first report in which a Penicillium gpd promoter is used for recombinant protein production. Our results open the way for the future development of a system for recombinant proteins expression in the biotechnologically important cheese-ripening fungus P. camemberti

The biosynthetic gene cluster for andrastin A in Penicillium roqueforti

© 2017 Rojas-Aedo, Gil-Durán, Del-Cid, Valdés, álamos, Vaca, García-Rico, Levicán, Tello and Chávez. Penicillium roqueforti is a filamentous fungus involved in the ripening of several kinds of blue cheeses. In addition, this fungus produces several secondary metabolites, including the meroterpenoid compound andrastin A, a promising antitumoral compound. However, to date the genomic cluster responsible for the biosynthesis of this compound in P. roqueforti has not been described. In this work, we have sequenced and annotated a genomic region of approximately 29.4 kbp (named the adr gene cluster) that is involved in the biosynthesis of andrastin A in P. roqueforti. This region contains ten genes, named adrA, adrC, adrD, adrE, adrF, adrG, adrH, adrI, adrJ and adrK. Interestingly, the adrB gene previously found in the adr cluster from P. chrysogenum, was found as a residual pseudogene in the adr cluster from P. roqueforti. RNA-mediated gene silencing of each of the ten genes resulted in s