Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

<p>Abstract</p> <p>Background</p> <p><it>Penicillium chrysogenum </it>converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the <it>penDE </it>gene. <it>In silico </it>analysis of the <it>P. chrysogenum </it>genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding <it>penDE </it>gene. We have termed this gene <it>ial </it>because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the <it>ial </it>gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.</p> <p>Results</p> <p>The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null <it>ial </it>mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities <it>in vivo</it>. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IAL^ARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in <it>Escherichia coli</it>. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive <it>in vitro</it>.</p> <p>Conclusion</p> <p>No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the <it>P. chrysogenum </it>IAL, the <it>A. nidulans </it>IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the <it>ial </it>gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in <it>A. nidulans</it>, the <it>ial </it>and <it>penDE </it>genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the <it>ial </it>and <it>penDE </it>genes, is also discussed.</p

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress

The lysine biosynthetic pathway has to supply large amounts of α-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G1534 to A1534 point mutation resulting in a Gly495 to Asp495 substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron-sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.Fil: Teves, Franco. Universidad de León; EspañaFil: Lamas Maceiras, Mónica. Universidad de León; EspañaFil: García Estrada, Carlos. Instituto de Biotecnología de León; EspañaFil: Casqueiro, Javier. Instituto de Biotecnología de León; España. Universidad de León; EspañaFil: Naranjo, Leopoldo. Universidad de León; EspañaFil: Ullán, Ricardo V.. Instituto de Biotecnología de León; EspañaFil: Scervino, Jose Martin. Instituto de Biotecnología de León; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Wu, Xiaobin. Instituto de Biotecnología de León; EspañaFil: Velasco Conde, Tania. Instituto de Biotecnología de León; EspañaFil: Martín, Juan F.. Instituto de Biotecnología de León; España. Universidad de León; Españ

Increased expression of two cDNAs encoding metallothionein‐like proteins during growth of Cicer arietinum epicotyls

The present study was undertaken to identify and characterize clones whose expres­sion increase during Cicer arietinum epicotyl growth. Two cDNAs encoding two different plant metallothionein (MT)-like proteins have been isolated from a cDNA library from epicotyls of Cicer arietinum L. cv. Castellana. The CanMT-1 deduced protein appears to have the typical structure of type I MT where ali Cys residues are in Cys-X-Cys motifs, while the CanMT-2 has the typical structure of type 2 MT having Cys-Cys and Cys-X-X-Cys motifs within the N-terminal domain. Both chickpea CanMTs are up-regulated during epicotyl growth, showing increased ex­pression in mature tissues, mostly CanMT-1, which is undetectable in young epi­cotyls. Accordingly, stem of chickpea plants displayed the highest leve! of CanMT-1 expression in the basal internode, with reduced growth, decreasing towards the apex. Osmotic stress by PEG, which inhibited growth, and ABA treatment induced the expression of MT-like genes, which points to a relationship between chickpea MTs and ABA-mediated stress response. Unlike CanMT-2, CanMT-1 is induced in chickpea epicotyls by cadmium indicating a different function for both clones. We conclude that these MT-like proteins, in particular CanMT-1, are regulated by the developmental stage and may participate in cell maturation processThis work was supported by grants from the Dirección General de Investigación Científicaca y Técnica (DGICYT), Spain (PB94-1395) and the Junta de Castilla y León (SA88/96)

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

Inactivation of the lys7 Gene, Encoding Saccharopine Reductase in Penicillium chrysogenum, Leads to Accumulation of the Secondary Metabolite Precursors Piperideine-6-Carboxylic Acid and Pipecolic Acid from α-Aminoadipic Acid

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active α-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1(−)) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3′-end region. P. chrysogenum SR1(−) lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1(−) was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1(−) was grown with l-lysine as the sole nitrogen source and supplemented with dl-α-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1(−) with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from α-aminoadipic acid and not from l-lysine catabolism

Penicillium chrysogenum as a fungal factory for feruloyl esterases

International audiencePlant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54-1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50-55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer. KEY POINTS: • Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity. • Overexpression of the ORFs produced a novel P. rubens strain with improved activity. • The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown