RESUMO
The knowledge about enzymes' compartmentalization and transport processes involved in the penicillin biosynthesis in Penicillium chrysogenum is very limited. The genome of this fungus contains multiple genes encoding transporter proteins, but very little is known about them. A bioinformatic search was made to find major facilitator supefamily (MFS) membrane proteins related to CefP transporter protein involved in the entry of isopenicillin N to the peroxisome in Acremonium chrysogenum. No strict homologue of CefP was observed in P. chrysogenum, but the penV gene was found to encode a membrane protein that contained 10 clear transmembrane spanners and two other motifs COG5594 and DUF221, typical of membrane proteins. RNAi-mediated silencing of penV gene provoked a drastic reduction of the production of the δ-(L-α-aminoadipyl-L-cysteinyl-D-valine) (ACV) and isopenicillin N intermediates and the final product of the pathway. RT-PCR and northern blot analyses confirmed a reduction in the expression levels of the pcbC and penDE biosynthetic genes, whereas that of the pcbAB gene increased. Localization studies by fluorescent laser scanning microscopy using Dsred and GFP fluorescent fusion proteins and the FM 4-64 fluorescent dye showed clearly that the protein was located in the vacuolar membrane. These results indicate that PenV participates in the first stage of the beta-lactam biosynthesis (i.e., the formation of the ACV tripeptide), probably taking part in the supply of amino acids from the vacuolar lumen to the vacuole-anchored ACV synthetase. This is in agreement with several reports on the localization of the ACV synthetase and provides increased evidence for a compartmentalized storage of precursor amino acids for non-ribosomal peptides. PenV is the first MFS transporter of P. chrysogenum linked to the beta-lactam biosynthesis that has been located in the vacuolar membrane.
Assuntos
Oligopeptídeos/biossíntese , Penicillium chrysogenum/metabolismo , beta-Lactamas/metabolismo , Northern Blotting , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT-DsRed and EGFP-SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.
Assuntos
Membranas Intracelulares/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Penicillium chrysogenum/metabolismo , Peroxissomos/metabolismo , Fenilacetatos/metabolismo , Transporte Biológico , Biologia Computacional , Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Membrana Transportadoras/genética , Penicilinas/biossíntese , Penicillium chrysogenum/genéticaRESUMO
The mechanisms of compartmentalization of intermediates and secretion of penicillins and cephalosporins in ß-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.
Assuntos
Acremonium/metabolismo , Cefalosporinas/biossíntese , Proteínas de Membrana/metabolismo , Penicilinas/metabolismo , Penicillium chrysogenum/genética , Peroxissomos/metabolismo , Sequência de Bases , Sistema Livre de Células , Primers do DNA , DNA Fúngico/isolamento & purificação , DNA Fúngico/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Penicillium chrysogenum/metabolismo , RNA Fúngico/isolamento & purificação , RNA Fúngico/metabolismo , Mapeamento por Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Acremonium/genética , Acremonium/metabolismo , Cefalosporinas/biossíntese , Proteínas Fúngicas/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Reporter/genética , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fases de Leitura Aberta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
NRPSs (non-ribosomal peptide synthetases) and PKSs (polyketide synthases) require post-translational phosphopantetheinylation to become active. This reaction is catalysed by a PPTase (4'-phosphopantetheinyl transferase). The ppt gene of Penicillium chrysogenum, encoding a protein that shares 50% similarity with the stand-alone large PPTases, has been cloned. This gene is present as a single copy in the genome of the wild-type and high-penicillin-producing strains (containing multiple copies of the penicillin gene cluster). Amplification of the ppt gene produced increases in isopenicillin N and benzylpenicillin biosynthesis. A PPTase-defective mutant (Wis54-PPT(-)) was obtained. It required lysine and lacked pigment and penicillin production, but it still synthesized normal levels of roquefortine. The biosynthesis of roquefortine does not appear to involve PPTase-mediated modification of the synthesizing enzymes. The PPT(-) mutant did not require fatty acids, which indicates that activation of the fatty acid synthase is performed by a different PPTase. Complementation of Wis54-PPT(-) with the ppt gene restored lysine biosynthesis, pigmentation and penicillin production, which demonstrates the wide range of processes controlled by this gene.
Assuntos
Proteínas de Bactérias/metabolismo , Lisina/biossíntese , Penicilinas/biossíntese , Penicillium chrysogenum/enzimologia , Processamento de Proteína Pós-Traducional , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Proteínas de Bactérias/genética , Northern Blotting , Southern Blotting , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Ácidos Graxos/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico/genética , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Indóis/metabolismo , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Piperazinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the beta-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.
Assuntos
Acremonium/genética , Cefalosporinas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Penicilinas/metabolismo , Penicillium chrysogenum/genética , Interferência de RNA , Acremonium/classificação , Acremonium/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Técnicas Genéticas , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Penicillium chrysogenum/metabolismo , Plasmídeos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Transformação Genética , beta-Lactamas/metabolismoRESUMO
The biosynthesis of the beta-lactam antibiotic penicillin is an excellent model for the study of secondary metabolites produced by filamentous fungi due to the good background knowledge on the biochemistry and molecular genetics of the beta-lactam producing microorganisms. The three genes (pcbAB, pcbC, penDE) encoding enzymes of the penicillin pathway in Penicillium chrysogenum are clustered, but no penicillin pathway-specific regulators have been found in the genome region that contains the penicillin gene cluster. The biosynthesis of this beta-lactam is controlled by global regulators of secondary metabolism rather than by a pathway-specific regulator. In this work we have identified the gene encoding the secondary metabolism global regulator LaeA in P. chrysogenum (PcLaeA), a nuclear protein with a methyltransferase domain. The PclaeA gene is present as a single copy in the genome of low and high-penicillin producing strains and is not located in the 56.8-kb amplified region occurring in high-penicillin producing strains. Overexpression of the PclaeA gene gave rise to a 25% increase in penicillin production. PclaeA knock-down mutants exhibited drastically reduced levels of penicillin gene expression and antibiotic production and showed pigmentation and sporulation defects, but the levels of roquefortine C produced and the expression of the dmaW involved in roquefortine biosynthesis remained similar to those observed in the wild-type parental strain. The lack of effect on the synthesis of roquefortine is probably related to the chromatin arrangement in the low expression roquefortine promoters as compared to the bidirectional pbcAB-pcbC promoter region involved in penicillin biosynthesis. These results evidence that PcLaeA not only controls some secondary metabolism gene clusters, but also asexual differentiation in P. chrysogenum.
Assuntos
Genes Reguladores , Indóis/metabolismo , Penicilinas/biossíntese , Penicillium chrysogenum/genética , Pigmentação/genética , Esporos Fúngicos/fisiologia , Sequência de Aminoácidos , Análise por Conglomerados , Biologia Computacional/métodos , Dosagem de Genes , Regulação da Expressão Gênica , Genes Fúngicos , Compostos Heterocíclicos de 4 ou mais Anéis/análise , Compostos Heterocíclicos de 4 ou mais Anéis/metabolismo , Indóis/análise , Dados de Sequência Molecular , Família Multigênica , Mutação , Proteínas Nucleares/química , Penicillium chrysogenum/metabolismo , Piperazinas/análise , Piperazinas/metabolismo , Plasmídeos , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The Acremonium chryrsogenum cefT gene encoding a membrane protein of the major facilitator superfamily implicated in the cephalosporin biosynthesis in A. chrysogenum was introduced into Penicillium chrysogenum Wisconsin 54-1255 (a benzylpenicillin producer), P. chrysogenum npe6 pyrG(-) (a derivative of Wisconsin 54-1255 lacking a functional penDE gene) and P. chrysogenum TA98 (a deacetylcephalosporin producer containing the cefD1, cefD2, cefEF and cefG genes from A. chrysogenum). RT-PCR analysis revealed that the cefT gene was expressed in P. chrysogenum strains. HPLC analysis of the culture broths of the TA98 transformants showed an increase in the secretion of deacetylcephalosporin C and hydrophilic penicillins (isopenicillin N and penicillin N). P. chrysogenum Wisconsin 54-1255 strain transformed with cefT showed increased secretion of the isopenicillin N intermediate and a drastic decrease in the benzylpenicillin production. Southern and northern blot analysis indicated that the untransformed P. chrysogenum strains contain an endogenous gene similar to cefT that may be involved in the well-known secretion of the isopenicillin N intermediate. In summary, the cefT transporter is a hydrophilic beta-lactam transporter that is involved in the secretion of hydrophilic beta-lactams containing alpha-aminoadipic acid side chain (isopenicillin N, penicillin N and deacetylcephalosporin C).
Assuntos
Acremonium/genética , Proteínas de Transporte/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Penicillium chrysogenum/genética , beta-Lactamas/metabolismo , Sequência de Bases , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Primers do DNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Penicillium chrysogenum oat1 gene, which encodes a class III omega-aminotransferase, was cloned and characterized. This enzyme converts lysine into 2-aminoadipic semialdehyde, and plays an important role in the biosynthesis of 2-aminoadipic acid, a precursor of penicillin and other beta-lactam antibiotics. The enzyme is related to ornithine-5-aminotransferases and to the lysine-6-aminotransferases encoded by the lat genes found in bacterial cephamycin gene clusters. Expression of oat1 is induced by lysine, ornithine and arginine, and repressed by ammonium ions. AreA-binding GATA and GATT sequences involved in regulation by ammonium, and an 8-bp direct repeat associated with arginine induction in Emericella (Aspergillus nidulans and Saccharomyces cerevisiae, were found in the oat1 promoter region. Deletion of the oat1 gene resulted in the loss of omega-aminotransferase activity. The null mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed reduced growth on lysine. Complementation of the null mutant with the oat1 gene restored normal levels of omega-aminotransferase activity and the ability to grow on ornithine, arginine and lysine. The role of the oat1 gene in the biosynthesis of 2-aminoadipic acid is discussed.