Penicillium chrysogenum
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PROVIDER: PRJNA112807 | ENA |
REPOSITORIES: ENA
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Project description:Penicillium chrysogenum was successfully engineered to produce a novel carbamoylated cephalosporin that can be used as a synthon for semi-synthetic cephalosporins. To this end, structural genes for Acremonium chrysogenum expandase/hydroxylase and Streptomyces clavuligerus carbamoyltransferase were expressed in a penicillinG high-producing strain of P. chrysogenum. Growth of the engineered strain in the presence of the side-chain precursor adipic acid resulted in production of adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA) and of several adipoylated pathway intermediates. A combinatorial chemostat-based transcriptome study, in which the ad7-ACCCA- producing strain and a strain lacking key genes in β-lactam synthesis were grown in the presence and absence of adipic acid, enabled the dissection of transcriptional responses to adipic acid per se and to ad7-ACCCA production. In chemostat cultures of both strains, adipic acid served as an additional carbon source. Transcriptome analysis supported an earlier proposal, based on 13C-labelling studies, that adipic acid catabolism in P. chrysogenum occurs via β-oxidation and enabled the identification of putative genes for enzymes involved in mitochondrial and peroxisomal β-oxidation pathways. Several of the genes that showed a specifically altered transcript level in ad7-ACCCA-producing cultures were previously implicated in oxidative stress responses. As strain improvement programmes lead to increased specific productivity and yields, a deeper understanding of these stress responses is likely to be important to also achieve high ad7-ACCCA titers with engineered strains of P. chrysogenum.
2009-01-05 | GSE12612 | GEO
Project description:Industrial production of penicillin G by Penicillium chrysogenum requires medium supplementation with the side chain precursor phenylacetate. However, P.chrysogenum grown in presence of phenylalanine as sole nitrogen source formed detectable extracellular amounts of phenylacetate and penicillin G. To get more insights in the metabolism implicated, chemostat-cultivation in presence of 13C9-phenylalanine were carried out. Quantification and modeling of the labeled metabolite pools indicated that phenylalanine was i) incorporated in nascent protein, ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation and iii) oxidized into tyrosine and subsequently assimilated in the homogentisate pathway. Comparative transcriptome analysis of phenylalanine and (NH4)2SO4 grown P.chrysogenum cultures enabled to identify two putative 2-oxo acid decarboxylases Pc13g9300 and Pc18g01490. Both cDNAs were cloned and expressed in the decarboxylase-free Saccharomyces cerevisiae CEN.PK711-7C (pdc1delta, 5delta, 6delta, aro10delta, thi3delta) strain that has lost the ability to grow on glucose as sole carbon source or on phenylalanine as sole nitrogen source. Only Pc13g09300 was able to restore growth on glucose and on phenylalanine, demonstrating that this gene encodes a dual substrate pyruvate, phenylpyruvate decarboxylase. This newly identified thiamine-dependent 2-oxo acid decarboxylase provides clues to explain the formation of phenylacetate via an Ehrlich-like pathway in P.chrysogenum. Penicillium chrysogenum DS17690 was grown in aerobic glucose limited chemostat at 25oC, pH 6.5 and a dilution rate of 0.03h-1 with either amonia or phenylalanine
2011-11-05 | E-GEOD-32097 | biostudies-arrayexpress
Project description:The recent discovery of a velvet complex containing several regulators of secondary metabolism in the model fungus Aspergillus nidulans raises the question whether similar type complexes direct fungal development in genera other than Aspergillus. Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, that we have named PcvelA and PclaeA. Data from array analysis using a ?PcvelA deletion strain indicate a significant role of PcvelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and HPLC quantifications of penicillin titres clearly show that both PcvelA and PclaeA play a major role in penicillin biosynthesis. Both regulators are further involved in different and distinct developmental processes. While PcvelA deletion leads to light independent conidial formation, dichotomous branching of hyphae and pellet formation in shaking cultures, a ?PclaeA strain shows a severe impairment in conidiophore formation in both the light and dark. Bimolecular fluorescence complementation assays finally provide evidence for a velvet-like complex in Penicillium chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators within filamentous ascomycetes. Transcriptomes of PcvelA- and PclaeA- deletion mutants were compared with expression data from recipient strain deltaPcku70 and reference strain P2niaD18 as a control
2010-06-17 | E-GEOD-18585 | biostudies-arrayexpress
Project description:The recent discovery of a velvet complex containing several regulators of secondary metabolism in the model fungus Aspergillus nidulans raises the question whether similar type complexes direct fungal development in genera other than Aspergillus. Penicillium chrysogenum is the industrial producer of the antibiotic penicillin, whose biosynthetic regulation is barely understood. Here we provide a functional analysis of two major homologues of the velvet complex in P. chrysogenum, that we have named PcvelA and PclaeA. Data from array analysis using a ΔPcvelA deletion strain indicate a significant role of PcvelA on the expression of biosynthesis and developmental genes, including PclaeA. Northern hybridization and HPLC quantifications of penicillin titres clearly show that both PcvelA and PclaeA play a major role in penicillin biosynthesis. Both regulators are further involved in different and distinct developmental processes. While PcvelA deletion leads to light independent conidial formation, dichotomous branching of hyphae and pellet formation in shaking cultures, a ΔPclaeA strain shows a severe impairment in conidiophore formation in both the light and dark. Bimolecular fluorescence complementation assays finally provide evidence for a velvet-like complex in Penicillium chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators within filamentous ascomycetes.
2010-06-17 | GSE18585 | GEO
Project description:Penicillium chrysogenum 4088766 Genome sequencing
| PRJNA210202 | ENA
Project description:Background: Microbial gene expression is to a large extend determined by environmental growth conditions. Differential gene expression analysis between two conditions has been frequently used to reveal regulatory networks and to assign physiological function to unknown genes. In nature, microorganisms cohabit however these interactions have been rarely studied and reproduced in laboratory set-up. Thus to quantitatively explore the genome-wide responses of microbial interaction, we co-cultivated Penicillium chrysogenum and Bacillus subtilis in chemostat culture. Results: Time course expression analysis of P. chrysogenum to co-cultivation with B. subtilis was carried out to understand the natural responses of P. chrysogenum to prokaryotes. Steady state chemostats of P. chrysogenum in non-B-lactam producing conditions was pulsed with B. subtilis and co-cultivation was followed for 72 hours. The dynamic physiological and transcriptional responses of P. chrysogenum in mixed culture were monitored. B. subtilis outcompeted growth of P. chrysogenum resulting in an increased B. subtilis biomass by more than three fold of its original size and a reduction in P. chrysogenum biomass to half of its original size after 72 h of mixed culture. Genes of the penicillin pathway, synthesis of the side-chain and precursors were overall unresponsive to the presence of B. subtilis. Moreover Penicillium polyketide synthase and nonribosomal peptide synthetase genes either remained silent or down-regulated, whereas genes responsible for protein synthesis, metabolism, energy conservation, respiration and transport were upregulated in the presence of B. subtilis. Among highly responsive genes, two putative B-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production in P. chrysogenum. Mutanase activity was not observed in pure cultures of P. chrysogenum and B. subtilis or when P. chrysogenum was co-cultured with B. subtilis supernatant or heat inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum pulsed with filter sterilized supernatants from mixed cultures P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that at least Pc12g07500 encoded an B-1,3 endoglucanase. Conclusion: Time course transcriptional profiling of P. chrysogenum revealed several differentially expressed genes during mixed culture, potentially reflecting interactions between the eukaryotic and the prokaryotic systems. M-oM-^AM-!-1,3 endoglucanase produced by P. chrysogenum against B. subtilis signals may have application in improving the efficacy of antibiotics by degrading exopolysacchride biofilms of pathogenic bacteria. The objective of the present study is to investigate the response of P. chrysogenum to co-cultivation with B. subtilis. To trigger an interaction specific behaviour, steady state chemostat of P. chrysogenum Wisconsin 54-1255 was pulsed with B. subtilis. The dynamic, transcriptional and physiological responses of P. chrysogenum in mixed culture were monitored and analyzed. Several differentially expressed genes potentially reflected interactions between the eukaryotic and the prokaryotic systems. To test whether any bacterial signaling molecules are responsible for differential expression of selected fungal genes, P. chrysogenum cultures were inoculated with supernatant of B. subtilis culture, supernatants from mixed culture and with heat-inactivated B. subtilis. The specific transcriptional responses identified using microarray was verified by analysis of fermentation broth and functional characterization by expression of selected genes in S. cerevisiae.
2014-05-12 | E-GEOD-53286 | biostudies-arrayexpress
Project description:Deletion of biosynthetic gene clusters in Penicillium chrysogenum DS68530
| PRJNA587307 | ENA
Project description:In filamentous fungi, secondary metabolism is often linked with developmental processes such as conidiation. In this study we analyzed the link between secondary metabolism and conidiation in the main industrial producer of the β-lactam antibiotic penicillin, the ascomycete Penicillium chrysogenum. Therefore, we generated mutants defective in two central regulators of conidiation, the transcription factors BrlA and StuA, respectively. Inactivation of both BrlA and StuA blocked conidiation and altered hyphal morphology during growth on solid media, as shown by light and scanning electron microscopy, but did not affect biomass production during liquid submerged growth. Genome-wide transcriptional profiling identified a complex StuA- and BrlA-dependent regulatory network, including genes previously shown to be involved in development and secondary metabolism. Remarkably, inactivation of StuA, but not BrlA, drastically down-regulated expression of the penicillin biosynthetic gene cluster during solid and liquid submerged growth. In agreement, penicillin V production was wild type-like in BrlA-deficient strains but 99 % decreased in StuA-deficient strains during liquid submerged growth as shown by HPLC analysis. Thus, among identified regulators of penicillin V production StuA has the most severe influence. Over-expression of StuA increased the transcript levels of BrlA and AbaA (another developmental regulator), de-repressed conidiation during liquid submerged growth, but did not affect penicillin V productivity. Taken together, these data demonstrate an intimate but not exclusive link between regulation of development and secondary metabolism in P. chrysogenum. Transcriptomes of PcbrlA- and PcstuA- deletion mutants were compared with expression data from recipient strain deltaPcku70 as a control Mycelia from the transformants and the reference strain were harvested at successive stages of development for RNA extraction and hybridization on Affymetrix microarrays.
2011-01-04 | E-GEOD-23326 | biostudies-arrayexpress
Project description:Penicillium chrysogenum NCPC10086 Genome sequencing and assembly
| PRJNA190642 | ENA