ΔMAT1-1-1 (Pc20g07800) vs. P2niaD18 (wild type)
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ABSTRACT:
ORGANISM(S): Penicillium chrysogenum
SUBMITTER: Kordula Becker
PROVIDER: E-GEOD-69016 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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:The multi-component global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions, two key components of the Velvet complex, on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigates the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited, aerobic conditions, relevant for industrial production of ?-lactams. The gene-deletion cassette for PcvelA or PclaeA was integrated in a hdfA mutant of the penicillin high-producing strain P. chrysogenum DS17690. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters which, in preliminary studies, led to a massive reduction of penicillin production in a PcvelA deletion mutant. The correct PcvelA and PclaeA deletion strains revealed a significant (up to 30 %) reduction of penicillin-G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of P. chrysogenum strains. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent response in the PcvelA? and PclaeA? mutants when grown in the absence of the penicillin-G side-chain precursor phenylacetic acid. 11 of these genes belonged to two small gene clusters (with 5 and 6 genes, respectively), one of which contains a gene with high homology to an aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi may be strongly context dependent Previous studies on the impact of the Velvet complex in P. chrysogenum were performed in prolonged batch cultures. Time course analysis revealed that the impact of PcvelA and PclaeA mutations was most pronounced after prolonged incubation {Hoff, 2010 6 /id}, but the physiological status of these cultures was not precisely defined. Industrial production of ?-lactam antibiotics is performed in sugar-limited, aerobic fed-batch cultures {Menezes, 1994 76 /id}. The aim of the present study is to investigate the impact of the Velvet complex on physiology, penicilllin production and transcriptional regulation under industrially relevant conditions. To this end, we studied the impact of PcvelA and PclaeA deletions in aerobic, glucose-limited chemostat cultures of the penicillin high-producing strain P. chrysogenum DS17690.
2011-12-06 | E-GEOD-31633 | biostudies-arrayexpress
Project description:In microbial production of non-catabolic products, a loss of production capacity upon long-term cultivation (for example, chemostat), a phenomenon called strain degeneration, is nearly always observed. In this study, a systems biology approach (monitoring changes from gene to produced flux) was used to study degeneration of penicillin production by Penicillium chrysogenum in ethanol-limited chemostat fermentations where the biomass specific penicillin production rate decreased 10-fold within 30 generations. Results showed that the copy number of penicillin gene clusters and expression levels of central metabolism showed little decrease. With respect to penicillin production, major changes were observed: a strong downregulation of the cysteine pathway in agreement with its nearly 10-fold flux reduction. Also, levels of ACVS and IPNS, two penicillin pathway enzymes, and the penicillin transport capacity decreased many fold. This indicates that degeneration is caused by changed regulation of post-translational modifications or an increased protein degradation rate of these proteins. Continued subcultivation of a degenerated culture resulted in partial recovery of the biomass specific penicillin production rate, however, it was still 5-fold lower than the peak biomass specific penicillin production rate. Prolonged chemostat cultivations up to 500 hours (30 generations) with ethanol as the sole limiting carbon source were performed because degeneration is found to be more pronounced with ethanol as a limiting sole carbon source compared to glucose. Measurements included genome level (number of penicillin gene clusters), genome-wide transcriptome, protein levels of penicillin pathway enzymes, number of peroxisomes (microbodies where part of the penicillin pathway occurs), metabolome (of central metabolism, nucleotides, penicillin pathway intermediates including intracellular PAA and PenG) and fluxome.
2011-06-10 | E-GEOD-24212 | biostudies-arrayexpress
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 potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed. On a condition that induces mating-type genes descrived in growth protocol section, each cogenic MAT1-1 and MAT1-2 mating-type strains were processed into RNA extraction and hybridization on Affymetrix microarrays. In a speculation that genes involved in putative mating process were reguated in mating-type dependent manner, we designed the cogenic strains that differs only in mating-type gene locus to analyze differential gene expression of MAT1-1 vs MAT1-2 strains.
2012-04-29 | E-GEOD-33542 | 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: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:Sexual identity of the human pathogenic mold Aspergillus fumigatus is determined by the mating-type idiomorphs MAT1-1 and MAT1-2 residing at the MAT locus. Given that the MAT1 gene products are DNA binding master regulators that govern fruiting body formation, the MAT1-driven transcriptomes of A. fumigatus were monitored by conditional overexpression of either MAT1 gene followed by RNA-seq analyses.
2018-10-09 | GSE113404 | GEO
Project description:The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the RIB40 reference strain. We now report the existence of a complimentary MAT1-2 gene and sequencing of an idiomorph region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of MAT1-1 and MAT1-2 genotype among 180 strains assayed including industrial tane-koji isolates. Strains used for sake and miso production showed a near 1:1 ratio of MAT1-1 and MAT1-2 mating-types, whereas strains used for soy sauce production showed a significant bias towards the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and comparisons were made of gene expression by DNA microarray and RT-PCR methodologies under conditions when MAT genes were expressed. 33 genes were found to be up-regulated greater than 10-fold in either the MAT1-1 host or MAT1-2 gene replacement strains relative to each other, showing that both MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating-type dependent manner, the first such report from a supposedly asexual fungus. MAT1-1 expression specifically up-regulated an a-pheromone precursor gene, but most genes affected were of unknown function. Results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.
2012-04-30 | GSE33542 | GEO
Project description:Comparison of transcriptional profiles of Mat1-/- Mouse embryonic fibroblasts to Mat1-/flox Mouse embryonic fibroblasts at 72h and 96h after Mat1 deletion.
2010-07-01 | E-MEXP-2348 | biostudies-arrayexpress