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Juan F. Martín - One of the best experts on this subject based on the ideXlab platform.
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Insight into the Genome of Diverse Penicillium Chrysogenum Strains: Specific Genes, Cluster Duplications and DNA Fragment Translocations.
International journal of molecular sciences, 2020Co-Authors: Juan F. MartínAbstract:Background: There are eighteen species within the Penicillium genus section chrysogena, including the original penicillin producers Penicillium notatum (Fleming strain) and Penicillium Chrysogenum NRRL 1951. Other wild type isolates of the Penicillium genus are relevant for the production of useful proteins and primary or secondary metabolites. The aim of this article is to characterize strain specific genes and those genes which are involved in secondary metabolite biosynthesis, particularly the mutations that have been introduced during the β-lactams strain improvement programs. Results: The available genomes of several classical and novel P. Chrysogenum strains have been compared. The first genome sequenced was that of the reference strain P. Chrysogenum Wis54-1255, which derives from the wild type P. Chrysogenum NRRL 1951; its genome size is 32.19 Mb and it encodes 12,943 proteins. Four chromosomes were resolved in P. Chrysogenum and P. notatum by pulse field gel electrophoresis. The genomes of three industrial strains have a similar size but contain gene duplications and truncations; the penicillin gene cluster copy number ranges from one in the wild type to twelve in the P. Chrysogenum ASP-E1 industrial strain and is organized in head to tail tandem repeats. The genomes of two new strains, P. Chrysogenum KF-25, a producer of antifungal proteins isolated from a soil sample, and P. Chrysogenum HKF2, a strain with carbohydrate-converting activities isolated from a sludge treatment plant, showed strain specific genes. Conclusions: The overall comparison of all available P. Chrysogenum genomes indicates that there are a significant number of strain-specific genes, mutations of structural and regulatory genes, gene cluster duplications and DNA fragment translocations. This information provides important leads to improve the biosynthesis of enzymes, antifungal agents, prebiotics or different types of secondary metabolites.
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Key role of LaeA and velvet complex proteins on expression of β-lactam and PR-toxin genes in Penicillium Chrysogenum: cross-talk regulation of secondary metabolite pathways
Journal of Industrial Microbiology & Biotechnology, 2017Co-Authors: Juan F. MartínAbstract:Penicillium Chrysogenum is an excellent model fungus to study the molecular mechanisms of control of expression of secondary metabolite genes. A key global regulator of the biosynthesis of secondary metabolites is the LaeA protein that interacts with other components of the velvet complex (VelA, VelB, VelC, VosA). These components interact with LaeA and regulate expression of penicillin and PR-toxin biosynthetic genes in P. Chrysogenum . Both LaeA and VelA are positive regulators of the penicillin and PR-toxin biosynthesis, whereas VelB acts as antagonist of the effect of LaeA and VelA. Silencing or deletion of the laeA gene has a strong negative effect on penicillin biosynthesis and overexpression of laeA increases penicillin production. Expression of the laeA gene is enhanced by the P. Chrysogenum autoinducers 1,3 diaminopropane and spermidine. The PR-toxin gene cluster is very poorly expressed in P. Chrysogenum under penicillin-production conditions (i.e. it is a near-silent gene cluster). Interestingly, the downregulation of expression of the PR-toxin gene cluster in the high producing strain P. Chrysogenum DS17690 was associated with mutations in both the laeA and velA genes. Analysis of the laeA and velA encoding genes in this high penicillin producing strain revealed that both laeA and velA acquired important mutations during the strain improvement programs thus altering the ratio of different secondary metabolites (e.g. pigments, PR-toxin) synthesized in the high penicillin producing mutants when compared to the parental wild type strain. Cross-talk of different secondary metabolite pathways has also been found in various Penicillium spp.: P. Chrysogenum mutants lacking the penicillin gene cluster produce increasing amounts of PR-toxin, and mutants of P. roqueforti silenced in the PR-toxin genes produce large amounts of mycophenolic acid. The LaeA-velvet complex mediated regulation and the pathway cross-talk phenomenon has great relevance for improving the production of novel secondary metabolites, particularly of those secondary metabolites which are produced in trace amounts encoded by silent or near-silent gene clusters.
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rna silencing in penicillium Chrysogenum and acremonium Chrysogenum validation studies using β lactam genes expression
Journal of Microbiological Methods, 2008Co-Authors: Ricardo V. Ullán, Fernando Teijeira, Ramiro P Godio, Inmaculada Vaca, Carlos Garciaestrada, Raul Feltrer, Katarina Kosalkova, Juan F. MartínAbstract: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.
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Expression of the Acremonium Chrysogenum cefT gene in Penicillum Chrysogenum indicates that it encodes an hydrophilic β-lactam transporter
Current genetics, 2008Co-Authors: Ricardo V. Ullán, Fernando Teijeira, Juan F. MartínAbstract: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).
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deacetylcephalosporin c production in penicillium Chrysogenum by expression of the isopenicillin n epimerization ring expansion and acetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Sonia Campoy, Javier Casqueiro, Francisco J Fernandez, Juan F. MartínAbstract:Summary Penicillium Chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different β-lactam antibiotics. We have constructed P. Chrysogenum strains expressing cefD1 , cefD2 , cefEF , and cefG genes cloned from Acremonium Chrysogenum . Northern analysis revealed that the four genes were expressed in P. Chrysogenum . The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth.
Ulrich Kück - One of the best experts on this subject based on the ideXlab platform.
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Transcriptome analysis of the two unrelated fungal \(\beta\)-lactam producers \(\textit {Acremonium Chrysogenum}\) and \(\textit {Penicillium Chrysogenum}\)
2017Co-Authors: Dominik Terfehr, Tim A. Dahlmann, Ulrich KückAbstract:\(\textbf {Background:}\) Cephalosporins and penicillins are the most frequently used \(\beta\)-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are \(\textit {Acremonium Chrysogenum}\) and \(\textit {Penicillium Chrysogenum}\), two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains. \(\textbf {Results:}\) Using the sequence of PcbC, a key enzyme of \(\beta\)-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In \(\textit {P. Chrysogenum}\), 748 genes showed differential expression, while 1572 genes from \(\textit {A. Chrysogenum}\) are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for \(\beta\)-lactam production. Other genes not essential for \(\beta\)-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator \(\it velvet\) from both production strains. We identified 567 \(\textit {P. Chrysogenum}\) and 412 \(\textit {A. Chrysogenum}\) Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including \(\beta\)-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains. \(\textbf {Conclusions:}\) The major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal \(\beta\)-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved \(\beta\)-lactam antibiotic titers during strain improvement in both fungi.
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two components of a velvet like complex control hyphal morphogenesis conidiophore development and penicillin biosynthesis in penicillium Chrysogenum
Eukaryotic Cell, 2010Co-Authors: Birgit Hoff, Hubert Kürnsteiner, Claudia Sigl, Ivo Zadra, Jens Kamerewerd, Rudolf Mitterbauer, Ulrich KückAbstract: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, which we have named P. Chrysogenum velA (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 high-performance liquid chromatography quantifications of penicillin titers clearly show that both PcVelA and PcLaeA play a major role in penicillin biosynthesis in a producer strain that underwent several rounds of UV mutagenesis during a strain improvement program. Both regulators are further involved in different 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 under both light and dark conditions. Bimolecular fluorescence complementation assays provide evidence for a velvet-like complex in P. Chrysogenum, with structurally conserved components that have distinct developmental roles, illustrating the functional plasticity of these regulators in genera other than Aspergillus.
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Development of an homologous transformation system for Acremonium Chrysogenum based on the beta-tubulin gene.
Current genetics, 1994Co-Authors: Claudia Nowak, Ulrich KückAbstract:Theβ−tubulin gene was isolated from the filamentous fungusAcremonium Chrysogenum using a heterologous gene probe to screen anA. Chrysogenum lambda library. Sequencing of theA. Chrysogenum gene revealed a mosaic gene which contains five exons and four intervening sequences. The exons encode for a polypeptide of 447 amino-acid residues which showed a high degree of similarity when compared with amino-acid sequences from β-tubulins of other eukaryotes. The introns are characterized by typical consensus sequences found in intervening sequences from other filamentous fungi. In-vitro mutagenesis of codon 167 of the β-tubulin gene resulted in the substitution of a phenylalanine by a tyrosine in the corresponding polypeptide sequence. The mutated gene was used successfully in the transformation and co-transformation ofA. Chrysogenum to benomyl resistance. The molecular analysis of transformants provided evidence that they contain the mutated β-tubulin gene in addition to the wild-type gene, as was proved by Southern-hybridization analysis and direct sequencing of PCR amplification products.
Roel A. L. Bovenberg - One of the best experts on this subject based on the ideXlab platform.
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Identification of a Polyketide Synthase Involved in Sorbicillin Biosynthesis by Penicillium Chrysogenum
Applied and environmental microbiology, 2016Co-Authors: Oleksandr Salo, Roel A. L. Bovenberg, Fernando Guzman-chavez, Marco Ries, Peter P. Lankhorst, Rob J. Vreeken, Arnold J. M. DriessenAbstract:ABSTRACT Secondary metabolism in Penicillium Chrysogenum was intensively subjected to classical strain improvement (CSI), the resulting industrial strains producing high levels of β-lactams. During this process, the production of yellow pigments, including sorbicillinoids, was eliminated as part of a strategy to enable the rapid purification of β-lactams. Here we report the identification of the polyketide synthase (PKS) gene essential for sorbicillinoid biosynthesis in P. Chrysogenum. We demonstrate that the production of polyketide precursors like sorbicillinol and dihydrosorbicillinol as well as their derivatives bisorbicillinoids requires the function of a highly reducing PKS encoded by the gene Pc21g05080 ( pks13 ). This gene belongs to the cluster that was mutated and transcriptionally silenced during the strain improvement program. Using an improved β-lactam-producing strain, repair of the mutation in pks13 led to the restoration of sorbicillinoid production. This now enables genetic studies on the mechanism of sorbicillinoid biosynthesis in P. Chrysogenum and opens new perspectives for pathway engineering. IMPORTANCE Sorbicillinoids are secondary metabolites with antiviral, anti-inflammatory, and antimicrobial activities produced by filamentous fungi. This study identified the gene cluster responsible for sorbicillinoid formation in Penicillium Chrysogenum, which now allows engineering of this diverse group of compounds.
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autophagy deficiency promotes β lactam production in penicillium Chrysogenum
Applied and Environmental Microbiology, 2011Co-Authors: Magdalena Bartoszewska, Jan A. K. W. Kiel, Roel A. L. Bovenberg, Marten Veenhuis, Ida J. Van Der KleiAbstract:We have investigated the significance of autophagy in the production of the β-lactam antibiotic penicillin (PEN) by the filamentous fungus Penicillium Chrysogenum. In this fungus PEN production is compartmentalized in the cytosol and in peroxisomes. We demonstrate that under PEN-producing conditions significant amounts of cytosolic and peroxisomal proteins are degraded via autophagy. Morphological analysis, based on electron and fluorescence microscopy, revealed that this phenomenon might contribute to progressive deterioration of late subapical cells. We show that deletion of the P. Chrysogenum ortholog of Saccharomyces cerevisiae serine-threonine kinase atg1 results in impairment of autophagy. In P. Chrysogenum atg1 cells, a distinct delay in cell degeneration is observed relative to wild-type cells. This phenomenon is associated with an increase in the enzyme levels of the PEN biosynthetic pathway and enhanced production levels of this antibacterial compound.
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Matching the proteome to the genome: the microbody of penicillin-producing Penicillium Chrysogenum cells
Functional & Integrative Genomics, 2009Co-Authors: Jan A. K. W. Kiel, Marco A. Berg, Fabrizia Fusetti, Bert Poolman, Roel A. L. Bovenberg, Marten Veenhuis, Ida J. KleiAbstract:In the filamentous fungus Penicillium Chrysogenum , microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. Chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. Chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. Chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. Chrysogenum microbodies.
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engineering of penicillium Chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor
Metabolic Engineering, 2009Co-Authors: Diana M Harris, Roel A. L. Bovenberg, Marco Van Den Berg, Ilja Westerlaken, Dick Schipper, Zita A Van Der Krogt, Andreas Karoly Gombert, John D Sutherland, Leonie M Raamsdonk, Jack T. PronkAbstract:Abstract 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, 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 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. Transcriptome analysis revealed that adipate 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.
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expression of the transporter encoded by the ceft gene of acremonium Chrysogenum increases cephalosporin production in penicillium Chrysogenum
Fungal Genetics and Biology, 2008Co-Authors: Jeroen G Nijland, Roel A. L. Bovenberg, Andriy Kovalchuk, Marco Van Den Berg, Arnold J. M. DriessenAbstract:By introduction of the cefEF genes of Acremonium Chrysogenum and the cmcH gene of Streptomyces clavuligerus, Penicillium Chrysogenum can be reprogrammed to form adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (ad7-ACCCA), a carbamoylated derivate of adipoyl-7-aminodeacetoxy-cephalosporanic acid. The cefT gene of A. Chrysogenum encodes a cephalosporin C transporter that belongs to the Major Facilitator Superfamily. Introduction of cefT into an ad7-ACCCA-producing P. Chrysogenum strain results in an almost 2-fold increase in cephalosporin production with a concomitant decrease in penicillin by-product formation. These data suggest that cephalosporin production by recombinant P. Chrysogenum strains is limited by the ability of the fungus to secrete these compounds.
Ricardo V. Ullán - One of the best experts on this subject based on the ideXlab platform.
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RNAi-Mediated Gene Silencing in the Beta-Lactam Producer Fungi Penicillium Chrysogenum and Acremonium Chrysogenum
Fungal Biology, 2014Co-Authors: Carlos García-estrada, Ricardo V. UllánAbstract:RNA silencing represents a powerful approach to test gene function, especially when essential genes with a lethal deletion phenotype are investigated. This phenomenon can be triggered by different systems. One of them gives rise to the formation of double-stranded RNA molecules by means of plasmid pJL43-RNAi (J Microbiol Methods 75:209–218, 2008), which has proven its efficiency in the beta-lactam producing fungi, Penicillium Chrysogenum and Acremonium Chrysogenum.
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Characterization of an autoinducer of penicillin biosynthesis in Penicillium Chrysogenum.
Applied and environmental microbiology, 2011Co-Authors: Jorge Martín, Ricardo V. Ullán, Carlos García-estrada, Angel Rumbero, Eliseo Recio, Silvia M. Albillos, Juan-francisco MartínAbstract:Filamentous fungi produce an impressive variety of secondary metabolites; many of them have important biological activities. The biosynthesis of these secondary metabolites is frequently induced by plant-derived external elicitors and appears to also be regulated by internal inducers, which may work in a way similar to that of bacterial autoinducers. The biosynthesis of penicillin in Penicillium Chrysogenum is an excellent model for studying the molecular mechanisms of control of gene expression due to a good knowledge of the biochemistry and molecular genetics of β-lactam antibiotics and to the availability of its genome sequence and proteome. In this work, we first developed a plate bioassay that allows direct testing of inducers of penicillin biosynthesis using single colonies of P. Chrysogenum. Using this bioassay, we have found an inducer substance in the conditioned culture broths of P. Chrysogenum and Acremonium Chrysogenum. No inducing effect was exerted by γ-butyrolactones, jasmonic acid, or the penicillin precursor δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine. The conditioned broth induced penicillin biosynthesis and transcription of the pcbAB, pcbC, and penDE genes when added at inoculation time, but its effect was smaller if added at 12 h and it had no effect when added at 24 h, as shown by Northern analysis and lacZ reporter studies. The inducer molecule was purified and identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as 1,3-diaminopropane. Addition of pure 1,3-diaminopropane stimulated the production of penicillin by about 100% compared to results for the control cultures. Genes for the biosynthesis of 1,3-diaminopropane have been identified in the P. Chrysogenum genome.
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rna silencing in penicillium Chrysogenum and acremonium Chrysogenum validation studies using β lactam genes expression
Journal of Microbiological Methods, 2008Co-Authors: Ricardo V. Ullán, Fernando Teijeira, Ramiro P Godio, Inmaculada Vaca, Carlos Garciaestrada, Raul Feltrer, Katarina Kosalkova, Juan F. MartínAbstract: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.
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Expression of the Acremonium Chrysogenum cefT gene in Penicillum Chrysogenum indicates that it encodes an hydrophilic β-lactam transporter
Current genetics, 2008Co-Authors: Ricardo V. Ullán, Fernando Teijeira, Juan F. MartínAbstract: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).
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deacetylcephalosporin c production in penicillium Chrysogenum by expression of the isopenicillin n epimerization ring expansion and acetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Sonia Campoy, Javier Casqueiro, Francisco J Fernandez, Juan F. MartínAbstract:Summary Penicillium Chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different β-lactam antibiotics. We have constructed P. Chrysogenum strains expressing cefD1 , cefD2 , cefEF , and cefG genes cloned from Acremonium Chrysogenum . Northern analysis revealed that the four genes were expressed in P. Chrysogenum . The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth.
Gang Liu - One of the best experts on this subject based on the ideXlab platform.
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improvement of the crispr cas9 mediated gene disruption and large dna fragment deletion based on a chimeric promoter in acremonium Chrysogenum
Fungal Genetics and Biology, 2020Co-Authors: Chang Chen, Yuanyuan Pan, Jiajia Liu, Chengbao Duan, Gang LiuAbstract:Abstract Acremonium Chrysogenum has been employed in the industrial production of cephalosporin C (CPC). However, there are still some impediments to understanding the regulation of CPC biosynthesis and improving strains due to the difficulty of genetic manipulation in A. Chrysogenum, especially in the CPC high-producing strain C10. Here, an improved CRISPR-Cas9 system was constructed based on an U6/tRNA chimeric promoter. Using this system, high efficiency for single gene disruption was achieved in C10. In addition, double loci were simultaneously targeted when supplying with the homology-directed repair templates (donor DNAs). Based on this system, large DNA fragments up to 31.5 kb for the yellow compound sorbicillinoid biosynthesis were successfully deleted with high efficiency. Furthermore, CPC production was significantly enhanced when the sorbicillinoid biosynthetic genes were knocked out. This study provides a powerful tool for gene editing and strain improvement in A. Chrysogenum.
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a myb transcription factor represses conidiation and cephalosporin c production in acremonium Chrysogenum
Fungal Genetics and Biology, 2018Co-Authors: Ying Wang, Yuanyuan Pan, Yanling Wang, Liangkun Long, Xiaoling Zhang, Gang LiuAbstract:Acremonium Chrysogenum is the industrial producer of cephalosporin C (CPC). We isolated a mutant (AC554) from a T-DNA inserted mutant library of A. Chrysogenum. AC554 exhibited a reduced conidiation and lack of CPC production. In consistent with it, the transcription of cephalosporin biosynthetic genes pcbC and cefEF was significantly decreased in AC554. Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed and sequence analysis indicated that a T-DNA was inserted upstream of an open reading frame (ORF) which was designated AcmybA. On the basis of sequence analysis, AcmybA encodes a Myb domain containing transcriptional factor. Observation of red fluorescent protein (RFP) tagged AcMybA showed that AcMybA is naturally located in the nucleus of A. Chrysogenum. Transcriptional analysis demonstrated that the AcmybA transcription was increased in AC554. In contrast, the AcmybA deleted mutant (ΔAcmybA) overproduced conidia and CPC. To screen the targets of AcmybA, we sequenced and compared the transcriptome of ΔAcmybA, AC554 and the wild-type strain at different developmental stages. Twelve differentially expressed regulatory genes were identified. Taken together, our results indicate that AcMybA negatively regulates conidiation and CPC production in A. Chrysogenum.
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acstua which encodes an apses transcription regulator is involved in conidiation cephalosporin biosynthesis and cell wall integrity of acremonium Chrysogenum
Fungal Genetics and Biology, 2015Co-Authors: Ying Wang, Jun Zhou, Yuanyuan Pan, Gang LiuAbstract:A transcriptional regulatory gene AcstuA was identified from Acremonium Chrysogenum. AcstuA encodes a basic helix-loop-helix protein with similarity to StuA which regulates the core developmental processes of Aspergillus nidulans. Like disruption of stuA in A. nidulans, deficiency of AcstuA blocked the conidiation of A. Chrysogenum through severely down-regulating the expression of AcbrlA and AcabaA which encode orthologs of the key fungal developmental regulators BrlA and AbaA. Disruption of AcstuA also drastically reduced cephalosporin production of A. Chrysogenum. In agreement, the transcriptions of pcbAB, pbcC, cefD1, cefD2, cefEF and cefG were remarkably decreased in the AcstuA disruption mutant (ΔAcstuA). In addition to defects in conidiation and cephalosporin biosynthesis, ΔAcstuA produced abnormal swollen and fragmented hyphal cells during fermentation. The phenotypic alterations of hyphal cells caused by AcstuA deletion were restored by supplementation of NaCl in the medium, indicating that the deficiency of AcstuA has an influence on the cell wall integrity of A. Chrysogenum. The transcriptions of two putative mannoprotein encoding genes Acmp2 and Acmp3 significantly reduced in ΔAcstuA, further indicating that cell wall integrity of the mutant is impaired. These results strongly suggested that AcstuA is involved in conidiation, cephalosporin production, hyphal fragmentation and cell wall integrity in A. Chrysogenum.