Dataset Information

A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum.

ABSTRACT: The Aspergillus nidulans velvet (veA) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the veA homologue AcveA from Acremonium chrysogenum, the major producer of the beta-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate that the nucleus-localized AcVEA polypeptide controls the transcriptional expression of six cephalosporin C biosynthesis genes. The most drastic reduction in expression is seen for cefEF, encoding the deacetoxycephalosporine/deacetylcephalosporine synthetase. After 120 h of growth, the cefEF transcript level is below 15% in both disruption strains compared to the wild type. These transcriptional expression data are consistent with results from a comparative and time-dependent high-performance liquid chromatography analysis of cephalosporin C production. Compared to the recipient, both disruption strains have a cephalosporin C titer that is reduced by 80%. In addition to its role in cephalosporin C biosynthesis, AcveA is involved in the developmentally dependent hyphal fragmentation. In both disruption strains, hyphal fragmentation is already observed after 48 h of growth, whereas in the recipient strain, arthrospores are not even detected before 96 h of growth. Finally, the two mutant strains show hyperbranching of hyphal tips on osmotically nonstabilized media. Our findings will be significant for biotechnical processes that require a defined stage of cellular differentiation for optimal production of secondary metabolites.

SUBMITTER: Dreyer J

PROVIDER: S-EPMC1907097 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum.

Dreyer Jacqueline J Eichhorn Heiko H Friedlin Ernst E Kürnsteiner Hubert H Kück Ulrich U

Applied and environmental microbiology 20070330 10

The Aspergillus nidulans velvet (veA) gene encodes a global regulator of gene expression controlling sexual development as well as secondary metabolism. We have identified the veA homologue AcveA from Acremonium chrysogenum, the major producer of the beta-lactam antibiotic cephalosporin C. Two different disruption strains as well as the corresponding complements were generated as a prelude to detailed functional analysis. Northern hybridization and quantitative real-time PCR clearly indicate tha ...[more]

PMID: 17400783

Similar Datasets

Project description:BACKGROUND:Autophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum. RESULTS:In this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (?Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ?Acatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ?Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation. CONCLUSIONS:Our results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.

Project description:BackgroundThe filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the β-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production.ResultsIn this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain.ConclusionsUsing comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.

Project description:β-lactam antibiotics are widely used in clinic. Filamentous fungus Acremonium chrysogenum is an important industrial fungus for the production of CPC, one of the major precursors of β-lactam antibiotics. Although its fermentation yield has been bred significantly over the past decades, little is known regarding molecular changes between the industrial strain and the wild type strain. This limits the possibility to improve CPC production further by molecular breeding. Comparative transcriptome is a powerful tool to understand the molecular mechanisms of CPC industrial high yield producer compared to wild type. A total of 57 million clean sequencing reads with an average length of 100 bp were generated from Illumina sequencing platform. 22,878 sequences were assembled. Among the assembled unigenes, 9502 were annotated and 1989 annotated sequences were assigned to 121 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) database. Furthermore, we compared the transcriptome differences between a high-yield and a wild-type strain during fermentation. A total of 4329 unigenes with significantly different transcription level were identified, among which 1737 were up-regulated and 2592 were down-regulated. 24 pathways were subsequently determined which involve glycerolipid metabolism, galactose metabolism, and pyrimidine metabolism. We also examined the transcription levels of 18 identified genes, including 11 up-regulated genes and 7 down-regulated genes using reverse transcription quantitative -PCR (RT-qPCR). The results of RT-qPCR were consistent with the Illumina sequencing. In this study, the Illumina sequencing provides the most comprehensive sequences for gene expression profile of Acremonium chrysogenum and allows de novo transcriptome assembly while lacking genome information. Comparative analysis of RNA-seq data reveals the complexity of the transcriptome in the fermentation of different yield strains. This is an important public information platform which could be used to accelerate the research to improve CPC production in Acremonium chrysogenum.

Dataset Information

A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum.

Publications

A homologue of the Aspergillus velvet gene regulates both cephalosporin C biosynthesis and hyphal fragmentation in Acremonium chrysogenum.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets