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.

Project description:Filamentous fungi are important producers of enzymes and secondary metabolites in the biotech industry. A relatively new but highly promising fungus for industrial production is Thermothelomyces thermophilus, which is closely related to the model fungus Neurospora crassa. Nevertheless, to fully exploit the potential of T. thermophilus, a deeper understanding of its biology is necessary. A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), regulated by various stimuli, including nutrient availability. Under certain submerged fermentation conditions, several filamentous fungi produce conidia, which can be detrimental to product expression. In this study, we utilized RNA-seq to map transcriptomic changes during conidia production under submerged conditions in T. thermophilus. We found that the essential genes for conidiation in Aspergillus sp. and N. crassa, fluG and fluffy (fl), are not essential for conidiation in T. thermophilus. However, we identified a transcription factor, res1, whose deletion resulted in a complete loss of conidia production under fermentation conditions. This deletion also led to increased biomass production without increasing enzyme secretion. Notably, the deletion of res1 did not affect spore production on solid media. Differential gene expression analysis between the wild type and the res1 deletion mutant revealed that res1 regulates a broad range of cellular and metabolic processes, including MAPK and spore production pathways. Overexpression of res1 caused a severe growth defect and early conidia production compared to the wild type. Using ChIP-seq, we identified 35 potential target genes of res1, including known conidiation-related genes such as acon3, MAPK genes, and transcription factors like cpc1, as well as genes with unknown functions. Overall, we identified res1 to be obligatory for submerged conidia production and lay the foundation for further work on the T. thermophilus conidiation pathway and its applications.

Dataset Information

Time-course gene expression data from deletion strains of Penicillium chrysogenum

Publications

Among developmental regulators, StuA but not BrlA is essential for penicillin V production in Penicillium chrysogenum.

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