Project description:Using in silico modelling, such Genome-Scale Metabolic Network (GSMN), represents a promising approach to predicting and understanding the potential for specialised metabolite production in a given organism. To address these questions, we reconstructed a new high-quality GSMN for the Penicillium rubens Wisconsin 54-1255 strain, a commonly used model organism. Our reconstruction, iPrub22, adheres to current convention standards and quality criteria, incorporating updated functional annotations, orthology searches with different GSMN templates, data from previous reconstructions, and manual curation steps targeting basal and specialised metabolites. With a MEMOTE score of 74% and a metabolic coverage of 45%, iPrub22 includes 5,464 metabolites interconnected by 5,919 reactions, of which 5,033 are supported by at least one genomic sequence. Of the metabolites present in iPrub22, 13% are categorised as belonging to specialised metabolism.
Project description:We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.
Project description:When considering mold prevention strategies, the environmental conditions in which fungi grow need to be taken into consideration. This environment is often characterized by a time-dependent relative humidity, and porous substrate. Growth has mainly been investigated in steady-state experiments. Therefore, the goal of this study is to understand the hyphal growth of Penicillium rubens on porous gypsum, under dynamic humidity conditions. Spores of P. rubens were inoculated on porous gypsum containing nutrients, and placed in a small incubation chamber, allowing for microscopic hyphal observation. The relative humidity in this chamber varied multiple times between a high (close to 100%) and low value (35%, 55%, or 75%). The hyphae reacted to a lowered relative humidity by an immediate growth stop and dehydration. When the relative humidity was increased again, the hyphae re-hydrated and three responses were found: regrowing after approximately 4 h, after a time equal to the germination time, or no regrowth at all. No substantial regrowth was found for fluctuations faster than 4 h. This time-scale was found for multiple decreases in relative humidity, and has been reported for the first time. KEY POINTS: • Hyphae restart growth after a characteristic time of approximately 4 h. • Relative humidity fluctuations of 3 h can suppress hyphal growth. • Hyphae do not regrow after a severe desiccation and short periods of high humidity.
Project description:Nonribosomal peptide synthetases (NRPS) are large multimodular enzymes that synthesize a diverse variety of peptides. Many of these are currently used as pharmaceuticals, thanks to their activity as antimicrobials (penicillin, vancomycin, daptomycin, echinocandin), immunosuppressant (cyclosporin) and anticancer compounds (bleomycin). Because of their biotechnological potential, NRPSs have been extensively studied in the past decades. In this review, we provide an overview of the main structural and functional features of these enzymes, and we consider the challenges and prospects of engineering NRPSs for the synthesis of novel compounds. Furthermore, we discuss secondary metabolism and NRP synthesis in the filamentous fungus Penicillium rubens and examine its potential for the production of novel and modified β-lactam antibiotics.
Project description:Penicillium chrysogenum is a commonly occurring mould in indoor environments and foods, and has gained much attention for its use in the production of the antibiotic penicillin. Phylogenetic analysis of the most important penicillin producing P. chrysogenum isolates revealed the presence of two highly supported clades, and we show here that these two clades represent two species, P. chrysogenum and P. rubens. These species are phenotypically similar, but extrolite analysis shows that P. chrysogenum produces secalonic acid D and F and/or a metabolite related to lumpidin, while P. rubens does not produce these metabolites. Fleming's original penicillin producing strain and the full genome sequenced strain of P. chrysogenum are re-identified as P. rubens. Furthermore, the well-known claim that Alexander Fleming misidentified the original penicillin producing strain as P. rubrum is discussed.