Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum

BACKGROUND: Penicillium chrysogenum is a filamentous fungus that is employed as an industrial producer of β-lactams. The high β-lactam titers of current strains is the result of a classical strain improvement program (CSI) starting with a wild-type like strain more than six decades ago. This involved extensive mutagenesis and strain selection for improved β-lactam titers and growth characteristics. However, the impact of the CSI on the secondary metabolism in general remains unknown. RESULTS: To examine the impact of CSI on secondary metabolism, a comparative genomic analysis of β-lactam producing strains was carried out by genome sequencing of three P. chrysogenum strains that are part of a lineage of the CSI, i.e., strains NRRL1951, Wisconsin 54-1255, DS17690, and the derived penicillin biosynthesis cluster free strain DS68530. CSI has resulted in a wide spread of mutations, that statistically did not result in an over- or underrepresentation of specific gene classes. However, in this set of mutations, 8 out of 31 secondary metabolite genes (20 polyketide synthases and 11 non-ribosomal peptide synthetases) were targeted with a corresponding and progressive loss in the production of a range of secondary metabolites unrelated to β-lactam production. Additionally, key Velvet complex proteins (LeaA and VelA) involved in global regulation of secondary metabolism have been repeatedly targeted for mutagenesis during CSI. Using comparative metabolic profiling, the polyketide synthetase gene cluster was identified that is responsible for sorbicillinoid biosynthesis, a group of yellow-colored metabolites that are abundantly produced by early production strains of P. chrysogenum. CONCLUSIONS: The classical industrial strain improvement of P. chrysogenum has had a broad mutagenic impact on metabolism and has resulted in silencing of specific secondary metabolite genes with the concomitant diversion of metabolism towards the production of β-lactams

Proteomics Shows New Faces for the Old Penicillin Producer Penicillium chrysogenum

Fungi comprise a vast group of microorganisms including the Ascomycota (majority of all described fungi), the Basidiomycota (mushrooms or higher fungi), and the Zygomycota and Chytridiomycota (basal or lower fungi) that produce industrially interesting secondary metabolites, such as β-lactam antibiotics. These compounds are one of the most commonly prescribed drugs world-wide. Since Fleming's initial discovery of Penicillium notatum 80 years ago, the role of Penicillium as an antimicrobial source became patent. After the isolation of Penicillium chrysogenum NRRL 1951 six decades ago, classical mutagenesis and screening programs led to the development of industrial strains with increased productivity (at least three orders of magnitude). The new “omics” era has provided the key to understand the underlying mechanisms of the industrial strain improvement process. The review of different proteomics methods applied to P. chrysogenum has revealed that industrial modification of this microorganism was a consequence of a careful rebalancing of several metabolic pathways. In addition, the secretome analysis of P. chrysogenum has opened the door to new industrial applications for this versatile filamentous fungus

Adding tools to the box: facilitating host strain engineering of Penicillium chrysogenum for the production of heterologous secondary metabolites

This thesis presents the adaption of the RNA-guided endonuclease Cas9 for engineering the genome of the ascomycete fungus P. chrysogenum, whereby the insertion of a donor DNA is greatly facilitated. The approach of delivering the Cas9 protein and the sgRNA as a preassembled ribonucleoprotein particle was further demonstrated in P. decumbens and led to verification of the Calbistrin BGC. This BGC was then selected as a proof-of-principle example for in vivo homologous recombination of a heterologous BGC into a BGC-reduced P. chrysogenum strain. Lastly, the Cas9-boosted homologous recombination frequency was utilized to demonstrate a time-saving approach for building and testing novel expression cassettes transcription factors such as an aldehyde-inducible promoter and protein degradation tags in P. chrysogenum. The genetic tools developed in this thesis speed up the strain construction approaches in this industrially relevant fungus. In the coming decade, precise, simultaneous-multi-loci-editing techniques of filamentous fungi will become routinely applied in research labs and researchers will increasingly consider fungi as a host organism for their purposes if the available toolbox is kept updated and reliable to apply

Molecular characterization of a fungal gene paralogue of the penicillin penDE gene of Penicillium chrysogenum

<p>Abstract</p> <p>Background</p> <p><it>Penicillium chrysogenum </it>converts isopenicillin N (IPN) into hydrophobic penicillins by means of the peroxisomal IPN acyltransferase (IAT), which is encoded by the <it>penDE </it>gene. <it>In silico </it>analysis of the <it>P. chrysogenum </it>genome revealed the presence of a gene, Pc13g09140, initially described as paralogue of the IAT-encoding <it>penDE </it>gene. We have termed this gene <it>ial </it>because it encodes a protein with high similarity to IAT (IAL for IAT-Like). We have conducted an investigation to characterize the <it>ial </it>gene and to determine the role of the IAL protein in the penicillin biosynthetic pathway.</p> <p>Results</p> <p>The IAL contains motifs characteristic of the IAT such as the processing site, but lacks the peroxisomal targeting sequence ARL. Null <it>ial </it>mutants and overexpressing strains indicated that IAL lacks acyltransferase (penicillin biosynthetic) and amidohydrolase (6-APA forming) activities <it>in vivo</it>. When the canonical ARL motif (leading to peroxisomal targeting) was added to the C-terminus of the IAL protein (IAL^ARL) by site-directed mutagenesis, no penicillin biosynthetic activity was detected. Since the IAT is only active after an accurate self-processing of the preprotein into α and β subunits, self-processing of the IAL was tested in <it>Escherichia coli</it>. Overexpression experiments and SDS-PAGE analysis revealed that IAL is also self-processed in two subunits, but despite the correct processing, the enzyme remained inactive <it>in vitro</it>.</p> <p>Conclusion</p> <p>No activity related to the penicillin biosynthesis was detected for the IAL. Sequence comparison among the <it>P. chrysogenum </it>IAL, the <it>A. nidulans </it>IAL homologue and the IAT, revealed that the lack of enzyme activity seems to be due to an alteration of the essential Ser309 in the thioesterase active site. Homologues of the <it>ial </it>gene have been found in many other ascomycetes, including non-penicillin producers. Our data suggest that like in <it>A. nidulans</it>, the <it>ial </it>and <it>penDE </it>genes might have been formed from a single ancestral gene that became duplicated during evolution, although a separate evolutive origin for the <it>ial </it>and <it>penDE </it>genes, is also discussed.</p