Fleming's penicillin producing strain is not Penicillium chrysogenum but P. rubens.
ABSTRACT: 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.
Project description:Antibiotics were derived originally from wild organisms and therefore understanding how these compounds evolve among different lineages might help with the design of new antimicrobial drugs. We report the draft genome sequence of Alexander Fleming's original fungal isolate behind the discovery of penicillin, now classified as Penicillium rubens Biourge (1923) (IMI 15378). We compare the structure of the genome and genes involved in penicillin synthesis with those in two 'high producing' industrial strains of P. rubens and the closely related species P. nalgiovense. The main effector genes for producing penicillin G (pcbAB, pcbC and penDE) show amino acid divergence between the Fleming strain and both industrial strains, whereas a suite of regulatory genes are conserved. Homologs of penicillin N effector genes cefD1 and cefD2 were also found and the latter displayed amino acid divergence between the Fleming strain and industrial strains. The draft assemblies contain several partial duplications of penicillin-pathway genes in all three P. rubens strains, to differing degrees, which we hypothesise might be involved in regulation of the pathway. The two industrial strains are identical in sequence across all effector and regulatory genes but differ in duplication of the pcbAB-pcbC-penDE complex and partial duplication of fragments of regulatory genes. We conclude that evolution in the wild encompassed both sequence changes of the effector genes and gene duplication, whereas human-mediated changes through mutagenesis and artificial selection led to duplication of the penicillin pathway genes.
Project description:Species classified in Penicillium sect. Chrysogena are primary soil-borne and the most well-known members are P. chrysogenum and P. nalgiovense. Penicillium chrysogenum has received much attention because of its role in the production on penicillin and as a contaminant of indoor environments and various food and feedstuffs. Another biotechnologically important species is P. nalgiovense, which is used as a fungal starter culture for the production of fermented meat products. Previous taxonomic studies often had conflicting species circumscriptions. Here, we present a multigene analysis, combined with phenotypic characters and extrolite data, demonstrating that sect. Chrysogena consists of 18 species. Six of these are newly described here (P. allii-sativi, P. desertorum, P. goetzii, P. halotolerans, P. tardochrysogenum, P. vanluykii) and P. lanoscoeruleum was found to be an older name for P. aethiopicum. Each species produces a unique extrolite profile. The species share phenotypic characters, such as good growth on CYA supplemented with 5 % NaCl, ter- or quarterverticillate branched conidiophores and short, ampulliform phialides (< 9 ?m). Conidial colours, production of ascomata and ascospores, shape and ornamentation of conidia and growth rates on other agar media are valuable for species identification. Eight species (P. allii-sativi, P. chrysogenum, P. dipodomyis, P. flavigenum, P. nalgiovense, P. rubens, P. tardochrysogenum and P. vanluykii) produce penicillin in culture.
Project description:Penicillium chrysogenum is a ubiquitous airborne fungus detected in every sampled region of the Earth. Owing to its role in Alexander Fleming's serendipitous discovery of Penicillin in 1928, the fungus has generated widespread scientific interest; however its natural history is not well understood. Research has demonstrated speciation within P. chrysogenum, describing the existence of four cryptic species. To discriminate the four species, we developed protocols for species-specific diagnostic PCR directly from fungal conidia. 430 Penicillium isolates were collected to apply our rapid diagnostic tool and explore the distribution of these fungi across the London Underground rail transport system revealing significant differences between Underground lines. Phylogenetic analysis of multiple type isolates confirms that the 'Fleming species' should be named Penicillium rubens and that divergence of the four 'Chrysogenum complex' fungi occurred about 0.75 million yr ago. Finally, the formal naming of two new species, Penicillium floreyi and Penicillium chainii, is performed.
Project description:To produce high levels of ?-lactams, the filamentous fungus Penicillium rubens (previously named Penicillium chrysogenum) has been subjected to an extensive classical strain improvement (CSI) program during the last few decades. This has led to the accumulation of many mutations that were spread over the genome. Detailed analysis reveals that several mutations targeted genes that encode enzymes involved in amino acid metabolism, in particular biosynthesis of l-cysteine, one of the amino acids used for ?-lactam production. To examine the impact of the mutations on enzyme function, the respective genes with and without the mutations were cloned and expressed in Escherichia coli, purified, and enzymatically analyzed. Mutations severely impaired the activities of a threonine and serine deaminase, and this inactivates metabolic pathways that compete for l-cysteine biosynthesis. Tryptophan synthase, which converts l-serine into l-tryptophan, was inactivated by a mutation, whereas a mutation in 5-aminolevulinate synthase, which utilizes glycine, was without an effect. Importantly, CSI caused increased expression levels of a set of genes directly involved in cysteine biosynthesis. These results suggest that CSI has resulted in improved cysteine biosynthesis by the inactivation of the enzymatic conversions that directly compete for resources with the cysteine biosynthetic pathway, consistent with the notion that cysteine is a key component during penicillin production.IMPORTANCE Penicillium rubens is an important industrial producer of ?-lactam antibiotics. High levels of penicillin production were enforced through extensive mutagenesis during a classical strain improvement (CSI) program over 70 years. Several mutations targeted amino acid metabolism and resulted in enhanced l-cysteine biosynthesis. This work provides a molecular explanation for the interrelation between secondary metabolite production and amino acid metabolism and how classical strain improvement has resulted in improved production strains.
Project description:Penicillium oxalicum strain 212 (PO212) is an effective biocontrol agent (BCA) against a large number of economically important fungal plant pathogens. For successful registration as a BCA in Europe, PO212 must be accurately identified. In this report, we describe the use of classical genetic and molecular markers to characterize and identify PO212 in order to understand its ecological role in the environment or host. We successfully generated pyrimidine (pyr-) auxotrophic mutants. In addition we also designed specific oligonucleotides for the pyrF gene at their untranslated regions for rapid and reliable identification and classification of strains of P.?oxalicum and P.?rubens, formerly P.?chrysogenum. Using these DNA-based technologies, we found that PO212 is a strain of P.?rubens, and is not a strain of P.?oxalicum. This work presents PO212 as the unique P.?rubens strain to be described as a BCA and the information contained here serves for its registration and commercialization in Europe.
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:BACKGROUND: Due to the importance of Penicillium chrysogenum holding in medicine, the genome of low-penicillin producing laboratorial strain Wisconsin54-1255 had been sequenced and fully annotated. Through classical mutagenesis of Wisconsin54-1255, product titers and productivities of penicillin have dramatically increased, but what underlying genome structural variations is still little known. Therefore, genome sequencing of a high-penicillin producing industrial strain is very meaningful. RESULTS: To reveal more insights into the genome structural variations of high-penicillin producing strain, we sequenced an industrial strain P. chrysogenum NCPC10086. By whole genome comparative analysis, we observed a large number of mutations, insertions and deletions, and structural variations. There are 69 new genes that not exist in the genome sequence of Wisconsin54-1255 and some of them are involved in energy metabolism, nitrogen metabolism and glutathione metabolism. Most importantly, we discovered a 53.7 Kb "new shift fragment" in a seven copies of determinative penicillin biosynthesis cluster in NCPC10086 and the arrangement type of amplified region is unique. Moreover, we presented two large-scale translocations in NCPC10086, containing genes involved energy, nitrogen metabolism and peroxysome pathway. At last, we found some non-synonymous mutations in the genes participating in homogentisate pathway or working as regulators of penicillin biosynthesis. CONCLUSIONS: We provided the first high-quality genome sequence of industrial high-penicillin strain of P. chrysogenum and carried out a comparative genome analysis with a low-producing experimental strain. The genomic variations we discovered are related with energy metabolism, nitrogen metabolism and so on. These findings demonstrate the potential information for insights into the high-penicillin yielding mechanism and metabolic engineering in the future.
Project description:We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple ?pex11 ?pex11B ?pex11C ?pex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production.
Project description:In studies on beta-lactam production by Penicillium chrysogenum, addition and omission of a side-chain precursor is commonly used to generate producing and non-producing scenarios. To dissect effects of penicillin-G production and of its side-chain precursor phenylacetic acid (PAA), a derivative of a penicillin-G high-producing strain without a functional penicillin-biosynthesis gene cluster (pcbAB-pcbC-penDE) was constructed. The copy number of this cluster was first reduced to one via spontaneous recombination. The remaining copy was removed by targeted deletion, thereby completely abolishing beta-lactam biosynthesis. In glucose-limited chemostat cultures of the high-producing and cluster-free strains, PAA addition caused a small reduction of the biomass yield, consistent with PAA acting as a weak-organic-acid uncoupler. A low rate of penicillin-G-independent PAA consumption indicated activity of a PAA-degrading pathway. Microarray-based analysis on chemostat cultures of the high-producing and cluster-free strains, grown in the presence and absence of PAA, showed that: (i) Absence of a penicillin gene cluster resulted in transcriptional upregulation of a gene cluster putatively involved in production of the secondary metabolite aristolochene and its derivatives, (ii) The homogentisate pathway for PAA catabolism is strongly transcriptionally upregulated in PAA-supplemented cultures (iii) Several genes involved in nitrogen and sulfur metabolism were transcriptionally upregulated under penicillin-G producing conditions only, suggesting a drain of amino-acid precursor pools. Furthermore, the number of candidate genes for penicillin transporters was strongly reduced, thus enabling a focusing of functional analysis studies. This study demonstrates the usefulness of combinatorial transcriptome analysis in chemostat cultures to dissect effects of biological and process parameters on transcriptional regulation. Overall design: Mutants of P. chrysogenum impaired in penicillin biosynthesis have been described. Most of these mutants were derived by random mutagenesis from the low-producing strain Wisconsin54-1255, which contains one copy of the penicillin biosynthesis cluster. Although these mutants were very useful for studying gene expression and gene/enzyme relationships, for identification of the factors important for penicillin-G production and PAA consumption a strain obtained from a high-producing strain background by targeted deletion is more beneficial. To this end we constructed a strain in which the tandem-repeated penicillin biosynthesis cluster was specifically deleted, whereas the strain background was retained. In such a way it is possible to identify, by a combinatorial approach, those genes affected by phenylacetic acid and those specifically important for penicillin-G biosynthesis.
Project description: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 DeltaPcvelA 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 DeltaPclaeA 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.