Proteome Analysis of the Penicillin Producer Penicillium chrysogenum

Abstract

Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites. Molecular & Cellular Proteomics 9:1182-1198, 2010.

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses

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Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness

“…Our SILAC-based LC-MS/MS study showed the average up-regulation to 1.85 fold for protein S100A11, down-regulation to 0.10 fold for WASL and 0.25 fold for PPP1R9B. In order to validate the protein level alteration we performed the semi-quantitative RT-PCR as a standard method to evaluate the transcription level of these proteins [13]. We observed the average of 1.75 and 2.1 fold over-expression of S100A11 in mRNA level in MDA-MB-231-ShA and MDA-MB-231-ShB respectively.…”

“…Upon completion of the cycling steps, a final extension at 72°C for 5 min was done for all of the reactions and then the reactions were stored at 4°C. The bands obtained after electrophoresis were quantified by densitometry and their intensities were normalized to that provided by the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) band (relative integral optical density (IOD)) as described before [13]. The average intensity value of the transcripts obtained from the negative control cells were set to 100%.…”

Proteome analysis reveals roles of L-DOPA in response to oxidative stress in neurons

“…Focusing of proteins (using BioRad IEF instrument), equilibration of the focused IPG strips and the 12.5% SDS-PAGE for the second dimension (carried out in an Ettan Dalt Six apparatus (GE Healthcare)) were performed as previously described [51]. Briefly, proteins were focused at 20°C according to the following program: 1 h, 0 V and 12 h, 30 V (rehydration); 30-min gradient to 10,000 V; up to 9 h, 10,000 V until 85 kV-h.…”