Proteomics and secretomics of Penicillium chrysogenummolecular characterization of proteins relevant for penicillin biosynthesis

Résumé

Eighty years after the discovery of penicillin, the interest of this ß-lactam antibiotic at industrial level is still remarkable. Despite the good background knowledge, little is known on how Penicillin chrysogenum became a good penicillin overproducer, and much of the molecular basis for improved productivity remains obscure. Some light has been shed on this issue after the recent publication of the P. chrysogenum genome (van den Berg et al., 2008). These authors reported that transcription of genes involved in biosynthesis of the amino acid precursors for penicillin biosynthesis, as well as of genes encoding microbody proteins was higher in the high-producing strain DS17690. However, full exploitation of P. chrysogenum requires the integration of knowledge from other -omics such as proteomics. Proteomics studies are one of the most powerful methods to evaluate the final result of gene expression and 2-DE has been the technique of choice to obtain a reference global picture of the proteomic and secretomic map. This technique has been successfully applied to other ascomycete fungi, such as Schizosaccharomyces pombe (Hwang et al., 2006), some Aspergilli (Melin et al., 2002; Kniemeyer et al., 2006; Vödisch et al., 2009) or Saccharomyces cerevisiae (Shevchenko et al., 1996; Bruckmann et al., 2009). But only Kiel and coworkers (2009) have performed a proteomics-based inventory of the proteins present in the microbody matrix of P. chrysogenum. Therefore, it was necessary to study in depth the cytosolic proteome and secretome of this important fungus. In this work, we have optimized the methods to extract the P. chrysogenum cytosolic proteins (proteome) as well as secreted protein to the culture (secretome). As a result, the cytosolic proteome reference map and secretome of this filamentous fungus has been developed. We have also characterized the relevant modifications produced during the industrial strain improvement program through the proteome analysis and of three P. chrysogenum strains: i) the wild-type strain NRRL 1951 isolated in Peoria, IL, ii) the genome reference strain Wisconsin 54-1255 (an improved but still low-producer) and iii) the high-producing strain AS-P-78 developed by Antibioticos S.A. (León, Spain).