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.
Highlights
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
We characterized the relevant modifications produced during the industrial strain improvement program through the proteome analysis of three P. chrysogenum strains: the wild-type strain NRRL 1951 isolated in Peoria, IL; the genome reference strain Wis 54-1255; and the high producer strain AS-P-78 developed by Antibioticos S.A
P. chrysogenum has been extensively mutated during the last decades to increase the penicillin titers, but despite the importance of this microorganism, the biochemical bases that have been modified during the strain improvement process remain poorly understood
Summary
Wisconsin; 2-DE, two-dimensional gel electrophoresis; CC, colloidal Coomassie; IPN, isopenicillin N; NL, non-linear; PMF, peptide mass fingerprinting; ddH2O, double distilled H2O; ACV, ␣-aminoadipyl-cysteinyl-valine. This is due to modifications in a microsomal cytochrome P450 monooxygenase (encoded by the pahA gene) that lead to a reduced degradation of phenylacetic acid and to penicillin overproduction (11) Another well characterized modification resulting from the industrial improvement process is the amplification of the genomic region that includes the three penicillin biosynthetic genes. Microbodies (peroxisomes), organelles involved in the final steps of the penicillin pathway catalyzed by the acyl-coenzyme A:isopenicillin N (IPN) acyltransferase and phenylacetyl-CoA ligase (14 –16), are known to be more abundant in high producer strains (14, 17) Despite this background knowledge, little is known regarding how P. chrysogenum became such a good penicillin overproducer, and much of the molecular basis for improved productivity remains obscure. We characterized the relevant modifications produced during the industrial strain improvement program through the proteome analysis of three P. chrysogenum strains: the wild-type strain NRRL 1951 isolated in Peoria, IL; the genome reference strain Wis 54-1255 (an improved but still low producer); and the high producer strain AS-P-78 developed by Antibioticos S.A
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