Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during the industrial strain improvement

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.

Fig. 1.

A, penicillin biosynthetic pathway. The schematic representation shows the genes and proteins involved in different steps of the biosynthesis of benzylpenicillin. The estimated mass for each enzyme of the pathway is also indicated. B, genealogy of some P. chrysogenum strains during industrial strain improvement programs. Thin arrows represent either steps of selection (without mutagenic treatment) or x-ray irradiation, UV irradiation, or nitrogen mustard treatment. Arrows between strains BL3-D10 and Wisconsin 54-1255 represents a total of nine selection steps. The thick arrows between the Wis 54-1255 strain and the high producer strains represent several selection steps in different industrial laboratories. The names of the strains used in this work are boxed. Penicillin titers are indicated on the right for some of these strains.

Fig. 2.

Reference map of cytosolic P. chrysogenum proteome and functional classification of identified proteins. A, proteins were separated by 2-DE using 18-cm wide range IPG strips (pH 3–10 NL) and 15% SDS-PAGE and stained with CC following the blue silver staining method. A total amount of 976 spots were resolved in this range and were analyzed by PMF and tandem mass spectrometry (see supplemental Table 1). Molecular masses are shown on the left. Assigned numbers over each spot correlate with those shown in supplemental Table 1. B, the 950 identified proteins (549 different proteins and isoforms) were classified according to their biological function.

Fig. 3.

Comparison of intracellular proteomes of wild-type NRRL 1951 and Wis 54-1255 strains. 2-DE gels of the intracellular proteomes of the NRRL 1951 and the Wis 54-1255 strains grown for 40 h under the same conditions are shown. Proteins were separated by 2-DE using 18-cm wide range IPG strips (pH 3–10 NL) and 12.5% SDS-PAGE and stained with CC following the blue silver staining method. The designation “N” is used for those spots overrepresented in the NRRL 1951 strain, and “Ws” is used for those spots overrepresented in the Wis 54-1255 strain. The spots differentially represented in each strain are numbered and correspond to those proteins listed in supplemental Table 3 (for the NRRL 1951 strain) and supplemental Table 4 (for the Wis 54-1255 strain).

Fig. 4.

Close-up view of spots differentially represented in either NRRL 1951 or Wis 54-1255 strains. Enlargements of gel portions containing the spots overrepresented in the gels of Fig. 3 are shown. The designation “N” is used for those spots overrepresented in the NRRL 1951 strain, and “Ws” is used for those spots overrepresented in the Wis 54-1255 strain. The number of those spots differentially represented is indicated and corresponds to those present in supplemental Table 3 (for the NRRL 1951 strain) and supplemental Table 4 (for the Wis 54-1255 strain).

Fig. 5.

Comparison of intracellular proteomes of Wis 54-1255 and high producer AS-P-78 strains. Representative 2-DE gels of the intracellular proteomes of the Wis 54-1255 and the AS-P-78 strains grown for 40 h under the same conditions are shown. Proteins were separated by 2-DE using 18-cm wide range IPG strips (pH 3–10 NL) and 12.5% SDS-PAGE and stained with CC following the blue silver staining procedure. The name “W” is used for those spots overrepresented in the Wis 54-1255 strain, whereas “A” is used for those spots overrepresented in the AS-P-78 strain. The number of each differentially represented spot is indicated and corresponds to those in supplemental Table 5 (for the Wis 54-1255 strain) and supplemental Table 6 (for the AS-P-78 strain).

Fig. 6.

Close-up view of spots differentially represented in either Wis 54-1255 or AS-P-78 strains. Enlargements of gel portions containing the spots overrepresented in the gels of Fig. 5 are shown. The designation “W” is used for those spots overrepresented in the Wis 54-1255 strain, and “A” is used for those spots overrepresented in the AS-P-78 strain. The number of each differentially represented spot is indicated and corresponds to those present in supplemental Table 5 (for the Wis 54-1255 strain) and supplemental Table 6 (for the AS-P-78 strain).

Fig. 7.

Transcriptional analysis of pcbC and penDE genes in Wis 54-1255 and AS-P-78 strains. A, electrophoresis of the RT-PCR products using RNA extracted at 40 and 60 h from cultures of the Wis 54-1255 strain (W) and the AS-P-78 strain (A) grown under the same conditions. The transcript of β-actin was used as control. The absence of contaminant DNA in the samples was tested by PCR (data not shown). B, the bands obtained after the electrophoresis were quantified by densitometry, and their intensity was normalized to that provided by the β-actin band (relative integral optical density (IOD)). The intensity values of the pcbC and penDE transcripts obtained from the Wis 54-1255 strain at 40 h were set to 100%.

Fig. 8.

Schematic representation of some pathways and networks modified during strain improvement program according to results obtained after proteome comparison of NRRL 1951, Wis 54-1255, and AS-P-78 strains. Font size is in concordance with the protein amount observed by CC staining strength in the comparative proteome analyses. αAA, α-aminoadipic acid; BDA1, 1,4-butanediol diacrylate esterase (putative β-lactamase); CBS, cystathionine β-synthase; CS, cysteine synthase; FDH, formate dehydrogenase; GR, glutathione reductase; GT, glutathione S-transferase; M, mitochondrion; MSD, methylmalonate-semialdehyde dehydrogenase; N, nucleus; P, peroxisome; PenG, benzylpenicillin; PPP, pentose phosphate pathway; QO, quinone oxidoreductase; TCAC, tricarboxylic acid cycle; TPP, thiamin pyrophosphate.