Matching the proteome to the genome: the microbody of penicillin-producing Penicillium chrysogenum cells.
In the filamentous fungus Penicillium chrysogenum, microbodies are essential for penicillin biosynthesis. To better understand the role of these organelles in antibiotics production, we determined the matrix enzyme contents of P. chrysogenum microbodies. Using a novel in silico approach, we first obtained a catalogue of 200 P. chrysogenum proteins with putative microbody targeting signals (PTSs). This included two orthologs of proteins involved in cephalosporin biosynthesis, which we demonstrate to be bona fide microbody matrix constituents. Subsequently, we performed a proteomics based inventory of P. chrysogenum microbody matrix proteins using nano-LC-MS/MS analysis. We identified 89 microbody proteins, 79 with a PTS, including the two known microbody-borne penicillin biosynthesis enzymes, isopenicillin N:acyl CoA acyltransferase and phenylacetyl-CoA ligase. Comparative analysis revealed that 69 out of 79 PTS proteins identified experimentally were in the reference list. A prominent microbody protein was identified as a novel fumarate reductase-cytochrome b5 fusion protein, which contains an internal PTS2 between the two functional domains. We show that this protein indeed localizes to P. chrysogenum microbodies.
Kiel
Jan A K W
JA
Molecular Cell Biology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, AA Haren, The Netherlands. J.A.K.W.Kiel@RUG.nl
van den Berg
Marco A
MA
Fusetti
Fabrizia
F
Poolman
Bert
B
Bovenberg
Roel A L
RA
Veenhuis
Marten
M
van der Klei
Ida J
IJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016681:Genome, Fungal; D008830:Microbodies / Q000378:metabolism* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D020543:Proteome; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D017931:DNA Primers; D004271:DNA, Fungal; D005656:Fungal Proteins; D010406:Penicillins; D021382:Protein Sorting Signals; D020543:Proteome; D011994:Recombinant Proteins