Overproduction of a single protein, Pc-Pex 11p, results in 2-fold enhanced penicillin production by Penicillium chrysogenum

Current industrial production of β-lactam antibiotics, using the filamentous fungus Penicillium chrysogenum, is the result of many years of strain improvement by classical mutagenesis. More efficient production strains showed significant increases in the number and volume fraction of microbodies in their cells, organelles that harbor key enzymes involved in the biosynthesis of β-lactam antibiotics. We have isolated the P. chrysogenum cDNA encoding Pc-Pex11p, a peroxin that is involved in microbody abundance. We demonstrate that overproduction of Pc-Pex11p in P. chrysogenum results in massive proliferation of tubular-shaped microbodies and a 2- to 2.5-fold increase in the level of penicillin in the culture medium. Notably, Pc-Pex11p-overproduction did not affect the levels of the enzymes of the penicillin biosynthetic pathway. Our results suggest that the stimulating effect of enhanced organelle numbers may reflect an increase in the fluxes of penicillin and/or its precursors across the now much enlarged microbody membrane

Biosynthesis of active pharmaceuticals: β-lactam biosynthesis in filamentous fungi

beta-lactam antibiotics (e.g. penicillins, cephalosporins) are of major clinical importance and contribute to over 40% of the total antibiotic market. These compounds are produced as secondary metabolites by certain actinomycetes and filamentous fungi (e.g. Penicillium, Aspergillus and Acremonium species). The industrial producer of penicillin is the fungus Penicillium chrysogenum. The enzymes of the penicillin biosynthetic pathway are well characterized and most of them are encoded by genes that are organized in a cluster in the genome. Remarkably, the penicillin biosynthetic pathway is compartmentalized: the initial steps of penicillin biosynthesis are catalyzed by cytosolic enzymes, whereas the two final steps involve peroxisomal enzymes. Here, we describe the biochemical properties of the enzymes of beta-lactam biosynthesis in P. chrysogenum and the role of peroxisomes in this process. An overview is given on strain improvement programs via classical mutagenesis and, more recently, genetic engineering, leading to more productive strains. Also, the potential of using heterologous hosts for the development of novel beta-lactam antibiotics and non-ribosomal peptide synthetase-based peptides is discussed

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: A sharp increase of the protein level induces the proliferation of numerous, small protein-import competent peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P-AOX.PEX3(7x)), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density(1.18 g cm(-3)) after sucrose gradient centrifugation of WT::P-AOX.PEX3(7x) homogenates, compared to normal peroxisomes (1.23 g cm(-3)). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P-AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. (C) 1997 John Wiley & Sons, Ltd