An engineered yeast efficiently secreting penicillin

• Published in: PloS one Vol. 4; no. 12; p. e8317
• Main Authors: Gidijala, Loknath, Kiel, Jan A K W, Douma, Rutger D, Seifar, Reza M, van Gulik, Walter M, Bovenberg, Roel A L, Veenhuis, Marten, van der Klei, Ida J
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.12.2009; Public Library of Science (PLoS)
• Subjects: Alcohol; Amides; Antibiotics; Bacillus subtilis; Baking yeast; Beta lactamases; beta-Lactams - metabolism; Biochemistry/Biocatalysis; Biological activity; Biological effects; Biology; Biosynthesis; Biotechnology; Biotechnology/Applied Microbiology; Biotechnology/Bioengineering; Cell Biology/Membranes and Sorting; Chromatography; Cytostatic agents; Drugs; Enzymes; Fermentation; Fungi; Gene Deletion; Gene expression; Genes, Fungal; Genetic Engineering; Genomics; Hansenula; Hansenula polymorpha; Immunosuppression; Industrial engineering; Industrial production; Isopenicillin N synthase; Lactams; Ligases; Localization; Manufacturing engineering; Mass spectrometry; Metabolites; Penicillin; Penicillins - metabolism; Penicillium chrysogenum; Penicillium chrysogenum - enzymology; Peptide Synthases - biosynthesis; Peptides; Peroxisomes; Peroxisomes - metabolism; Pichia - cytology; Pichia - genetics; Pichia - metabolism; Pichia - ultrastructure; Protein Transport; Proteins; Quantitative analysis; Saccharomyces cerevisiae; Sampling techniques; Scientific imaging; Sfp gene; Subcellular Fractions - ultrastructure; Synthetic peptides; Valine; Yeast; β-Lactam antibiotics; Netherlands
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0008317

This study aimed at developing an alternative host for the production of penicillin (PEN). As yet, the industrial production of this beta-lactam antibiotic is confined to the filamentous fungus Penicillium chrysogenum. As such, the yeast Hansenula polymorpha, a recognized producer of pharmaceuticals, represents an attractive alternative. Introduction of the P. chrysogenum gene encoding the non-ribosomal peptide synthetase (NRPS) delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) in H. polymorpha, resulted in the production of active ACVS enzyme, when co-expressed with the Bacillus subtilis sfp gene encoding a phosphopantetheinyl transferase that activated ACVS. This represents the first example of the functional expression of a non-ribosomal peptide synthetase in yeast. Co-expression with the P. chrysogenum genes encoding the cytosolic enzyme isopenicillin N synthase as well as the two peroxisomal enzymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL) resulted in production of biologically active PEN, which was efficiently secreted. The amount of secreted PEN was similar to that produced by the original P. chrysogenum NRRL1951 strain (approx. 1 mg/L). PEN production was decreased over two-fold in a yeast strain lacking peroxisomes, indicating that the peroxisomal localization of IAT and PCL is important for efficient PEN production. The breakthroughs of this work enable exploration of new yeast-based cell factories for the production of (novel) beta-lactam antibiotics as well as other natural and semi-synthetic peptides (e.g. immunosuppressive and cytostatic agents), whose production involves NRPS's.