title: Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene.
abstract: We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host-vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine beta-lyase. With this new host-vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein-PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL-SKL produced in H. polymorpha displays normal enzymatic activities.
authors.affiliation: Eukaryotic Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB) University of Groningen, Haren, The Netherlands.
authors.forename: Loknath
authors.initials: L
authors.lastname: Gidijala
authors.forename: Ida J
authors.initials: IJ
authors.lastname: van der Klei
authors.forename: Marten
authors.initials: M
authors.lastname: Veenhuis
authors.forename: Jan A K W
authors.initials: JA
authors.lastname: Kiel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003066:Coenzyme A Ligases / Q000235:genetics* / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008190:Lyases / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010406:Penicillins / Q000096:biosynthesis*; D010408:Penicillium chrysogenum / Q000201:enzymology*; D020675:Peroxisomes / Q000201:enzymology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D010406:Penicillins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D008190:Lyases; C019965:cystathionine beta-lyase; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase