title: Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case.
abstract: Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.
authors.affiliation: Technical University of Denmark, Department of Systems Biology, Center for Microbial Biotechnology, Building 223, 2800 Kgs. Lyngby, Denmark.
authors.forename: Bjarne G
authors.initials: BG
authors.lastname: Hansen
authors.forename: Bo
authors.initials: B
authors.lastname: Salomonsen
authors.forename: Morten T
authors.initials: MT
authors.lastname: Nielsen
authors.forename: Jakob B
authors.initials: JB
authors.lastname: Nielsen
authors.forename: Niels B
authors.initials: NB
authors.lastname: Hansen
authors.forename: Kristian F
authors.initials: KF
authors.lastname: Nielsen
authors.forename: Torsten B
authors.initials: TB
authors.lastname: Regueira
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
authors.forename: Kiran R
authors.initials: KR
authors.lastname: Patil
authors.forename: Uffe H
authors.initials: UH
authors.lastname: Mortensen
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D005810:Multigene Family; D009173:Mycophenolic Acid / Q000378:metabolism; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D048630:Polyketide Synthases / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D048630:Polyketide Synthases; D009173:Mycophenolic Acid