Hansen, Bjarne G.;
Salomonsen, Bo;
Nielsen, Morten T.;
Nielsen, Jakob B.;
Hansen, Niels B.;
Nielsen, Kristian F.;
Regueira, Torsten B.;
Nielsen, Jens;
Patil, Kiran R.;
Mortensen, Uffe H.
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
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Media type:
E-Article
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
Contributor:
Hansen, Bjarne G.;
Salomonsen, Bo;
Nielsen, Morten T.;
Nielsen, Jakob B.;
Hansen, Niels B.;
Nielsen, Kristian F.;
Regueira, Torsten B.;
Nielsen, Jens;
Patil, Kiran R.;
Mortensen, Uffe H.
imprint:
American Society for Microbiology, 2011
Published in:Applied and Environmental Microbiology
Language:
English
DOI:
10.1128/aem.01768-10
ISSN:
0099-2240;
1098-5336
Origination:
Footnote:
Description:
<jats:title>ABSTRACT</jats:title>
<jats:p>
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
<jats:named-content content-type="genus-species">Aspergillus nidulans</jats:named-content>
. 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
<jats:italic>mpaC</jats:italic>
gene from
<jats:named-content content-type="genus-species">Penicillium brevicompactum</jats:named-content>
to
<jats:named-content content-type="genus-species">A. nidulans</jats:named-content>
. In parallel, we produced defined mutant derivatives of
<jats:italic>mpaC</jats:italic>
, and the combined analysis of
<jats:named-content content-type="genus-species">A. nidulans</jats:named-content>
strains expressing
<jats:italic>mpaC</jats:italic>
or mutated
<jats:italic>mpaC</jats:italic>
genes unequivocally demonstrated that
<jats:italic>mpaC</jats:italic>
indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.
</jats:p>