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.

Biosynthesis of Terpenoid Natural Products in Fungi

“…Analysis based on co-expression arrays was only recently applied to natural product gene cluster analysis in A. nidulans [28]. Comprehensive deep transcriptome analysis of fungi grown under conditions that activate a broad range of natural products pathways [29,39,247,248] should enable accurate prediction of complete pathways, including satellite or superclusters of split pathways known from fungi [57,[249][250][251].…”

Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum

“…In summary, the performed experiments show that mpaC is a key gene involved in biosynthesis of MPA by P. brevicompactum, and our results set the groundwork for future experiments to elucidate the remaining intriguing biosynthetic steps in MPA biosynthesis. The accompanying manuscript by Hansen et al (24) conclusively shows that the polyketide synthase MpaC catalyzes the producion of 5-methylorsellinic acid. A thorough study of several of the catalytic steps in MPA biosynthesis, like the prenyl transfer, heterocyclization, and chain cleavage steps, may result in a deeper understanding of the biosynthesis of this valuable immunosuppressant.…”

Accurate prediction of secondary metabolite gene clusters in filamentous fungi

“…Specifically, sequences upstream and downstream of the gene to be deleted were amplified by PCR using primers containing a uracil residue (Table S2). The two PCR fragments were simultaneously inserted into the PacI/Nt.BbvCI USER cassette of pU20002A by USER cloning (54,55). As a result, AFpyrG is now flanked by the two PCR fragments to complete the gene targeting substrate.…”