Establishing a New Methodology for Genome Mining and Biosynthesis of Polyketides and Peptides through Yeast Molecular Genetics

Abstract

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (5–20 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.

An In Planta -Expressed Polyketide Synthase Produces ( R )-Mellein in the Wheat Pathogen Parastagonospora nodorum

“…A similar convergent evolution of bacterial and fungal type I PKSs has previously been observed in orsellinic acid biosynthesis. While the fungal orsellinic acid synthases are typical fungal nonreducing PKSs (41)(42)(43), the bacterial orsellinic acid synthases are related to bacterial iterative type I PKSs (homologous to fungal PR-PKSs) and are likely to have evolved from ancestral bacterial 6MSASs via mutations in the KR domain (44)(45)(46). From a protein engineering perspective, the convergent evolution of MLNSs implies that there is more than one way in nature to modify the chain length specificity and ketoreduction regioselectivity of iterative PKSs.…”

Effective Use of Heterologous Hosts for Characterization of Biosynthetic Enzymes Allows Production of Natural Products and Promotes New Natural Product Discovery

“…This technique, which we termed "overlap-extension PCR-yeast homologous recombination (ExRec)," allowed reliable preparation of a S. cerevisiae expression plasmid containing the full-length, intron-free open reading frame of a target large fungal gene synthesized from a pool of total mRNA. 16) Next, the expression vector was introduced into an engineered S. cerevisiae strain SCKW5, whose genome is modified to include a G418 resistance marker kanMX, a malonyl-CoA synthetase gene matB and a phosphopantetheinyl transferase gene npgA. 16) Expression of the foreign gene was checked by Western blotting analysis, and biosynthesis of the corresponding nonribosomal peptide compound was examined.…”

“…16) Next, the expression vector was introduced into an engineered S. cerevisiae strain SCKW5, whose genome is modified to include a G418 resistance marker kanMX, a malonyl-CoA synthetase gene matB and a phosphopantetheinyl transferase gene npgA. 16) Expression of the foreign gene was checked by Western blotting analysis, and biosynthesis of the corresponding nonribosomal peptide compound was examined. The culture of pKW5022 (Afu6g12080)/SCKW5 contained peptide products 37 (Chart 9A) and 49 (Chart 13) with approximate yields of 0.4 and 1.8 mg/L of culture, respectively.…”

“…Once mRNA can be obtained, the corresponding cDNA can be prepared in a streamlined fashion even for a polyketide synthase (PKS) or a nonribosomal peptide synthetase (NRPS) gene, which can exceed 20 kilobases (kb) in length. 16) Availability of cDNA allows transfer of the gene into a convenient host, such as yeast, to achieve heterologous production of a biosynthetic enzyme for detailed analysis of the reaction mechanism. If sufficient genes from the cluster can be transferred to yeast, it becomes possible to biosynthesize the otherwise inaccessible new compounds heterologously.…”

Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis

“…1). The PKSs include five from A. nidulans shown to produce orsellinic acid or derivatives (Ahuja et al, 2012), a PKS from Cladonia grayi hypothesized to be responsible for production of orsellinic acid in route to the synthesis of the orcinol depsidone grayanic acid (Armaleo et al, 2011), and a PKS from Coprinopsis cinerea shown in Saccharomyces cerevisiae to produce orsellinic acid (Ishiuchi et al, 2012). The resulting genealogy resolved the 32 PKSs into three major clades each with 100% bootstrap support.…”