Understanding and exploiting fungal PKS-NRPS biosynthesis in Pyricularia and Magnaporthe species

The fungus Pyricularia oryzae is a major pathogen of rice (Oryzae sativa) that causes substantial loss of yields every year. Remarkably, certain rice strains carrying the Pi33 gene are resistant to strains of P. oryzae encoding a PKS-NRPS called the avirulence conferring enzyme1 (ACE1). By heterologous expression studies in Aspergillus oryzae, more insights into the early biosynthetic steps of this ACE1 metabolite were obtained. O-Methyl-L-tyrosine 71 was identified as the preferred substrate for the adenylation domain of the ACE1 NRPS, indicating that the OME1 gene (O-methyltransferase) belongs to the ACE1 cluster. However, even after all biosynthetic genes of the ACE1 cluster were co-expressed; the produced compound was 88 - a shunt intermediate. In-vitro enzyme assays confirmed that reduction of the aldehyde 90 to the alcohol 88 is catalysed by a native A. oryzae enzyme at an early biosynthetic step; thus stalling the biosynthesis. Ectopic expression of BC1 (transcription factor from the ACE1 BGC) in P. oryzae did not lead to the production of ACE1-related compounds. However, the compound hinnulin A 105 was formed, which belongs to the class of DHN-melanins. A putative partial BGC potentially involved in the biosynthesis of 105 was validated by RT-PCR and a possible biosynthetic pathway was proposed. Another cytochalasan BGC was previously demonstrated to be responsible for the production of pyrichalasin H 50 in Magnaporthe grisea and now revealed to be homologous to the ACE1 BGC. Thus, the 50 BGC was used as model system to elucidate the function of two potential key enzymes in cytochalasan biosynthesis: an αβ-hydrolase (HYD) and a putative Diels-Alderase (pDA). Targeted gene deletion experiments in M. grisea revealed that the HYD PyiE is involved in early steps during the biosynthesis of 50, but its exact role remained elusive. The pDA PyiF was shown to be involved in the intramolecular [4+2] cycloaddition forming 72. Complementation studies with ORF3 from the ACE1 BGC indicated a similar catalytic function of both enzymes. By recombinant gene expression studies in E. coli soluble ORFZ (HYD) and ORF3 (pDA) protein was obtained. In-vitro assays are underway to gain further insights into their biosynthetic role