Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae.

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.

Rational domain swaps decipher programming in fungal highly reducing polyketide synthases and resurrect an extinct metabolite.

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.

Nongenetic reprogramming of a fungal highly reducing polyketide synthase.

The biosynthesis of the fungal metabolite tenellin from Beauveria bassiana CBS110.25 was investigated in the presence of the epigenetic modifiers 5-azacytidine and suberoyl bis-hydroxamic acid and under conditions where individual genes from the tenellin biosynthetic gene cluster were silenced. Numerous new compounds were synthesized, indicating that the normal predominant biosynthesis of tenellin is just one outcome out of a diverse array of possible products. The structures of the products reveal key clues about the programming selectivities of the tenellin polyketide synthase.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase.

The tenS gene encoding tenellin synthetase (TENS), a 4239-residue polyketide synthase nonribosomal-peptide synthetase (PKS-NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M-2-3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C-terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans-acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.