Gene_locus Report for: aspcl-CCSE

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. Here we investigate the role of the highly conserved alpha-beta -hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the alpha-beta -hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined .

Tailoring enzymes in cytochalasan biosynthesis are relatively promiscuous. Exploiting this property, we deduced the function of four cryptic cytochrome P450 monooxygenases via heterologous expression of six cytochrome P450-encoding genes, originating from Hypoxylon fragiforme and Pyricularia oryzae, in pyrichalasin H deltaP450 strains. Three cryptic cytochrome P450 enzymes (HffD, HffG, and CYP1) restored pyrichalasin H production in mutant strains, while CYP3 catalyzed a site-selective epoxidation leading to the isolation of three novel cytochalasans.

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.

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. Here we investigate the role of the highly conserved alpha-beta -hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the alpha-beta -hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined .

Tailoring enzymes in cytochalasan biosynthesis are relatively promiscuous. Exploiting this property, we deduced the function of four cryptic cytochrome P450 monooxygenases via heterologous expression of six cytochrome P450-encoding genes, originating from Hypoxylon fragiforme and Pyricularia oryzae, in pyrichalasin H deltaP450 strains. Three cryptic cytochrome P450 enzymes (HffD, HffG, and CYP1) restored pyrichalasin H production in mutant strains, while CYP3 catalyzed a site-selective epoxidation leading to the isolation of three novel cytochalasans.

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.

Cytochalasins are a group of fungal secondary metabolites with diverse structures and bioactivities, including cytochalasin E produced by Aspergillus clavatus, which is a potent anti-angiogenic agent. Here, we report the identification and characterization of the cytochalasin gene cluster from A. clavatus NRRL 1. As a producer of cytochalasin E and K, the genome of A. clavatus was analyzed and the -30 kb ccs gene cluster was identified based on the presence of a polyketide synthase-nonribosomal peptide synthetases (PKS-NRPS) and a putative Baeyer-Villiger monooxygenase (BVMO). Deletion of the central PKS-NRPS gene, ccsA, abolished the production of cytochalasin E and K, confirming the association between the natural products and the gene cluster. Based on bioinformatic analysis, a putative biosynthetic pathway is proposed. Furthermore, overexpression of the pathway specific regulator ccsR elevated the titer of cytochalasin E from 25mg/L to 175 mg/L. Our results not only shed light on the biosynthesis of cytochalasins, but also provided genetic tools for increasing and engineering the production.