Strobilurin biosynthesis in Basidiomycete fungi

Abstract

Strobilurins from fungi are the inspiration for the creation of the β-methoxyacrylate class of agricultural fungicides. However, molecular details of the biosynthesis of strobilurins have remained cryptic. Here we report the sequence of genomes of two fungi that produce strobilurins and show that each contains a biosynthetic gene cluster, which encodes a highly reducing polyketide synthase with very unusual C-terminal hydrolase and methyltransferase domains. Expression of stpks1 in Aspergillus oryzae leads to the production of prestrobilurin A when the fermentation is supplemented with a benzoyl coenzyme A (CoA) analogue. This enables the discovery of a previously unobserved route to benzoyl CoA. Reconstruction of the gene cluster in A. oryzae leads to the formation of prestrobilurin A, and addition of the gene str9 encoding an FAD-dependent oxygenase leads to the key oxidative rearrangement responsible for the creation of the β-methoxyacrylate toxophore. Finally, two methyltransferases are required to complete the synthesis.

O-Methylation steps during strobilurin and bolineol biosynthesis

“…The vector pTYGS-niaD was used to clone either str2 or str3 (see ESI †). In combination with previously constructed plasmids pTYGS-arg-stPKS, pTYGS-ade-str11-str8-str10 and pTYGS-met-str9, 6 all four plasmids were used to transform A. oryzae strain NSAR1, which harbours four auxotrophic lesions on its genome allowing the introduction of up to four DNA fragments in parallel. [10][11][12] Heterologous expression of str3…”

“…Both compounds were identied by comparing retention time and mass fragmentation to previously isolated standards, followed by isolation and NMR analysis. 6 Compounds 11 and 4 coelute but detailed analysis of the mass spectrum (ES + mode) of the peak eluting at 7. (Fig.…”

“…5 The strobilurin fungicides are estimated to make up 25% of the fungicide market and 6.7% of the total crop protection market (worth $3.4 billion in 2015). 6 More than 20 different strobilurin natural products are known, with the b-methoxyacrylate toxophore as their common structural element. A very close relation of 1 is the formally reduced congener bolineol 4 which was isolated as a cometabolite of strobilurins from Bolinea lutea, an apparent ascomeycete.…”

“…7 The producing fungus was recently renamed as Strobilurus lutea as sequencing data revealed that it is a basidiomycete. 6 Isotopic feeding experiments showed that strobilurin A 1 is a polyketide. 8 The biosynthetic steps to strobilurin A 1 have been reported recently by our group (Scheme 1).…”

“…The rearrangement step is catalysed by the FAD dependent oxygenase Str9 as shown both in vivo and in vitro. 6 In order to complete the biosynthesis to 1, two O-methyltransferases (O-MeT) Str2 and Str3 are required. 6 O-Methyltransferases catalyse the transfer of a methyl group from Sadenosyl-L-methionine to a hydroxyl or carboxyl group of the acceptor molecule to form methyl ether or methyl ester derivatives, respectively.…”