Aryl-Aldehydes in Fungal Polyketides: Discovery and Characterization of Novel Biosynthesis Pathways

Fungal polyketides are an important class of natural products, which leads to the development of numerous pharmaceuticals such as blockbusting drug lovastatin. Genome sequencing of various fungal strains reveals that each strain contains many more polyketide and/or non-ribosomal peptide (NRP) biosynthesis pathways than what has been reported. These unknown or “cryptic” pathways present both great opportunities and challenges. An aryl-aldehyde is a common feature in fungal polyketides. It can be retained in the final product such as 3-(2,4-dihydroxy-6-methylbenzyl)-orsellinaldehyde, or serves as an active intermediate for further modifications like in asperfuranone biosynthesis. On the genetic and biochemical level, all previously characterized aryl-aldehydes in fungal polyketides are generated via a reducing domain (R domain), which is attached to the end of a non-reducing polyketide synthase (NR-PKS). However, we discovered and characterized a second mechanism for generating aryl-aldehydes via cloning and heterologous expression of NR-PKS and NRPS-like genes in Saccharomyces cerevisiae. We first cloned and expressed NR-PKS (ATEG_03629) and NRPS-like protein (ATEG_03630) from Aspergillus terreus. We discovered that ATEG_03629 alone can produce 2,4-dihydroxy-5,6-dimethyl benzoic acid and the ATEG_03629_03630 two genes cassette can produce 2,4-dihydroxy-5,6-dimethyl benzaldehyde. Because NRPS-like gene contains an activation domain (A domain), an ACP domain and a R domain, we proposed that 2,4-dihydroxy-5,6-dimethyl benzoic acid produced from NR-PKS is first activated and attached to the ACP domain via adenylation, and then reduced by the R domain to generate aryl-aldehydes. Further genome mining of all sequenced fungal strains shows that such NR-PKS and NRPS-like pairs can be found in over 20 different fungal strains, which indicates such novel mechanism is common and highly conserved in all fungi. Therefore, we expanded our cloning and expression effort towards three additional similar gene clusters from Aspergillus nidulans, Aspergillus terreus and Penicillium marneffei. Our results show that the same mechanism was also used in these gene clusters, although products with different methylation patterns on the aromatic ring are produced from different pathways.