Application of biolgical and chemical approaches to generate new and diverse fungal natural products

Full title: 

Application of biolgical and chemical approaches to generate new and diverse fungal natural products

Author: 

Somoza, Amber Dorothy

Year: 

2012

Reference type: 

Thesis

ISBN/ISSN: 

9781267701909

Abstract: 

Filamentous fungi are prolific producers of bioactive secondary metabolites. Recent genome sequencing reveals fungi harbor more secondary metabolites than are currently known. Exploration of fungal secondary metabolism is more attractive today with recent advancements in genomics and molecular biology. Efficient gene-targeting technology is a powerful tool used to mine the genome for novel secondary metabolites and identify the genes in the biosynthetic pathway. Furthermore, this technology can be applied to engineering pathways to generate unnatural natural products. Integration of biosynthetic engineering with chemical synthesis can introduce greater structural diversity into natural products, a promising avenue for discovering therapeutic drugs. The current work describes strategies that utilize the strengths of biosynthetic engineering and chemical synthesis to generate novel fungal natural products.Genome mining efforts in Aspergillus nidulans revealed the novel azaphilone polyketide, asperfuranone; the first azaphilone with its biosynthetic pathway elucidated. Azaphilone natural products are structurally diverse and exhibit a variety of biological activities. We hypothesized the asperfuranone pathway can be reengineered to synthesize a putative intermediate to the azaphilone natural product, sclerotiorin, and apply chemical synthesis to access additional azaphilone compounds both natural and unnatural. Our strategy uses gene-targeting technology to replace the transcription factor, afoA , promoter with an inducible promoter and to knock out afoD , a gene that encodes for a hydroxylase, with the aim of leveraging the biosynthetic machinery to overproduce the putative intermediate, dimethyloctadiene benzaldehyde. Structural modifications utilizing synthetic chemistry transform the advanced intermediate into unnatural azaphilones in 2-3 steps. They were evaluated for biological activity against lipoxygenase-1 and provided a structure-activity relationship.To illustrate the potential of introducing diversity and generating novel compounds using a single biosynthetic pathway, a domain swapping strategy was applied to the pathway of asperfuranone. Our strategy to replace the loading domain of the native polyketide synthase (PKS), AfoE, with the loading domain of sterigmatocystin PKS would select for a different starter unit and alter the structure of asperfuranone. Identification of the linking regions between domains and utilization of gene-targeting technology allowed for the successfully assembly of the hybrid PKS. The engineered A. nidulans strain produced an unnatural naphthoquinone compound, revealing the hybrid PKS altered the polyketide backbone. Moreover, this work also suggested two domains within the non-reduced PKS are chiefly responsible for controlling chain length of the polyketide. Altogether, a single fungal biosynthetic pathway can be easily manipulated in multiple ways to produce potentially new bioactive metabolites highlighting the utility and value of combining chemical synthesis with biotechnology.