Reconstruction of the biosynthetic pathway for the core fungal polyketide scaffold rubrofusarin in Saccharomyces cerevisiae

Abstract

BackgroundFungal polyketides include commercially important pharmaceuticals and food additives, e.g. the cholesterol-lowering statins and the red and orange monascus pigments. Presently, production relies on isolation of the compounds from the natural producers, and systems for heterologous production in easily fermentable and genetically engineerable organisms, such as Saccharomyces cerevisiae and Escherichia coli are desirable. Rubrofusarin is an orange polyketide pigment that is a common intermediate in many different fungal biosynthetic pathways.ResultsIn this study, we established a biosynthetic pathway for rubrofusarin in S. cerevisiae. First, the Fusarium graminearum gene encoding polyketide synthase 12 (PKS12) was heterologously co-expressed with the Aspergillus fumigatus gene encoding phosphopantetheinyl transferase (npgA) resulting in production of YWA1. This aromatic heptaketide intermediate was converted into nor-rubrofusarin upon expression of the dehydratase gene aurZ from the aurofusarin gene cluster of F. graminearum. Final conversion into rubrofusarin was achieved by expression of the O-methyltransferase encoding gene aurJ, also obtained from the aurofusarin gene cluster, resulting in a titer of 1.1 mg/L. Reduced levels of rubrofusarin were detected when expressing PKS12, npgA, and aurJ alone, presumably due to spontaneous conversion of YWA1 to nor-rubrofusarin. However, the co-expression of aurZ resulted in an approx. six-fold increase in rubrofusarin production.ConclusionsThe reconstructed pathway for rubrofusarin in S. cerevisiae allows the production of a core scaffold molecule with a branch-point role in several fungal polyketide pathways, thus paving the way for production of further natural pigments and bioactive molecules. Furthermore, the reconstruction verifies the suggested pathway, and as such, it is the first example of utilizing a synthetic biological “bottom up” approach for the validation of a complex fungal polyketide pathway.

Fusarium Mycotoxins and Their Role in Plant–Pathogen Interactions

“…The biosynthetic pathway, depending on the PKS12 gene and proceeding via nor-rubrofusarin, which is dimerized by a laccase, was elucidated in F. graminearum and F. pseudograminearum [137][138][139]. It was recently partly reconstituted in yeast [140]. Extracellular reduction potential is necessary for the extracellular dimer formation [141].…”

Genome Mining for Fungal Secondary Metabolic Gene Clusters

“…From an engineering perspective, a great deal of information has already been accumulated regarding the structure and function of PKS/NRPS enzymes, with numerous recent reports highlighting the potential for engineering PKSs and also NRPSs from Ascomycetes such as Fusarium and Aspergillus [92,93] and Basidiomycetes such as Ustilago maydis and Suillus grevillei [15,17]. Ma et al [92] conducted a detailed characterization of the lovastatin nonaketide synthase LovB; a highly reducing PKS catalyzing the production of dihydromonacolin L. Extensive in vitro analyses, as well as production from Saccharomyces cerevisiae and substrate feeding experiments provided the authors with a detailed understanding of LovB structure and function.…”

Quantifying intracellular metabolites in yeast using a matrix with minimal interference from naturally occurring analytes

“…Thus, determining the intracellular metabolite pool sizes helps in defining the metabolic state of the cell and in identifying the bottlenecks in precursor supply, as possible targets for metabolic engineering. In that respect, Aspergillus and Saccharomyces are widely used cell factories for production of polyketide-based secondary metabolites [4,5]. Polyketides are biosynthesized from acetyl coenzyme A (Ac-CoA), 1 malonyl coenzyme A (Mal-CoA), and NADPH.…”