Studies of the Production of Fungal Polyketides in Aspergillus nidulans by Using Systems Biology Tools

Abstract

Many filamentous fungi produce polyketide molecules with great significance as human pharmaceuticals; these molecules include the cholesterol-lowering compound lovastatin, which was originally isolated from Aspergillus terreus. The chemical diversity and potential uses of these compounds are virtually unlimited, and it is thus of great interest to develop a well-described microbial production platform for polyketides. Using genetic engineering tools available for the model organism Aspergillus nidulans, we constructed two recombinant strains, one expressing the Penicillium griseofulvum 6-methylsalicylic acid (6-MSA) synthase gene and one expressing the 6-MSA synthase gene and overexpressing the native xylulose-5-phosphate phosphoketolase gene (xpkA) for increasing the pool of polyketide precursor levels. The physiology of the recombinant strains and that of a reference wild-type strain were characterized on glucose, xylose, glycerol, and ethanol media in controlled bioreactors. Glucose was found to be the preferred carbon source for 6-MSA production, and 6-MSA concentrations up to 455 mg/liter were obtained for the recombinant strain harboring the 6-MSA gene. Our findings indicate that overexpression of xpkA does not directly improve 6-MSA production on glucose, but it is possible, if the metabolic flux through the lower part of glycolysis is reduced, to obtain quite high yields for conversion of sugar to 6-MSA. Systems biology tools were employed for in-depth analysis of the metabolic processes. Transcriptome analysis of 6-MSA-producing strains grown on glucose and xylose in the presence and absence of xpkA overexpression, combined with flux and physiology data, enabled us to propose an xpkA-msaS interaction model describing the competition between biomass formation and 6-MSA production for the available acetyl coenzyme A.Natural products from microbial, plant, marine, and even mammalian sources have traditionally been a major drug source and continue to play a significant role in today's drug discovery environment. Approximately 40% of new drugs introduced between the mid-1980s and 1990s originated from natural molecules, whereas 9 of the top 20 small-molecule drugs introduced in 1999 were developed from natural products (13). Polyketide natural products play an important role in the treatment of a wide range of human physiological disorders. They are widely used as antimicrobial (erythromycin, rifamycin, tetracycline), antifungal (amphotericin B), immunosuppressant (tacrolimus [TK506], rapamycin), and anticancer (doxorubicin, epothilone, geldanamycin) agents, as well as cholesterol-lowering agents (lovastatin). Current chemical technologies are in most cases unable to generate commercial quantities of most polyketide compounds. As a result, the natural sources of these compounds, usually bacteria and fungi, are employed as biocatalysts for large-scale production. Additionally, in the past two decades heterologous expression of polyketide genes has become possible by the explosive growth in the cloning and seq...

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