Production of secondary metabolites from acetyl Co-A precursors in bacterial and fungal hosts

This dissertation investigates two classes of secondary metabolites: isoprenoids and polyketides.;In Part 1, we consider the heterologous production of a plant isoprenoid---the malarial drug precursor amorphadiene---in the bacterium Escherichia coli. Metabolic flux analysis is used to investigate the changes to E. coli's metabolism resulting from the incorporation of a non-native pathway that draws on E. coli's supply of acetyl-CoA to produce amorphadiene. We have built a system for flux analysis that involves (1) compilation of a biochemical network representing the central metabolism and heterologous pathways in E. coli; (2) chemostat cultivation of the strains; (3) measurements of biomass components and secreted metabolites; (4) isotopomer measurements using 13C-labeled glucose as a carbon source; and (5) a solving program to calculate intracellular metabolic fluxes.;In general, the strain engineered to produce amorphadiene demonstrates decreased fluxes to protein, lipids, acetate, and carbohydrate relative to the control strain carrying only empty vectors. This may mean that acetyl-CoA is being redirected from lipid biosynthesis and acetate secretion to amorphadiene production in the amorphadiene producing strain, or it may be that the amorphadiene producer is growing at a slower rate than the control strain because of the burden of heterologous protein production or the toxicity of prenylphosphate intermediates. The observed differences may simply be the expected growth-rate-dependent differences.;The fatty acid distribution is different between the amorphadiene producer and the control strain. Cyclopropane fatty acids are produced at very low levels in the amorphadiene producer relative to the control strain. The reason for this difference is unknown. It may be that S-adenosyl-L-methionine becomes limiting in the amorphadiene producing strain as a result of increased demand for tRNA methylation. Alternatively, it may be that amorphadiene, a hydrocarbon, affects the fluidity of the cell membrane, and the cell changes its fatty acid profile to compensate. It may also be a growth-rate-dependent observation. Preliminary microarray studies supporting these observations are discussed.;In Part 2, we consider the production of norsolorinic acid (NOR), a precursor to the toxic, carcinogenic polyketide, sterigmatocystin (ST), in the filamentous fungus Aspergillus nidulans. (Abstract shortened by UMI.)...