Metabolic engineering of Escherichia coli for the production of plant phenylpropanoid derived compounds

More than 200,000 plant natural products (PNPs) are currently known. As many of them have favorable effects in the treatment of various diseases such as cancer, diabetes and heart diseases, PNPs are of high interest for the development of new pharmaceutical drugs. A major obstacle during drug development is the limited availability of most PNPs. In comparison to purification from the respective plant sources or costly chemical synthesis, the microbial production of these compounds could provide sufficient quantities from inexpensive substrates. The main goal of this thesis was the development of a microbial production platform for the synthesis of pinosylvin, a stilbene found in the heartwood of pine trees (e. g. Pinus strobus and Pinus sylvestris) in Escherichia coli from L-phenylalanine. The first step in the biosynthetic pathway is the deamination of L-phenylalanine to trans-cinnamic acid, which is catalyzed by a phenylalanine ammonia lyase. The resulting acid is subsequently activated by a 4-coumarate-CoA ligase yielding the coenzyme A thioester trans-cinnamoyl-CoA. A stilbene synthase catalyzes the successive condensation of three malonyl-CoA molecules with transcinnamoyl- CoA and the cyclization of the resulting linear tetraketide intermediate to form the stilbene pinosylvin. Initially, two different variants for each of the three enzymes towards pinosylvin were selected on the basis of their biochemical characteristics. After the construction of several pathway variants and optimization of gene expression, pinosylvin concentrations of up to 3 mg/L were detected by HPLC-MS analysis of ethyl acetate extracts from culture supernatants, showing that the plant pathway was functional in E. coli. Analysis of precursor availability and a comparative analysis of intracellular levels of pathway intermediates and product identified limited malonyl-CoA availability and low activity of the pine-derived stilbene synthase as key bottlenecks. By increasing malonyl-CoA availability through addition of the fatty acid synthase inhibitor cerulenin and by in vivo evolution of the stilbene synthase from Pinus strobus for enhanced activity in E. coli, the pinosylvin titer could be increased up to 70 mg/L. A further increase up to 91 mg/L was achieved by supplementation of the precursor L-phenylalanine to the medium.