From Biofuels to Natural Products: Microbial Metabolic Engineering for Chemical Production

This work covers three metabolic engineering projects falling into the general categories of pathway optimization, strain optimization, and pathway construction. The first project explores the functionality of isopropyl beta-D-1-thiogalactopyranoside (IPTG) inducible promoters for the control of heterologous gene expression in the freshwater cyanobacterium Synechoccocus elongatus PCC 7942 (7942). Initial experiments for the project revealed that the occurrence of unrepressed gene expression in the absence of an inducer was actually caused by the particular pathway genes of the previously established 2,3-butanediol (23BD) production pathway rather than by the choice of promoter itself. Tight control of heterologous gene expression for the production pathway was successfully achieved via the rearrangement of operon order to remove the effect of hidden constitutive promoters within pathway genes.;This same 23BD production pathway was adapted for use in the marine cyanobacterium Synechoccocus sp. PCC 7002 (7002) in the second project described here. 7002 has attracted much interest in the metabolic engineering community as a new host strain due to its rapid growth rate and increased tolerance to heat, light, and salt. After screening different systems for control of inducible gene expression, gene arrangements, and culturing conditions, production of 23BD was achieved in 7002 for the first time with a titer of 1.6 g/L after 16 days.;The final project described here outlines the design and beginnings of the construction of an engineered biosynthetic pathway for the production of the tropane alkaloid scopolamine in the model host organism Escherichia coli. Scopolamine is a muscarinic antagonist in high worldwide demand for a range of treatments, but which suffers from a limited supply from the traditional natural product sources of plant extraction and total chemical synthesis. In this new engineered pathway design the starting materials for the scopolamine ester are derived from both chemical and biological syntheses in an effort to draw from the strengths of each strategy. The bulk of the work done for this project concerned the activation of a critical pathway intermediate into a coenzyme A thioester. Given the lack of a previously characterized enzyme for this step, work was done to screen and design an enzyme capable of doing so. Despite several rounds of screening a new enzyme was not found, but the work done nevertheless has set a precedent for future endeavors in this area.