Epathoptimize: A Combinatorial Approach for Transcriptional Balancing of Metabolic Pathways

Recent advancements in synthetic biology give scientists the ability to balance a variety of factors involved in gene regulation to improve product titers, yields, and productivities. Using a library of isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible mutant T7 promoters of varied strength, a combinatorial method was developed for transcriptional balancing of the violacein pathway. Violacein is a potent bioactive compound that has been shown to have various antitumor, antifungal, and anticancer properties. The violacein pathway is also an excellent model for exploratory metabolic engineering efforts into pathway regulation and control due to many colorful intermediate and side products allowing for easy analysis and strain comparison. Upon screening approximately 4% of the initial library, several high-titer mutants were discovered that resulted in up to a 63-fold improvement over the control strain. With further fermentation optimization, titers were improved to 1829±46 mg/L, a 2.6-fold improvement in titer and a 30-fold improvement in productivity over previous literature reports. Future applications of this technology towards improving the microbial production of biofuels, pharmaceuticals, and commodity chemicals could expedite the transition from the laboratory to the marketplace.