Approaches to Improve the Titers of Natural Products in E. coli

J. Andrew Jones, Wenqin He, Robert J. Linhardt, Mattheos A. G. Koffas 

Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Troy, NY 12180

Metabolic engineering has enabled the intelligent design of microbial strains for the high-titer production of many chemicals ranging from biofuels to high-value pharmaceutical precursors. Here we present several recently published methods used to improve the titers of biologically interesting molecules in E. coli. First, we present our work on the development and optimization of polycultures (three or more strains in co-culture) for the extension of the flavonoid branch pathway in vivo. This technology has enabled, for the first time, the de novo production of flavan-3-ols in E. coli. Utilizing a computationally guided optimization approach, we were able to demonstrate up to a 970-fold improvement over previously published monoculture titers.

Second, we will present our method for combinatorial transcriptional optimization, ePathOptimize. Applying the traditional Design-Build-Test-Learn cycle we were able to fine-tune the genetic optimization of the violacein pathway. Using this approach for genetic optimization, coupled with traditional shake flask fermentation optimization, we were able to achieve titers of 1.8 g/L violacein, a 2.6-fold improvement over previous benchmarks in E. coli. Additionally, we will highlight the power of this technology through optimization of an exogenous methanol utilization pathway in E. coli enabling growth on methanol and, for the first time, flux from methanol to high-value specialty products in vivo.

Finally, we will demonstrate optimized production of the polysaccharide, chondroitin, through modification of transcriptional polycistronic operon configurations of the three-gene production pathway. After scale-up, 2.4 g/L of chondroitin was produced in a dissolved oxygen-stat fed batch bioreactor. Expanding upon this work, efforts to successfully express and purify human chondroitin 4-sulfotransferase (C4ST1) have been fruitful, resulting in high-level conversion of chondroitin to chondroitin sulfate type-A in vitro. These technologies are leading the way towards enabling industrially relevant titers, yields, and productivities for a variety of biologically interesting natural products.