Scale Up of a Novel Method to Maximize Malonyl-CoA in Escherichia Coli

The instability of oil’s price along with its limited availability and impact on the environment motivate the search for an alternative feedstock that can sustain the profitability of chemical companies. Industrial biotechnology can promote renewable sources of energy and products by using microorganisms to produce a wide range of chemical compounds. The three-carbon metabolite, malonyl-CoA, can serve as a precursor to a variety of industrial chemicals. The major hurdle with using malonyl-CoA in industry is that its intracellular concentration in Escherichia Coli is very low. Previous attempts to increase the intracellular level of malonyl-CoA have ranged from genetic engineering of proteins involved in fatty acid biosynthesis to adding inhibitors of enzymes. Tatiana Mello, a graduate researcher at Louisiana State University, recently developed a clean, low-cost method that increases the level of malonyl-CoA in E. Coli [1]. At 5 mL, the method produced flaviolin, and indicator of malonyl-CoA at 68 mM/gWW with a titer of 1.053 g/L. The first goal of this project was to scale up the method by three orders of magnitude from a shake flask (milliliters) to a benchtop bioreactor (liters). At the 5L scale, E. Coli produced 74.1 mM/gDCW of flaviolin with a titer of 0.04 g/L. Compared to the production and titer at the 5 mL scale, the 5 L experiments showed similar target chemical production per unit biomass, but significantly lower target chemical concentration per batch. This suggests that the individual cells are producing similar amounts of the target chemical, but that the cells are not reproducing to similar concentrations. Another interesting finding at the 5 mL scale was the effect of CO2 concentration on malonyl-CoA production. Unpublished findings by Mello, at the 5mL scale, showed that an optimum concentration of approximately 1.6 mM CO2 exists where flaviolin production is significantly increased. This trend was investigated at the 5 L scale, but was not observed. The production of industrial chemicals is the end goal of this work, so experiments were also done to produce 3-hydroxypropionic acid (3-HP), a precursor to acrylics, from E. Coli. Qualitative results from thin layer chromatography were promising for this application of the method