Metabolic engineering for production of valuable chemicals based on Escherichia coli strains designed in silico

Xiaolin Zhang, Cellular and Molecular Biology Graduate Program, University of Wisconsin - Madison, Madison, WI and Jennifer L. Reed, Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI

Optimizing production of a specific chemical usually involves increasing synthesis of the precursors of the chemical in bacteria. Pyruvate is a starting compound for synthesizing a variety of biofuels and chemicals. A high-yield pyruvate producing strain has great potential for creating strains to produce a variety of valuable chemicals. Guided by a genome-scale metabolic model of Escherichia coli, we identified different strategies for enhancing the production of pyruvate from glucose. The targeted gene deletions minimize acetyl-CoA production, undesired product (acetate and lactate) formation, and NAD(P)H formation. We constructed a number of strains and six of them achieved yields of more than 0.88 g pyruvate per g of glucose (90% theoretical yield) under aerobic conditions.
Pyruvate is a precursor for synthesis of ethanol. To produce ethanol, pyruvate formate-lyase (PflB) was deleted, and pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (AdhB) from Zymomonas mobilis were over- expressed in the engineered pyruvate strains. These genetically modified strains fermented glucose to ethanol with a yield of 0.42 g ethanol per g of glucose (~80% of theoretical yield).
In addition to producing native metabolites in E. coli, pyruvate can be used to make non-native metabolites when the necessary enzymes are expressed in E. coli to enable their synthesis. For this purpose, we computationally identified what non-native metabolites could be made from pyruvate and which exogenous reactions need to be added. The developed high-producing pyruvate strains can be subsequently adapted to generate strains capable of producing other important chemicals.