P135: Metabolic engineering of E. coli to minimize byproduct formate during succinate production by heterologous overexpression of a NAD+-dependent formate dehydrogenase

Succinate has been recognized by the US Department of Energy as one of the top 12 building block chemicals. Various succinate producing microorganisms and processes have been reported.  However, most of the current strains show byproduct formation (formate, acetate, pyruvate, malate, etc.) that contributes to an increase in the cost of succinate purification. Previously, we constructed E. coli SBS550MG(pHL413Km) for producing high succinate with a maximum theoretical yield of 1.6 mol/mol. This strain also produces about 15 mM of byproduct formate along with succinate. In this study, we describe reengineering of SBS550MG(pHL413Km) to minimize byproduct formate through heterologous overexpression of a NAD⁺-dependent formate dehydrogenase of Candida boidinii that coverts 1 mole of formate into 1 mole of NADH and CO₂. The new strain retains the reducing power of formate resulting in a more robust strain with a much faster average succinate production rate and reduced byproduct formate. The functionality of the newly engineered strain was demonstrated in anaerobic shake flask fermentations and bioreactor cultures. In an anaerobic fed-batch bioreactor process, succinate productivity and byproduct formate concentration of the parent strain SBS550MG(pHL413Km) were 1 g/L/h and 17 mM, respectively. Whereas, the succinate productivity and byproduct formate concentration of the reengineered strain SBS550MG(pHL413KF1) were 2 g/L/h and 0-3 mM, respectively. Moreover, we also demonstrated an increase in succinate yield using externally supplemented formate in shake flask fermentations. Reduced levels of byproduct formate in succinate fermentation broth would significantly reduce the cost associated with downstream processing.