Enzyme-Assisted Microbial Electrosynthesis of Poly(3-hydroxybutyrate) via CO2 Bioreduction by Engineered Ralstonia eutropha

Microbial electrosynthesis (MES) is a promising technology to reduce carbon dioxide using inward electron transfer mechanisms to synthesize value-added chemicals with microorganisms as electrocatalysts and electrons from cathodes as reducing equivalents. To enhance CO2 assimilation in Ralstonia eutropha, a formate dehydrogenase (FDH) assisted MES system was constructed, in which FDH catalyzed the reduction of CO2 to formate in the cathodic chamber. Formate served as the electron carrier to transfer electrons derived from cathodes into R eutropha. To enable efficient formation of formate from CO2, neutral red (NR) was used to facilitate the extracellular regeneration of NADH, the cofactor of FDH. Meanwhile, NR also played an essential role as electron shuttle to directly deliver electrons from cathodes into R. eutropha to increase the level of intracellular reducing equivalents, thus facilitating the efficiency of MES. On the other hand, the Calvin Benson Bassham (CBB) cycle was further engineered by the heterologous expression of the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in R eutropha, which strengthened the CBB pathway for CO, fixation. Upon application of the cathode potential at -0.6 V (vs Ag/AgCI) in the MES system with the genetically engineered R. eutropha, 485 +/- 13 mg/L poly(3-hydroxybutyrate) (PHB) was obtained, which was similar to 3 times that synthesized by the control (165 +/- 8 mg/L), i.e., the wild-type R. eutropha in the absence of FDH and NR.