Exceeding the theoretical fermentation yield in mixotrophic Rubisco-based engineered Escherichia coli

Rubisco-based engineered Escherichia toll MZLFB (E. coli BL21(DE3) Delta zwf, Delta ldh, Delta frd) containing heterologous phosphoribulokinase (Prk) and Ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was constructed for the mixotrophic growth. However, in situ CO2 recycling was hindered by clogs of pyruvate during glucose metabolism, which consequently resulted in an insufficient regeneration of NAD(+) through the pflB-mediated ethanol production. Recombinant plasmid pLOI295 (encodes pyruvate decarboxylase and alcohol dehydrogenase II, referred to as the Pdc-based carbon tap valve (CTV) for convenience) was introduced into E. coli. MZLFB CTV to bypass the pflB-mediated ethanol production. Results show that while the C-2/C-1 ratio (i.e., the molar ratio of ethanol and acetate to formate and total CO2) for parental strain MZLFB was 1.0 +/- 0.1, the C-2/C-1 for MZLFB + CTV increased to 1.6 +/- 0.1. This indicates that the Pdc-based CTV enhanced the performance of in situ CO2 recycling. By simultaneously utilizing glucose and CO2, the fermentation product yield of MZLFB + CTV exceeded the normal theoretical yield and reached 2.2 +/- 0.0 (mol/mol). In silico analysis shows that 61% of the glucose consumption went through the Rubisco-based engineered pathway when the CTV was equipped. Also shown are the average CO2 consumption rate of 55.3 mg L-1.h(-1) and an average ethanol production rate of 144.8 mg L-1.h(-1). The conversion of CO2 to ethanol through the Rubisco-based engineered pathway and the Pdc-based carbon tap valve is important for mixotrophic growth, since these two modules serve as the energy sink to achieve intracellular energy balance. Also, during mixotrophic growth, ATP production from a certain percentage (39% in this study) of the EMP pathway activity is needed for mixotrophic growth.