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

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Physiology

Background:

• Engineered Escherichia coli (E. coli) strains are developed for mixotrophic growth, utilizing both organic carbon and CO2.
• Initial Rubisco-based E. coli strains faced limitations in CO2 recycling due to pyruvate metabolism and insufficient NAD+ regeneration.
• The pflB-mediated ethanol production pathway was identified as a bottleneck for efficient NAD+ regeneration.

Purpose of the Study:

• To enhance in situ CO2 recycling in Rubisco-based E. coli for improved mixotrophic growth.
• To overcome limitations in NAD+ regeneration by bypassing the pflB-mediated ethanol production pathway.
• To increase fermentation product yield by simultaneously utilizing glucose and CO2.

Main Methods:

• Construction of a Rubisco-based engineered E. coli strain (MZLFB) with heterologous phosphoribulokinase (Prk) and Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).
• Introduction of a recombinant plasmid pLOI295 encoding pyruvate decarboxylase and alcohol dehydrogenase II (Pdc-based carbon tap valve, CTV) into E. coli MZLFB.
• In silico analysis to determine metabolic flux distribution and experimental measurement of CO2 consumption and ethanol production rates.

Main Results:

• The introduction of the Pdc-based CTV into E. coli MZLFB significantly increased the C-2/C-1 ratio from 1.0 ± 0.1 to 1.6 ± 0.1, indicating enhanced CO2 recycling.
• The engineered strain MZLFB + CTV achieved a fermentation product yield exceeding the theoretical yield, reaching 2.2 ± 0.0 (mol/mol).
• In silico analysis revealed that 61% of glucose consumption proceeded through the Rubisco-based pathway with the CTV, alongside an average CO2 consumption rate of 55.3 mg/L·h and ethanol production rate of 144.8 mg/L·h.

Conclusions:

• The Pdc-based CTV effectively enhances in situ CO2 recycling and NAD+ regeneration in Rubisco-based engineered E. coli.
• Simultaneous utilization of glucose and CO2 through the engineered pathway leads to significantly improved fermentation yields.
• The Rubisco-based pathway coupled with the Pdc-based CTV serves as a crucial energy sink for intracellular energy balance during mixotrophic growth.