Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• Phosphoenolpyruvate (PEP) is crucial for anaerobic succinate and malate production.
• Inactivating PEP/carbohydrate phosphotransferase systems (PTS) increases PEP but reduces glucose utilization.
• Enhancing glucose uptake is key for efficient anaerobic fermentation.

Purpose of the Study:

• To improve anaerobic glucose utilization rate for efficient succinate and malate production in Escherichia coli.
• To investigate the combinatorial effect of galactose permease (galP) and glucokinase (glk) gene expression modulation.
• To develop engineered regulatory parts for precise gene expression control.

Main Methods:

• Constructed libraries of artificial regulatory parts (promoters, mRNA stabilizing regions) using λ-Red recombination.
• Modulated galP and glk gene expression in a PTS-inactivated E. coli strain using a one-step recombination method.
• Evaluated glucose utilization rates under anaerobic conditions.

Main Results:

• Individual modulation of galP or glk increased glucose utilization rates, with rates positively correlating to gene expression strength.
• Combinatorial modulation of galP and glk exhibited a synergistic effect, significantly boosting glucose utilization.
• The highest achieved glucose utilization rate was 1.64 g/L/h, a tenfold increase over the PTS(-) strain.

Conclusions:

• Engineered regulatory parts enable effective modulation of galP and glk expression.
• Synergistic enhancement of glucose utilization is achievable through combinatorial gene expression strategies.
• The developed E. coli strains are promising for efficient anaerobic production of succinate and malate.