A GTP-driven central carbon metabolism in the cellulolytic bacterium Ruminiclostridium cellulolyticum

Area of Science:

• Microbiology
• Biochemistry
• Metabolic Engineering

Background:

• The anaerobic bacterium Ruminiclostridium cellulolyticum exhibits unusual preferences for guanosine triphosphate (GTP) over adenosine triphosphate (ATP) in key enzymes like hexokinase and galactokinase.
• Phosphofructokinase in this bacterium is pyrophosphate-dependent, further suggesting a non-canonical central carbon metabolism pathway.

Purpose of the Study:

• To characterize the nucleotide preferences of all kinases in the central carbon metabolism pathway of R. cellulolyticum.
• To elucidate the primary energy currency driving this bacterium's core metabolic processes.
• To investigate the metabolic flexibility and potential for engineering this pathway.

Main Methods:

• Biochemical characterization of individual kinases involved in central carbon metabolism.
• Determination of preferred nucleoside triphosphate (NTP) or nucleoside diphosphate (NDP) substrates for each enzyme.
• In vivo reciprocal exchange experiments involving GTP-dependent hexokinase and ATP-dependent glucokinase between R. cellulolyticum and Escherichia coli.

Main Results:

• Kinases in the initial steps of the pathway, which consume NTPs, were found to be GTP-dependent.
• Kinases in downstream steps, which generate NTPs, showed no significant nucleotide preference.
• Cellular GTP levels in R. cellulolyticum are comparable to ATP levels, supporting a GTP-driven metabolism.
• Engineered strains with exchanged hexokinases maintained glucose and disaccharide catabolism, demonstrating pathway adaptability.

Conclusions:

• The central carbon metabolism of R. cellulolyticum is predominantly driven by GTP, not ATP.
• This finding highlights unexpected diversity and flexibility in bacterial central metabolic pathways.
• The study provides a basis for metabolic engineering of anaerobic bacteria.