Targeted cofactor quantification in metabolically engineered E. coli using solid phase extraction and hydrophilic...

Area of Science:

• Metabolic Engineering
• Microbial Physiology
• Analytical Chemistry

Background:

• Accurate quantification of cellular energy and redox cofactors is crucial for understanding and optimizing engineered microbial strains.
• Current methods for intracellular cofactor measurement often lack comprehensive coverage, reproducibility, sensitivity, or specificity.

Purpose of the Study:

• To develop and validate a novel SPE-HILIC/MS approach for the simultaneous determination of six key cofactors (ATP, ADP, NAD, NADH, NADP, NADPH) in Escherichia coli.
• To compare the performance of the new approach against conventional enzymatic assays.
• To apply the method for profiling cofactor balance shifts in engineered E. coli strains with varying isobutanol production.

Main Methods:

• Solid-phase extraction (SPE) coupled with hydrophilic interaction liquid chromatography-mass spectrometry (HILIC/MS).
• Simultaneous quantification of adenosine triphosphate (ATP), adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), and their reduced forms (NADH, NADPH).
• Analysis of polar metabolites involved in central carbon metabolism.

Main Results:

• The SPE-HILIC/MS approach demonstrated excellent linearity, precision, and metabolite recovery, confirming its reliability for targeted cofactor quantification.
• The new method outperformed conventional enzymatic assays in terms of sensitivity and specificity.
• Analysis of engineered E. coli strains revealed that optimal energy fitness and highest isobutanol yield were associated with specific cofactor balances modulated by transhydrogenase and NAD(+) kinase activity.
• The SPE enrichment step also enabled the identification of 39 additional groups of polar metabolites.

Conclusions:

• The developed SPE-HILIC/MS method provides a robust and superior alternative for simultaneous intracellular cofactor quantification in microbial systems.
• This approach offers valuable insights into metabolic flux and energy balance in engineered strains, facilitating strain improvement.
• The study highlights the importance of cofactor balance in optimizing metabolic pathways for enhanced production of biofuels like isobutanol.