Metabolic Profiling Analysis of Cytochrome B5 Production in E. Coli N4830-1 Using GC-MS

Introduction: Recombinant protein production is pivotal across diverse industries, necessitating efforts to enhance both quantity and quality. Objectives: We employed gas chromatography coupled with mass spectrometry (GC-MS) to investigate the metabolic effects of producing mammalian cytochrome b ₅ in the bacterial host E. coli N4830-1. The model system studied involved the cyt b ₅ gene being introduced on plasmids into the host with varying copy numbers (0-6) under a λP _L heat-sensitive promoter. Methods: Metabolic profiling involved GC-MS analysis with quality assessments using pooled QCs and multivariate chemometric analysis. This approach revealed specific metabolic features (identified to Level 2 of the Metabolomics Standards Initaive) correlating significantly with different cultivation conditions and variable copy numbers of the cyt b ₅ gene among the examined bacterial strains. Results: Our study shows that CYT b ₅ production imposed a substantial energetic burden, reflected by depletion of metabolites associated with the tricarboxylic acid (TCA) cycle, glycolysis/gluconeogenesis, pentose phosphate pathway, and glyoxylate metabolism, indicating increased ATP demand. Concurrent reductions in unsaturated fatty acids and changes in lipid-related metabolites suggest membrane remodeling in response to temperature induction (needed to initiate production of CYT b ₅ ), as well as metabolic stress. Alterations in glyoxylate shunt and pentose phosphate pathway intermediates further indicate metabolic reprogramming to maintain carbon homeostasis, while significant changes in nucleotide- and amino acid-related metabolites suggest impacts on biosynthetic capacity associated with recombinant expression and gene copy number variation. Conclusion: Overall, these findings demonstrate that CYT b ₅ production induces coordinated metabolic adjustments affecting energy-related metabolic pathways, bacterial cell wall and membrane biosynthesis, nucleotide metabolisms and bacterial stress responses, providing valuable insights that may be useful for the optimisation of recombinant protein production processes.