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 b5 in the bacterial host E. coli N4830-1. The model system studied involved the cyt b5 gene being introduced on plasmids into the host with varying copy numbers (0-6) under a λPL 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 b5 gene among the examined bacterial strains. Results: Our study shows that CYT b5 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 b5), 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 b5 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.