Metabolic profiling analysis of cytochrome b₅ production in E. coli N4830-1 using GC-MS

Recombinant protein production is pivotal across diverse industries, necessitating efforts to enhance both quantity and quality.

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 λP_L heat-sensitive promoter.

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 Initiative ) correlating significantly with different cultivation conditions and variable copy numbers of the cyt b₅ gene among the examined bacterial strains.

Our study shows that CYT b₅ production imposed a substantial energetic burden, reflected by depletion of metabolites associated with the tricarboxylic acid 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.

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.