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

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Abstract 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 Initiative ) 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 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.

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Related topics: Microbial Metabolic Engineering and Bioproduction · Bacterial Genetics and Biotechnology · Cancer Research and Treatments