Inactivating FruR global regulator in plasmid-bearing Escherichia coli alters metabolic gene expression and improves growth rate

• Published in: Journal of biotechnology Vol. 131; no. 3; pp. 261 - 269
• Main Authors: Ow, Dave Siak-Wei, Lee, Raymond Ming-Yung, Nissom, Peter Morin, Philp, Robin, Oh, Steve Kah-Weng, Yap, Miranda Gek-Sim
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 15-09-2007; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biotechnology; Catabolite repressor/activator; Cell Proliferation; Escherichia coli; Escherichia coli - physiology; Escherichia coli Proteins - biosynthesis; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fructose repressor; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - physiology; Genetic Enhancement - methods; Global transcriptional regulator; Growth rate recovery; Metabolic engineering; Plasmid metabolic burden; Plasmids - genetics; Protein Engineering - methods; Recombinant Proteins - biosynthesis; Repressor Proteins - genetics; Repressor Proteins - metabolism; PBS; Catabolite repressor/activator; Growth rate recovery; Global transcriptional regulator; slpm; qRT-PCR; FruR; PTS; P; Fructose repressor; 2DE; Cra; TCA; Plasmid metabolic burden; LB; fruR cells; Metabolic engineering; WT; Growth rate; Transcription; Escherichia coli; Regulator; Gene expression; Inactivation; Metabolism; Fructose; Optimization; Plasmid; Bacteria; Enterobacteriaceae
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2007.07.508

The introduction of plasmids into
Escherichia coli is known to impose a metabolic burden, which diminishes the growth rate. This effect could arise from perturbation of the central metabolic pathways, which supply precursors and energy for macromolecule synthesis. We knocked out a global regulator of central metabolism, FruR (also called Cra), to assess its phenotypic effect in
E. coli carrying plasmids. During bioreactor runs, a higher specific growth rate of 0.91
h
−1 was observed for the plasmid-bearing fruR knockout (P+ fruR) cells compared to its parental plasmid-bearing wildtype (P+ WT) cells (0.75
h
−1), while both the plasmid-free cells displayed similar growth rates (1.0
h
−1, respectively). To investigate gene expression changes possibly related to the growth rate recovery, quantitative reverse transcriptase PCR and 2DE proteomic studies were performed. In P+ fruR cells, expression of enzymes involved in sugar catabolism, glycolysis and transcription/translation processes were upregulated, while those related to gluconeogenesis, tricarboxylic acid cycle and stress response were downregulated. Our findings demonstrate that the inactivation of FruR global regulator in recombinant
E. coli alters metabolic gene expression and significantly reduces growth retardation from the burden of maintaining a plasmid. This study represents the first attempt to explore the role of a global regulatory gene on plasmid metabolic burden.