mRNA stability and plasmid copy number effects on gene expression from an inducible promoter system

Abstract

The effects of mRNA stability and plasmid copy number on gene expression in Escherichia coli were evaluated by constructing multicopy (pMB1-based) and low-copy (F-based) plasmids containing an arabinose-inducible promoter system, the lacZ reporter gene, and mRNA-stabilizing 5′ hairpin structures. Product formation and cell growth were evaluated under a number of inducer concentrations. The introduction of a 5′ hairpin into the untranslated region of the mRNA resulted in significantly higher gene expression from the multicopy plasmids at low inducer concentrations and increased gene expression from the low-copy plasmids across all inducer concentrations investigated. With high inducer concentrations, expression from high-copy plasmids significantly slowed cell growth, whereas expression from the low-copy plasmids had little effect on growth rate. At inducer concentrations between 1 × 10⁻⁴ and 4 × 10⁻⁴%, the productivity of low-copy plasmids containing the 5′-hairpin was equal to or greater than that from multicopy plasmids. Together, these two gene expression strategies may find important use in metabolic engineering and heterologous gene expression. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:666–672, 1998.