Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants

Several biotechnological processes rely on the utilization of high-copy-number plasmids for heterologous gene expression, and understanding the interactions between plasmid DNA and bacterial hosts is highly relevant for bioprocess optimization. We assessed metabolic modifications and physiological changes exerted by expression of a plasmid-encoded alcohol-acetaldehyde dehydrogenase from Leuconostoc mesenteroides (adhE ( Lm )) in Escherichia coli redox mutants.

Plasmid pET( Lm ), a pBluescript II KS(-)-derivative carrying adhE ( Lm ), was introduced in E. coli CT1061 [arcA creC(Con)]. This recombinant was able to attain a higher ethanol concentration in glycerol cultures compared to the parental strain. pBluescript II KS(-) was rapidly lost in 72-h bioreactor cultures (7.8 +/- 1.2% of plasmid-bearing cells), while pET( Lm ) was present in 92.4 +/- 7.2% of the cells.

In E. coli CT1061 carrying pBluescript II KS(-) the plasmid copy number steadily diminished in bioreactor cultures to reach 334 +/- 45 copies per chromosome at 72 h, while pET( Lm ) was stably maintained, reaching 498 +/- 18 copies per chromosome at the end of the cultivation. Plasmid pETOmega( Lm ), bearing a defective copy of adhE ( Lm ) interrupted by cat, reached 293 +/- 62 copies per chromosome, implying a functional role of adhE ( Lm ) on plasmid maintenance.

The intracellular NADH/NAD(+) content suggest that regeneration of oxidized co-factors by the heterologous bioreaction might play a relevant role in plasmid maintenance.