Isolation of mutants of Escherichia coli lacking NAD- and NADP-linked malic enzyme activities

When Escherichia coli utilizes C₄-dicarboxylic acids as sole sources of carbon and energy for growth, phosphoenolpyruvate is synthesized by means of phosphoenolpyruvate carboxykinase or by sequential action of malic enzyme and phosphoenolpyruvate synthetase. Chemical mutagenesis of a strain of E. coli K-12 lacking phosphoenolpyruvate carboxykinase activity yielded a mutant which grows very slowly on malate-mineral medium and lacks NAD-linked malic enzyme activity as well as phosphoenolpyruvate carboxykinase activity. A revertant of this double mutant which possesses elevated levels of NADP-linked malic enzyme activity grows well on malate-mineral medium. Further mutagenesis of this revertant produced a mutant which grows very slowly on malate-mineral medium and lacks NADP-linked malic enzyme activity as well as phosphoenolpyruvate carboxykinase and NAD-linked malic enzyme activities.