Molecular cloning and over-expression of the glyoxylate bypass operon from Escherichia coli ML308

A recombinant plasmid carrying an 11 kb restriction-endonuclease-ClaI fragment of genomic DNA from Escherichia coli ML308 was constructed. This plasmid complements an aceA mutation. The plasmid encodes the structural genes of the glyoxylate bypass operon, namely malate synthase A (aceB), isocitrate lyase (aceA) and isocitrate dehydrogenase kinase/phosphatase (aceK), as judged by overexpression of enzyme activities and transcription/translation experiments in vitro. Subcloning confirmed that expression of the aceK gene is essential for growth on acetate.