Construction and expression of hybrid plasmids containing the Escherichia coli glyA genes

The Escherichia coli glyA gene, encoding serine transhydroxymethylase (STHM), has been cloned in the plasmid vector pACYC184. The recombinant plasmid (pGS1) contains a 13 kb EcoRI insert. Genetic and biochemical experiments indicate that the region controlling STHM synthesis is present on the insert. Strains bearing multi-copy plasmid vectors carrying the glyA gene overproduce the enzyme from 17- to 26-fold. The glyA gene was identified on the insert by analyzing a set of plasmids derived from pGS1 that carry random insertions of the transposable kanamycin resistance element Tn5. Cloning of segments of the original insert into the plasmid pBR322 established that a 2.5 kb SalI-BclI fragment carries the glyA gene. A physical map of this fragment is presented.