AbstractPenicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics.Escherichia coliATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinantE. coliHB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. TheE. colistrain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: anE. coliATCC 11105 mutant (E. coliG133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity ofE. coliG271. The host strain,E. coliG133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) intranson the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production byE. coliG133, PGA production by its transformant (E. coliG271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation ofE. coliG133 with a pBR322 derivative containing theHindlllfragment of the PGA gene, showed that the performance ofE. coliG271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficientE. coliHB101 derivatives brought some light on the mechanism of regulation of the PGA gene. © 1993 John Wiley&Sons, Inc