Abstract: Site directed mutagenesis was performed at the start codon ATG of the regulatory gene of the penicillin G acylase operon ( pacR ) in E.coli HB101 (pPA6). The CAT was substituted by CTC. A plasmid pPA52 was constructed containing the mutant at the start codon ATG of pacR . The pPA52 was transformed into E.coli HB101. The regulation of penicillin G acylase gene ( pac ) expression from the mutant strain was studied. It can be founded that the posttranslational modifications of PAC in E.coli HB101 (pPA52) is similar to that in E.coli HB101 (pPA6). Although pacR translation was deleted from E.coli HB101 (pPA52), the expression of pac is still induced by PAA. The activities of PAC in E.coli HB101 (pPA52) is more than that in E.coli HB101(pPA6), with 0.05%PAA, 0.2%PAA or without PAA. The expression of pac is repressed by glucose in E.coli HB101 (pPA6), not in E.coli HB101 (pPA52). Site directed mutagenesis of pacR at start codon has improved production of PAC.