Expression of penicillin G acylase from the cloned pac gene of Escherichiacoli ATCC11105 - Effects of pacR and temperature

The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G a cylase (PGA). Within the pac gene, there is a regulatory gene pacR, which i s transcribed in the opposite direction. Site-directed mutagenesis was perf ormed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused P GA expression to be constitutive rather than inductive in two strains (E. c oli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant i nduction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pa c is preserved in the pacR translation-initiation mutant and the optimum te mperature of PGA production was 22 degreesC in both wild type and mutant. A t a higher temperature of 37 degreesC, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by we stern blot analysis. Our investigations confirmed the hypothesis of pacR-me diated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA prec ursor polypeptide.