MOLECULAR-CLONING AND EXPRESSION IN DIFFERENT MICROBES OF THE DNA ENCODING PSEUDOMONAS-PUTIDA U PHENYLACETYL-COA LIGASE - USE OF THIS GENE TO IMPROVE THE RATE OF BENZYLPENICILLIN BIOSYNTHESIS IN PENICILLIUM-CHRYSOGENUM

The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of t he pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant protei ns were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresp onding to the phenylacetyl-CoA ligase previously purified by us (Marti nez-Blanco, H., Reglero, A. Rodriguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene i n Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defi ned medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in th e quantities of benzylpenicillin accumulated in the broths (between 1. 8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precurs or, phenylacetyl-CoA This report describes the sequence of a phenylace tyl-CoA ligase for the first time and provides direct evidence that th e expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for imp roving the biosynthetic machinery of this fungus.