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 the 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 proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in 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 defined 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 the 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 precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.
Miñambres
B
B
Departamento de Bioquímica y Biología Molecular, Facultades de Veterinaria y Biología, Universidad de León, 24007, León, España.
Martínez-Blanco
H
H
Olivera
E R
ER
García
B
B
Díez
B
B
Barredo
J L
JL
Moreno
M A
MA
Schleissner
C
C
Salto
F
F
Luengo
J M
JM
D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003066:Coenzyme A Ligases / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010400:Penicillin G / Q000378:metabolism*; D010408:Penicillium chrysogenum / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D000249:Adenosine Monophosphate; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase; D010400:Penicillin G