Abstract
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.
Highlights
The gene encoding phenylacetyl-CoA ligase, 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
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
We showed that P. putida U could be cultured in minimal medium containing phenylacetic acid (PA)1 as the sole carbon source [4] and that the degradation of this compound was carried out through a newly discovered catabolic pathway involving the participation of a phenylacetyl-CoA ligase enzyme, which under aerobic conditions, catalyzes the activation of PA to PA-CoA
Summary
Materials—Oligonucleotide primers were synthesized by ISOGEN Bioscience BV (Netherlands). E. coli strain NM538 [20] was used for generation and amplification of a P. putida genomic library. The different strains used in the experiments described below were cultured in the media and conditions reported elsewhere [4, 23]. DNA Amplification—When required, DNA amplification was carried out by the polymerase chain reaction (PCR). Purification, Partial Sequencing, and Characterization of the Recombinant PCLs—PCLs obtained from the different recombinant strains were purified from cell-free extracts following the procedure previously reported [4]. In Vitro Coupling of the Different Recombinant PCL Proteins with Acyltransferase Purified from P. chrysogenum—The reproduction in vitro of the last step in the biosynthetic pathway of benzylpenicillin was carried out as reported previously [32]. Analysis of the reaction products was performed by HPLC [33, 34] or by bioassay against Micrococcus luteus [19]
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