The PalkBFGHJKL promoter is under carbon catabolite repression control in Pseudomonas oleovorans but not in Escherichia coli alk+ recombinants.
The alk genes are located on the OCT plasmid of Pseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans and Escherichia coli alk+ recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads. Pseudomonas putida GPo12 is a P. oleovorans derivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently in Pseudomonas and in E. coli strains. These results also indicate that PalkBFGHJKL, the Palk promoter, might be useful in attaining high expression levels of heterologous genes in E. coli grown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.
Institut für Biotechnologie, Swiss Federal Institute of Technology (ETH), ETH Hönggerberg, HPT, 8093 Zürich, Switzerland.
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Staijen
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Marcionelli
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Witholt
D000473:Alkanes / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism; D002874:Chromosome Mapping; D003227:Conjugation, Genetic; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
D000473:Alkanes; D002264:Carboxylic Acids; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A; D005990:Glycerol