Development of biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes inducible in aerobic and anaerobic conditions.
We developed biocatalysts carrying naphthalene dioxygenase and dihydrodiol dehydrogenase genes cloned from plasmid pN3 of Pseudomonas fluoresceins N3 involved in naphthalene degradation, as an alternative approach to the production of hydroxylated compounds by chemical synthesis. Naphthalene dioxygenase is responsible for hydroxylation of the hydrocarbon into the corresponding 1,2-dihydro-1,2-dihydroxy derivative and dihydrodiol dehydrogenase is involved in the subsequent transformation into the 1,2-dihydroxy derivative. The first reaction strictly requires the presence of oxygen, essential for the dioxygenation reaction, while the second one can also be performed in anaerobic conditions that are optimal to avoid the easy oxidation of bioconversion products. Consequently, we constructed biocatalysts carrying the genes responsible for the biotransformation of hydrocarbons, inducible under aerobic and anaerobic conditions. We cloned the dioxygenase gene under its promoter, inducible by salicylic acid and the dihydrodiol dehydrogenase under the Pnar promoter of Escherichia coli, inducible by nitrate, in a nitrogen atmosphere, in order to develop biological systems with the possibility of controlling the expression of the cloned genes by the shift from aerobic to anaerobic conditions. Bioconversion experiments performed in aerobic conditions showed dihydrodiol production and dehydrogenase repression; as soon as cultures were switched to nitrogen, dihydrodiol dehydrogenation with an efficient production of 1,2-dihydroxyderivatives was observed.
Department of Genetics and Biology of Microorganisms, University of Milan, Italy.
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PD
Gennaro
E
E
Galli
F
F
Orsini
F
F
Pelizzoni
G
G
Sello
G
G
Bestetti
D000332:Aerobiosis; D000693:Anaerobiosis; D002384:Catalysis; D003001:Cloning, Molecular; D003470:Culture Media; D049308:Dioxygenases; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005818:Genetic Engineering; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism; D009281:Naphthalenes / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D011995:Recombination, Genetic
D003470:Culture Media; D009097:Multienzyme Complexes; D009281:Naphthalenes; C031721:naphthalene; D010088:Oxidoreductases; D010105:Oxygenases; D049308:Dioxygenases; C038703:naphthalene dioxygenase; C048338:trans-1,2-dihydrobenzene-1,2-diol dehydrogenase