Cloning of Catechol 2,3-Dioxygenase Gene and Construction of a Stable Genetically Engineered Strain for Degrading Crude Oil
Biology Department, Northwestern University, Xi’an, China.
strain BNF1 was isolated to degrade aromatic hydrocarbons efficiently and use phenol as a main carbon and energy source to support its growth. Catechol 2,3-dioxygenase was found to be the responsible key enzyme for the biodegradation of aromatic hydrocarbons. Catechol 2,3-dioxygenase gene was cloned from plasmid DNA of P. putida
strain BNF1. The nucleotide basesequence of a 924 bp segment encoding the catechol 2,3-dioxygenase (C23O) was determined. This segment showed an open reading frame, which encoded a polypeptide of 307 amino acids. C23O
gene was inserted into NotI-cut transposon vector pUT/mini-Tn5 (Kmr) to get a novel transposon vector pUT/mini-Tn5-C23O
. With the helper plasmid PRK2013, the transposon vector pUT/mini-Tn5-C23O
was introduced into one alkanes degrading strain Acinetobacter
sp. BS3 by triparental conjugation, and then the C23O
gene was integrated into the chromosome of Acinetobacter
sp. BS3. And the recombinant BS3-C23O,
which could express catechol 2,3-dioxygenase protein, was obtained. The recombinant BS3-C23O
was able to degrade various aromatic hydrocarbons and n-alkanes. Broad substrate specificity, high enzyme activity, and the favorable stability suggest that the BS3-C23O
was a potential candidate used for the biodegradation of crude oil.
Keywoards: Catechol 2,3-dioxygenase; Biodegradation; pUT/mini-Tn5; Genetically engineered microorganisms.