CLONING AND CHARACTERIZATION OF STYRENE CATABOLISM GENES FROM PSEUDOMONAS-FLUORESCENS ST

A gene bank from Pseudomonas fluorescens ST was constructed in the bro ad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW3 40. Identification of recombinant cosmids containing the styrene catab olism genes was performed by screening transconjugants for growth on s tyrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of thr ee enzymatic activities: a monooxygenase activity encoded by a 3-kb Ps tI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxys tyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escheric hia coli clones containing the 3-kb PstI-EcoRI fragment were able to t ransform styrene into epoxystyrene, and those containing the 2.3-kb Ba mHI fragment converted epoxystyrene into phenylacetaldehyde or, only i n the presence of glucose, into 2-phenylethanol. The three genes appea r to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the c ontrol of its own promoter, whereas the expression of the other two ge nes was dependent on the presence of an external vector promoter.