CLONING AND EXPRESSION OF THE GENES ENCODING THE PROPENE MONOOXYGENASE FROM XANTHOBACTER, PY2

Xanthobacter Py2 grows on propene as sole carbon source, converting pr opene to propene oxide (epoxypropane) using an alkene-specific monooxy genase, as the first step in catabolism. Four mutants, NZ1-4, with a p ropene(-) propene oxide(+) phenotype were isolated by 1-methyl-3-nitro -1-nitrosoguanidine mutagenesis or by enrichment with the suicide subs trate vinylidene chloride, and were shown to have lost the ability to convert alkenes to epoxides. All four mutants were complemented by a n umber of clones of Xanthobacter Py2 chromosomal DNA in the broad-host- range cosmid pLAFR5, some of which appeared to be non-overlapping. Rep resentatives of the different clones obtained were transferred into Xa nthobacter autotrophicus JW33 and one, pNY2, the most frequently isola ted clone, was shown to express an inducible, fully functional propene monooxygenase. Subcloning revealed that all four mutants were complem ented by a 2.4-kb EcoRI-PstI fragment situated at one end of the cosmi d insert. However, activity in X. autotrophicus JW33 could only be exp ressed from pNY2, containing the complete insert (25 kb), suggesting a large operon or some form of long-range control. pNY2 failed to expre ss in E. coli. In X. autotrophicus JW33 [pNY2] at least three new poly peptides were evident after induction with propene compared with a con trol carrying only the cosmid pLAFR5.