Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST

Abstract

A gene bank from Pseudomonas fluorescens ST was constructed in the broad-host-range cosmid pLAFR3 and mobilized into Pseudomonas putida PaW340. Identification of recombinant cosmids containing the styrene catabolism genes was performed by screening transconjugants for growth on styrene and epoxystyrene. Transposon mutagenesis and subcloning of one of the selected genome fragments have led to the identification of three enzymatic activities: a monooxygenase activity encoded by a 3-kb PstI-EcoRI fragment and an epoxystyrene isomerase activity and an epoxystyrene reductase activity encoded by a 2.3-kb BamHI fragment. Escherichia coli clones containing the 3-kb PstI-EcoRI fragment were able to transform styrene into epoxystyrene, and those containing the 2.3-kb BamHI fragment converted epoxystyrene into phenylacetaldehyde or, only in the presence of glucose, into 2-phenylethanol. The three genes appear to be clustered and are probably encoded by the same DNA strand. In E. coli, expression of the epoxystyrene reductase gene was under the control of its own promoter, whereas the expression of the other two genes was dependent on the presence of an external vector promoter.

Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST

“…Functional analysis of styA and styB. We previously demonstrated that the 3-kb PstI-EcoRI fragment of pTPE30 conferred on E. coli transformants the ability to convert styrene into epoxystyrene (26). Our sequence data show that StyA, which is weakly homologous to POBA (48) and NAHG (50), is located approximately 900 bp downstream of the PstI restriction site.…”

“…We determined the sequence of a 4,377-bp region (indicated by a heavy bar in Fig. 1B) and identified the genes encoding enzymes involved in the first steps of styrene catabolism (26). Computer analysis of the sequence showed the presence of four complete open reading frames, named styA, styB, styC, and styD, localized on one DNA strand and of two open reading frames, orf1 and orf2, localized on the opposite strand.…”

“…Metabolite analysis. Analysis of epoxystyrene biotransformation products was performed by reverse-phase high-performance liquid chromatography as previously described (26), with a Waters Bondapak C 18 column (3.9 by 300 mm) eluted with acetonitrile-water (50:50) at a flow rate of 1 ml/min, and compounds were detected by determination of A 254 . Quantitation was done on the basis of standard curves obtained with the authentic compounds.…”

“…The genes involved in the first steps of styrene degradation were found to be chromosomally encoded, and we were able to localize the gene(s) for the styrene monooxygenase responsible for the conversion of styrene into epoxystyrene in a 3-kb PstI-EcoRI fragment, while Escherichia coli cells transformed with a 2.3-kb BamHI fragment were able to convert epoxystyrene into phenylacetaldehyde and 2-phenylethanol, suggesting that epoxystyrene isomerase and epoxystyrene reductase activities could be encoded by this fragment (26).…”

“…Two main routes of aerobic styrene breakdown have been proposed: the first involves oxidation of the lateral chain to phenylacetate (3, 17, 30, 36-38, 41, 42), and the second involves initial oxidation of the aromatic nucleus (2,6,16,46,47). In a previous publication (26), we reported the cloning of the styrene catabolic genes from P. fluorescens ST and demonstrated that this strain degrades styrene by oxidation of the lateral chain.…”