In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis
- 341 Downloads
The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.
KeywordsBiocatalysis Directed evolution Epoxidation Styrene monooxygenase
This work was supported by the Science Foundation Ireland (grant no. 04/IN3/B581). We thank Dr. Derek Boyd and Dr. Narain Sharma, Queens University Belfast, for the synthesis of 1S-2R-indene oxide.
- Beltrametti F et al (1997) Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Appl Environ Microbiol 63:2232–2239Google Scholar
- Di Gennaro P, Colmegna P, Galli E, Sello G, Pelizzioni F, Bestetti G (1999) A new biocatalyst for production of optically pure aryl epoxides by styrene monooxygenase from Pseudomonas fluorescens ST. Appl Environ Microbiol 65:2794–2797Google Scholar
- Lindahl E, Hess B, van der Spoel D (2001) GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 7:306–317Google Scholar
- O'Connor KE, Buckley CM, Hartmans S, Dobson ADW (1995) Possible regulatory role for nonaromatic carbon sources in styrene degradation by Pseudomonas putida CA-3. Appl Environ Microbiol 61:544–548Google Scholar
- O'Connor KE, Dobson ADW, Hartmans S (1997) Indigo formation by microorganisms expressing styrene monooxygenase activity. Appl Environ Microbiol 63:4287–4291Google Scholar
- O'Leary ND, O'Connor KE, Duetz W, Dobson ADW (2001) Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3. Microbiol 147:211–218Google Scholar
- Panke S, Witholt B, Schmid A, Wubbolts MG (1998) Towards a biocatalyst for (S)-styrene oxide production: characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl Environ Microbiol 64:2032–2043Google Scholar
- Panke S, Meyer A, Huber CM, Witholt B, Wubbolts MG (1999) An alkane-responsive expression system for the production of fine chemicals. Appl Environ Microbiol 65:2324–2332Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
- Stratagene (2005) GeneMorph II random mutagenesis kit: instruction manual. Stratagene, La JollaGoogle Scholar
- Velasco A, Alonso S, Garcıa JL, Perera J, Diaz E (1998) Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2. J Bacteriol 180:1063–1071Google Scholar