Characterization of Styrene Catabolic Genes of Pseudomonas putida SN1 and Construction of a Recombinant Escherichia coli Containing Styrene Monooxygenase Gene for the Production of (S)-Styrene Oxide

  • Park Mi-So (Department of Chemical and Biochemical Engineering,Pusan National University) ;
  • Bae Jong-Won (Department of Chemical and Biochemical Engineering,Pusan National University) ;
  • Han Ju-Hee (Department of Chemical and Biochemical Engineering,Pusan National University) ;
  • Lee Eun-Yeol (Institute for Environmental Technology and Industry, Pusan National University) ;
  • Lee Sun-Gu (Department of Chemical and Biochemical Engineering,Pusan National University) ;
  • Park Sung-Hoon (Department of Chemical and Biochemical Engineering,Pusan National University)
  • Published : 2006.07.01

Abstract

Some Pseudomonas species can grow on styrene as a sole carbon and energy source. From the new isolate Pseudomonas putida SN1, the genes for styrene catabolism were cloned and sequenced. They were composed of four structural genes for styrene monooxygenase (styA and styB), styrene oxide isomerase (styC), and phenylacetaldehyde dehydrogenase (styD), along with two genes for the regulatory system (styS and styR). All the genes showed high DNA sequence (91% to 99%) and amino acid sequence (94% to 100%) similarities with the corresponding genes of the previously reported styrene-degrading Pseudomonas strains. A recombinant Escherichia coli to contain the styrene monooxygenase from the SN1 was constructed under the control of the T7 promoter for the production of enantiopure (S)-styrene oxide, which is an important chiral building block in organic synthesis. The recombinant E. coli could convert styrene into an enantiopure (S)-styrene oxide (ee >99%) when induced by IPTG The maximum activity was observed as 140 U/g cell, when induced with 1 mM IPTG at $15^{\circ}C$.

Keywords

References

  1. Beltrametti, F., A. M. Marconi, G. Bestetti, C. Colombo, E. Galli, M. Ruzzi, and E. Zennaro. 1997. Sequencing and functional analysis of styrene catabolism genes from Pseudomonas fluorescens ST. Appl. Environ. Microbiol. 63: 2232-2239
  2. Besse, P. and H. Veschambre. 1994. Chemical and biological synthesis of chiral epoxides, Tetrahedron 50: 8885-8927 https://doi.org/10.1016/S0040-4020(01)85362-X
  3. Banryx, F. 1999. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: 411-421 https://doi.org/10.1016/S0958-1669(99)00003-8
  4. Choi, K. O., S. H. Song, and Y. J. Yoo. 2004. Permeabilization of Ochrobactrum anthropi SY509 cells with organic solvents for whole cell biocatalyst. Biotechnol. Bioprocess Eng. 9: 147-150 https://doi.org/10.1007/BF02942284
  5. Duetz, W. A., J. B. Beilen, and B. Witholt. 2001. Using proteins in their natural environment: Potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis. Curr. Opin. Biotechnol. 12: 419-425 https://doi.org/10.1016/S0958-1669(00)00237-8
  6. Harayama S., M. Kok, and E. L. Neidle. 1992. Functional and evolutionary relationships among diverse oxygenases. Annu. Rev. Microbiol. 46: 565-601 https://doi.org/10.1146/annurev.mi.46.100192.003025
  7. Holland, H. L. and H. K Weber. 2000. Enzymatic hydroxylation reactions. Curr. Opin. Biotechnol. 11: 547-553 https://doi.org/10.1016/S0958-1669(00)00142-7
  8. Kim, J., H. W. Ryu, D. J. Jung, T. H. Lee, and K.-S. Cho. 2005. Styrene degradation in a polyurethane biofilter inoculated with Pseudomonas sp. IS-3. J. Microbiol. Biotechnol. 15: 1207-1213
  9. Lee, N., J. M. Lee, K. H. Min, and D. Y. Kwon. 2003. Purification and characterization of 2,3-dihydroxybiphenyl 1,2-dioxygenase from Comamonas sp. SMN4. J. Microbiol. Biotechnol. 13: 487-494
  10. Lim, H. K., S. U. Lee, S. I. Chung, K. H. Jung, and J. H. Seo. 2004. Induction of the T7 promoter using lactose of production of recombinant plasminogen kringle 1-3 in Escherichia coli. J. Microbiol. Biotechnol. 14: 225-230
  11. Marconi, A. M., F. Beltrametti, G. Bestetti, F. Solinas, M. Ruzzi, E. Galli, and E. Zennaro. 1996. Cloning and characterization of styrene catabolism genes from Pseudomonas fluorescens ST. Appl. Environ. Microbiol. 62: 121-127
  12. Munthali, M. T., K. N. Timmis, and E. Diaz. 1996. Restricting the dispersal of recombinant DNA: Design of a contained biological catalyst. Bio/Technology 14: 189-191 https://doi.org/10.1038/nbt0296-189
  13. Neidhardt, C. F., L. J. Ingraham, and M. Schaechter. 1990. Physiology of the Bacterial Cell: A Molecular Approach, pp. 4. Sinauer Associates, Inc. Sunderland, Massachusetts, U.S.A
  14. Nothe, C. and S. Hartmans. 1994. Formation and degradation of styrene oxide stereoisomers by different microorganisms. Biocatalysis 10: 219-225 https://doi.org/10.3109/10242429409065231
  15. O'Leary, N. D., K. E. O'Conner, W. Duetz, and A. D. W. Dobson. 2001. Transcriptional regulation of styrene degradation in Pseudomonas putida CA-3. Microbiology 147: 973-979 https://doi.org/10.1099/00221287-147-4-973
  16. Otto, K., K. Hofstetter, M. Rothlisberger, B. Witholt, and A. Schmid. 2004. Biochemical characterization of StyAB from Pseudomonas sp. strain VLB120 as a two-component flavindiffusible monooxygenase. J. Bacteriol. 186: 5292-5302 https://doi.org/10.1128/JB.186.16.5292-5302.2004
  17. Panke, S., A. Meyer, C. M. Huber, B. Witholt, and M. G. Wubbolts. 1999. An alkane-responsive expression system for the production of fine chemicals. Appl. Environ. Microbiol. 65: 2324-2332
  18. Panke, S., B. Witholt, A. Schmid, and M. G. Wubbolts. 1998. Towards a biocatalyst for (S)-styrene oxide production: Characterization of the styrene degradation pathway of Pseudomonas sp. strain VLB120. Appl. Environ. Microbiol. 64: 2032-2043
  19. Panke, S., M. G. Wubbolts, A. Schmid, and B. Witholt. 2000. Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase. Biotechnol. Bioeng. 69: 91-100 https://doi.org/10.1002/(SICI)1097-0290(20000705)69:1<91::AID-BIT11>3.0.CO;2-X
  20. Panke, S., V. de Lorenzo, A. Kaiser, B. Witholt, and M. G. Wubbolts. 1999. Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous twoliquid-phase applications. Appl. Environ. Microbiol. 65: 5619-5623
  21. Park, M. S., J. H. Han, S. S. Yoo, E. Y. Lee, S. G. Lee, and S. Park. 2005. Degradation of styrene by a new isolate Pseudomonas putida SN1. Kor. J. Chem. Eng. 22: 418-424 https://doi.org/10.1007/BF02719421
  22. Park, N. S., J. S. Myeong, H.-J. Park, K. Han, S.-N. Kim, and E.-S. Kim. 2005. Characterization and culture optimization of regiospecific cyclosporine hydroxylation in rare Actinomycetes species. J. Microbiol. Biotechnol. 15: 188- 191
  23. Sambrook, J., E. F. Fritsch, and T. Maniatis. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A
  24. Swaving, J. and J. A. M. de Bont. 1998. Microbial transformation of epoxide. Enzyme Microb. Technol. 22: 19-26 https://doi.org/10.1016/S0141-0229(97)00097-5
  25. Swartz, J. R. 2001. Advances in Escherichia coli production of therapeutic proteins. Curr. Opin. Biotechnol. 12: 195-201 https://doi.org/10.1016/S0958-1669(00)00199-3
  26. Van Beilen, J. B., M. G. Wubbolts, and B. Witholt. 1994. Genetics of alkane oxidation by Pseudomonas oleovorans. Biodegradation 5: 161-174 https://doi.org/10.1007/BF00696457
  27. Velasco, A., S. Alonso, J. L. Garcia, J. Perera, and E. Diaz. 1998. Genetic and functional analysis of the styrene catabolic cluster of Pseudomonas sp. strain Y2. J. Bacteriol. 180: 1063-1071
  28. Wubbolts, M. G., P. Reuvekamp, and B. Witholt. 1994. Efficient production of optically active styrene epoxidation in two-liquid phase cultures. Enzyme Microb. Technol. 16: 608-615 https://doi.org/10.1016/0141-0229(94)90127-9