Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Synthesis of L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS) by Thermostable L-Threonine Aldolase Expressed in Corynebacterium glutamicum R

Corynebacterium glutamicum에서 발현된 L-Threonine Aldolase를 이용한 파킨슨병 치료제 L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS)의 합성

  • Baik, Sang-Ho (Department of Food Science and Human Nutrition, and Research Institute of Human Ecology, Chonbuk National University)
  • 백상호 (전북대학교 생활과학대학 식품영양학과 & 인간생활과학연구소)
  • Published : 2008.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

In order to examine efficient L-threo-2,3-Dihydroxyphenylserine (L-threo-DOPS) synthesis process using whole cell biocatalyst, a thermostable L-threonine aldolase (L-TA), which cloned from Streptomyces coelicolor A3(2) and improved for stability, was expressed in a Corynebacterium glutamicum R strain. The constructed Corynebacterium expression vector, pCG-H44(1) successfully expressed L-TA in C. glutamicum R strain, but showed very low expression level. In order to improve the expression level, the expression vector named pCG-H44(2) was reconstructed by eliminating 1 nucleotide between SD sequence and start codon of L-TA. The pCG-H44(2) vector plasmid was able to overexpress L-TA approximately 3.2 times higher than pCG-H44(1) in C. glutamicum R strain (CGH-2). When the whole cell of CGH-2 was examined in a repeated batch system, L-threo-DOPS was successfully synthesized with a yield of 4.0 mg/ml and maintain synthesis rate constantly after 30 repeated batch reactions for 130 h.

Erro-prone PCR에 의해서 열안정성이 향상된 Streptomyces coelicolor A(3) 유래의 L-threonine aldolase를 Corynebacterium glutamicum R에서 과잉발현시키기 위하여 Corynebacterium용 vector plasmid인 pCRB1의 SD배열과 개시코든사이의 1염기를 제거한 고발현용 vector plasmid인 pCG-H44(2)를 구축하였다. pCG-H (2)에 의해서 형질전환된 C. glutamicum R 균주(CGH44-2)에서 L-TA를 발현시킨 결과, 기존의 Corynebacterium용 vector plasmid인 pCRB1(CGH44-1)보다 L-TA의 발현량이 높았다. L-threo-DOPS의 합성을 위한 최적조건은 $30^{\circ}C$, 0.1 M cirtric acid buffer(pH 7.0)이었으며, 0.1% TritonX-100를 첨가하였을 경우 보다 높은 활성을 보였다. 최적조건하에서 CGH44-2를 whole cell biocatalyst로 이용한 반복회분식반응에서 재조합대장균을 숙주로 이용한 경우보다 재조합Corynebacterium을 이용하였을 경우, 목적하는 L-threo-DOPS의 합성이 안정적으로 이루어졌다.

Keywords

References

  1. Baik, S. H., H. Yoshioka, H. Yukawa, and S. Harayama. 2007. Synthesis of L-threo-3,4-dihydroxyphenylserine (Lthreo- DOPS) with thermostabilized low-specific L-threonine aldolase from Streptomyces coelicolor A3(2). J. Microbiol. Biotechnol. 17: 721-727
  2. Gold, L., D. Pribnow, T. Schnerder, S. Shinedling, B. S. Singer, and G. Stormo. 1981. Translational initiation in procaryotes. Annu. Rev. Microbiol. 35: 365-403 https://doi.org/10.1146/annurev.mi.35.100181.002053
  3. Goldstein, D. S. 2006. L-Dihydroxyphenylserine (L-DOPS): a norepinephrine prodrug. Cardiovasc. Drug Rev. 24: 189- 203 https://doi.org/10.1111/j.1527-3466.2006.00189.x
  4. Greenberg, W. A., A. Varvak, S. R.Hanson, K. Wong, H. J. Huang, P. Chen, and M. J. Burk. 2004. Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. Proc. Natl. Acad. Sci. USA 101: 5788-5793
  5. Hannemann, F., C. Virus, and R. Bernhardt. 2006. Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases. J. Biotechnol. 124: 172-181 https://doi.org/10.1016/j.jbiotec.2006.01.009
  6. Herbert, R. B., B. Wilkinson, G. J. Ellames, and E. K. Kunec. 1993. Streospecific lysis of a range of $\beta$-hydroxy-$\alpha$-amino acids catalyzed by a novel aldolase from S. amakusaensis. J. Chem. Soc. Chem. Commun. 205-206
  7. Hui, A., J. Hayflick, K. Dinkelspiel, and H. A. de Boer. 1984. Mutagenesis of the three base pair preceding the start codon of the b-galactosidase mRNA and its effect on translation in E. coli. EMBO. J. 3: 623-629
  8. Karasek, M. A. and D. M. Greenberg. 1957. Studies on the properties of threonine aldolases. J. Biol. Chem. 227: 191-205
  9. Kataoka, M., M. Ikemi, T. Morikawa, T. Miyoshi, K. Nishi, M. Wada, H. Yamada, and S. Shimizu. 1997. Isolation and characterization of D-threonine aldolase, a pyridoxal-5'- phosphate-dependent enzyme from Arthrobacter sp. DK-38. Eur. J. Biochem. 248: 385-393 https://doi.org/10.1111/j.1432-1033.1997.00385.x
  10. Kumagai, H., T. Nagatae, H. Yoshida, and H. Yamada. 1972. Threonine aldolase from Candida humicola: purification, crystallization and properties. Biochim. Biophys. Acta. 258: 779-790 https://doi.org/10.1016/0005-2744(72)90179-9
  11. Kurusu, Y., M. Kainuma, M. Inui, Y. Satoh, and H. Yukawa. 1990. Electroporation-transformation system for Corynebacteria by auxotrophic complementation. Agric. Biol. Chem. 54: 443-447 https://doi.org/10.1271/bbb1961.54.443
  12. Liu, J. Q., T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, and H. Yamada. 1998. Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity Lthreonine aldolase from Escherichia coli. Eur. J. Biochem. 255: 220-226 https://doi.org/10.1046/j.1432-1327.1998.2550220.x
  13. Liu, J. Q., S. Ito, T. Dairi, N. Itoh, S. Shimizu, and H. Yamada. 1998. Low-specific L-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to $\beta$-hydroxy-$\alpha$-amino acid synthesis. Appl. Microbiol. Biotechnol. 49: 702-708 https://doi.org/10.1007/s002530051235
  14. Liu, J. Q., T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, and H. Yamada. 2000. Diversity of microbial threonine aldolases and their application. J. Mol. Catal. B: Enz. 10: 107-115 https://doi.org/10.1016/S1381-1177(00)00118-1
  15. Liu, J. Q., M. Odani, T. Yasuoka, T. Dairi, N. Itoh, M. Kataoka, S. Shimizu, and H. Yamada. 2000. Gene cloning and overexpression of low-specific D-threonine aldolase from Alcaligenes xylosoxidans and its application for production of a key intermediate for parkinsonism drug. Appl. Microbiol. Biotechnol. 54: 44-51 https://doi.org/10.1007/s002539900301
  16. Liu, J. Q., S. Nakata, T. Dairi, H. Misono, S. Shimizu, and H. Yamada. 1997. GLY1 gene of Saccharomyces cerevisiae encodes a low-specific L-threonine aldolase that catalyzes cleavage of L-allo-threonine and L-threonine to glycine: expression of the gene in Escherichia coli and purification and characterization of the enzyme. Eur. J. Biochem. 245: 289-293 https://doi.org/10.1111/j.1432-1033.1997.00289.x
  17. Nielsen, T. B. and J. A. Reynolds. 1978. Measurements of molecular weights by gel electrophoresis. Methods Enzymol. 48: 3-10 https://doi.org/10.1016/S0076-6879(78)48003-6
  18. Ohashi, N., S. Nagata, K. Ishizumi, and K. Maeshima. 1984. Process for producing threo-3(3,4-dihydroxyphenyl)serine. European patent 0084928
  19. Ra, K. S., H. S. Baik, Y. S. Lee, and J. W. Choi. 2000. Effect of random Shine-Dalgarno sequence on the expression of bovine growth hormone gene in Escherichia coli. Kor. J. Life Sci. 10: 422-430
  20. Roberto P., L. Roberto, T. Lucia, C. John, P. Maria, and M. Enrico. 1991. DL-allothreonine aldolase in rat liver. Biochem. Soc. Trans. 19: 346-347 https://doi.org/10.1042/bst019346s
  21. Schmid A., J. S. Dordick, B. Hauer, A. Kiener, M. G. Wubbolts, and B. Witholt. 2001. Industrial Biocatalysis today and tomorrow. Nature 409: 258-268 https://doi.org/10.1038/35051736
  22. Schoemaker H. E., D. Mink, and M. G. Wubbolts. 2003. Dispelling the myths - biocatalysis in industrial synthesis. Science 14: 1694-1697
  23. Schirch, L. V. and T. Gross. 1968. Serine transhydroxymethylase: identification as the threonine and allothreonine aldolase. J. Biol. Chem. 243: 5651-5655
  24. Shepard, M. G., E. Yelverton, and D. Y. Goeddel. 1982. Increased synthesis in E. coli of fibroblast and leukocyte interferons through alterations in ribosome binding sites. DNA. 1: 123-131
  25. Straathof A. J., S. Panke, and A. Schmid. 2002. The production of fine chemicals by biotransformation. Curr. Opin. Biotechnol. 13: 548-556 https://doi.org/10.1016/S0958-1669(02)00360-9