KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Development of L-Threonine Producing Recombinant Escherichia coli using Metabolic Control Analysis

대사 조절 분석 기법을 이용한 L-Threonine 생산 재조합 대장균 개발

  • Published : 2007.02.28
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Abstract

New strain development strategy using kinetic models and metabolic control analysis was investigated. In this study, previously reported mathematical models describing the enzyme kinetics of intracellular threonine synthesis were modified for mutant threonine producer Escherichia coli TF5015. Using the modified models, metabolic control analysis was carried out to identify the rate limiting step by evaluating the flux control coefficient on the overall threonine synthesis flux exerted by individual enzymatic reactions. The result suggested the production of threonine could be enhanced most efficiently by increasing aspartate semialdehyde dehydrogenase (asd) activity of this strain. Amplification of asd gene in recombinant strain TF5015 (pCL-$P_{aroF}$-asd) increased the threonine production up to 23%, which is much higher than 14% obtained by amplifying aspartate kinse (thrA), other gene in threonine biosynthesis pathway.

대사 공학을 이용한 생산 균주 개발의 핵심 기술은 원하는 대사산물을 과량으로 얻기 위하여 기존의 대사회로에서 제거, 증폭, 변경을 시켜야 할 유전자를 선정하는 것이다. 대사조절 분석 기법은 대사 흐름이 특정 효소의 활성에 따라 어떻게 변하는지를 예측하는 기술이다. 본 논문에서는 대장균의 threonine 생합성 효소 반응 kinetic model과 대사조절 분석 기법을 이용하여 threonine 생합성 flux를 가장 효과적으로 증가시키기 위하여 활성 증가가 필요한 효소가 aspartate semialdehyde dehyogenase라는 것을 밝혔다. 이러한 결과를 확인하기 위하여 asd가 과발현된 vector와 threonine 생합성 경로의 다른 효소인 aspartate kinase를 coding하는 thrA를 과발현 시키는 vector를 제작하여 threonine 생산 균주인 TF5015에 형질전환하여 threonine 농도를 측정하였다. Flask 배양결과 대사조절 분석 기법으로 확인된 유전자 asd를 과발현시킬 경우가 생합성 경로의 다른 유전자를 과발현시킨 경우보다 더 높은 threonine 농도의 증가를 보였다. 이러한 연구 결과들은 효소 반응 kinetic model과 대사조절 분석 기법을 이용하여 원하는 product를 효율적으로 생산할 수 있는 생산 균주를 제작할 수 있게 할 것이다.

Keywords

References

  1. Raab, R. M., Tyo, K., and G. Stephanopoulos (2005), Metabolic engineering, Adv. Biochem. Eng. Biotechnol. 100, 1-17 https://doi.org/10.1007/b136411
  2. Lee, S. Y., Lee, D. Y., and T. Y. Kim (2005), Systems biotechnology for strain improvement, Trends Biotechnol. 23(7), 349-358 https://doi.org/10.1016/j.tibtech.2005.05.003
  3. Heijnen, J. J., van Gulik, W. M., Shimizu, H., and G. Stephanopoulos (2004), Metabolic flux control analysis of branch points: an improved approach to obtain flux control coefficients from large perturbation data, Metab. Eng. 6(4), 391-400 https://doi.org/10.1016/j.ymben.2004.07.002
  4. Visser, D. and J. J. Heijnen (2002), The mathematics of metabolic control analysis revisited, Metab. Eng. 4(2) , 114-23 https://doi.org/10.1006/mben.2001.0216
  5. Wiechert, W. and K. Noh (2005), From stationary to instationary metabolic flux analysis, Adv. Biochem. Eng. Biotechnol. 92, 145-72
  6. Yang, C., Hua, Q., and K. Shimizu (1999), Development of a kinetic model for L lysine blosynthesis in Corynebacterium glutamicum and its application to metabolic control analysis, J. Biosci. Bioeng. 88(4), 393-403 https://doi.org/10.1016/S1389-1723(99)80216-3
  7. Chassagnole, C., Rais, B., Quentin, E., Fell, D. A., and J. P. Mazat (2001), An integrated study of threonine pathway enzyme kinetics in Escherichia coli, Biochem. J. 356(Pt 2), 415-23 https://doi.org/10.1042/0264-6021:3560415
  8. Rais, B., Chassagnole, C., Letellier, T., Fell, D. A., and J. P. Mazat (2001), Threonine synthesis from aspartate in Escherichia coli cell free extracts: pathway dynamics, Biochem. J. 356(Pt 2), 425-32 https://doi.org/10.1042/0264-6021:3560425
  9. Ikeda, M. (2003), Amino acid production processes, Adv. Biochem. Eng. Biotechnol. 79, 1-35 https://doi.org/10.1007/3-540-45989-8_1
  10. Lee, J., Lee, D. E., Lee, B. U., and H. S. Kim (2003), Global Analyses of Transcriptomes and Proteomes of a Parent Strain and an Threonine Overproducing Mutant Strain, J. Bacteriol. 9, 5442-.5451
  11. Blattner, F. R., Plunkett, G 3rd, Bloch, C. A., Perna, N. T., Burland, V., Riley. M., Collado Vides, J., Glasner , J. D., Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A., Goeden, M. A., Rose, D. J., Mau, B., and Y. Shao (1997), The complete genome sequence of Escherichia coli K-12, Science 277 (5331), 1453-74 https://doi.org/10.1126/science.277.5331.1453
  12. Lerner, C. G. and M. Inouye (1990), Low copy number plasmids for regulated low level expression of cloned genes in Escherichia coli with blue/white insert screening capability, Nucleic Acids Res. 18, 4631 https://doi.org/10.1093/nar/18.15.4631
  13. Sumbrook. J. and D. W. Russell (2001), Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  14. Olivier, B. G. and J. L. Snoep (2004), Web based kinetic modelling using JWS Online, Bioinformatics 20, 2143-2144 https://doi.org/10.1093/bioinformatics/bth200