KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
권호 표지

Production of Enantiomerically Pure [R]-3-Hydroxybutyric acid by Metabolically Engineered Escherichia coli with Inducible System

Inducible System을 이용한 재조합 대장균으로부터 광학적으로 순수한 [R]-3-Hydroxybutyric acid 생산

  • 이영 ;
  • 최종일 (한국과학기술원 생명화학공학과 및 생물공정연구센터) ;
  • 이상엽 (한국과학기술원 생명화학공학과 및 생물공정연구센터, 한국과학기술원 바이오시스템학과)
  • Published : 2004.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

Inducible system을 이용하여 (R)-hydroxybutyric acid (R3HB)를 생산하는 재조합 대장균을 개발하였다. Ralstonia eutropha에서 유래한 PHB depolymerase 유전자를 inducible promoter에 의하여 발현되게 만들고, PHB 생합성 유전자가 있는 vector에 cloning하였다. PHB 생합성 유전자와 depolymerase 유전자를 가지고 있는 재조합 대장균을 배양하여 먼저 PHB를 축적시킨 후에 depolymerase를 발현시켜 배지 내로 분비된 R3HB를 확인하였다. 그 결과 축적된 PHB의 대부분은 발현된 depolymerase에 의하여 분해되었으며, depolymerase의 발현 이후에도 재조합 대장균은 PHB를 축적하고 분해하여 R3HB의 농도는 배양시간에 따라 증가하였다. 이러한 결과는 inducible depolymerase를 갖는 재조합 대장균을 이용하여 높은 농도의 R3IB를 얻을 수 있다는 것을 보여준다.

Keywords

References

  1. Biotechnol. Bioeng. v.49 Bacterial polyhydroxyalkanoates Lee, S. Y. https://doi.org/10.1002/(SICI)1097-0290(19960105)49:1<1::AID-BIT1>3.3.CO;2-1
  2. Microbiol. Mol. Biol. Rev. v.63 Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic Madison, L. L.;G. W. Huisman
  3. Trends Biotechnol. v.16 Bacterial and other biological systems for polyester production Steinbuchel, A.;B. Fuchtenbusch https://doi.org/10.1016/S0167-7799(98)01194-9
  4. FEMS Microbiol. Lett. v.128 Diversity of bacterial polyhydroxyalkanoic acids Steinbuchel, A.;H. E. Valentin https://doi.org/10.1111/j.1574-6968.1995.tb07528.x
  5. Org. Synth. v.71 Direct degradation of the biopolymer poly[(R)-3-hydroxybutyric acid] to (R)-3-hydroxybutanoic acid and its methyl ester Seebach, D.;A. K. Beck;R. Breitschuh;K. Job
  6. Chem. Lett. v.1985 A synthetic approach to (+)-thienamycin from methylene (R)-3-hydroxybutanoate Chiba, T.;T. Nakai
  7. Liebigs Ann. Chem. v.1987 Total synthesis of (+)-colletodiol from (S,S)-tartarate and (R)-3-hydroxybutanoate Schnurrenberger, P.;E. Hungerbuhler;D. Seebach https://doi.org/10.1002/jlac.198719870821
  8. Ullmann's Encyclopedia of Industrial Chemistry(5th ed.) Hydroxycarboxylic acid, aliphatic Miltenberger, K.;H. Aktiengesellschaft;H.-J. Arpe(ed.);E. Biekert(ed.);H. T. Davis(ed.);W. Gerharz(ed.);H. Gerrens(ed.);W. Keim(ed.);J. L. McGuire(ed.);A. Mitsutani(ed.);H. Pilat(ed.);Sri C. Reece(ed.);D. P. Sheetz(ed.); H. E. Simmons(ed.);E. Weise(ed.);R. Wirtz(ed.);H.-R. Wuthrich(ed.)
  9. Enzyme Microb. Technol. v.27 Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate Lee, Y.;S. H. Park;I. T. Lim;K. Han;S. Y. Lee https://doi.org/10.1016/S0141-0229(00)00146-0
  10. J. Bacteriol. v.183 Cloning of an intracellular poly[d(-)-3-hydroxybutyrate] depolymerase gene from Ralstonia eutropha H16 and characteriazation of the gene product Saegusa, H.;M. Shiraki;C. Kanai;T. Saito https://doi.org/10.1128/JB.183.1.94-100.2001
  11. Biotechnol. Bioeng. v.65 Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals Lee, S. Y.;Y. Lee;F. Wang https://doi.org/10.1002/(SICI)1097-0290(19991105)65:3<363::AID-BIT15>3.0.CO;2-1
  12. Appl. Environ. Microbiol. v.69 Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(-)-hydroxycarboxylic acids Lee, S. Y.;Y. Lee https://doi.org/10.1128/AEM.69.6.3421-3426.2003
  13. Appl. Environ. Microbiol. v.64 Cloning of the Alcaligenes latus polyhydroxyalkanoate biosynthesis genes and use of these genes for the enhanced production of Poly(3-hydroxybutyrate) in Escherichia coli Choi, J.;S. Y. Lee;K. Han
  14. Biotechnol. Bioeng. v.44 Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid), and morphological changes Lee, S. Y.;K. M. Lee;H. N. Chang;A. Steinbuchel https://doi.org/10.1002/bit.260441110
  15. A laboratory manual (2nd ed.) Molecular cloning Sambrook, J.;E. F. Fritsch;T. Maniatis