Enhanced PHB Accumulation in Photosystem- and Respiration-defective Mutants of a Cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803의 에너지 대사 결함 돌연변이 균주에서의 Poly(3-hydroxybutyrate) 축적량 증진

  • Kim Soo-Youn (Department of Microbiology, Chungnam National University) ;
  • Choi Gang Guk (Department of Microbiology, Chungnam National University) ;
  • Park Youn Il (Department of Biology, Chungnam National University) ;
  • Park Young Mok (Biomolecular Research Team, Korea Basic Science Institute) ;
  • Yang Young Ki (Department of Genetic Engineering, Chosun University) ;
  • Rhee Young Ha (Department of Microbiology, Chungnam National University)
  • 김수연 (충북대학교 미생물학과) ;
  • 최강국 (충북대학교 미생물학과) ;
  • 박연일 (충남대학교 생물학과) ;
  • 박영목 (한국기초지원연구원 프로테옴분석팀) ;
  • 양영기 (조선대학교 유전공학과) ;
  • 이영하 (충북대학교 미생물학과)
  • Published : 2005.03.01


Photoautotrophic bacteria are promising candidates for the production of poly(3-hydroxybutyrate) (PHB) since they can address the critical problem of substrate costs. In this study, we isolated 25 Tn5-inserted mutants of the Synechocystis sp. PCC 6803 which showed enhanced PHB accumulation compared to the wild-type strain. After 5-days cultivation under nitrogen-limited mixotrophic conditions, the intracellular levels of PHB content in these mutants reached up to $10-30\%$ of dry cell weight (DCW) comparable to $4\%$ of DCW in the wild-type strain. Using the method of inverse PCR, the affected genes of the mutants were mapped on the completely known genome sequence of Synechocystis sp. PCC 6803. As a result, the increased PHB accumulation in 5 mutants were found to be resulted from defects of genes coding for NADH-ubiquinone oxidoreductase, O-succinylbenzoic-CoA ligase, photosystem II PsbT protein or histidine kinase, which are involved in photosystem in thylakoid inner membrane of the cell. The values of $NAD(P)H/NAD(P)^+$ ratio in the cells of these mutants were much higher than that of the wild-type strain as measured by using pulse-amplitude modulated fluorometer, suggesting that PHB synthesis could be enhanced by increasing the level of cellular NAD(P)H which is a limiting substrate for NADPH-dependent acetoacetyl-CoA reductase. From these results, it is likely that NAD(P)H would be a limiting factor for PHB synthesis in Synechocystis sp. PCC 6803.


  1. 박연일. 1993. 배추 잎의 무기인산 결핍에 따른 틸라코이드막 전자전달능의 변화. 이학박사학위논문, 서울대학교, 대한민국
  2. Arifio, X. and J. Julio. 1995. Effect of sulfur starvation on the morphology and ultrastructure of the cyanobacterium Gloeocapsasp. PCC6909. Arch. Microbiol. 163, 447-453 https://doi.org/10.1007/BF00272134
  3. Braunegg, G., G. Lefebvre, and K.F. Genser. 1998. Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects. J. Biotechnol. 65, 127-161 https://doi.org/10.1016/S0168-1656(98)00126-6
  4. Hein, S., H. Tran, and A. Steinbüchel. 1998. Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria. Arch. Microbiol. 170, 162-170 https://doi.org/10.1007/s002030050629
  5. Howitt, C.A. and W.F.J. Vermass. 1998. Quinol and cytochrome oxidase in the cyanobacterium Synechocystis sp. PCC6803. Biochemistry 37, 17944-17951 https://doi.org/10.1021/bi981486n
  6. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N. Miyajima, M. Sugiura, S. Sasamoto, T. Kimura, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, T. Wada, A. Watanabe, M. Tamda, M. Yauda, and S. Tabata. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3, 109-136 https://doi.org/10.1093/dnares/3.3.109
  7. Kang, H.O., C.W. Chung, H.W. Kim, Y.B. Kim, and Y.H. Rhee. 2001. Cometabolic biosynthesis of copolyesters consisting of 3-hydroxyvalerate and medium-chain-length 3-hydroxyalkanoates byPseudomonas sp. DSY-82. Antonie van Leeuwenhoek 80, 185-191 https://doi.org/10.1023/A:1012214029825
  8. Kim, G.J., Y.K. Yang, and Y.H. Rhee. 2001. Economic consideration of poly(3-hydroxybutyrate) production by fed-batch culture of Ralstonia eutropha KHB 8862. Kor. J. Microbiol. 37: 92-99
  9. Kim, Y.B., D.Y. Kim, and Y.H. Rhee. 1999. PHAs produced by Pseudomonas putida and Pseudomonas oleovorans grown with n-alkanoic acids containing aromatic groups. Macromolecules 32, 6058-6064 https://doi.org/10.1021/ma982033t
  10.  Lee, I.Y., M.K. Kim, H.N. Chang, and Y.H. Park. 1995. Regulation of poly-$\beta$-hydroxybutyrate biosynthesis by nicotinamide nucleotide in Alcaligenes eutrophus. FEMS Microbiol. Lett. 131, 35-39
  11.  Lim, S.J., Y.M. Jung, H.D. Shin, and Y.H. Lee. 2002. Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon. J. Biosci. Bioeng. 93, 543-549 https://doi.org/10.1016/S1389-1723(02)80235-3
  12.  Lyu, J.Y. 2002. Cytochrome aa3 oxidase of the cyanobacterium Synechocystis sp. PCC6803 is a main terminal oxidase for the oxidation of reduced plastoquinone in the dark. M.Sc. thesis, Chungnam National University, Korea
  13.  Madison, L.L. and G.W. Huisman. 1999. Metabolic engineering of poly(3-hydorxyalkanoates): from DNA to plastic. Microbiol. Mol. Biol. Rev. 63, 21-53
  14.  Massimo, V., S. Claudio, M. Ricardo, and E. Alba. 1990. Occurrence of poly-$\beta$-hydorxybutyrate in Spirulina species. J. Bacteriol.172, 2791-2792 https://doi.org/10.1128/jb.172.5.2791-2792.1990
  15.  Ogawa, T. 1991. A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential to inorganic carbon transport of Synechocystis sp. PCC6803. Proc. Natl. Acad. Sci. USA 88, 4275-4279
  16.  Rippka, R., J. Deruelles, J.B. Waterbury, M. Herdman, and R.J. Stanier. 1979. Genetic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 3, 1-61
  17.  Schmetterer, G. 1995. Genetic analysis of cyanbacteria, p. 409-435. In D.A. Bryant (ed), The molecular biology of cyanobacteria. Kluwer Academic Publishers, London
  18.  Schneider, D., S. Berry, P. Seidler, and M. Rogner. 2001. A regulatory role of the PetM subunit in a cyanobacteria cytochrome b6f complex. J. Biol. Chem. 76, 16780-16785
  19.  Steinbüchel, A. 2001. Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Micromol. Biosci. 1, 1-24 https://doi.org/10.1002/1616-5195(200101)1:1<1::AID-MABI1>3.0.CO;2-B
  20.  Taroncher-Oldenburg, G., K. Nishina, and G. Stephanopoulos. 2000. Identification and analysis the polyhydroxyalkanoate-specific $\beta$-ketothiolase and acetoacetyl coenzyme A reductase gene in the cyanobacterium Synechocystis sp. PCC6803. Appl. Environ. Microbiol. 66, 4440-4448 https://doi.org/10.1128/AEM.66.10.4440-4448.2000
  21. Wu, C.F., Q.Y. Wu, and Z.Y. Shen. 2001. Accumulation of poly-$\beta$-hydroxybutyrate in cyanobacterium Synechocystis sp. PCC6803. Bioresource Technol. 76, 85-90 https://doi.org/10.1016/S0960-8524(00)00099-7