Korean Chemical Engineering Research

The Korean Institute of Chemical Engineers (한국화학공학회)
권호 표지
DOI QR코드

DOI QR Code

Inhibition of poly 3-hydroxybutyrate (PHB) synthesis by phaR deletion in Methylobacterium extorquens AM1

메탄올자화균 Methylobacterium extorquens AM1의 phaR 유전자 결실을 통한 poly 3-hydroxybutyrate (PHB) 생합성 억제

  • Kim, Yujin (Department of Chemical and Biomolecular Engineering, Sogang University) ;
  • Lee, Kwanghyun (Department of Life Sciences, Sogang University) ;
  • Kim, Hyeonsoo (Department of Chemical and Biomolecular Engineering, Sogang University) ;
  • Cho, Sukhyeong (C1 Gas Refinery R&D Center, Sogang University) ;
  • Lee, Jinwon (Department of Chemical and Biomolecular Engineering, Sogang University)
  • 김유진 (서강대학교 화공생명공학과) ;
  • 이광현 (서강대학교 생명과학과) ;
  • 김현수 (서강대학교 화공생명공학과) ;
  • 조숙형 (서강대학교 C1 Gas Refinery 사업단) ;
  • 이진원 (서강대학교 화공생명공학과)
  • Received : 2017.01.03
  • Accepted : 2017.02.13
  • Published : 2017.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

Methylotrophy is able to use reduced one-carbon compound, such as methanol and methylamine, as a sole carbon source. Methylobacterium extorquens AM1 is the most extensively studied methylotroph utilizing serine-isocitrate lyase cycle. Because the Poly 3-hydroxybutyrate (PHB) synthesis pathway in M. extorquens AM1 is likely to interlink with EMCP (ethylmalonyl-CoA pathway), glyoxylate, and TCA cycles, regulation of PHB production is needed to produce EMCP-derived acid or TCA acids. To adjust carbon flux to PHB production, PhaR, which seems to have function of regulator of PHB synthesis and acetyl-CoA flux, was knocked out in M. extorquens AM1 by using markerless gene deletion methods. As a result, PHB granules were remarkably reduced in the knockout strain ${\Delta}phaR$ compared to parental strain. Although lag phase was extended for 12h, ${\Delta}phaR$ showed similar cell growth and methanol consumption rate compared to wild type.

메탄올자화균이란 일탄소 화합물인 메탄올을 주탄소원 및 에너지원으로 이용할 수 있는 미생물을 말한다. Methylobacterium extorquens AM1은 serine cycle을 탄소대사경로로 이용하는 메탄올자화균 중에서도 가장 많이 연구가 진행된 균주이다. M. extorquens AM1의 poly 3-hydroxybutyrate (PHB) cycle은 EMCP (ethylmalonyl-CoA pathway), glyoxylate regeneration cycle, TCA cycle과 연결되어 있으며 EMCP 유래 유기산 또는 TCA 유기산을 생산하기 위해서는 PHB cycle로 흐르는 carbon flux의 차단이 필요하다. 이를 위해서 PHB 합성과 acetyl-CoA flux의 조절유전자로 알려져 있는 PhaR 유전자를 markerless gene deletion 방법을 이용해서 M. extorquens AM1에서 knockout했다. 결과적으로, knockout 균주인 ${\Delta}phaR$에서 야생종 대비 확연히 PHB granule이 줄어든 것이 확인되었다. Lag phase가 약 12 h 늦어졌지만, ${\Delta}phaR$은 야생종과 비슷한 세포성장과 메탄올소비 경향을 보임을 확인하였다.

Keywords

References

  1. Hou, C.-T., Methylotrophs: Microbiology, biochemistry, and genetics, CRC pp. 1-53(1984).
  2. Faust, U. and Pr Fa, H., "Biomass from Methane and Methanol," Biotechnology. VCH Weinheim 3, 84(1991).
  3. Kim, P., Kim, J.-H. and Oh, D.-K., "Improvement in Cell Yield of Methylobacterium sp. by Reducing the Inhibition of Medium Components for Poly-${\beta}$-hydroxybutyrate Production," World J. Microbiol. Biotechnol., 19, 357(2003). https://doi.org/10.1023/A:1023969629568
  4. Schrader, J., Schilling, M., Holtmann, D., Sell, D., Villela Filho, M., Marx, A. and Vorholt, J. A., "Methanol-based Industrial Biotechnology: Current Status and Future Perspectives of Methylotrophic Bacteria," Trends Biotechnol., 27, 107(2009). https://doi.org/10.1016/j.tibtech.2008.10.009
  5. Lidstrom, M. E., Murrell, J. C. and Dalton, H., (Ed.), The genetics and molecular biology of methanol-utilizing bacteria: Methane and Methanol Utilizers, Springer US, 183-206(1992).
  6. Anthony, C., The Biochemistry of Methylotrophs, Academic Press, London, United Kingdom (1982).
  7. Senior, P. J. and Windass, J., "The ICI Single Cell Protein Process," J. Biotechnol. Lett., 2(5), 205-210(1980). https://doi.org/10.1007/BF01209434
  8. Asenjo, J. A. and Suk, J. S., J. Fem Technol., 64, 271-2789(1986). https://doi.org/10.1016/0385-6380(86)90118-4
  9. Chistoserdova, L., "Modularity of Methylotrophy, Revisited," Environ. Microbiol., 13, 2603-2622(2011). https://doi.org/10.1111/j.1462-2920.2011.02464.x
  10. Salem, A. R. and Quayle, J. R., "Mutants of Pseudomonas AM1 that Require Glycollate or Glyoxylate for Growth on Methanol and Ethanol," Biochem. J., 124, 74(1971). https://doi.org/10.1042/bj1240074P
  11. Chistoserdova, L., Chen, S. W., Lapidus, A. and Lidstrom, M. E., "Methylotrophy in Methylobacterium extorquens AM1 from a Genomic Point of View," J. Bacteriol., 185(10), 2980-2987(2003). https://doi.org/10.1128/JB.185.10.2980-2987.2003
  12. Chistoserdova, L., Metabolism of formaldehyde in M. extorquens AM1: Microbial Growth on C1 Compounds, Springer Netherlands, 16-24(1996).
  13. Marx, C. J. and Lidstrom, M. E., "Development of Improved Versatile Broad-host-range Vectors for Use in Methylotrophs and Other Gram-negative Bacteria," Microbiology, 147(8), 2065-2075 (2001). https://doi.org/10.1099/00221287-147-8-2065
  14. http://www.integratedgenomics.com/genomereleases.html#list6.
  15. Anderson, A. J. and Dawes, E. A., "Occurrence, Metabolism, Metabolic Role, and Industrial Uses of Bacterial Polyhydroxyalkanoates," Microbiol Rev., 54(4), 450-472(1990).
  16. Ginige, M. P., Hugenholtz, P., Daims, H., Wagner, M., Keller, J., and Blackall, L. L., "Use of Stable-isotope Probing, Full-cycle rRNA Analysis, and Fluorescence in situ Hybridization-microautoradiography to Study a Methanol-fed Denitrifying Microbial Community," Appl. Environ. Microbial., 70(1), 588-596(2004). https://doi.org/10.1128/AEM.70.1.588-596.2004
  17. Korotkova, N., Chistoserdova, L. and Lidstrom, M. E., "Poly-${\beta}$- hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11, gap20, and phaR," J. Bacteriol., 184(22), 6174-6181(2002). https://doi.org/10.1128/JB.184.22.6174-6181.2002
  18. Korotkova, N. and Lidstrom, M. E., "Connection Between Poly-${\beta}$-Hydroxybutyrate Biosynthesis and Growth on $C_1$ and $C_2$ Compounds in the Methylotroph Methylobacterium extorquens AM1," J. Bacteriol., 183(3), 1038-1046(2001). https://doi.org/10.1128/JB.183.3.1038-1046.2001
  19. Van Dien, S. J., Strovas, T. and Lidstrom, M. E., "Quantification of Central Metabolic Fluxes in the Facultative Methylotroph Methylobacterium extorquens AM1 Using $^{13}C$-label Tracing and Mass Spectrometry," Biotechnol. Bioeng., 84, 45-55(2003). https://doi.org/10.1002/bit.10745
  20. Schafer, A., Tauch, A., Jager, W., Kalinowski, J., Thierbach, G., and Puhler, A., "Small Mobilizable Multi-purpose Cloning Vectors Derived from the Escherichia coli Plasmids pK18 and pK19: Selection of Defined Deletions in the Chromosome of Corynebacterium glutamicum," Gene, 145(1), 69-73(1994). https://doi.org/10.1016/0378-1119(94)90324-7
  21. Kim, H. G., Han, G. H., Eom, C. Y. and Kim, S. W., "Isolation and Taxonomic Characterization of a Novel type I Methanotrophic Bacterium," J. Bacteriol., 46(1), 45-50(2008).
  22. Degelau, A., Scheper, T., Bailey, J. E. and Guske, C., "Fluorometric Measurement of Poly-${\beta}$ Hydroxybutyrate in Alcaligenes eutrophus by Flow Cytometry and Spectrofluorometry," Appl Microbiol Biotechnol., 42(5), 653-657(1995). https://doi.org/10.1007/BF00171939
  23. Spiekermann, P., Rehm, B. H., Kalscheuer, R., Baumeister, D., and Steinbüchel, A., "A Sensitive, Viable-colony Staining Method Using Nile Red for Direct Screening of Bacteria that Accumulate Polyhydroxyalkanoic Acids and Other Lipid Storage Compounds," Arch Microbiol., 171(2), 73-80(1999). https://doi.org/10.1007/s002030050681
  24. Lee, O. K., Hur, D. H., Nguyen, D. T. N. and Lee, E. Y., "Metabolic Engineering of Methanotrophs and Its Application to Production of Chemicals and Biofuels from Methane," Biofuels, Bioprod. Bioref. (2016).
  25. Lee, W., "Selection of Medium Components by Plackett-Burman Design for Cell Growth of a Newly Isolated Methylobacterium sp. WJ4," Korean Chem. Eng. Res., 54(6), 812-816(2016). https://doi.org/10.9713/kcer.2016.54.6.812