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

김유진; 이광현; 김현수; 조숙형; 이진원; Yujin Kim; Kwanghyun Lee; Hyeonsoo Kim; Sukhyeong Cho; Jinwon Lee

Korean Chemical Engineering Research, Vol.55, No.3, 363-368, June, 2017

DOI10.9713/kcer.2017.55.3.363 Export Citation

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

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

김유진¹, 이광현², 김현수¹, 조숙형³, 이진원^{1, 3, †}

¹서강대학교 화공생명공학과, 04107 서울특별시 마포구 백범로 35
²서강대학교 생명과학과, 04107 서울특별시 마포구 백범로 35
³서강대학교 C1 Gas Refinery 사업단, 04107 서울특별시 마포구 백범로 35

Yujin Kim¹, Kwanghyun Lee², Hyeonsoo Kim¹, Sukhyeong Cho³, and Jinwon Lee^{1, 3, †}

¹Department of Chemical and Biomolecular Engineering, Sogang University, 35, Baekbeom-ro, Mapo-gu, Seoul, 04107, Korea
²Department of Life Sciences, Sogang University, 35, Baekbeom-ro, Mapo-gu, Seoul, 04107, Korea
³C1 Gas Refinery R&D Center, Sogang University, 35, Baekbeom-ro, Mapo-gu, Seoul, 04107, Korea

E-mail:

초록

메탄올자화균이란 일탄소 화합물인 메탄올을 주탄소원 및 에너지원으로 이용할 수 있는 미생물을 말 다. Methylobacterium extorquens AM1은 serine cycle을 탄소대사경로로 이용하는 메탄올자화균 중에 도 가장 많이 연구 가 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 유기을 생산하기 위해 서는 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 ΔphaR에서 야생종 대비 확연히 PHB granule이 줄어든 것이 확인되었다. Lag phase가 약 12 h 늦어졌지만, ΔphaR은 야생종과 비슷한 세포성장과 메탄올소비 경향을 보임을 확인하였다.

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 ΔphaR compared to parental strain. Although lag phase was extended for 12h, ΔphaR showed similar cell growth and methanol consumption rate compared to wild type.

Keywords:PhaR;PHB;Methylobacterium extorquens AM1;poly(3-hydroxybutyrate) granule formation

[References]

Hou CT, Methylotrophs: Microbiology, biochemistry, and genetics, CRC pp. 1-53(1984).
Faust U, Pr Fa H, Biotechnology. VCH Weinheim, 3, 84 (1991)
Kim P, Kim JH, Oh DK, World J. Microbiol. Biotechnol., 19, 357 (2003)
Schrader J, Schilling M, Holtmann D, Sell D, Villela Filho M, Marx A, Vorholt JA, Trends Biotechnol., 27, 107 (2009)
Lidstrom ME, Murrell JC, Dalton H, (Ed.), The genetics and molecular biology of methanol-utilizing bacteria: Methane and Methanol Utilizers, Springer US, 183-206(1992).
Anthony C, The Biochemistry of Methylotrophs, Academic Press, London, United Kingdom (1982).
Senior PJ, Windass J, J. Biotechnol., 2(5), 205 (1980)
Asenjo JA, Suk JS, J. Fem Technol., 64, 271 (1986)
Chistoserdova L, Environ. Microbiol., 13, 2603 (2011)
Salem AR, Quayle JR, Biochem. J., 124, 74 (1971)
Chistoserdova L, Chen SW, Lapidus A, Lidstrom ME, J. Bacteriol., 185(10), 2980 (2003)
Chistoserdova L, Metabolism of formaldehyde in M. extorquens AM1: Microbial Growth on C1 Compounds, Springer Netherlands, 16-24(1996).
Marx CJ, Lidstrom ME, Microbiology, 147(8), 2065 (2001)
http://www.integratedgenomics.com/genomereleases.html#list6.
Anderson AJ, Dawes EA, Microbiol Rev., 54(4), 450 (1990)
Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J, Blackall LL, Appl. Environ. Microbiol., 70(1), 588 (2004)
Korotkova N, Chistoserdova L, Lidstrom ME, J. Bacteriol., 184(22), 6174 (2002)
Korotkova N, Lidstrom ME, J. Bacteriol., 183(3), 1038 (2001)
Van Dien SJ, Strovas T, Lidstrom ME, Biotechnol. Bioeng., 84(1), 45 (2003)
Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A, Gene, 145(1), 69 (1994)
Kim HG, Han GH, Eom CY, Kim SW, J. Bacteriol., 46(1), 45 (2008)
Degelau A, Scheper T, Bailey JE, Guske C, Appl. Microbiol. Biotechnol., 42(5), 653 (1995)
Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A, Arch. Microbiol., 171(2), 73 (1999)
Lee OK, Hur DH, Nguyen DTN, Lee EY, “Metabolic Engineering of Methanotrophs and Its Application to Production of Chemicals and Biofuels from Methane,” Biofuels, Bioprod. Bioref. (2016).
Lee W, Lee J, Korean Chem. Eng. Res., 54(6), 812 (2016)

[1] Hou CT, Methylotrophs: Microbiology, biochemistry, and genetics, CRC pp. 1-53(1984).

[2] Faust U, Pr Fa H, Biotechnology. VCH Weinheim, 3, 84 (1991)

[3] Kim P, Kim JH, Oh DK, World J. Microbiol. Biotechnol., 19, 357 (2003)

[4] Schrader J, Schilling M, Holtmann D, Sell D, Villela Filho M, Marx A, Vorholt JA, Trends Biotechnol., 27, 107 (2009)

[5] Lidstrom ME, Murrell JC, 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 PJ, Windass J, J. Biotechnol., 2(5), 205 (1980)

[8] Asenjo JA, Suk JS, J. Fem Technol., 64, 271 (1986)

[9] Chistoserdova L, Environ. Microbiol., 13, 2603 (2011)

[10] Salem AR, Quayle JR, Biochem. J., 124, 74 (1971)

[11] Chistoserdova L, Chen SW, Lapidus A, Lidstrom ME, J. Bacteriol., 185(10), 2980 (2003)

[12] Chistoserdova L, Metabolism of formaldehyde in M. extorquens AM1: Microbial Growth on C1 Compounds, Springer Netherlands, 16-24(1996).

[13] Marx CJ, Lidstrom ME, Microbiology, 147(8), 2065 (2001)

[14] http://www.integratedgenomics.com/genomereleases.html#list6.

[15] Anderson AJ, Dawes EA, Microbiol Rev., 54(4), 450 (1990)

[16] Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J, Blackall LL, Appl. Environ. Microbiol., 70(1), 588 (2004)

[17] Korotkova N, Chistoserdova L, Lidstrom ME, J. Bacteriol., 184(22), 6174 (2002)

[18] Korotkova N, Lidstrom ME, J. Bacteriol., 183(3), 1038 (2001)

[19] Van Dien SJ, Strovas T, Lidstrom ME, Biotechnol. Bioeng., 84(1), 45 (2003)

[20] Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A, Gene, 145(1), 69 (1994)

[21] Kim HG, Han GH, Eom CY, Kim SW, J. Bacteriol., 46(1), 45 (2008)

[22] Degelau A, Scheper T, Bailey JE, Guske C, Appl. Microbiol. Biotechnol., 42(5), 653 (1995)

[23] Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A, Arch. Microbiol., 171(2), 73 (1999)

[24] Lee OK, Hur DH, Nguyen DTN, Lee EY, “Metabolic Engineering of Methanotrophs and Its Application to Production of Chemicals and Biofuels from Methane,” Biofuels, Bioprod. Bioref. (2016).

[25] Lee W, Lee J, Korean Chem. Eng. Res., 54(6), 812 (2016)