Development of Cre-lox based multiple knockout system in Deinococcus radiodurans R1

Sun-Wook Jeong; Jung Eun Yang; Seonghun Im; Yong Jun Choi

Korean Journal of Chemical Engineering, Vol.34, No.6, 1728-1733, June, 2017

DOI10.1007/s11814-017-0082-5 Export Citation

Development of Cre-lox based multiple knockout system in Deinococcus radiodurans R1

Sun-Wook Jeong, Jung Eun Yang¹, Seonghun Im², and Yong Jun Choi^†

School of Environmental Engineering, University of Seoul, Seoul 02504, Korea
¹Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea
²Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahak-ro 125, Yuseong-gu, Daejeon 34141, Korea

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The extremophilic bacterium Deinococcus radiodurans R1 has been considered as an attractive microorganism due to its remarkable tolerance to various external stresses. Considering the nature of D. radiodurans R1, it has potential as a platform microorganism for industrial applications, including biorefinery and bioremediation process. However, D. radiodurans R1 is well known for its hard genetic manipulation. Thus, much effort has been made to develop efficient genetic engineering tools for making D. radiodurans R1 suitable for industrial platform microorganism. Although a plasmid-based single gene knockout method has been reported, development of multiple gene knockout system has not yet been reported. Here we report, for the first time, Cre-lox based rapid and efficient multiple knockout method for metabolic engineering of D. radiodurans R1. Also, deletion of dr0053 gene was successfully achieved within seven days to make biofilm overproducing strain.

Keywords:Deinococcus radiodurans R1;Cre-lox;Multiple Knockout Method;Genetic Engineering;Biofilm Production

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[1] Kumar RR, Prassad S, Indian J. Microbiol., 51, 403 (2011)

[2] Singh S, Kang SH, Mulchandani A, Chen W, Curr. Opin. Biotechnol., 19, 437 (2008)

[3] Esther J, Sukla LB, Pradhan N, Panda S, Korean J. Chem. Eng., 31, 1 (2015)

[4] Roh C, Kang CK, Lloyd JR, Korean J. Chem. Eng., 32(9), 1720 (2015)

[5] Stephanopoulos G, ACS Synth. Biol., 1, 514 (2012)

[6] Vikramaditya VG, Mey MD, Lim CG, Ajikumar PK, Stephanopoulos G, Metab. Eng., 14, 233 (2012)

[7] Makarova KS, Aravind L, Wolf YI, Tatusov RL, Minton KW, Koonin EV, Daly MJ, Microbiol. Mol. Biol. Rev., 65, 44 (2001)

[8] Shashidhar R, Kumar SA, Misra HS, Bandekar JR, Can. J. Microbiol., 56, 195 (2010)

[9] Grimsley JK, Masters CI, Clark EP, Minton KW, Int. J. Radiat. Biol., 60, 613 (1991)

[10] Brim H, McFarlan SC, Fredrickson JK, Minton KW, Zhai M, Wackett LP, Daly MJ, Nat. Biotechnol., 18, 85 (2000)

[11] Brim H, Osborne JP, Kostandarithes HM, Fredrickson JK, Wacklett LP, Daly MJ, Microbiology, 152, 2469 (2006)

[12] Abremski K, Hoess R, Sternberg N, Cell, 32, 1301 (1983)

[13] Hoess R, Abremski K, J. Mol. Biol., 181, 351 (1985)

[14] Ohba H, Satoh K, Yanagisawa T, Narumi I, Gene, 363, 133 (2005)

[15] Meima R, Lidstrom ME, Appl. Environ. Microbiol., 66, 3856 (2000)

[16] Nguyen HH, de la Tour CB, Toueille M, Vannier F, Sommer S, Servant P, Mol. Microbiol., 73, 240 (2009)

[17] Satoh K, Tu Z, Ohba H, Narumi I, Plasmid, 62, 1 (2009)

[18] Appukuttan D, Seo HS, Jeong SW, Im SH, Joe MH, Song DS, Choi JJ, Lim SY, PLoS One, 10, e0118275 (2015).

[19] O’Toole GA, Pratt LA, Watnick PI, Newman DK, Weaver VB, Kolter RG, Methods Enzymol., 310, 91 (1999)

[20] Boles BR, Horswill AR, PLoS Pathog., 4, e1000052 (2008).

[21] Moseley BE, Setlow JK, Proc. Natl. Acad. Sci. U.S.A., 61, 176 (1968)

[22] Lambert JM, Bonger RS, Kleerebezem M, Appl. Environ. Microbiol., 73, 11262 (2007)

[23] Dixit R, Wasiullah, Malaviya D, Pandiyan K, Singh UB, Sahu A, Shukla R, Singh BP, Rai JP, Sharma PK, Lade H, Paul D, Sustainability, 7, 2189 (2015)

[24] Kolari M, Nuutinen J, Salkinoja-Salonen MS, J. Ind. Microbiol. Biotechnol., 27, 343 (2001)

[25] Slade D, Radman M, Microbiol. Mol. Biol. Rev., 75, 133 (2011)

[26] Lemee L, Peuchant E, Clerc M, Brunner M, Pfander H, Tetrahedron, 53, 919 (1997)

[27] Gerber E, Bernard R, Castang S, Chabot N, Coze F, Dreux-Zigha A, Hauser E, Hivin P, Joseph P, Lazarelli C, Letellier G, Olive J, Leonetti JP, J. Appl. Microbiol., 119(1), 1 (2015)