[논문]Development and Characterization of Expression Vectors for Corynebacterium glutamicum

Lee, Jinho

doi:10.4014/jmb.1310.10032

Development and Characterization of Expression Vectors for Corynebacterium glutamicum 원문보기

Journal of microbiology and biotechnology, v.24 no.1, 2014년, pp.70 - 79

Lee, Jinho (Department of Food Science and Biotechnology, Kyungsung University)

Abstract ▼ AI-Helper

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

주제어

AI 본문요약
AI-Helper

* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.

가설 설정

glutamicum with two different vectors for over-production of valuable metabolites or proteins. I expect that the developed expression vector systems will apply to the study of genetics, physiology, and metabolic engineering of C. glutamicum.

제안 방법

In this study, I report the construction of expression vector systems for C. glutamicum with three types of promoters, P_tac, P_sod, and P_ilvC, which are known to function in C. glutamicum, and verified its capability through the analyses of quantitative reverse transcription PCR (qRT-PCR) and protein expression using green fluorescent protein (GFPuv). Furthermore, I developed novel expression vector systems in which the conserved SD sequence of C.
Native SDS-PAGE was performed as follows. Loading samples of native state were prepared by adding a loading dye (0.21 M Tris-HCl, pH 8.45, 11% glycerol, 0.8% SDS, 0.004% Coomassie blue G, 0.004% phenol red) into crude extracts and incubated for 2h at 37℃. After running the SDS-PAGE, the gel was washed three times with 10 mM Tris-HCl buffer (pH 8.

대상 데이터

2). The resulting purified fragments cut by XbaI and EcoRI were then cloned into the XbaI/EcoRI-cleaved pCXT20 in which P_tacwas removed to produce pCXS35, pCXI43, and pCXI45, respectively (Fig. 3).

성능/효과

In conclusion, I developed expression vector systems for C. glutamicum with three types of promoters and their derivatives, which are known to function in C. glutamicum, and characterized each promoter’s capability at the transcriptional and translational levels by analyses of qRTPCR, GFP fluorescence, and SDS-PAGE.

참고문헌 (41)

Becker J, Wittmann C. 2012. Bio-based production of chemicals, materials and fuels - Corynebacterium glutamicum as versatile cell factory. Curr. Opin. Biotechnol. 23: 631-640.

상세보기
Becker J, Klopprogge C, Zelder O, Heinzle E, Wittmann C. 2005. Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl. Environ. Microbiol. 71: 8587- 8596.

상세보기
Becker J, Zelder O, Hafner S, Schroder H, Wittmann C. 2011. From zero to hero-design-based systems metabolic engineering of Corynebacterium glutamicum for L-lysine production. Metab. Eng. 13: 159-168.

상세보기
Berg L, Lale R, Bakke I, Burroughs N, Valla S. 2009. The expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5'-untranslated part of mRNA. Microb. Biotechnol. 2: 379-389.

상세보기
Billman-Jacobe H, Wang L, Kortt A, Steward D, Radford A. 1995. Expression and secretion of heterologous proteases by Corynebacterium glutamicum. Appl. Environ. Microbiol. 61: 1610-1613.

상세보기
Cortay JC, Negre D, Galinier A, Duclos B, Perriere G, Cozzone AJ. 1991. Regulation of the acetate operon in Escherichia coli: purification and functional characterization of the IclR repressor. EMBO J. 10: 675-679.

상세보기
Date M, Itaya H, Matsui H, Kikuchi Y. 2006. Secretion of human epidermal growth factor by Corynebacterium glutamicum. Lett. Appl. Microbiol. 42: 66-70.

상세보기
de Smit MH, van Duin J. 1994. Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data. J. Mol. Biol. 244: 144-150.

상세보기
Hanssler E, Muller T, Palumbo K, Patek M, Brocker M, Kramer R, Burkovski A. 2009. A game with many players: control of gdh transcription in Corynebacterium glutamicum. J. Biotechnol. 142: 114-122.

상세보기
Hermann T. 2003. Industrial production of amino acids by coryneform bacteria. J. Biotechnol. 104: 155-172.

상세보기
Ikeda M, Nakagawa S. 2003. The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl. Microbiol. Biotechnol. 62: 99-109.

상세보기
Jager W, Schafer A, Puhler A, Labes G, Wohlleben W. 1992. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J. Bacteriol. 174: 5462-5465.

상세보기
Jakoby M, Ngouoto-Nkili CE, Burkovski A. 1999. Construction and application of new Corynebacterium glutamicum vectors. Biotechnol. Tech. 13: 437-441.
Jana S, Deb JK. 2005. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67: 289-298.

상세보기
Kalinowski J, Bathe B, Bartels D, Bischoff N, Bott M, Burkovski A, et al. 2003. The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of L-aspartate-derived amino acids and vitamins. J. Biotechnol. 104: 5-25.

상세보기
Khlebnikov A, Risa O, Skaug T, Carrier TA, Keasling JD. 2000. Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture. J. Bacteriol. 182: 7029-7034.

상세보기
Kim HJ, Kim TH, Kim Y, Lee HS. 2004. Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J. Bacteriol. 186: 3453-3460.

상세보기
Kohlstedt M, Becker J, Wittmann C. 2010. Metabolic fluxes and beyond - systems biology understanding and engineering of microbial metabolism. Appl. Microbiol. Biotechnol. 88: 1065- 1075.

상세보기
Komarova AV, Tchufistova LS, Dreyfus M, Boni IV. 2005. AU-rich sequences within 5' untranslated leaders enhance translation and stabilize mRNA in Escherichia coli. J. Bacteriol. 187: 1344-1349.

상세보기
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-( $\Delta\Delta$ CT) method. Methods 25: 402-408.

상세보기
Martin JF, Barreiro C, Gonzalez-Lavado E, Barriuso M. 2003. Ribosomal RNA and ribosomal proteins in corynebacteria. J. Biotechnol. 104: 41-53.

상세보기
Nesvera J, Patek M. 2011. Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl. Microbiol. Biotechnol. 90: 1641-1654.

상세보기
Neuner A, Heinzle E. 2011. Mixed glucose and lactate uptake by Corynebacterium glutamicum through metabolic engineering. Biotechnol. J. 6: 318-329.

상세보기
Park JU, Jo JH, Kim YJ, Chung SS, Lee JH, Lee HH. 2008. Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes. J. Microbiol. Biotechnol. 18: 639-647.

원문보기 상세보기
Park YS, Seo SW, Hwang S, Chu HS, Ahn JH, Kim TW, et al. 2007. Design of 5'-untranslated region variants for tunable expression in Escherichia coli. Biochem. Biophys. Res. Commun. 356: 136-141.

상세보기
Patek M, Eikmanns BJ, Patek J, Sahm H. 1996. Promoters from Corynebacterium glutamicum: cloning, molecular analysis and search for a consensus motif. Microbiology 142: 1 2 97- 1309.

상세보기
Ravasi P, Peiru S, Gramajo H, Menzella HG. 2012. Design and testing of a synthetic biology framework for genetic engineering of Corynebacterium glutamicum. Microb. Cell Fact. 11: 147-157.

상세보기
Romasi EF, Lee J. 2013. Development of indole-3-acetic acidproducing Escherichia coli by functional expression of IpdC, AspC, and Iad1. J. Microbiol. Biotechnol. 23: 1726-1736.

원문보기 상세보기
Salim K, Haedens V, Content J, Leblon G, Huygen K. 1997. Heterologous expression of the Mycobacterium tuberculosis gene encoding antigen 85A in Corynebacterium glutamicum. Appl. Environ. Microbiol. 63: 4392-4400.

상세보기
Salis HM, Mirsky EA, Voigt CA. 2009. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27: 946-950.

상세보기
Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Santamaria R, Gil JA, Mesas JM, Martin JF. 1984. Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum. J. Gen. Microbiol. 130: 2237-2246.
Seo SW, Yang J, Jung GY. 2009. Quantitative correlation between mRNA secondary structure around the region downstream of the initiation codon and translational efficiency in Escherichia coli. Biotechnol. Bioeng. 104: 611-616.

상세보기
Suzuki N, Inui M, Yukawa H. 2007. Site-directed integration system using a combination of mutant lox sites for Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 77: 871-878.

상세보기
Tauch A, Puhler A, Kalinowski J, Thierbach G. 2003. Plasmids in Corynebacterium glutamicum and their molecular classification by comparative genomics. J. Biotechnol. 104: 27-40.

상세보기
Tsuchiya M, Morinaga Y. 1988. Genetic control systems of Escherichia coli can confer inducible expression of cloned genes in coryneform bacteria. Nat. Biotechnol. 6: 428-430.

상세보기
van der Rest ME, Lange C, Molenaar D. 1999. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Appl. Microbiol. Biotechnol. 52: 541-545.

상세보기
Vasco-Cardenas MF, Banos S, Ramos A, Martin JF, Barreiro C. 2013. Proteome response of Corynebacterium glutamicum to high concentration of industrially relevant C4 and C5 dicarboxylic acids. J. Proteomics 85: 65-88.

상세보기
Vasicova P, Patek M, Nesvera J, Sahm H, Eikmanns B. 1999. Analysis of the Corynebacterium glutamicum dapA promoter. J. Bacteriol. 181: 6188-6191.

상세보기
Wendisch VF. 2003. Genome-wide expression analysis in Corynebacterium glutamicum using DNA microarrays. J. Biotechnol. 104: 273-285.

상세보기
Yim SS, An SJ, Kang M, Lee J, Jeong KJ. 2013. Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol. Bioeng. 110: 2959-2969.

상세보기

저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

내보내기 구분	파일저장 인쇄 메일전송
구성항목	기본정보 상세정보 관리번호, 논문명, 저널/프로시딩명, 저자 , 발행년, 권, 호, 시작페이지, 끝페이지, 발행기관 관리번호, 논문명, 대등논문명, 저자 , 저널/프로시딩명, 발행기관, 발행년, 발행언어, 권, 호, 시작페이지, 끝페이지, ISBN, ISSN, 주제분야, 키워드, 초록(한글), 초록(영문), 저자(소속기관)
저장형식	Text(ASCII format) Excel format RefWorks Direct Export RIS format (for Reference Manager, ProCite, EndNote), Scholar's Aids, Mendeley
메일정보	받는사람 (필수) @ 보내는사람 (선택) @ 제목 내용 KISTI 검색결과 이메일 서비스
안내	총 건의 자료가 검색되었습니다. 다운받으실 자료의 인덱스를 입력하세요. (1-10,000) 검색결과의 순서대로 최대 10,000건 까지 다운로드가 가능합니다. 데이타가 많을 경우 속도가 느려질 수 있습니다.(최대 2~3분 소요) 다운로드 파일은 UTF-8 형태로 저장됩니다. 파일의 내용이 제대로 보이지 않을실 때는 웹브라우저 상단의 보기 -> 인코딩 -> 자동선택 여부를 확인하십시오. ~ Text(ASCII format) Excel format

연합인증