利用增强型绿色荧光蛋白研究不同启动子在乳酸克鲁维酵母中的功能

doi:10.13560/j.cnki.biotech.bull.1985.2017-0049

摘要/Abstract

摘要： 以增强型绿色荧光蛋白基因（egfp）为报告基因评价不同启动子在乳酸克鲁维酵母（Kluyveromyces lactis）中的转录功能,寻找高效表达外源蛋白的启动子。以改造后载体pKLAC1*为基础,PCR克隆来源于K. lactis、酿酒酵母（Saccharomyces cerevisiae）和Spathaspora passalidarum中7个不同的启动子,采用重叠延伸PCR分别与egfp融合后插入pKLAC1*,Bst XI线性化重组表达载体后电转化K. lactis GG799获得重组菌,利用特异性引物PCR扩增法快速筛选获得不同启动子调控下的单拷贝整合重组菌,通过定性和定量分析绿色荧光蛋白（EGFP）的表达,比较不同启动子的转录活性。荧光显微镜观察结果表明,K. lactis来源的pKladh4、pKlmal22,S. cerevisiae来源的pScgal7、pScgpd1,S. passalidarum来源的pSpmal1、pSpmal6、pSpgal1均成功调控EGFP的表达。荧光分光光度计定量测定结果表明,重组菌在pKladh4、pScgal7、pScgpd1调控下荧光强度分别为2.82、2.92和4.74,且在相应的诱导条件下转录能力分别提高了13%、57%和22%,均高于当前应用最广泛的强启动子pKllac4（荧光强度为2.13）。探究了不同来源启动子在K. lactis中的功能,pKladh4、pScgal7、pScgpd1具有高效起始转录能力,其中pScgpd1在K. lactis中的应用为首次报道。

关键词: 乳酸克鲁维酵母, 启动子, 增强型绿色荧光蛋白, 单拷贝整合, 荧光强度

Abstract: In order to seek the promoters efficiently expressing heterologous protein,the transcription functions of different promoters were evaluated using Enhanced Green Fluorescent Protein gene（egfp）as a reporter gene in Kluyveromyces lactis. Seven different promoters from K. lactis,Saccharomyces cerevisiae and Spathaspora passalidarum were cloned by PCR,fused to egfp respectively by overlap extension PCR and cloned into the reformed vector pKLAC1*. The recombinant expression plasmids were linearized by Bst XI and electro-transformed into K. lactis GG799. Further,the single-copy integrated recombinants regulated by different promoters were screened by PCR with specific primers,and the functions of different promoters were compared after qualitative and quantitative analysis of EGFP. The results of fluorescence microscopy showed that the expression of EGFP was regulated by the pKladh4 and pKlmal22 from K. lactis,pScgal7 and pScgpd1 from S. cerevisiae,as well as pSpmal1,pSpmal6 and pSpgal1 from S. passalidarum. The quantitative results of fluorospectrophotometer revealed that the fluorescence intensity of EGFP regulated by pKladh4,pScgal7 and pScgpd1 was 2.82,2.92 and 4.74,respectively;moreover,under the condition of the corresponding induction,the expression of EGFP was improved 13%,57% and 22%,respectively. These promoters were much stronger than pKllac4（2.13）that was the most widely used currently. Conclusively,this work probed the functions of promoters from different organisms in K. lactis,and the pKladh4,pScgal7 and pScgpd1 presented efficient initial transcription. It is the first time to report the application of pScgpd1 in K. lactis.

Key words: Kluyveromyces lactis, promoter, EGFP, single-copy integration, fluorescence intensity

孙海烨, 张梁, 李由然, 顾正华, 丁重阳, 石贵阳. 利用增强型绿色荧光蛋白研究不同启动子在乳酸克鲁维酵母中的功能[J]. 生物技术通报, 2017, 33(6): 197-206.

SUN Hai-ye, ZHANG Liang, LI You-ran, GU Zheng-hua, DING Zhong-yang, SHI Gui-yang. Study on the Function of Different Promoters in Kluyveromyces lactis by Enhanced Green Fluorescent Protein[J]. Biotechnology Bulletin, 2017, 33(6): 197-206.

参考文献

[1] Idiris A, Tohda H, Kumagai H, et al. Engineering of protein secretion in yeast：strategies and impact on protein production[J]. Applied Microbiology and Biotechnology, 2010, 86（2）：403-417.
[2] Swinkels BW, van Ooyen AJJ, Bonekamp FJ. The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression[J]. Antonie van Leeuwenhoek, 1993, 64（2）：187-201.
[3] Gellissen G, Hollenberg CP. Application of yeasts in gene expression studies：a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis-a review[J]. Gene, 1997, 190（1）：87-97.
[4] Van Ooyen AJJ, Dekker P, Huang M, et al. Heterologous protein production in the yeast Kluyveromyces lactis[J]. FEMS Yeast Research, 2006, 6（3）：381-392.
[5] Dujon B, Sherman D, Fischer G, et al. Genome evolution in yeasts[J]. Nature, 2004, 430（6995）：35-44.
[6] Spohner SC, Schaum V, Quitmann H, et al. Kluyveromyces lactis：An emerging tool in biotechnology[J]. Journal of Biotechnology, 2016, 222：104-116.
[7] Heintzman ND, Ren B. The gateway to transcription：identifying, characterizing and understanding promoters in the eukaryotic genome[J]. Cellular and Molecular Life Sciences, 2007, 64（4）：386-400.
[8] Juven-Gershon T, Kadonaga JT. Regulation of gene expression via the core promoter and the basal transcriptional machinery[J]. Developmental Biology, 2010, 339（2）：225-229.
[9] Alper H, Fischer C, Nevoigt E, et al. Tuning genetic control through promoter engineering[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102（36）：12678-12683.
[10] Niedenthal RK, Riles L, Johnston M, et al. Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast[J]. Yeast, 1996, 12（8）：773-786.
[11] 吴沛桥, 巴晓革, 胡海, 等. 绿色荧光蛋白 GFP 的研究进展及应用[J]. 生物医学工程研究, 2009, 28（1）：83-86.
[12] Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein（GFP）[J]. Gene, 1996, 173（1）：33-38.
[13] Saliola M, Mazzoni C, Solimando N, et al. Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumin in Kluyveromyces lactis[J]. Applied and Environmental Microbiology, 1999, 65（1）：53-60.
[14] Mazzoni C, Saliola M, Falcone C. Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis[J]. Molecular Microbiology, 1992, 6（16）：2279-2286.
[15] Maggi RG, Govind NS. Regulated expression of green fluorescent protein in Debaryomyces hansenii[J]. Journal of Industrial Microbiology and Biotechnology, 2004, 31（7）：301-310.
[16] 蒋慧慧, 李丰功, 陆毅, 等. 三种酵母启动子在毕赤酵母中的功能比较[J]. 中国生物工程杂志, 2011, 31（5）：60-68.
[17] Leifso KR, Williams D, Hintz W E. Heterologous expression of cyan and yellow fluorescent proteins from the Kluyveromyces lactis KlMAL21-KlMAL22 bi-directional promoter[J]. Biotechnology Letters, 2007, 29（8）：1233-1241.
[18] Bergkamp RJM, Kool IM, Geerse RH, et al. Multiple-copy integra-tion of the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis[J]. Current Genetics, 1992, 21（4-5）：365-370.
[19] Madhavan A, Sukumaran RK. Promoter and signal sequence from filamentous fungus can drive recombinant protein production in the yeast Kluyveromyces lactis[J]. Bioresource Technology, 2014, 165：302-308.
[20] Read JD, Colussi PA, Ganatra MB, et al. Acetamide selection of Kluyveromyces lactis cells transformed with an integrative vector leads to high-frequency formation of multicopy strains[J]. Applied and Environmental Microbiology, 2007, 73（16）：5088-5096.
[21] 刘波, 马清钧, 吴军. 乳酸克鲁维酵母表达外源蛋白研究进展[J]. 生物技术通讯, 2007, 18（6）：1039-1042.
[22] 许强, 张梁, 李由然, 等. 腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达[J]. 微生物学通报, 2016, 43（11）：2341-2352.
[23] 唐瑞琪, 熊亮, 白凤武, 等. 酿酒酵母人工杂合启动子与天然启动子活性比较[J]. 生物技术通报, 2017, 33（1）：120-128.
[24] Tanaka R, Ishibashi M, Tokunaga H, et al. Secretion of hen egg white lysozyme from Kluyveromyces lactis[J]. Bioscience, Biotechnology, and Biochemistry, 2000, 64（12）：2716-2718.
[25] Fermiñán E, Domínguez A. Heterologous protein secretion directed by a repressible acid phosphatase system of Kluyveromyces lactis：characterization of upstream region-activating sequences in the KIPHO5 Gene[J]. Applied and Environmental Microbiology, 1998, 64（7）：2403-2408.
[26] Laplaza JM, Torres BR, Jin YS, et al. Sh ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis[J]. Enzyme and Microbial Technology, 2006, 38（6）：741-747.
[27] 范贺超. 新型木糖利用酵母的评价及其遗传表达系统构建[D]. 无锡：江南大学, 2015.