启动子和质粒对酵母细胞青蒿二烯产量的影响

中国生物工程杂志

2013, Vol. 33

Issue (8): 17-24

研究报告

启动子和质粒对酵母细胞青蒿二烯产量的影响

王思佳, 丁明珠, 元英进

系统生物工程教育部重点实验室天津大学化工学院制药工程系天津 300072

The Impact of Promoters and Vectors on the Amorphadiene Production in Yeast Cells

WANG Si-jia, DING Ming-zhu, YUAN Ying-jin

Key Laboratory of Systems Biology, Ministry of Education;Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

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摘要： 为了研究并优化不同启动子和质粒类型对酵母细胞青蒿二烯产量的影响,对携带游离型质粒的酿酒酵母人工细胞进行了四种启动子替换,对携带整合型质粒和着丝粒型质粒的人工细胞进行了强启动子和弱启动子替换。利用GC-TOF/MS检测并比较以上几株人工细胞的发酵产物浓度。结果表明,所构建的人工细胞均能生产青蒿二烯,其中携带着丝粒型质粒的人工细胞SyBE_001243(W-tH-20[pRS316/TDH/ADS])产量最高,为44.7 mg/L。进一步对比以上结果发现,发酵液中青蒿二烯的浓度与启动子的强弱呈正相关;对每种底盘细胞,发酵液中青蒿二烯的浓度与导入的外源模块中质粒类型有关;且对于强启动子,携带不同类型质粒的人工细胞发酵液中青蒿二烯的浓度差异较大,而对于弱启动子,携带不同类型质粒的人工细胞发酵液中青蒿二烯的浓度均较低,浓度差异较小。

关键词： 青蒿二烯; 青蒿素; 酵母人工细胞; 合成生物学

Abstract: To investigate the impact of different promoters and vectors on the amorphadiene production in yeast cells, four promoters are replaced in the yeast cells with free replicated plasmid; a strong promoter and a weak promoter are replaced in the yeast cells with integrated or centromeric plasmid. GC-TOF/MS analysis is utilized to detect and compare fermentation products concentration of the above cells. The results show that all of the constructed yeast cells can produce amorphadiene, and the cells with centromeric plasmid SyBE_001243(W-tH-20[pRS316/TDH/ADS]) is with the highest yield of 44.7mg/L. Further comparison of the foregoing results show that concentration of the amorphadiene in the fermentation broth is positively correlated to the strength of the promoters. Moreover, for each chassis cell, concentration of the amorphadiene in the fermentation broth is related to the type of plasmid of the introduced module. And for the strong promoter, concentration of the amorphadiene in the fermentation broth is in large differences among different plasmid types of engineered cells. For the weak promoter, the concentration is in small differences among different plasmid types of engineered cells, which is much lower than the strong promoter.

Key words: Amorphadiene Artimesinin Engineered yeasts Synthetic biology

收稿日期: 2013-05-10 出版日期: 2013-08-25

ZTFLH:

基金资助: 国家 "863"计划资助项目(2012AA02A701)

通讯作者: 丁明珠ding_mingzhu@tju.edu.cn E-mail: ding_mingzhu@tju.edu.cn

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引用本文:

王思佳, 丁明珠, 元英进. 启动子和质粒对酵母细胞青蒿二烯产量的影响[J]. 中国生物工程杂志, 2013, 33(8): 17-24.

WANG Si-jia, DING Ming-zhu, YUAN Ying-jin. The Impact of Promoters and Vectors on the Amorphadiene Production in Yeast Cells. China Biotechnology, 2013, 33(8): 17-24.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I8/17

[1] 孔建强,程克棣,王丽娜,等. HMG-CoA还原酶和FPP合酶基因拷贝数对紫穗槐-4,11-二烯酵母工程菌产量的影响. 药学学报,2007,42(12):1314-1319. Kong J Q, Cheng K D, Wang L N, et al. Increase of copy number of HMG-CoA reductase and FPP synthase genes improves the amorpha-4,11-diene production in engineered yeast. Acta Pharmaceutica Sinica,2007,42(12):1314-1319.
[2] 刘夺,杜瑾,赵广荣,等.合成生物学在医药及能源领域的应用. 化工学报,2011,62(9):2391-2397. Liu D, Du J, Zhao G R, et al. Applications of synthetic biology in medicine and energy.CIESC Journal,2011,62(9):2391-2397.
[3] 孔建强,黄勇,沈君豪,等. 紫穗槐-4,11-二烯合酶及其代谢工程研究进展. 药学学报,2009,44 (12):1320-1327. Kong J Q, Huang Y, Shen J H, et al. Recent advances in the study of amorpha-4,11-diene synthase and its metabolic engineering . Acta Pharmaceutica Sinica,2009,44 (12):1320-1327.
[4] Martin V J J, Pitera D J, Withers S T, et al. Engineering a mevalonale pathway in Escherichia coli for production of terpenoids.Nat Biotechnol,2003,21(7):796-803.
[5] Newman J D, Marshall J, Chang M, et al. High-level production of amorpha4,11-diene in a two-phase partitioning bioreactor of metabolically engineered Escherichia coli. Biotech Bioeng,2006,95(4):684-691.
[6] Lindahl A L, Olsson M E, Mercke P, et al.Production of the artemisinin precursor amorpha-4,11-diene by engineered Saccharomyces cerevisiae.Biokchnol Lett,2006,28(8):571-580.
[7] Ro D K, Paradise E M, Ouellet M, et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast.Nature,2006,440(13):940-943.
[8] Alper H, Fischer C, Nevoigt E, et al. Tuning genetic control through promoter engineering. PNAS,2005,102(36):12678-12683.
[9] Nevoigt E, Kohnke J, Fischer C R, et al.Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol,2006,72(8):5266-5273.
[10] Chenfeng L, Jeffries T. Shuffling of promoters for multiple genes to optimize xylose fermentation in an engineered Saccharomyces cerevisiae strain.Appl Environ Microbiol,2007 ,73(19):6072-6077.
[11] Lee T I, Rinaldi N J, Robert F, et al. Transcriptional regulatory networks in Saccharomyces cerevisiae.Science,2002,298:799-804.
[12] 孔建强,沈君豪,黄勇,等.酵母工程菌制备紫穗槐-4,11-二烯的研究. 药学学报,2009,44 (11):1297-1303. Kong J Q, Shen J H, HuangY, et al. Production of amorpha-4,11-diene in engineered yeasts. Acta Pharmaceutica Sinica,2009,44 (11):1297-1303.
[13] Adams A.酵母遗传学实验方法指南.北京:科学出版社,2000. Adams A. Methods in Yeast Genetics. Beijing:Science Press,2000.
[14] Gietz R D, Schiestl R H, Willems A R, et al. Studies on the transformation of intact yeast cells by the LiAc/ss-DNA/PEG procedure . Yeast,1995,11(4):355-360.
[15] Wallaart T E, Bouwmeester H J, Hille J, et al. Amorpha-4,11-diene synthase:cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta,2001,212:460-465.
[16] Ro D K, Ouellet M, Paradise E M, et al. Induction of multiple pleiotropic drug resistance genes in yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. BMC Biotechnology, 2008,8:83.
[17] Mumberg D, Muller R, Funk M. Regulatable promoters of Saccharomyces cerevisiae:comparison of transcriptional activity and their use for heterologous expression.Nucleic Acids Research,1994,22(25):5767-5768.
[18] Ohto C, Muramatsu M, Obata S, et al. Overexpression of the gene encoding HMG-CoA reductase in Saccharomyces cerevisiae for production of prenyl alcohols. Appl Microbiol Biotechnol ,2009,82:837-845.
[19] Kong J Q, Wang W, Wang L N. et al. The improvement of amorpha-4,11-diene production by a yeast-conform variant. Journal of Applie Microbiology,2009,106:941-951.
[20] 贾云婧,赵鹃,丁明珠,等. 青蒿二烯功能模块与酵母底盘的适配性研究.高校化学学报,2014,2. Jia Y J, Zhao J, Ding M Z. et al. Fitness of amorphadiene production functional modules and yeast chassis. Chiemical Journal of Chinese Universities,2014,2.
[21] Muntendam R, Melillo E, Ryden A, et al. Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts.Appl Microbiol Biotechnol,2009,84:1003-1019.
[22] Engelsa B, Dahmb P, Jennewein S. Metabolic engineering of taxadiene biosynthesis in yeast as a first step towards taxol(paclitaxel) production. Metabolic Engineering,2008,10:201-206.
[23] Dai Z B, Liu Y, Huang L Q, et al. Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2012,109(11):2845-2853.

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