[1]龚映雪,台艳,肖文娟,等.酵母多基因表达载体在纤维素生物转化中应用[J].深圳大学学报理工版,2010,27(1):82-87.
 GONG Ying-xue,TAI Yan,XIAO Wen-juan,et al.Construction of Saccharomyces cerevisiae integrated expression vector and its application in cellulose bioconvesion[J].Journal of Shenzhen University Science and Engineering,2010,27(1):82-87.
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酵母多基因表达载体在纤维素生物转化中应用()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第27卷
期数:
2010年1期
页码:
82-87
栏目:
环境与能源
出版日期:
2010-01-31

文章信息/Info

Title:
Construction of Saccharomyces cerevisiae integrated expression vector and its application in cellulose bioconvesion
文章编号:
1000-2618(2010)01-0082-06
作者:
龚映雪台艳肖文娟王峻梅唐根云全艳彩刘泽寰
暨南大学生命与健康工程研究院,广州510632
Author(s):
GONG Ying-xueTAI YanXIAO Wen-juanWANG Jun-meiTANG Gen-yunQUAN Yan-caiand LIU Ze-huan
Institutes of Life and Health Engineering,Jinan University,Guangzhou 510632 ,P. R. China
关键词:
基因工程可再生能源纤维素生物转化酿酒酵母多基因共表达绿色木霉内切葡聚糖酶纤维二糖水解酶
Keywords:
genetic engineeringrenewable energy resourcecellulosebioconversionSaccharomyces cerevisiae multigene co-expressionTrichoderma viride endoglucanasecellobiohydrolase
分类号:
Q 78;X 382.1
文献标志码:
A
摘要:
构建含酿酒酵母组成型强启动子PGK、G418抗性基因及rDNA片段的整合型载体pScIKP,利用rDNA多个同源重组位点,将外源基因以多拷贝整合到酵母染色体上,无需诱导即可持续表达;利用载体位于表达盒两端同尾酶,可插入多个基因表达盒,实现多基因稳定共表达.为检验共表达情况,反转录从绿色木霉中获得纤维素酶基因eg3和cbh2, 克隆并转化获得重组酵母菌株S.cerevisiae-ec. 该重组酵母能降解羧甲基纤维素形成水解圈;用羧甲基纤维素还原糖法和滤纸酶活力法测定酶活力,其最适温度和最适pH值与所表达单酶相似,表明共表达未影响两种酶的生物学特性;且双酶具有协同作用,能更有效降解非结晶纤维素.pScIKP载体能成功用于多个外源基因共表达和产物协同作用的研究,为构建能直接降解纤维素的酿酒酵母菌株,实现纤维素可再生能源的生物利用奠定了基础.
Abstract:
To construct a Saccharomyces cerevisiae integrated expression vector,the 3-phosphoglycerate kinase (pgk) promoter and terminator,the G418 resistance gene and the S.cerevisiae rDNA sequence were cloned into a backbone vector pBluescript II SK(-) to generate a shuttle vector pScIKP.This vector can integrate foreign genes into the yeast genome with multiple copies by homologous recombination,and constitutively express these genes without induction.Inserting several expression cassettes into the vector is a simple process that facilitates multiple genes co-expression.To test the co-expression ability,two cellulase genes endoglucanase 3 and cellobiohydrolase 2 were obtained from Trichoderma viride AS3.3711 by RT-PCR and inserted into pSCIK in order to generate the co-expression vector pScIKP-ec.The S.cerevisiae-ec transformants could stably express eg3 and cbh2 with the highest CMCase activity of 4.60 U/mL and the filter paper assay activity of 0.70 U/mL.These co-expressed enzymes were able to degrade phosphoric acid-swollen cellulose more efficiently than eg3 or cbh2 alone.These results demonstrate that the S.cerevisiae integrated vector pScIKP can co-express multiple foreign genes and be applied to the studying of the synergism of co-expression products.This system can also be used to construct S.cerevisiae, which has the ability to express multiple cellulases and directly degrade cellulosic materials.

参考文献/References:


[1] Zhang J,Tian S,Zhang Y,等.构建重组酿酒酵母表达融合蛋白和转化木糖、葡萄糖生成乙醇的研究[J].应用生物化学和生物技术,2008,150(2):185-192.(英文版)
[2] Maya D,Quintero M J,de la Cruz Muoz-Centeno M,等.酿酒酵母应用基因控制系统[J].生物技术通讯,2008,30(6):979-987.(英文版)
[3]Van Wvk J P,Mohulatsi M.里氏木霉纤维素酶生物降解废纸[J].生物资源技术,2003,86(1):21-23.(英文版)
[4]Lopes T S,Klootwijk J,Veenstra A E,等.酿酒酵母rDNA高拷贝整合:一种新的高表达载体[J].基因,1989,79(2):199-206.(英文版)
[5]McNabb D S,Reed R,Marciniak R A.用双色荧光分析系统进行酿酒酵母基因表达快速定量[J].真核细胞,2005,4(9):1539-1549.(英文版)
[6]Sambrook J,Fritsch E F,Maniatis T.分子克隆实验指南[M].第2版,纽约:美国冷泉港实验室出版社,1989.(英文版)
[7]Thompson J R,Register E,Curotto J,等.酵母细胞电击转化法的改进[J].酵母,1998,14(6):565-571.(英文版)
[8]霍克克,虞兰兰,陈新杰,等.酵母高稳定载体的构建及人-αA干扰素在酵母中的高表达和分泌[J].中国科学,1992,9:922-929.
[9]Lee J H,Lim M Y,Kim M J,等.细菌木糖内切酶和木霉葡萄糖内切酶在酿酒酵母中的持续共表达[J].微生物学技术杂志,2007,17(12):2076-2080.(英文版)
[10]Den Haan R,Rose S H,Lynd L R,等.无定型纤维素在重组酿酒酵母中的水解和发酵[J].代谢工程,2007,9(1):87-94.(英文版)
[11]Wang Y,Shi W L,Liu X Y,等.工业酿酒酵母菌株中木糖代谢途径的建立[J].生物技术通讯,2004,26(11):885-890.(英文版)
[12]Lopes T S,de Wijs I J,Steenhauer S I,等.酿酒酵母rDNA高拷贝整合影响有丝分裂稳定性的因素[J].酵母,1996,12(5):467-477.(英文版)
[13]武志强,贾耐兵,李娜,等.酵母整合型载体的构建及其功能分析.生物学通报,2008,43:47-50.


[1]Zhang J,Tian S,Zhang Y,et al.Construction of a recombinant S.cerevisiae expressing a fusion protein and study on the effect of converting xylose and glucose to ethanol[J].Applied Biochemistry and Biotechnology,2008,150(2):185-192.
[2]Maya D,Quintero M J,de la Cruz Muoz-Centeno M,et al.Systems for applied gene control in Saccharomyces cerevisiae[J].Biotechnol Lett,2008,30(6):979-987.
[3]Van Wvk J P,Mohulatsi M.Biodegradation of wastepaper by cellulase from Trichoderma viride[J].Bioresource Technology,2003,86(1):21-23.
[4]Lopes T S,Klootwijk J,Veenstra A E,et al.High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae:a new vector for high-level expression[J].Gene,1989,79(2):199-206.
[5]McNabb D S,Reed R,Marciniak R A.Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae[J].Eukaryot Cell,2005,4(9):1539-1549.
[6]Sambrook J,Fritsch E F,Maniatis T.Molecular Cloning:A Laboratory Manual[M].2nd edition.New York:Cold Spring Harbor Laboratory Press,1989.
[7]Thompson J R,Register E,Curotto J,et al.An improved protocol for the preparation of yeast cells for transformation by electroporation[J].Yeast,1998,14(6):565-571.
[8]HUO Ke-ke,YU Lan-lan,CHEN Xin-jie,et al.Construction of high stable Saccharomyces cerevisiae vector and hlFN-αA expression[J].Science in China,1992,9:922-929.(in Chinese)
[9]Lee J H,Lim M Y,Kim M J,et al.Constitutive coexpression of Bacillus endoxylanase and Trichoderma endoglucanase genes in Saccharomyces cerevisiae[J].Journal of Microbiology and Biotechnology,2007,17(12):2076-2080.
[10]Den Haan R,Rose S H,Lynd L R,et al.Hydrolysis and fermentation of amorphous cellulose by recombinant Saccharomyces cerevisiae[J].Metabolic Engineering,2007,9(1):87-94.
[11]Wang Y,Shi W L,Liu X Y,et al.Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae[J].Biotechnology Letters,2004,26(11):885-890.
[12]Lopes T S,de Wijs I J,Steenhauer S I,et al.Factors affecting the mitotic stability of high-copy-number intergration into the ribosomal DNA of Saccharomyces cerevisiae[J].Yeast,1996,12(5):467-477.
[13]WU Zhi-qiang,JIA Nai-bing,LI Na,et al.Construction of Saccharomyces cerevisiae integrated vector and functional analysis[J].Bulletin of Biology,2008,43:47-50.(in Chinese)

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备注/Memo

备注/Memo:
收稿日期:2009-09-18;修回日期:2009-12-23
基金项目:新世纪优秀人才支持计划基金资助项目(NCET-05-0745);珠海市科技攻关资助项目(PC20071080)
作者简介:龚映雪(1978-),女(汉),湖北省宜昌市人,暨南大学副研究员、博士.E-mail:tyxgong@jnu.edu.cn
通讯作者:刘泽寰(1972-),男(汉),暨南大学副教授、博士.E-mail:zhliu@jnu.edu.cn
更新日期/Last Update: 2010-02-06