Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2011, Vol. 31 Issue (7): 91-97    
研究报告     
代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株
张晓阳1, 杜风光1, 池小琴2, 王品美2, 郑道琼2, 吴雪昌2
1. 河南天冠企业集团有限公司车用生物燃料技术国家重点实验室 南阳 473000;
2. 浙江大学生命科学学院微生物研究所 杭州 310058
Construction of Saccharomyces cerevisiae Strains Improved Stress Tolerance and Ethanol Fermentation Performance through Metabolic Engineering and Genome Recombination
ZHANG Xiao-yang1, DU Feng-guang1, CHI Xiao-qin2, WANG Pin-mei2, ZHENG Dao-qiong2, WU Xue-chang2
1. State Key Laboratory of Motor Vehicle Biofuel Technolog, Tianguan Group Co., Ltd, Nanyang 473000, China;
2. Institute of Microbiology, College of Life Science, Zhejiang University, Hangzhou 310058, China
 全文: PDF(887 KB)   HTML
摘要：

将酿酒酵母海藻糖代谢工程与全基因组重组技术相结合,改良工业酿酒酵母菌株的抗逆性和乙醇发酵性能。对来源于二倍体出发菌株Zd4的两株优良单倍体Z1和Z2菌株进行杂交获得基因组重组菌株Z12,并对Z1和Z2先进行(1)过表达海藻糖-6-磷酸合成酶基因 (TPS1) ,(2)敲除海藻糖水解酶基因 (ATH1), (3)同时过表达 TPS1和敲除ATH1, 经此三种基因工程操作后再进行杂交获得代谢工程菌株的全基因组重组菌株Z12ptps1、Z12 Δath1和Z12pTΔA。与亲株Zd4相比,Z12及结合代谢工程获得的菌株在高糖、高乙醇浓度与高温条件下生长与乙醇发酵性能都有不同程度的改进。对比研究结果表明:在高糖发酵条件下,同时过表达 TPS1和敲除ATH1 的双基因操作工程菌株胞内海藻糖积累、乙醇主发酵速率和乙醇产量相对于亲株的提高幅度要大于只过表达 TPS1,或敲除ATH1 的工程菌。结合了全基因组重组后获得的二倍体工程菌株Z12pTΔA,与原始出发菌株Zd4及重组子Z12相比,主发酵速率分别提高11.4%和6.3%,乙醇产量提高7.0%和4.1%,与其胞内海藻糖含量高于其它菌株、在胁迫条件下具有更强耐逆境能力相一致。结果证明,海藻糖代谢工程与杂交介导的全基因组重组相结合,是提高酿酒酵母抗逆生长与乙醇发酵性能的有效策略与技术途径。
关键词： 酿酒酵母;  代谢工程;  基因组重组;  乙醇发酵;  逆境    
Abstract:

Some environmental stresses, i.e., high osmotic stress, fluctuating temperature and ethanol concentration, will greatly influence the viability and capability of the yeast S.cerevisiae strains during ethanol fermentation. In this study, we constructed a series of yeast strains with improved stress tolerances and ethanol fermentation performance through trehalose metabolism engineering combined with hybridization-based whole genome recombination. Firstly, two haploid strains , Z1 and Z2 isolated from the diploid strain Zd4, were engineered to enhance the intracellular trehalose by (1) overexpression of trehalose-6-phosphate synthase gene TPS1 (Z1ptps1 and Z2ptps1), (2) deletion of acidic trehalase gene ATH1 (Z1Δath1 and Z2Δath1), and (3) TPS1 overexpression combined with ATH1 deletion (Z1pTΔA and Z2pTΔA). We then obtained four recombination strains (Z12, Z12ptps1, Z12Δath1 and Z12 pTΔA) through the hybridization of Z1 and Z2, and their engineered strains. The results of high-gravity fermentation (270 g/L glucose) showed that TPS1 overexpression combined with ATH1 deletion had a distinct advantage in the improvement of stress tolerance over the single genetic manipulation. Compared to the original strain Zd4 and Z12, the strain Z12pTΔA(the hybrid of Z1pTΔA and Z2pTΔA) improved the fermentation rate by 11.4% and ethanol yield by 7.0%, while the strain Z12 without metabolic engineering only increased the main fermentation rate by 4.8% and ethanol yield by 2.8%. These improvements of fermentation performance consisted with their tolerances of the constructed strains under the conditions with osmotic pressure, high temperature and high concentration of ethanol. The combination of trehalose metabolic engineering and genome recombinant technology could effectively improve the stress tolerance and the ethanol fermentation performance of the industrial S.cerevisiae strains is demonstrated, and an innovative strategy for industrial yeast breeding is proposed.
Key words: Saccharomyces cerevisiae    Metabolic engineering    Genome recombination    Ethanol fermentation    Stress
收稿日期: 2011-05-30 出版日期: 2011-07-25
ZTFLH:  Q786  
基金资助:

国家"863"计划资助项目(2008AA10Z338-02)
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
张晓阳
杜风光
池小琴
王品美
郑道琼
吴雪昌

引用本文:

张晓阳, 杜风光, 池小琴, 王品美, 郑道琼, 吴雪昌. 代谢工程与全基因组重组构建酿酒酵母抗逆高产乙醇菌株[J]. 中国生物工程杂志, 2011, 31(7): 91-97.

ZHANG Xiao-yang, DU Feng-guang, CHI Xiao-qin, WANG Pin-mei, ZHENG Dao-qiong, WU Xue-chang. Construction of Saccharomyces cerevisiae Strains Improved Stress Tolerance and Ethanol Fermentation Performance through Metabolic Engineering and Genome Recombination. China Biotechnology, 2011, 31(7): 91-97.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/91


[1] Yamada R, Taniguchi N, Tanaka T, et al. Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnology for Biofuels, 2011, 4(1): 8.

[2] 赵心清, 白凤武, 李寅. 系统生物学和合成生物学研究在生物燃料生产菌株改造中的应用. 生物工程学报, 2010, 26(7):880-887. Zhao X Q, Bai F W, Li Y. Chinese Journal of Biotechnology, 2010, 26(7):880-887.

[3] Li H, Wang H L, Du J, et al. Trehalose protects wine yeast against oxidation under thermal stress. World Journal of Microbiology and Biotechnology, 2010, 26(6): 969-976.

[4] Mahmud S A, Nagahisa K, Hirasawa T, et al. Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae . Yeast, 2009, 26(1): 17-30.

[5] 林贝, 赵心清, 张秋美, 等. 絮凝酵母海藻糖合成酶基因 TPS1 启动子区的克隆和乙醇胁迫下启动子活性的变化. 生物工程学报, 2010, 26(7):1014-1018. Lin B, Zhao X Q, Zhang Q M, et al. Chinese Journal of Biotechnology, 2010, 26(7):1014-1018.

[6] 吕烨, 肖冬光, 和东芹, 等. 酵母海藻糖酶缺失突变株的构建及其耐性. 微生物学报, 2008, 48(010):1301-1307. Lv Y, Xiao D G, He D Q, et al. Acta Microbiologica Sinica, 2008, 48(010):1301-1307.

[7] Zheng D Q, Wu X C, Tao X L, et al. Screening and construction of Saccharomyces cerevisiae strains with improved multi-tolerance and bioethanol fermentation performance. Bioresource Technology, 2011, 102(3): 3020-3027.

[8] Zheng D Q, Wu X C, Wang P M, et al. Drug resistance marker-aided genome shuffling to improve acetic acid tolerance in Saccharomyces cerevisiae . Journal of Industrial Microbiology & Biotechnology, 2011, 38(3): 415-422.

[9] Wang P M, Wu X C, Chi X Q, et al. Development and application of RAPD-SCAR markers to identify intra-species hybrids of industrial Saccharomyces cerevisiae . World Journal of Microbiology and Biotechnology, 2010, 27(1): 185-188.

[10] Guldener U, Heck S, Fielder T, et al. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Research, 1996, 24(13): 2519-2524.

[11] Gietz R D,Woods R A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods in Enzymology, 2002, 350: 87-96.

[12] Streeter J G,Gomez M L. Three enzymes for trehalose synthesis in Bradyrhizobium cultured bacteria and in bacteroids from soybean nodules. Applied and Environmental Microbiology, 2006, 72(6): 4250-4255.

[13] Jules M, Guillou V, Francois J, et al. Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae . Applied and Environmental Microbiology, 2004, 70(5): 2771-2778.

[14] Kim J, Alizadeh P, Harding T, et al. Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications. Applied and Environmental Microbiology, 1996, 62(5): 1563-1569.

[15] Garre E, Perez-Torrado R, Gimeno-Alcaniz J V, et al. Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae . FEMS Yeast Research, 2009, 9(1): 52-62.
[1] 马宁,王汉杰. 光遗传学在细菌生产调控中的应用进展[J]. 中国生物工程杂志, 2021, 41(9): 101-109.
[2] 苗轶男,李敬知,王帅,李春,王颖. 萜烯生物合成中关键酶的研究进展*[J]. 中国生物工程杂志, 2021, 41(6): 60-70.
[3] 董曙馨,秦磊,李春,李珺. 利用转录因子工程重塑代谢网络实现细胞工厂高效生产[J]. 中国生物工程杂志, 2021, 41(4): 55-63.
[4] 薛志勇,代红生,张显元,孙艳颖,黄志伟. 表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*[J]. 中国生物工程杂志, 2021, 41(11): 32-39.
[5] 李媛媛,李妍,曹英秀,宋浩. 黄素介导的胞外电子转移研究与工程改造*[J]. 中国生物工程杂志, 2021, 41(10): 89-99.
[6] 石鹏程, 纪晓俊. 酵母系统表达人表皮生长因子研究进展 *[J]. 中国生物工程杂志, 2021, 41(1): 72-79.
[7] 闫伟欢,黄统,洪解放,马媛媛. 丁醇在大肠杆菌中的生物合成研究进展*[J]. 中国生物工程杂志, 2020, 40(9): 69-76.
[8] 薛艳婷,吴胜波,徐程杨,袁博鑫,杨书鹃,刘家亨,乔建军,朱宏吉. 群体感应在动态代谢调控中的研究进展 *[J]. 中国生物工程杂志, 2020, 40(6): 74-83.
[9] 岑黔鸿,高彤,任怡,雷涵. 重组酿酒酵母表达幽门螺杆菌VacA蛋白及其免疫原性分析*[J]. 中国生物工程杂志, 2020, 40(5): 15-21.
[10] 刘金丛,刘雪,於洪建,赵广荣. 微生物合成根皮素及其糖苷研究进展 *[J]. 中国生物工程杂志, 2020, 40(10): 76-84.
[11] 章小毛,郭敬涵,洪解放,陆海燕,丁娟娟,邹少兰,范寰. UPRE-lac Z为报告基因评价酵母UPR响应初步研究 *[J]. 中国生物工程杂志, 2020, 40(10): 1-9.
[12] 胡妍,李辉,何承文,朱婧,谢志平. 酵母亚细胞结构分离效率评估菌株的构建 *[J]. 中国生物工程杂志, 2020, 40(10): 10-23.
[13] 马雅婷,刘珍宁,刘雪,於洪建,赵广荣. 微生物异源合成植物异喹啉生物碱的新进展 *[J]. 中国生物工程杂志, 2019, 39(11): 123-131.
[14] 张正坦,朱婧,谢志平. 酿酒酵母全基因组SNARE蛋白的亚细胞定位研究 *[J]. 中国生物工程杂志, 2019, 39(10): 44-57.
[15] 陆海燕,李佳蔓,孙思凡,章小毛,丁娟娟,邹少兰. CRISPR - Cas9系统介导的工业酵母营养缺陷型菌株构建 *[J]. 中国生物工程杂志, 2019, 39(10): 67-74.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会