1172 微生物学通报 2008, Vol.35, No.8 [2], ;,,, (Saccha- romyces cerevisiae),,,,, [3] γ- GSH1 γ-, - [4],, ADH2 ADH II (alcohol dehydrogenase II),, [5] ADH2 G

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1 微生物学通报 AUG 20, 2008, 35(8): 1171~1175 Microbiology 2008 by Institute of Microbiology, CAS 研究报告低乙醇脱氢酶 Ⅱ 活性的抗老化啤酒 1 蔡勇酵母工程菌的构建 1 母茜 2 王肇悦 2 张博润 1* 晏本菊 ( ) ( ) 摘要 : 采用自克隆技术, 破坏啤酒酵母工业菌株 YSF31 的 ADH2 基因, 在 ADH2 基因位点插入来源于 YSF31 的编码 γ- 谷氨酰半胱氨酸合成酶的 GSH1 基因和铜抗性筛选标记 CUP1 基因通过铜抗性筛选转化子, 经 PCR 和乙醇脱氢酶 (ADH ) 活性测定验证, 获得了 1 株啤酒酵母工程菌 10 P 麦芽汁发酵实验显示, 自克隆菌株的乙醇脱氢酶活性是受体菌的 65%, 谷胱甘肽含量比受体菌 YSF31 的高 34% 其他发酵指标并没有发生明显改变由于 DNA 操作过程中没有外源基因介入, 因此啤酒酵母工程菌为生物安全的自克隆菌株, 具有重要的应用价值关键词 : 啤酒酵母工业菌株, 谷胱甘肽, 乙醇脱氢酶, 自克隆 Construction of Self-cloning Industrial Brewing Yeast with High-glutathione Production and Low-ADH II Enzyme Activity CAI Yong 1 MU Qian 1 WANG Zhao-Yue 2 ZHANG Bo-Run 2 YAN Ben-Ju 1* (1. College of Life Sciences, Sichuan Agricultural University, Ya an ) (2. Institute of Microbiology, Chinese Academy of Science, Beijing ) Abstract: Self-cloning strains of industrial brewing yeast were constructed by integrating Saccharomyces cerevisiae genes, γ-glutamylcysteine synthetase gene (GSH1) and copper resistant gene (CUP1) into the locus of alcohol dehydrogenase II (ADH II) gene (ADH2). The self-cloning strains were selected for their resistance to CuSO 4 and identified by PCR amplification. The results of ADH II and glutathione (GSH) assay from fermentation with the self-cloning strains in 500ml conical flask showed that ADH II activity decreased to 65% and GSH content was 1.3 fold compared with that of host yeasts. The self-cloning strains do not contain any heterologous DNA; they may be more acceptable to the public. Keywords: Industrial brewing yeast, Glutathione, Alcohol dehydrogenase II, Self-cloning (CH 3 CHO) [1] 5 mg/l~12 mg/l 3 mg/l, * 通讯作者 :Tel: ; : yanbenju@sicau.edu.cn 收稿日期 : ; 接受日期 :

2 1172 微生物学通报 2008, Vol.35, No.8 [2], ;,,, (Saccha- romyces cerevisiae),,,,, [3] γ- GSH1 γ-, - [4],, ADH2 ADH II (alcohol dehydrogenase II),, [5] ADH2 GSH1 ADH2, 1, 1 材料与方法 1.1 菌株和质粒 (Escherichia coli)dh5α YSF31 YEp352(amp R URA3) / CUP1 pycup GSH1 pgf 培养基和工具酶 [6] YEPD [7] T4 DNA DNA ( ) 1.3 遗传操作 [6] DNA [7], [8] 1.4 引物合成与 PCR 扩增 ADH2 [9] P1 5 -CAAGAATTCAGCACAACAATACCAGTCC G-3 P2 5 -GGCAAGCTTAACATTGATGATACC CTGGG-3 EcoR Hind YSF31 DNA, PCR PCR s; 56 1 min, 72 2 min, 30 ; min 1.5 自克隆菌株的构建酵母菌转化 :, 6 mmol/l CuSO 4 YEPD PCR 验证自克隆菌株 : PCR 1 PCR 25 μl, 2 μl 10 PCR buffer, 1.2 mmol/l MgCl2, 200 μmol/l dntp, 400 ng DNA, 0.2 μmol/l, 1.5 U Taq 94 5 min; s, 55 1 min, 72 3 min, 30 ; min Primers CUP1-1 CUP1-2 GSH1-1 GSH1-2 表 1 引物列表 Table 1 List of primers Sequence 5 3 CGCTATACGTGCATATGTTC ATCTGTTGTACTATCCGCTT ATCTCATATTGACTTCCTT TACAAGATCTAACAGGAGCA 1.6 遗传稳定性分析 YEPD, 5 ml YEPD ( ), 28, 24 h 1, 5, YEPD, , 4 h, YEPD 6 mmol/l CuSO 4 YEPD, h, CuSO 谷胱甘肽含量测定 [10] GSH 1.8 乙醇脱氢酶 Ⅱ 活性测定 [11]

3 : 小试发酵实验 5 ml, h, ml, h, 270 ml, d 2 结果 2.1 重组质粒的构建 pacg YSF31 DNA, PCR 1.7 kb ADH2 EcoR Hind PCR, YEp352 EcoR Hind pya Sac Sal pycup CUP1, Sal Sph pgf-2 GSH1, pya Sac Sph Pacg ( 1) pacg ( 2) ADH2 0.5 kb DNA CUP1 GSH1 Fig. 1 图 1 重组质粒 pacg 的构建 Construction of recombinant plasmid pacg 2.2 酵母菌的转化与筛选 PvuⅡ pacg kb DNA CUP1 - GSH1, ADH2 5 3 DNA YSF31, ADH2 2.3 转化子的验证 PCR 扩增验证 : 1 ADH2 CUP1 GSH1, ADH2 DNA, CUP1-1/CUP1-2, GSH1-1/GSH1-2, CUP1-1/GSH1-1 PCR, CUP1 GSH1 1.1 kb 4.2 kb, CUP1-1/GSH kb, ( 3) CUP1 GSH1

YEPD, 2.5 自克隆菌株的小型发酵实验, 4, ( 4), 8 YSF31 34 GSH1 ADH2, ADH II, 65-N ( 2) CO 2, 图 3 受体菌和转化子的 PCR 分析 Fig.

4 1174 微生物学通报 2008, Vol.35, No.8 图 2 质粒 pacg 的酶切验证图 Fig. 2 Digestion patterns of plasmid pacg M: DNA marker; 1: EcoRⅤ ; 2: ScaⅠ ; 3: SalⅠ ; 4: NcoⅠ ; 5: EcoRⅠ M: DNA marker; 1: EcoR Ⅴ ; 2: Sca Ⅰ ; 3: Sal Ⅰ ; 4: Nco Ⅰ ; 5: EcoRⅠ 100 YEPD 6 mmol/l CuSO 4 YEPD, 2.5 自克隆菌株的小型发酵实验, 4, ( 4), 8 YSF31 34 GSH1 ADH2, ADH II, 65-N ( 2) CO 2, 图 3 受体菌和转化子的 PCR 分析 Fig. 3 PCR analysis of self-cloning strains and their hosts M: DNA marker; 1,2,5,6 : CUP1-1/ CUP1-2; 3,7 : GSH1-1/ GSH1-2; 4,8, CUP1-1/ GSH1-1; 1,5 : ; 2~4 : YSF31 DNA; 6~8 : DNA M: DNA marker; Primers for lane 1,2,5,6: CUP1-1/CUP1-2; Primers for lane 3,7: GSH1-1/GSH1-2; lane 4,8, CUP1-1/GSH1-1; DNA templates for lane 1,5: Negative control; DNA templates for lane 2-4, DNA of YSF31; DNA templates for lane 6-8: DNA of self-cloning strain ADHⅡ 酶活测定 : [10],, 65 ADH2 2.4 遗传稳定性分析, 100,, 100 YEPD, 6 mmol/l CuSO 4 YEPD 图 4 自克隆菌株和受体菌的谷胱甘肽含量和 ADHⅡ 酶活比较 Fig. 4 GSH content and ADH II activity of the self-cloning strain and host 表 2 发酵指标的检测 Table 2 Fermentation profiles Profiles YSF31 Self-cloning strain CO 2 weight reducing (g/l) Flocculation (%) α-n-amino acid assimilation (%) Real degree fermentation (%) 讨论, ADH2 DNA CUP1 GSH1, 1 ADH, GSH, ADH2 GSH1, GSH GSH

5 : 1175,, GSH,, GSH, GSH, ADH2, ADH,,, CUP1 - GSH1 YSF31, 参考文献 [1] Margalith PZ. Flavor Microbiology. Springfield, Illinois: Charles C Thomas Publishers, 1981, pp [2] Adams MR, Moss MO. Food Microbiology. Cambridge, UK: Royal Society of Chemistry, 1995, p.290. [3] Orna CH, Gisela S. Roles of the glutathione- and thioredoxin- dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Ann Rev Microbiol, 2000, 54: [4] Fu XY, He XP, Guo XN, et al. Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae. Biotechnology Letters, 2004, 26(5): [5] Michael GS, Shelley GD, Michael S, et al. Microbial synergy via an ethanol- triggered pathway. Mol Cell Biol, 2004, 24(9): [6] Sambrook J, Fritseh EF, Maniatis T. Molecular cloning- a laboratory manual. 3 rd Ed. New York: Cold Spring Harbor Laboratory Press, 2001, p.33. [7] Burke D, Dawson D, Stearns T. Methods in yeast genetics-a cold spring harbor laboratory course manual. New York: Cold Spring Harbor Laboratory Press, 2000, pp [8] Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet, 1989, 16(5-6): [9] Russell DW, Smith M, Williamson VM, et al. Nucleotide sequence of the yeast alcohol dehydrogenase II gene. J Biol Chem, 1983, 258(4): [10],,,.., 2000, 20(1): [11] Denis CL, Ciriacy M, Young ET. A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae. J Mol Biol, 1981, 148(4): 稿件书写规范论文中有关正斜体的约定 ( ),,,,, BamH Hind EcoR Msp Sau3A 3,,,,,

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇