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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  2021, Vol. 41 Issue (11): 32-39    DOI: 10.13523/j.cb.2106049
研究报告     
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*
薛志勇,代红生,张显元,孙艳颖,黄志伟()
东华大学化学化工与生物工程学院 生态纺织教育部重点实验室 上海 201620
Effects of Vitreoscilla Hemoglobin Gene on Growth and Intracellular Oxidation State of Saccharomyces cerevisiae
XUE Zhi-yong,DAI Hong-sheng,ZHANG Xian-yuan,SUN Yan-ying,HUANG Zhi-wei()
Key Lab of Science & Technology of Eco-textile (Ministry of Education), College of Chemistry & Chemical Engineering and Biotechnology,Donghua University, Shanghai 201620, China
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摘要：

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。
关键词： 透明颤菌血红蛋白;  酿酒酵母;  生长抑制;  细胞过氧化    
Abstract:

Heterologous expression of Vitreoscilla hemoglobin gene vgb in a variety of research and industrial fermentation bacteria can solve the problem of dissolved oxygen rate in high-density fermentation. Saccharomyces cerevisiae is a classic eukaryotic model and has an important application value in the fermentation industry. However, the impact of vgb in Saccharomyces cerevisiae on cell growth is unclear. In this study, a heterologous expression plasmid YEplac195-ADH1pr-vgb containing Vistreoscilla hemoglobin gene vgb was constructed with Adh1 as promoter, and transformed into Saccharomyces cerevisiae BY4741. Growth sensitivity test showed that the heterologous expression of vgb inhibited the growth of the strain in both fermentation and non fermentation carbon sources. Then, we found the accumulation of reactive oxygen species (ROS), the change of membrane permeability and lipid peroxidation in S. cerevisiae cells overexpressing vgb by DCFH-DA and PI staining and lipid peroxidation products assay. The results showed that the overexpression of vgb changed the oxidation state in cells, promoted the accumulation of reactive oxygen (ROS), and oxidative stress caused the growth inhibition of the strain.
Key words: Vitreoscilla hemoglobin    Saccharomyces cerevisiae    Growth inhibition    Oxidative stress
收稿日期: 2021-06-28 出版日期: 2021-12-01
ZTFLH:  Q819  
基金资助: * 中央高校基本科研业务费专项资金(2232021G)
通讯作者: 黄志伟     E-mail: zhiweih@dhu.edu.cn
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薛志勇
代红生
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孙艳颖
黄志伟

引用本文:

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.

XUE Zhi-yong,DAI Hong-sheng,ZHANG Xian-yuan,SUN Yan-ying,HUANG Zhi-wei. Effects of Vitreoscilla Hemoglobin Gene on Growth and Intracellular Oxidation State of Saccharomyces cerevisiae. China Biotechnology, 2021, 41(11): 32-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2106049    或    https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I11/32

Primer Purpose Primer sequence Template
F1 vgb cloning AACTGCAGACCCTCATGTTAGACCAG pUC8:16
R1 GGGGTACCCAAGGCACACCTGAAGAC Process biochemistry 41 (2006)930-934
F2 ADH1 promoter ACGCCAAGCTTACCCTTTTTCCATTTGCCATC Genomic DNA
R2 GTCGACCTGCAGCATTGTATATGAGATAGTTGATTG
F3 vgb RT-PCR GATATGGGTCGCCAAGAAT Yeast cell with plasmids
R3 ATCTGCTTCCACTTGAATA
表1  用于构建重组质粒YEplac195-ADH1pr-vgb的PCR引物
图1  YEplac195-ADH1pr-vgb构建及在酿酒酵母细胞中的表达
图2  过表达vgb在发酵碳源条件下对细胞生长的影响
图3  过表达vgb在非发酵碳源条件下对酵母细胞生长的影响
图4  过表达vgb促进酵母细胞活性氧积累
图5  vgb过表达危害细胞膜的完整性和引起胞内脂质过氧化
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