Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression
Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression
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DOI：
10.1007/s00253-016-8069-4
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发表时间：
2017-04-01
影响因子：
5
通讯作者：
Ehrenreich, Armin
中科院分区：
文献类型：
--
作者：
Mientus, Markus;Kostner, David;Ehrenreich, Armin
醋酸菌因其能够以区域和立体选择性方式不完全氧化多种碳水化合物、醇和相关化合物而被用于生物技术。这些反应由膜结合脱氢酶 (mDH) 催化，通常具有广泛的底物谱。在本研究中，对氧化葡糖杆菌 621H 的 6 个 mDH 的启动子进行了表征。使用乙醇脱氢酶的组成型启动子和肌醇脱氢酶的葡萄糖抑制启动子构建穿梭载体系统，以在缺乏mDH的多缺失菌株G.oxydans BP.9中实现mDH的全功能表达。该系统用于表达 G. oxydans 621H 的每个 mDH，以便单独表征它们氧化的底物。在 55 种测试化合物中，醇脱氢酶氧化 30 种底物，多元醇脱氢酶氧化 25 种底物。醇脱氢酶的底物谱大部分与醛脱氢酶重叠，部分与多元醇脱氢酶重叠。因此，我们能够解析 G. oxydans 621H 主要 mDH 的重叠底物光谱。所描述的方法还可用于来自其他乙酸细菌或宏基因组的 mDH 使用的底物的表达和详细表征。
Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.