Mientus Markus, Kostner David, Peters Björn, Liebl Wolfgang, Ehrenreich Armin
Lehrstuhl für Mikrobiologie Technische Universität München, Emil-Ramann-Str. 4, 85354, Freising, Germany.
Appl Microbiol Biotechnol. 2017 Apr;101(8):3189-3200. doi: 10.1007/s00253-016-8069-4. Epub 2017 Jan 7.
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.
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、醇类及相关化合物的能力而被用于生物技术领域。这些反应由膜结合脱氢酶（mDHs）催化，这些酶通常具有广泛的底物谱。在本研究中，对氧化葡萄糖杆菌621H的六种mDHs的启动子进行了表征。利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型启动子构建了一个穿梭载体系统，用于在缺乏其mDHs的多缺失菌株氧化葡萄糖杆菌BP.9中实现mDHs的全功能表达。该系统用于表达氧化葡萄糖杆菌621H的每种mDH，以便分别表征它们氧化的底物。在55种测试化合物中，乙醇脱氢酶氧化了30种底物，多元醇脱氢酶氧化了25种。乙醇脱氢酶的底物谱与醛脱氢酶有很大重叠，与多元醇脱氢酶有部分重叠。因此，我们能够解析氧化葡萄糖杆菌621H主要mDHs的重叠底物谱。所描述的方法也可用于表达和详细表征来自其他醋酸菌或宏基因组的mDHs所使用的底物。
