Institute of Microbiology and Biotechnology, Meckenheimer Allee 168, 53115, Bonn, Germany.
Appl Microbiol Biotechnol. 2016 Dec;100(23):9967-9978. doi: 10.1007/s00253-016-7680-8. Epub 2016 Jun 23.
Gluconobacter (G.) oxydans is able to incompletely oxidize various sugars and polyols for the production of biotechnologically important compound. Recently, we have shown that the organism produces and accumulates mannitol as compatible solute under osmotic stress conditions. The present study describes the role of two cytoplasmic mannitol dehydrogenases for osmotolerance of G. oxydans. It was shown that Gox1432 is a NADP-dependent mannitol dehydrogenase (EC 1.1.1.138), while Gox0849 uses NAD as cofactor (EC 1.1.1.67). The corresponding genes were deleted and the mutants were analyzed for growth under osmotic stress and non-stress conditions. A severe growth defect was detected for Δgox1432 when grown in high osmotic media, while the deletion of gox0849 had no effect when cells were exposed to 450 mM sucrose in the medium. Furthermore, the intracellular mannitol content was reduced in the mutant lacking the NADP-dependent enzyme Gox1432 in comparison to the parental strain and the Δgox0849 mutant under stress conditions. In addition, transcriptional analysis revealed that Gox1432 is more important for mannitol production in G. oxydans than Gox0849 as the transcript abundance of gene gox1432 was 30-fold higher than of gox0849. In accordance, the activity of the NADH-dependent enzyme Gox0849 in the cell cytoplasm was 10-fold lower in comparison to the NADPH-dependent mannitol dehydrogenase Gox1432. Overexpression of gox1432 in the corresponding deletion mutant restored growth of the cells under osmotic stress, further strengthening the importance of the NADP-dependent mannitol dehydrogenase for osmotolerance in G. oxydans. These findings provide detailed insights into the molecular mechanism of mannitol-mediated osmoprotection in G. oxydans and are helpful engineering strains with improved osmotolerance for biotechnological applications.
氧化葡萄糖菌（G.）能够不完全氧化各种糖和多元醇，用于生产具有生物技术重要性的化合物。最近，我们已经表明，该生物体在渗透胁迫条件下产生和积累甘露醇作为相容溶质。本研究描述了两种细胞质甘露醇脱氢酶对氧化葡萄糖菌耐渗性的作用。结果表明，Gox1432 是一种 NADP 依赖性甘露醇脱氢酶（EC 1.1.1.138），而 Gox0849 使用 NAD 作为辅助因子（EC 1.1.1.67）。删除相应的基因，并分析突变体在渗透胁迫和非胁迫条件下的生长情况。当在高渗培养基中生长时，Δgox1432 表现出严重的生长缺陷，而当细胞在培养基中暴露于 450 mM 蔗糖时，gox0849 的缺失没有影响。此外，与亲本菌株和应激条件下的Δgox0849 突变体相比，缺乏 NADP 依赖性酶 Gox1432 的突变体的细胞内甘露醇含量降低。此外，转录分析表明，Gox1432 比 Gox0849 对氧化葡萄糖菌中甘露醇的产生更为重要，因为基因 gox1432 的转录丰度是 gox0849 的 30 倍。相应地，与 NADPH 依赖性甘露醇脱氢酶 Gox1432 相比，细胞细胞质中 NADH 依赖性酶 Gox0849 的活性低 10 倍。在相应的缺失突变体中过表达 gox1432 恢复了细胞在渗透胁迫下的生长，进一步证实了 NADP 依赖性甘露醇脱氢酶对氧化葡萄糖菌耐渗性的重要性。这些发现为氧化葡萄糖菌中甘露醇介导的渗透保护的分子机制提供了详细的见解，并有助于工程菌株提高生物技术应用中的耐渗性。
