Biological Science Laboratories, Kao Corporation, 2606 Akabane Ichikai, Haga, Tochigi 321-3497, Japan.
Microb Cell Fact. 2012 Jun 8;11:74. doi: 10.1186/1475-2859-11-74.
Bacillus subtilis genome-reduced strain MGB874 exhibits enhanced production of exogenous extracellular alkaline cellulase Egl-237 and subtilisin-like alkaline protease M-protease. Here, we investigated the suitability of strain MGB874 for the production of α-amylase, which was anticipated to provoke secretion stress responses involving the CssRS (Control secretion stress Regulator and Sensor) system.
Compared to wild-type strain 168, the production of a novel alkaline α-amylase, AmyK38, was severely decreased in strain MGB874 and higher secretion stress responses were also induced. Genetic analyses revealed that these phenomena were attributable to the decreased pH of growth medium as a result of the lowered expression of rocG, encoding glutamate dehydrogenase, whose activity leads to NH3 production. Notably, in both the genome-reduced and wild-type strains, an up-shift of the external pH by the addition of an alkaline solution improved AmyK38 production, which was associated with alleviation of the secretion stress response. These results suggest that the optimal external pH for the secretion of AmyK38 is higher than the typical external pH of growth medium used to culture B. subtilis. Under controlled pH conditions, the highest production level (1.08 g l(-1)) of AmyK38 was obtained using strain MGB874.
We demonstrated for the first time that RocG is an important factor for secretory enzyme production in B. subtilis through its role in preventing acidification of the growth medium. As expected, a higher external pH enabled a more efficient secretion of the alkaline α-amylase AmyK38 in B. subtilis. Under controlled pH conditions, the reduced-genome strain MGB874 was demonstrated to be a beneficial host for the production of AmyK38.
枯草芽孢杆菌基因组减毒株 MGB874 表现出增强的外源细胞外碱性纤维素酶 Egl-237 和枯草菌素样碱性蛋白酶 M-蛋白酶的生产。在这里，我们研究了菌株 MGB874 生产α-淀粉酶的适宜性，预计这会引发涉及 CssRS（控制分泌应激调节剂和传感器）系统的分泌应激反应。
与野生型菌株 168 相比，新型碱性α-淀粉酶 AmyK38 的产量在 MGB874 中严重降低，并且还诱导了更高的分泌应激反应。遗传分析表明，这些现象归因于生长培养基 pH 值降低，这是由于谷氨酸脱氢酶 rocG 的表达降低所致，其活性导致 NH3 的产生。值得注意的是，在基因组减毒株和野生型菌株中，通过添加碱性溶液来提高外部 pH 值均可改善 AmyK38 的生产，这与缓解分泌应激反应有关。这些结果表明，AmyK38 分泌的最佳外部 pH 值高于用于培养枯草芽孢杆菌的典型生长培养基的外部 pH 值。在控制 pH 值的条件下，使用 MGB874 菌株获得了最高的 AmyK38 生产水平（1.08g/L）。
我们首次证明 RocG 通过其在防止生长培养基酸化中的作用，是枯草芽孢杆菌中分泌酶生产的重要因素。如预期的那样，较高的外部 pH 值使碱性α-淀粉酶 AmyK38 在枯草芽孢杆菌中的分泌更有效率。在控制 pH 值的条件下，已证明基因组减毒株 MGB874 是生产 AmyK38 的有益宿主。
