Expression of degQ gene and its effect on lipopeptide production as well as formation of secretory proteases in Bacillus subtilis strains

<em>Bacillus subtilis</em> is described as a promising production strain for lipopeptides. In case of <em>B. subtilis</em> strains JABs24 and DSM10^T, surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho-transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In <em>B. subtilis</em> 168, a point mutation in the <em>degQ</em> promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14-fold reduced expression. This effect on <em>degQ</em> expression and the associated impact on lipopeptide formation was examined for <em>B. subtilis</em> JABs24, a lipopeptide-producing derivative of strain 168, and <em>B. subtilis</em> wild-type strain DSM10^T, which has a native <em>degQ</em> expression. Based on stimulatory effects of the DegU regulator on secretory protease formation, the impact of <em>degQ</em> expression on extracellular protease activity was additionally investigated. To follow the impact of <em>degQ</em>, a deletion mutant was constructed for DSM10^T, while a natively expressed <em>degQ</em> version was integrated into strain JABs24. This allowed strain-specific quantification of the stimulatory effect of <em>degQ</em> expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10^T. While an unaffected <em>degQ</em> expression reduced surfactin production in JABs24 about 25%, a 6-fold increase in plipastatin was observed. In contrast, <em>degQ</em> deletion in DSM10^T increased surfactin titer by 3-fold but decreased plipastatin production by 5-fold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.枯草芽孢杆菌（Bacillus subtilis）是一类极具应用前景的脂肽类化合物（lipopeptides）生产宿主菌株。针对枯草芽孢杆菌JABs24与DSM10^T菌株，二者均可合成表面活性素（surfactin）与脂肽菌素（plipastatin）。脂肽的生物合成受多种调控因子协同调控，其中包括DegU应答调控蛋白（DegU response regulator）。多效调控因子DegQ可激活DegS传感器激酶（DegS sensor kinase）介导的磷酸化激活通路，进而增强DegU的调控活性。在枯草芽孢杆菌168菌株中，degQ基因启动子区域发生的点突变会导致该基因转录表达水平下调，对应的报告菌株检测结果显示，该突变株的degQ基因表达量仅为野生型的1/14。本研究以脂肽生产菌株168的衍生株JABs24，以及携带天然degQ表达水平的野生型枯草芽孢杆菌DSM10^T菌株为研究对象，探究了degQ基因表达异常对自身的调控效应，以及由此带来的脂肽合成变化。鉴于DegU应答调控蛋白对分泌型蛋白酶合成具有激活效应，本研究同时考察了degQ基因表达对菌株胞外蛋白酶活性（extracellular protease activity）的影响。为明确degQ基因的调控作用，本研究为DSM10^T菌株构建了degQ基因缺失突变株（deletion mutant），并将天然表达型degQ基因整合至JABs24菌株中。该遗传操作实现了两菌株的特异性定量分析，明确了degQ基因表达对JABs24与DSM10^T菌株中脂肽菌素（plipastatin）合成的激活效应，以及对表面活性素（surfactin）合成的抑制效应。在JABs24菌株中，维持degQ基因正常表达可使表面活性素合成量下降约25%，但可使脂肽菌素合成量提升6倍。与之相反，在DSM10^T菌株中敲除degQ基因可使表面活性素效价提升3倍，但脂肽菌素合成量下降至原始水平的1/5。此外，尽管本研究检测到胞外蛋白酶活性存在显著差异，但并未观察到发酵培养过程中表面活性素与脂肽菌素的合成量出现下降。