【摘要】 刺糖多孢菌(Saccharopolyspora spinosa)是重要的工业微生物,可产生绿色广谱的生物杀虫剂—多杀菌素(spinosad)。目前我国多杀菌素单位发酵产量较低,为了更有效改造多杀菌素高产菌株,需对刺糖多孢菌次级代谢调控机制进行研究。群感效应是微生物通过级联网络调控自身次级代谢和/或形态分化的一种调控机制,而刺糖多孢菌中的群感效应研究未见报道。本研究发现刺糖多孢菌发酵液提取物可诱导γ-丁酸内酯(γ-butyrolactone,GBL)指示菌株产生色素;在培养72 h添加其发酵液提取物的水复溶物可使多杀菌素产量提高22.6%,提示刺糖多孢菌可产生GBL类似信号分子,存在群感效应类似级联调控系统。生物信息学分析表明,刺糖多孢菌中存在1个GBL受体同源蛋白SsbR(Saccharopolyspora spinosaγ-butyrolactone receptor)和3个中心转录调控因子同源蛋白(Saccharopolyspora spinosa central transcription activator,SstA)SstA1、SstA2和SstA3。利用qRT-PCR检测发酵过程中刺糖多孢菌群感效应关键级联调控同源基因的表达情况,结果表明,ssbR和sstAs的表达水平变化趋势与灰色链霉菌(Streptomyces griseus)中adpA(A-factor dependent protein)被ArpA(A-factor receptor protein)阻遏调控的模型相似。同时,刺糖多孢菌中3个靶标蛋白同源基因griR(grixazone regulator)、sprA(serine protease A)、ssgA(sporulation of Streptomyces griseus A)在发酵中的表达水平变化趋势和sstAs一致,提示这3个基因可能受SstAs的激活调控。本研究为深入探讨刺糖多孢菌的群感效应调控机制提供了理论基础。更多还原

【Abstract】 Saccharopolyspora spinosa(S. spinosa) is an important industrial microorganism known for spinosad production, which is a kind of biological insecticides with high efficiency, broad spectrum and low toxicity. At present, the yield of spinosad is low for industrial production in China. In order to improve the yield of spinosad, it is necessary to gain insight into the regulatory mechanisms and networks of spinosad biosynthesis in S. spinosa. Studies have shown that quorum sensing affects morphological differentiation and/or secondary metabolism in microorganism through cascade regulation. However, no report on the quorumsensing regulation mechanism of S. spinosa has been published yet. Present bioassay found that fermentation broth extract of S. spinosa could induce γ-butyrolactone(GBL)/receptor indicator strain Streptomyces coelicolor M145 to produce pigment, and the spinosad yield increased by 22.6% through adding water extract of its fermentation broth, which indicates that there is GBL/receptor cascade regulation of quorum sensing in S. spinosa. Bioinformatics analysis showed that S. spinosa had one GBL receptor homologous protein SsbR(Saccharopolyspora spinosa γ-butyrolactone receptor) and 3 central transcription regulators homologous proteins SstA1, SstA2, SstA3(Saccharopolyspora spinosa central transcription activator). Real-time quantitative PCR(qRT-PCR) was used to detect the expression of key homologous genes related to cascade regulation. The expression levels of ssbR and sstAs during the fermentation conformed to model that the expression of sstAs was repressed by SsbR. In addition, the expression levels of 3 target proteins homologous genes griR(grixazone regulator), sprA(serine protease A) and ssgA(sporulation of Streptomyces griseus A) in S. spinosa were the same as that of sstAs, which suggests that these 3 genes may be activated by SstAs. This study provides a reference for the further research of the quorum sensing regulation mechanism of S. spinosa.更多还原