环腺苷酸受体蛋白基因的过表达对刺糖多孢菌生长和多杀菌素合成的影响

Impact on Strain Growth and Spinosad Biosynthesis by Overexpression of Cyclic AMP Receptor Protein Gene in Saccharopolyspora spinosa

【目的】利用全局性调控因子环腺苷酸受体蛋白(cyclic AMP receptor protein,Crp)基因在刺糖多孢菌(Saccharopolyspora spinosa)中的过量表达,研究其对刺糖多孢菌生长及形态方面的影响,促进多杀菌素的生物合成。【方法】PCR扩增环腺苷酸受体蛋白基因(crp),通过限制性酶切、连接方法构建中间载体pOJ260-cm-PermE-crp,使crp置于红霉素增强型启动子PermE的控制下;PCR扩增PermE-crp基因片段,利用Red/ET同源重组技术将PermE-crp亚克隆至实验室保藏的大肠杆菌-链霉菌穿梭载体pUC-spn上,构建成Crp表达载体pUC-spn-PermE-crp。然后,采用接合转移方法将重组载体pUC-spn-PermE-crp导入野生型刺糖多孢菌中,通过单交换同源重组将其整合至刺糖多孢菌染色体上,以染色体上整合了原始质粒pUC-spn的刺糖多孢菌作为对照菌株。利用PCR扩增阿伯拉霉素抗性基因及目的基因的方法鉴定阳性接合子;观察工程菌株及对照菌株在不同固体培养基中的生长及菌落形态差异,同时比较工程菌株及对照菌株在液体培养基中的生长情况,测定生长曲线。借用扫描电子显微镜观察工程菌株及对照菌株的菌丝体形态;采用高效液相色谱检测工程菌株及对照菌株中多杀菌素生物合成情况。【结果】采用分子生物学方法成功构建了Crp重组表达载体pUC-spn-PermE-crp,并通过接合转移导入到刺糖多孢菌中;PCR检测结果显示,工程菌株作为模板扩增出长约2 kb的cm-PermE-crp目的条带,说明crp已成功整合到刺糖多孢菌染色体上,并且获得的重组工程菌株S.spinosa-Crp遗传性能稳定。在BHI培养基和ISP-2培养基上,工程菌株S.spinosa-Crp孢子萌发及形成速率与对照菌株S.spinosa-1相比均有所延迟,但在R6培养基上两者生长速率及菌落形态无明显差异。与对照菌株S.spinosa-1相比,液体培养基中工程菌株S.spinosa-Crp二次生长现象消失,且生物量略高于S.spinosa-1。扫描电子显微镜结果显示工程菌株菌丝片段化程度较高,分支较少,有利于提高工程菌株S.spinosa-Crp发酵过程中的溶氧水平。摇瓶发酵结果表明,工程菌株中的多杀菌素产量与对照菌株相比提高了128%。【结论】crp的过量表达对刺糖多孢菌的菌丝形态及生长产生一定影响,并有效促进刺糖多孢菌中多杀菌素的生物合成,为通过其他正调控基因的超量表达促进多杀菌素的生物合成奠定了基础。

[Objective] The objective of this study is to promote spinosad biosynthesis in Saccharopolyspora spinosa and to study the effects on aspects of strain growth and morphological development through overexpression of Streptomyces global regulatory factor cyclic AMP receptor protein gene （crp）. [Method] crp was amplified by PCR, and an intermediate vector pOJ260-cm-PermE-Crp was constructed by restriction enzymes digestion and ligation, in which crp was placed under the control of erythromycin enhanced promoter PermE. The PermE-Crp frangment was amplified from pOJ260-cm-PermE-crp and subcloned into Escherichia coli-Streptomyces shuttle vector pUC-spn stored in authors＇ lab by Red/ET homologous recombination technology, generating Crp expression vector pUC-spn-Pe^mE-Crp. Then, The vector pUC-spn-PermE-Crp was introduced into S. spinosa by conjugal transfer, and integrated into the chromosome via single-cross homologous recombination, and the strain whose chromosome was integrated by original plasmid pUC-spn was used as control strain in this study. The apramycin resistant gene and the target gene were amplified by PCR to confirme positive transconjugants. The morphological comparison of the engineering strain S. spinosa-Crp and the control strain S. spinosa-1 on different media were observed. Growth curves were compared between S. spinosa-Crp and S. spinosa-1 in liquid medium. The mycelial morphologies between S. spinosa-Crp and S. spinosa-1 were observed by scanning electron microscopy and the spinosad production of S. spinosa was detected by high performance liquid chromatography. [ Result ] A vector pUC-spn-PermE-crp expressing Crp was constructed successfully by molecular biology method, and was transferred into S. spinosa by conjugation. PCR detection results exhibited that the 2 kb long target band em-Pe^mFCrp could be amplified in engineering strain S. spinosa-Crp, suggesting that crp was integrated into chromosome of S. spinosa successfully. The recombinant engineering strain S. spinosa-Crp abtained was genetically stable. On BHI and ISP-2 media, the spore germination and formation rate of engineering strain S. spinosa-Crp delayed compared to the control strain S. spinosa-1, however, the spore germination and formation rate didn＇t show significant difference between S. spinosa-Crp and S. spinosa-1 in R6 medium. Compared to the control strain of S. spinosa-1, the secondary growth phenomenon of engineering strains S. spinosa-Crp disappeared cultured in liquid medium, and its biomass became higher. Under the scanning electron microscopy it was showed that the engneering strain had a higher degree of mycelial fragments and less branches, which can improve the level of dissolved oxygen in S. spinosa-Crp in the process of fermentation. Shaking flask fermentation results revealed that spinosad yield of the engineering strain increased by 128% than that of the control strain. [ Conclusion ] Overexpression of crp had a certain effect on the mycelial morphology and growth of S. spinosa, and effectively promoted the biosynthesis of spinosad, which lay an important foundation for improvming spinosad production by overexpression of other positive regulatory genes.