紫芝倍半萜合酶GsSTPS2可溶性表达条件的探索

Study on soluble expression conditions of sesquiterpene synthase GsSTPS2 from Ganoderma sinense

关键词:  紫芝  倍半萜合酶  功能验证  HS-SPME-GC-MS分析

Key words:  Ganoderma sinense  sesquiterpene synthase  characterization  HS-SPME-GC-MS

摘要:

[目的] 探索可溶性较差蛋白紫芝倍半萜合酶GsSTPS2的表达方法。[方法] 更换不同的表达载体和表达菌株，通过对不同菌体密度、温度、时间、IPTG浓度的筛选优化GsSTPS2的诱导表达条件，在此基础上采用顶空固相微萃取法（HS-SPME）与气质联用技术（GC-MS）相结合分析重组大肠杆菌细胞培养液中的挥发性成分，鉴定GsSTPS2在大肠杆菌体内的催化活性。[结果] 在初始菌液OD₆₀₀为0.8，诱导温度为18℃，诱导时间为12h，IPTG终浓度为1mmol/L的培养条件下重组蛋白pET32a-GsSTPS2在Rosetta（DE3）中的可溶性表达量最高，达到28.07%。GsSTPS2可催化生成倍半萜碳氢化合物δ-杜松烯、杜松-3，5-二烯、β-杜松烯和顺-衣兰油-4（15），5-二烯及倍半萜含氧衍生物异香叶醇，荜澄茄油烯醇和τ-依兰油醇，其中异香叶醇作为GsSTPS2的主产物之一，具有杀白蚁、抗蠕虫和植物生长调节作用，有良好的应用价值和前景。[结论] 本研究为可溶性较差或者易形成包涵体蛋白的表达提供了参考，为通过合成生物学方法生产异香叶醇奠定了基础。

Abstract:

[Objective] To explore the expression methods of sesquiterpene synthase GsSTPS2 from Ganoderma sinense. [Methods] Different expression vectors and strains were replaced,and the induced expression conditions of GsSTPS2 were optimized by screening at different OD₆₀₀,temperatures,time and IPTG concentration. On this basis, the volatile components in the cell culture medium of recombinant Escherichia coli were analyzed by headspace solid phase microextraction(HS-SPME) and gas chromatography-mass spectrometry(GC-MS). [Results] The soluble expression of GsSTPS2 was the highest,reaching 28.07%,under the conditions of initial bacterial solution OD₆₀₀ of 0.8,induction temperature of 18℃,induction time of 12 h,and final IPTG concentration of 1 mmol/L. GsSTPS2 catalyzed the production of sesquiterpene hydrocarbons Cadina-3,5-diene,β-Cadinene,cis-Muurola-4(15),5-diene and δ-Cadinene and sesquiterpene oxygenated derivatives gleenol,epicubenol and τ-muurolol,among which gleenol,as one of the main products of GsSTPS2,has the effects of killing termites,anti-helminth and plant growth regulation,and has good application value and prospects. [Conclusion] This study provides a reference for the expression of proteins with poor solubility or easy to form inclusion bodies,and lays a foundation for the production of gleenol by synthetic biological methods.