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

[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OD_(600)为0.8,诱导温度为18℃,诱导时间为12 h,IPTG终浓度为1 mmol/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。

[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 OD_(600),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 andδ-Cadinene and sesquiterpene oxygenated derivatives gleenol,epicubenol andτ-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.