Burkholderia sp.IDO3中靛蓝合成基因的克隆表达及其合成特性  

Cloning, expression and application of an indigo-producing gene from Burkholderia sp. IDO3

作　　者：刘紫嫣[1] 马桥[1] 由胜男 张旭旺[1,2] 张西迪 殷庆鑫 李炫莹 厉舒祯 张照婧 曲媛媛[1] 

机构地区：[1]大连理工大学环境学院工业生态与环境工程教育部重点实验室,辽宁大连116024 [2]大连理工大学食品与环境学院工业生态与环境工程教育部重点实验室,辽宁盘锦124000

出　　处：《微生物学通报》2017年第11期2634-2643,共10页Microbiology China

基　　金：国家自然科学基金项目(No.51508068);新世纪优秀人才支持计划项目(No.NCET-13-0077);中央高校基本科研业务费专项项目(No.DUT14YQ107);高等学校学科创新引智计划资助项目(No.B13012)~~

摘　　要：【目的】克隆和表达靛蓝合成基因,并将其用于靛蓝合成研究。【方法】对菌株Burkholderia sp.IDO3中靛蓝合成基因进行克隆和大肠杆菌异源表达,构建能合成蓝色色素的基因工程菌。利用液相色谱和质谱对产物进行分析,采用单因素法对培养温度、转速、培养基成分等进行优化,并考察优化条件下的靛蓝合成曲线。【结果】构建了一株重组大肠杆菌E.coli IND_AB,该菌株能够在LB培养基生长的过程中合成蓝色色素,产物分析表明该色素为靛蓝;菌株IND_AB在30°C和150 r/min条件下能在LB培养基中合成22.9 mg/L靛蓝,优化培养条件后产量达到25.4 mg/L;优化LB培养基各组分浓度后产量可提高到35.1 mg/L;外加50.0 mg/L吲哚或0.1 g/L色氨酸后靛蓝产量可分别提高到57.7 mg/L和64.4 mg/L,相比初始产量提高了152.0%和181.2%;靛蓝合成曲线表明在添加吲哚或色氨酸的培养基中,菌株IND_AB前6 h没有靛蓝生成,6-15 h为靛蓝合成加速期,18 h达到产量平衡。【结论】重组大肠杆菌IND_AB可用于生物合成高纯度靛蓝,为靛蓝的微生物合成提供了有效的基因资源。[Objective] An indigo-producing gene was cloned and expressed, and its application in indigo production was investigated. [Methods] An indigo-producing gene from Burkholderia sp. IDO3 was cloned and heterogeneously expressed in Escherichia coli BL21（DE3）. Recombinant strain was able to produce blue pigments. HPLC and LC/MS were applied to analyze the reaction product. The conditions for indigo production, such as rotary speed, temperature, and growth medium components were optimized, and indigo-producing curve was also determined under the optimal conditions. [Results] Recombinant strain IND_AB was successfully constructed and could produce blue pigment in LB medium. Product analysis revealed the blue pigment was indigo. Strain IND_AB could generate 22.9 mg/L indigo when incubated at 30 ℃and 150 r/min. The indigo yield was improved to 25.4 mg/L and 35.1 mg/L after optimization of environmental parameters and medium components. The indigo yield was subsequently enhanced to 57.7 mg/L and 64.4 mg/L with the addition of 50 mg/L indole and 0.1 g/L tryptophan, which rose by 152.0% and 181.2% compared with initial yield. Time-course analysis indicated that there was no indigo production in the previous 6 h, and indigo was exponentially produced between 6-15 h and reached the maximum at 18 h. [Conclusion] Recombinant strain IND_AB could produce relatively pure indigo, and present study provided efficient genetic resource for indigo microbial production.

关 键 词：靛蓝合成基因 吲哚 色氨酸 微生物合成 

分 类 号：Q78[生物学—分子生物学] Q936