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

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。

关键词:靛蓝合成基因，吲哚，色氨酸，微生物合成

Abstract:

[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 °C 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.

Key words:Indigo-producing gene, Indole, Tryptophan, Microbial synthesis