产绿原酸内生菌的分离及其绿原酸合成途径关键基因的克隆和功能研究

Isolation of chlorogenic acid-producing endophyte and cloning of a key gene of chlorogenic acid synthetic pathway

摘要:

【目的】分离产绿原酸的内生菌并对其绿原酸合成途径的一种关键酶基因进行克隆和功能研究。【方法】采用表面消毒法从金银花中分离内生菌，以高效液相色谱(HPLC)和液相色谱-质谱联用(LC-MS)筛选确定产绿原酸的内生菌，克隆、表达该内生菌的组氨酸解氨基酶(HAL)并进行酶活测定。【结果】从金银花根中分离到一株产绿原酸的内生细菌RP1，同时在该内生菌发酵液中检测到了绿原酸代谢的中间物肉桂酸。对RP1的分子鉴定表明该内生菌为枯草芽孢杆菌(Bacillus subtilis)。对RP1的HAL基因进行克隆和原核表达，酶活测定表明该酶具有HAL和PAL (苯丙氨酸解氨基酶)双功能，其PAL活性产生的肉桂酸与LC-MS检测的结果一致。【结论】推测该内生菌可能利用其HAL的苯丙氨酸解氨基活性将苯丙氨酸的催化产物肉桂酸导入苯丙烷途径，从而产生绿原酸。

Abstract:

[Objective] To isolate a chlorogenic acid (CGA)-producing endophyte, and to study the function of a key gene of the CGA synthetic pathway from the endophyte. [Methods] Endophytes were isolated from Flos Lonicerae japonicae by surface sterilization. The endophytic bacterium producing CGA was screened and confirmed by HPLC and LC-MS. Histidine ammonia lyase (HAL) gene of the endophytic bacterium was cloned and expressed in Escherichia. coli and enzyme activity of the recombinant HAL was determined. [Results] An endophytic bacterium RP1 isolated from the root of Flos Lonicerae japonicae was confirmed to produce CGA. Moreover, cinnamate, an intermediate of CGA synthetic pathway, was detected from RP1 fermentation broth. RP1 was identified as Bacillus subtilis through 16S rRNA sequence analysis. The recombination HAL protein had the dual function of HAL and PAL (phenylalanine ammonia lyase). Cinnamate produced by PAL activity of recombination protein agreed with the result of LC-MS. [Conclusion] Presumably endophytic RP1 laded cinnamate, produced from phenylalanine by the PAL activity of HAL, to the phenylpropanoid metabolic pathway to produce CGA.