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

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.