Endophytic Bacteria As Contributors To Theanine Production In Camellia Sinensis

Tea,is consumed widely because of its rich secondary metabolites and unique flavor characteristics.The specific accumulation of theanine has become one of the hotspots of secondary metabolites in tea,but the only gene reported so far theanine biosynthesis in tea plants is CsTS1.It is endophytes participate in the synthesis of secondary metabolites in the host that has been reported in other plant species.There are abundant endophytes in tea plants,but its role in the synthesis of theanine in tea plants is still unclear.Therefore,we used tea plantlets with different varieties as test materials.Based on the isolation and identification of endophytic bacteria in tea plants,the ability and synthesis mechanism of endophytes to synthesize theanine and the role of γ-glutamyltranspeptidase gene(γ-GGT)from tea and endophytes in the anabolism of theanine were preliminarily investigated.The main research work and achievements in this study were summarized as follows:1)Plantlets of the tea cultivars ’Shuchazao’,’Wuniuzao’,’Longjing No.43’,’Yunkang No.10’ and C.oleifera ’Changlin No.4’ were grown in vitro,were used to isolate endophytic bacteria.Morphology and 16S rDNA identification showed that the endophytes from C.sinensis are belong to Rhodanobacteraceae Luteibacter spp.and the endophytes from C.oleifera are Bacillus safensis.2)The mixed endophytes CsE-SCZm,CsE-WNZm,CsE-LJm,CsE-YKm separated from different tea varieties ’Shuchazao’,’Wuniuzao’,’Longjing No.43’ and ’Yunkang No.10’respectively.The mixed endophytic CoE-CLm isolated from the oil tea variety ’Changlin No.4’ was cultured in vitro,and the final concentration was 20 mM glutamine and 20 mM ethylamine hydrochloride,and culture was continued for 7 days.The results showed that the total production of theanine in CsE-WNZm culture was 6.45 mg/L,followed by CsE-SCZm of 4.87 mg/L,CsE-LJm of 4.18 mg/L,and CsE-YKm of 3.73 mg/L.Taking CsE-SCZm as an example,the extracellular secretion efficiency of the theanine synthesized by the endophyte was further examined.The results showed that more than 90%of the theanine synthesized by endophytes could be secreted independently of the cells.The production of theanine was not detected in the culture solution and the cells of the endophytic bacteria CoE-CLm.3)Cloning,heterologous expression and enzyme activity analysis of GGT and GS(TS)from C.sinensis and endophytic bacteria.The results showed that the specific activity of the purified recombinant proteins rCsE7GGT and rCsE8GGT from the endophyte CsE7 and CsE8,with 20 mM Gln and 50 mM ethylamine hydrochloride as substrate were 431.86μkat/μg and 299.17μkat/μg,respectively,and the specific activity of rCsGGT2 and rCsGGT4 from tea plant were 1.36μkat/μg and 0.94μkat/μg,respectively.The data showed that the in vitro enzyme activities of endophytic rCsE7GGT were 317.54 and 459.43 times of rCsGGT2 and rC sGGT4,respectively,and the in vitro enzyme activities of endophytic rCsE8GGT were 219.98 times and 311.64 times of rCsGGT2 and rCsGGT4,respectively.The specific activity of the recombinant protein rCsTS 1 from tea plant,with 20 mM Glu and 50 mM ethylamine hydrochloride as substrate was 0.80μkat/μ,g,and the recombinant proteins rCsE7GS and rCsE8GS from the endophyte CsE7 and CsE8,were 0.33μkat/μg and 0.49μkat/μg,respectively.Which showed that the recombinant proteins,both rCsTS1 from C.sinensis and rCsE7GS and rCsE8GS from the endophytes,have the ability to convert glutamic acid(Glu)and ethylamine hydrochloride to theanine in vitro,but the enzyme activity of rCsE7GS and rCsE8GS was significantly lower than that of CsTS1 in tea plants.4)The endophytic strain CsE7 from C.sinensis could convert glutamine and anyone of 18 different amino acids into theanine was analyzed.The results showed that the strain CsE7 had the highest donversion efficiency to lysine(31.87μg/L),which was about 3 times that of other amino acids including alanine(9.67μg/L).