Construction Of Engineered Bacteria Of Glutamine Synthetase (CsGS1-3) From Camellia Sinensis

Glutamine,the scientific name 2-amino-4-carbamoylbutyric acid,is the main form of nitrogen transport in plants.It is involved in the metabolism of ammonia in plants and plays an important role in plant growth and development.Glutamine synthetase(GS)is the major pathway for ammonia assimilation in plants(GS/GOGAT),and catalyzes the assimilation of NH4+to glutamine.GS is a key enzyme in nitrogen metabolism,and previous studies have shown that the γ-glutamyl transfer activity of GS can catalyze the transglutaminyl reaction of glutamine and ethylamine to obtain theanine.Therefore,as a heuristic point,the function of the GS from Camellia sinensis was identified.In this paper CsGS1-3(GenBank accession no.AB117934.1)was successfully cloned from the roots of Camellia sinensis and ligated to the shuttle expression vector pZ8-1.Then the recombinant plasmid was transformed into Corynebacteriuo glutamicum ATCC 13032c by electrotransformation and the enzyme activity of CsGS was verified.Then CsGS1-3 was cloned into the expression vector pET-32a(+)and pGEX-4T-1,than transferred into E.coli for enzyme activity verification and enzymatic properties studies.The specific research results are as follows:(1)In this study,CsGS1-3,a gene encoding GS,the deduced protein of CsGS1-3 consisted of 357 amino acids,had a molecular weight of 39.21 kDa,and had a theoretical isoelectric point of 5.47 was successfully cloned from the roots of Camellia sinensis.Phylogenetic tree analysis indicated that the GSs in Camellia sinensis exhibited homologies of 10%-83%with other GS at the amino acid level.(2)The target gene was ligated to the shuttle expression vector pZ8-1 and sequenced.After verification,the recombinant pZS-CsGS1-3 was transformed into Corynebacterium glutamicum ATCC 13032c by electrotransformation used for construct an engineered strain which could synthesize theanine,and the expression of C.glutamicum strain ATCC 13032/pZ8-CsGS1-3 was analyzed by SDS-PAGE and the enzyme activity was detected.(3)The target gene was ligated to the expression vector pET-32a(+)and pGEX-4T-1,then transformed into E.coil(BL21).Then the recombinants were induced with IPTG and the enzyme activity was detected.Then analyzed the synthesized products with different substrates added in.The results showed that PET-32a(+)-CsGS1-3 could synthesize theanine and glutamine by adding different substrates,while PGEX-CsGS1-3 could only synthesize glutamine.After optimization of conditions,the the activity of two different recombinants was compared.The results showed that the activity of pGEX-CsGS1-3 was higher than that of pET-32a(+)-CsGS1-3,but show a lower expression level compared to pET-32a(+)-CsGS1-3,which was not applicable the future experiments.(4)Under the optimal conditions,the content of glutamine and theanine synthesis was 4.52 mg/mL and 0.071 mg/mL,respectively.Then sodium glutamate:ethylamine hydrochloride:hydroxylamine hydrochloride were added in a ratio of 9:4:2,and it was found that both glutamine and theanine were produced,that the concentration of the obtained glutamine(3.9 mg/mL)was approximately 113.5 times higher than theanine(0.0341 mg/mL).(5)Then purified the target protein and the purified protein can only synthesize glutamine,but show a lower activity level compared to the crude protein,it indicated that CsGS1-3 showed a higher activity of glutamine synthetase than theanine synthetase.