Cloning, Heterologous Expression And Crystallization Of Terpene Synthase Genes In Streptomyces Sp. LZ35

Terpenoids, with widely physiological and ecological functions as well as medical va lues, are the most numerous and structurally diverse family of natural products. Terpene sy nthase is the key enzyme in the biosynthetic pathway of terpenoids, cyclizing the substra te to generate terpenoids. Terpene synthases in different species have specific protein stru ctures and catalytic mechanism, which results in structural variety and bioactivity diversit y of terpenoids. Research on the protein expression and crystal structure of terpene synth ase gene will help to discover new biosynthetic mechanism of terpenoids and study meta bolism regulation and protein engineering.Streptomyces sp. LZ35was a marine strain producing various antibiotics, which was isolated from the intertidal zone soil of Jimei in Xiamen. By the whole genome sequenci ng and bioinformation analysis of Streptomyces sp. LZ35, it contained four terpene synth ase gene, consisting of one diterpene synthase gene, two sesquiterpene synthase gene and one triterpene synthase gene.The in-depth research was carried out about diterpene synthase gene cyc. Firstly, wit h molecular methods, the CYC native protein and Se-Met derivative protein heterologous expression systems were constructed and got high purified targeted protein for the scree ning of crystallization conditions. The CYC native crystal and Se-Met protein crystal wer e obtained after optimization. Native and Se-Met derivative crystals were diffracted to1.75and2.6A resolutions, respectively. The crystal structure of diterpene synthase was so lved; Secondly, we attempted to study co-crystallization of CYC protein with substrate an alague10-F-GGPP in order to find out the catalytis sites; Thirdly, the cyc gene overexpr ession system with conjugal transfer to mutant Streptomyces sp. LZ35was congstructed to improve the production of natural diterpenoids. After extraction and purification of fer mentation product, the new diterpenoid Heng-1was detected by HPLC.This thesis also did preliminary research on two sesquiterpene synthase gene:pentale ne synthase gene pen and geosmin synthase gene geo. The protein expression synstem an d purification procedure were builted with the same method of diterpene synthase. Pental ene synthase (PEN) had done screening crystallization without getting crystal; Geosmin s ynthase (GEO) had not expressed soluble protein. We would do more purificaiton and cr ystallization screening of sesquiterpene synthases.In conclusion, with methods of melocular biology and proteomics, we successfully s olved the crystal structure of diterpene synthase. This was the first crystal structure of di terpene synthase of bacteria origin. The co-crystallization, enzyme acvtivity in vitro and verification of gene overexpression are still needed to do further studies.