| Salidroside is a potent glycosides drug extracted from Rhodiola rosea L., which is formed by dehydration condensation reaction between uridine diphosphoglucose and tyrosol. Salidroside has the functions of anti-aging, anti-tumor, anti-fatigue, anti-radiation, enhancing immunity, protecting cardiovascular and so on. The supply of salidroside is far from enough in the international market, for the relatively low yield (0.01～0.6%, DW) of salidroside in wild-type Rhodiola rosea L., which greatly limits the commercialization of the drug. Although salidroside could be synthesized chemically, it is limited to be put into production due to complex reaction route, low total yield and complex experimental equipment. With the successful cloning of genes involved in the biosynthesis of salidroside, genetic engineering is thought to be one of the most promising approaches to enhance the production of salidroside. To insight into salidroside biosynthesis at molecular level and provide new target genes for metabolic engineering of salidroside biosynthesis pathway, the full-length cDNA of tyrosine decarboxy gene involved in the metabolic pathway of salidroside was cloned by RACE method, then characterized and functionally identified, which was named RcTYDC, in n order to increase salidroside content in Rhodiola crenulata by using genetic engineering method.TYDC is an essential enzyme of the salidroside biosynthesis pathway, which catalyzes the conversion of L-Tyrosine into tyramine (4-Hydroxy-phenethylamine). The full-length cDNA of TYDC was cloned and characterized for the first time from R. crenulata. The new cDNA was named as RcTYDC. The full-length cDNA of RcTYDC was 1657 bp containing a 1473 bp open reading frame (ORF) encoding a polypeptide of 490 amino acids with a calculated molecular mass of 54.6 kDa and an isoelectric point of 5.55.Comparative and bioinformatic analyses revealed that RcTYDC had extensive homology with TYDCs from other plant species (45%-51% identities). RcTYDC contained the typical TYDC catalytic activity domain. A phylogenetic tree of TYDCs was constructed from different organisms including plants, algaes, bacteria and archaea. The result demonstrated that TYDCs were derived from an ancestor gene and evolved into three groups including plants, algaes, bacteria and archaea TYDC groups, and RcTYDC belonged to plant TYDCs'family. Furthermore, RcTYDC had the closest genetic relationship with TYDCs of Zea mays and Oryza sativa. The cloning and characterization of RcTYDC will be helpful to understand more about the role of TYDC involved in the salidroside biosynthesis at the molecular level.To verificat the function of RcTYDC, the plasmid pET28-RcTYDC carrying RcTYDC coding sequence was transformed into the E. coli strain Rosetta, which was able to express heterologous protein. The 53KD restructuring RcTYDC protein was synthesized in Rosetta, which was extracted and purified with urea, then renatured by dialysising. The reaction with L-tyrosine as substrate, pyridoxal-5-phosphate hydrate as coenzyme was carried out to detect the catalytic activity of restructuring RcTYDC protein. HPLC detected that reaction product tyramine was produced, which showed that this study has acquired RcTYDC with the activity and function of tyrosine decarboxy.Tyrosine decarboxy (TYDC) is an important enzyme in salidroside biosynthesis pathway. To increase the yeild of salidroside in R. crenulata through metabolic pathway engineering, the recombined plasmid p1304+-RcTYDC was introduced into Agrobacterium tumefaciens strain EHA105 to generate engineered strain. This study also successfully established aseptic seedlings and regeneration system of Rhodiola crenulata. This study on metabolic regulation of salidroside biosynthesis has laid a foundation for the future development of Chinese herbal medicine metabolic pathways engineering. As salidroside biosynthesis pathway related genes were cloned in succession, using metabolic engineering methods to enhance salidroside content is becoming possible.
|
PDF Full Text Request