Cloning, prokaryotic expression, and functional identification of β-amyrin synthase cDNA of Psammosilene tunicoides / 中草药

ABSTRACT

Objective: To clone, express, and characterize the full-length cDNA of β-amyrin synthase provided an important basis for the study on the key role in the biosynthetic pathway of triterpenoid saponins and secondary metabolism engineering applied to Psammosilene tunicoides. Methods: The full-length cDNA fragment of P. tunicoides was isolated by the method of RT-PCR and rapid amplification of cDNA ends (RACE). The fragment was transformed into Escherichia coli expression strain BL21, which was induced by IPTG, and the crude recombinant enzyme was purified from E. coli cell. In the presence of 2, 3-oxidosqualene and other substances, 2, 3-oxidosqualene was converted into β-amyrin efficiently. The catalytic product of P. tunicoides β-amyrin synthase was detected by high-performance liquid chromatography (HPLC) and identified as β-amyrin. Results: The full-length cDNA fragment of P. tunicoides is 2 882 bp and contains an open reading frame of 2 284 bp nucleotides, which codes for 760 amino acids. Conclusion: The full-length cDNA can produce β-amyrin synthase by prokaryotic expression. The expression product has the catalytic activity of 2, 3-oxidosqualene into β-amyrin, which would provide an important basis for the secondary metabolism engineering to P. tunicoides.