Polymorphism of β-amyrin synthase gene (β-AS) influence the accumulation of triterpenes in licorice

Licorice is one of the most-frequently-used Chinese herbs. Glycyrrhizic acid (GA), as the most important marker component in licorice, is biosynthesized by mevalonic acid (MVA) pathway and regulated by many key enzymes. Among them, β-amyrin synthase (β-AS) is responsible for the triterpene skeleton formation. In this paper, we tried to find out the influence of β-AS gene polymorphism on the GA biosynthesis. The contents of 18α-glycyrrhizic acid (18α-GA) and 18β-glycyrrhizic acid (18β-GA) in 60 licorice samples were assayed by HPLC. For the β-AS gene polymorphism analysis, six samples with high GA content and seven samples with low GA content were selected. Thirteen β-AS cDNA sequences were obtained and eleven haplotypes were determined. The full length of these β-AS cDNA sequences is 2289 bp, with a complete open reading frame (ORF), encoding 762 amino acid residues. Haplotype-3 was the major haplotype in the group with high GA content, while haplotype-8 was the major haplotype in the group with low GA content. The missense mutation sites between haplotype-3 and haplotype-8 were G/C at 104 bp and 106 bp, resulting in the mutations of serine/threonine at the 35th amino acid residue site and alanine/proline at the 36th amino acid residue site. The yeast expression vectors, GS115-BAS-3 and GS115-BAS-8, containing haplotype-3 or haplotype-8 were constructed, and later transformed into Pichia pastoris GS115 by electrotransformation, respectively. The catalytic efficiency of protein BAS-3 and BAS-8 expressed in the different recombinant P. pastoris GS115 strains were analyzed by GC–MS. It was showed that compared with BAS-8, BAS-3 had a higher catalytic activity. The relative expression analysis showed that there was a positive correlation between the expression level of β-AS gene and the content of GA. We hope this paper will provide a basis for functional gene studies involved in the biosynthetic pathway of GA and high-quality cultivated licorice selection in the future.