Gene cloning and expression of a triterpene synthase from alangium lamarckii

Abstract

Triterpenes belong to the natural product group of terpenoid compounds with structural diversity mostly found in higher plants. The compounds are formed by cyclisation of 2,3-oxidosqualene which is catalyzed by triterpene synthase. Triterpenes from Alangium lamarckii are known to be present in the leaves such as friedelin, β- sitosterol and stigmasterol. Therefore, the leaf part was used in this study as a source of triterpene synthase mRNA. Five triterpene synthase clones, designated AlOSC1, AlOSC3, AlOSC4, AlOSC5 and AlOSC6, were introduced with yeast consensus sequence (HAMAMA) upstream of the sequence and were changed nucleotide codons of the second amino acid from TGG to TCC for increasing expression in yeast. In this study, the samples were classified into 2 groups, (1) TGG group, in which the yeast consensus sequence was introduced up-stream to AlOSC6 and the nucleotide codon at amino acid position 2 was TGG, and (2) TCC group, in which the yeast consensus sequence was introduced up-stream to AlOSC6 and the nucleotide codon at amino acid position 2 was TCC. All the introduced clones were induced to express in Saccharomyces cerevisae strain GIL 77, a lanosterol synthase auxotroph. It was found that the sample no.35, pYES2-AlOSC6 construct from the group 1, which showed 80% nucleotide identity to the multifunctional triterpene synthase (CrAS) from Catharanthus roseus producing 4 triterpenes, namely taraxasterol, β-amyrin, Ѱ-taraxasterol and an unknown triterpene mono-alcohol. These triterpenes could also be detected in hexane extracts of A. lamarckii leaves. Moreover, it was found that the samples of (1)-pYES2-AlOSC6 group were able to produce triterpene mono-alcohols related to the sample no.35. In contrast, pYES2-AlOSC6 samples in the group 2 were not found to produce the triterpene mono-alcohols. These results suggested that changing of second amino acid codon, TGG which encodes for tryptophan, to be TCC which encodes for serine had the effect on the protein structure of this multifunctional triterpene synthase and caused no production of triterpene mono-alcohols.