Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, “ambrein synthase,” for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8–15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid β-induced apoptosis, were discovered using the synthesized ambrein.

Functional genomics approach to the study of triterpene biosynthesis

The Arabidopsis thaliana genome-sequencing project has identified the presence of 13 oxidosqualene cyclase homologs in this plant. In addition to the already identified clones, namely, CAS1 cycloartenol synthase, LUP1 lupeol synthase, and YUP8H12R.43 multifunctional triterpene synthase, two new cDNAs of the putative oxidosqualene cyclase genes, F1019.4 and T30F21.16, were obtained by polymerase chain reaction (PCR) and functionally expressed in yeast. Liquid chromatography/mass spectrometry (LC/MS) analysis led to the identification of some of their reaction products. Interestingly, except for CAS1 for sterol biosynthesis of primary metabolism, so-far-obtained all triterpene synthases of this plant are multifunctional, producing more than one cyclization product. A feeding experiment of 13C-labeled acetate with LUP1 lupeol synthase transformant demonstrated the stereospecific water addition to lupenyl cation intermediate, yielding 3β,20 dihydroxylupane, which accounts for the multiproduct nature of this synthase.