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

Abstract

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.

Efforts toward Ambergris Biosynthesis

“…The resulting double mutant BmeTC Y167A/D373C produced (+)-ambrein from squalene via 8α-hydroxypolypoda-13,17,21-triene with a yield of 21.5%, approximately 10-fold higher than that of BmeTC D373C which produced (+)-ambrein from squalene via 3-deoxyachilleol A (2.2%). 25 However, 3-deoxyachilleol A was accumulated as a byproduct during the biocatalysis of BmeTC Y167A/D373C . To accelerate squalene conversion to 8αhydroxypolypoda-13,17,21-triene and meanwhile to reduce 3deoxyachilleol A accumulation, a dual enzyme system was constructed by coupling the wild-type BmeTC and BmeT-C Y167A/D373C (Figure 2), showing 20 times higher yield than using BmeTC D373C alone (46.0% vs. 2.2%).…”

“…To further improve the reaction efficiency of mutant BmeTC D373C , alanine screening was conducted for the six residues presumed to be located near the intermediates during the reaction based on the modeling structure of BmeTC. The resulting double mutant BmeTC Y167A/D373C produced (+)-ambrein from squalene via 8α-hydroxypolypoda-13,17,21-triene with a yield of 21.5%, approximately 10-fold higher than that of BmeTC D373C which produced (+)-ambrein from squalene via 3-deoxyachilleol A (2.2%) . However, 3-deoxyachilleol A was accumulated as a byproduct during the biocatalysis of BmeTC Y167A/D373C .…”

“…However, 3-deoxyachilleol A was accumulated as a byproduct during the biocatalysis of BmeTC Y167A/D373C . To accelerate squalene conversion to 8α-hydroxypolypoda-13,17,21-triene and meanwhile to reduce 3-deoxyachilleol A accumulation, a dual enzyme system was constructed by coupling the wild-type BmeTC and BmeTC Y167A/D373C (Figure ), showing 20 times higher yield than using BmeTC D373C alone (46.0% vs. 2.2%) . The authors anticipated the replacement of BmeTC D373C with this dual enzyme system in the above-mentioned engineered P. pastoris strain may lead to an (+)-ambrein titer of 2 g·L –1 .…”

From Ambergris to (−)-Ambrox: Chemistry Meets Biocatalysis for Sustainable (−)-Ambrox Production

“…A recent report described the cyclization of squalene to ambrein in E. coli with a two-enzyme system based on BmeTC only, comprising wild-type BmeTC and a novel “ambrein synthase” (BmeTC variant) . Photooxidation of ambrein produced volatiles in 8–15% yield.…”

“…Depending on the treatment, (−)-ambrox accounted for up to 25.5% of the volatiles. This two-enzyme system enabled yields 20 times higher than with the best system described so far, and because it can be produced in Pichia, an ambrein titer of 2 g L –1 was anticipated . Biosynthetic ambrein may be a significant alternative to the limited supply of jetsam ambergris, which is also subject to restrictions on its use because it comes from an endangered animal species.…”