Overview
Ambergris and Sandalwood Oil are both classic and precious fragrance fixatives since centuries before. They are famous for their extraordinary abilities of prolonging the longevity and richness of fragrance products like perfumes. The current ways of ambergris and sandalwood oil production have is not sustainable, and environmentally unfriendly.
In order to respond to the high demand of these two substances and protect the Inscence Culture in Asia, we have come up with a solution.
Our project aims to produce ambrein -- the major component of ambergris, and santalol -- the major component of sandalwood oil using S.cerevisiae. The final product obtained will be provided to daily chemical industries, letting the economical and high-quality fixatives become more accessible to all.
Figure 1. Overall design of Aroma Anchor
What is Fragrance Fixative?
It is fragrance fixative that makes perfume last longer in perfume industry. Fragrance fixative is a substance with high molecular weight that can reduce the vapour pressure of other raw materials inside a perfume, and as a consequence, makes the perfume last longer.
These compounds equalize vapor pressures and limit the instability of fragrance facets, prolonging the longevity of the raw materials found in any perfume's essential oils. [1]
In order to satisfy consumers' expectations of the lasting ability the perfumes possess, a fixative is necessarily added into the perfume. In addition to prolonging the longevity, fragrance fixatives improve the overall quality of a perfume by balancing different scent notes. It also enhance the richness of perfume, which can make the scent more complex.
Figure 2. Schematic diagram of bottled essence [2]
Fixtatives Now
Due to the accelerated expansion in the cosmetics and skin care industry and the rising standard of living in emerging economies etc, the global market for fragrance fixatives in 2020 was estimated at USD 1.25 billion million and is expected to mushroom by the year, 2030 at a compound annual growth rate (CAGR) of 4.3 % during the forecast period. [2, 3]
According to research, natural sources and chemical synthesis are two major sources to produce fragrance fixatives.
Natural plant fixatives such as sandalwood, benzoin, labdanum, and storax, and animal products such as ambergris, castoreum, and musk are commonly used. Despite the high quality and optionality of the products, the process of obtaining the substances usually comes with a high cost and low sustainability.
Meanwhile, chemical synthesized products such as synthetic musk (white musk), certain crystalline high boiling spice compounds, polybasic acids, and esters are popular. [3] However, the high energy requirement for the synthesis and the low quality of the products has led to a concern.
The drawbacks in today’s fragrance fixative production has made the products either unable to meet the need for research and development of new fragrances in the future, or develop unsutainable pattern of consumption. Therefore, we aim to use the technology of synthetic biology to solve this dilemma.
Figure 3. Comparison of traditional fragrance fixatives extraction processes
Ambergris and Ambrein
In the 1st century, ambergris was first found by fishermen in China. Ambergris was discovered to be another type of fragrance and brought as tribute to the emperor. This fragrance become known as the Dragon's Vomit.
The word "ambergris" comes from the Old French "ambre gris" or "grey amber". The addition of "grey" came about when, in the Romance languages the sense of the word "amber" was extended to Boltic amber (fossil resin), as white or yellow amber (ambre jaune), from as early as the late 13th century.
Ambrein is the major component of ambergris. It is odorless, but after exposure to sunlight, seawater, and air, it becomes ambrox, which is the best-known fragrance of ambergris. As a fixative, however, ambrein is more efficient than ambrox since it has a larger molecular structure capable of holding other fragrance molecules on any surface. [5]
The primary source of ambergris is the excretion of sperm whales. When sperm whales consume giant cuttlefish and are unable to digest their bones, and if these indigestible things reach the gastrointestinal tract, sperm whales secrete a waxy substance that wraps around them. After they are expelled from the body of sperm whales, they need to float in the sea for months or years before finally forming ambergris.
As the hunting of sperm whales is banned worldwide, the supply of ambergris declined dramatically. The result is a high price for ambergris, roughly $10,000 per pound [6].
Figure 4. Typical odor formation mechanism of Ambergris[5]
Sandalwood Oil and Santalol
Santalol is a fragrant molecule extracted from sandalwood. Traditional sandalwood oils are extremely dependent on the age of the tree Santalum Album. The older the tree, the better oil it can be made into[7]. Santalol also attains a large molecular size which allows it to be a decent fixative.
Traditional sandalwood oil gets its scent primarily from two compounds, α-santalol and β-santalol. The α form of the aromatic alcohol generally dominates at around 55% of the mix, while the β version at around 25% and other related compounds making up the balance.[8]
It is sometimes used to accompany floral notes such as freesia. Mature sandalwood trees weigh around 100 kg, of which 20 kg is the aromatic heartwood, which in turn produces between 600-700mL of oil per tree. In New Caledonia, there is an awareness of the need to conserve this precious sandalwood, with a policy of sustainable exploitation. With the establishment of nurseries, there is a strong desire to preserve this precious sandalwood for future generations. For the protection of this species of tree and the efficiency of santalol production, we decided to find a better way to produce santalol.
Due to the requirement of sandalwood growth for sandalwood, natural sandalwood oil is expensive, at a price of $20 per milliliter. Santalol is also available through chemical synthesis, yet still costly, at approximately $25 per g.[9,10]
Figure 5. Aroma Molecular Composition in Sandalwood Oil [8]
Limitation of current Ambergris and Sandalwood Oil industry
Sandalwood, mainly distributed in the tropical regions of India and the Indonesian islands of Timor and Sumba, demands a considerable land area to grow. The necessity of specific temperature control and water source demands, growing sandalwood suitable for sandalwood oil production is difficult, and it also requires a large number of natural resources. Adding to this, to grow sandalwood with sandalwood oil, the tree has to grow at least for at least 30 years. To protect the sandalwood, many regions has set restrictions to the logging of this tree. What follows the gradually decreasing number of sandalwood is the reducing accessibility of sandalwood oil, and the fading asian inscence culture.
Figure 6. The growth stages of sandalwood made into sandalwood oil
Sperm whale, the source of ambergris plays an important role in the marine ecosystem, recycling nutrients from the depths to the phonic zone. In the past, people obtained ambergris by hunting sperm whales, which has done great damage to the sperm whales and the environment undersea with a long-term impact affecting the present. Although hunting would be more efficient than collecting ambergris by the shore, the quality and quantity of ambergris contained in the whales are always unknown and unstable. The hunting sharpened the high scarcity of ambergris. This chastening history is not only meant to be lamented, but we should also change this situation.
Figure 7. Sperm whales have been brutally killed to obtain the ambergris that may exist inside their bodies
S. cerevisiae terpenoids factory
Saccharomyces cerevisiae is chosen to be the chassis to produce santalol and ambrein, by virtue of its self-contained Mevalonate pathway (MVA pathway). Through the MVA pathway, Acetyl-CoA can be turned into many terpenoids precursor, such as Geranyl pyrophosphate (GPP), Farnesyl pyrophosphate (FPP), and Squalene. Based on the MVA pathway in S. cerevisiae, many researchers have successfully utilised such organism to produce essence ingredients (e.g. Linalool, Limonene) from these precursors.[11]
Figure 8. MVA pathway in Saccharomyces cerevisiae[11]
Overall Design
Here comes our project, producing an "aroma anchor" consists of ambrein and santalol, which can be utilised by perfumers as fixatives added to perfume. The Aroma Anchor will perform to anchor the aroma molecules within fragrant products to prolong the longevity of such products, as well as enriching the scents. Through which way the greatest perfumers can produce the long-lasting perfumes desired by consumers.
Our project consists mainly of three cycles: (1) Overexpress tHMG1 and IDI1 to construct terpenoids' producer. (2) Heterologously express ClSS, CYP736A167, SaCPR2 to construct santalol producer. (3) HeAfter regulating metabolic flow by the optimization of synthesis pathway and inhibit of divided pathway, the yield of santalol ambrein would reach higher and thus lower the production cost. Then we will provide excellent and economical fixative ingredients extracted from the cell culture, supplying the perfume industry in production process. The sustainable, environmental-friendly and economical manufacturing technology we established is expected to bring a brand new reform to the essence industry.
Figure 9. Biosynthetic pathway for santalol (A) and ambrein (B) in engineered S. cerevisiae. Notes: The red star indicates that the corresponding gene is overexpressed. The genes with green or blue type represent their heterologous expression in yeast. tHMG1: Truncated HMG1 with the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase; IDI1: IPP isomerase; IPP: Isopentenyl pyrophosphate; DMAPP: Dimethylallyl pyrophosphate; FPP: Farnesyl diphosphate; DPP1: Diacylglycerol pyrophosphate phosphatase 1; LPP1:Lipid phosphate phosphatase 1; ClSS: α-Santalene Synthase from Clausena lansium; SaCPR2: cytochrome P450 reductases found in S. album; CYP736A167: a cytochrome P450 from S. album. BmeTC: Tetraprenyl-β-curcumene Cyclase from Bacillus megaterium.
Benefiting from synthetic biology, we believe a future, a future when buying perfumes containg ambrein and santalol isn't a privilege of the rich, a future when general consumers are able to buy the long-lasting perfumes they desire. Such future is realised by synthetic biology, by the power it brought to human society. Therefore, this is the mission of us being genetic engieers, this is the meaning of the existence of this discipline--Synthetic biology project is responsible and good for the world.
References
[1] Wan-Yu Lu, Wei-Cheng Lin, et al. Determination of the ambience duration of lavender essential oil with three perfume fixatives using the thermokinetics approach. Journal of Thermal Analysis and Calorimetry, 2022, 147: 7551-7561.
[2] https://www.alphaaromatics.com/blog/fragrance-fixatives/
[3] https://www.bmvfragrances.com/productcategory/fixative-perfumery-base
[4] https://www.grandviewresearch.com/blog/fragrance-fixatives-market-size-outlook
[5] Cristiano, L.; Guagni, M. Zooceuticals and Cosmetic Ingredients Derived from Animals. Cosmetics 2022, 9, 13. https://doi.org/10.3390/cosmetics9010013.
[6]Meares, Hadley. “In Search of Ambergris, a Highly Prized Slurry of Squid Beaks and Whale Feces.” Atlas Obscura, 2022. www.atlasobscura.com/articles/ambergris-chanel-ingredient.
[7] What Is Sandalwood Oil? How It Is Extracted. sandalwoodcompany.com/extraction.html.
Das, Kuntal Kanti. “Sandalwood (Santalum Album) Oils.” Essential Oils in Food Preservation, Flavor and Safety, 2016, https://doi.org/10.1016/b978-0-12-416641-7.00082-1.
[8] Chonglong Wang. (2015) Shaking up ancient scents: Insights into santalol synthesis in engineered Escherichia coli. Process Biochemistry. https://www.sciencedirect.com/science/article/pii/S1359511315002457#bib0695.
[9]https://shop.home-essential-oils.com/products/doterra-sandalwood-essential-oil?variant=28741064687690&FORM=SSAPC1
[10]https://www.chemicalbook.com/Price/SANTALOL.htm#:~:text=SANTALOL%20%2450,-%20%242468.4
[11]Govinda Ramnath Navale. (2020) Improved production of epi-cedrol and santalene by fusion protein expresssion: Stability study and cyclization mechanism of epi-cedrol biosynthesis. National Chemical Laboratory (Ph.D. Thesis in India). https://dspace.ncl.res.in/xmlui/handle/20.500.12252/5904.