Team:BNDS China/Description
Project Inspiration and Description
The city in which we live, Beijing, is a place steeped in ancient history and traditional culture. The museums here, especially the renowned Palace Museum and the Capital Museum, hold vast collections of precious artworks and antiquities. In recent years, the popularity of creative products of the Palace Museum and the broadcast of documentaries such as Masters in the Forbidden City and Every Treasure Tells A Story raise a fever about cultural heritage antiquities and their conservation across China. Exploring the process of repairing these artifacts, we noticed that the materials used by the restorers sometimes posed difficulties: some are expensive, some are hard to attain, and some others could cause severe allergic reaction to workers . With our rather convenient access to museums, we met with the restores and an antiquity collector to learn more about the applications of different materials and the obstacles they often encountered during the conservation process. Ultimately, we decided to devote ourproject this year to produce artificial lacquer and carminic acid, two essential materials utilized in the restoration of Chinese traditional lacquerware and painting.
Raw Lacquer
Raw lacquer is intensively used in Asian lacquerware and other artifacts (including furniture, jewelry, and musical instruments) to not only increase the resistance to heat and corrosion but also add gloss. It is also useful in architecture and lacquer paintings. Raw lacquer is derived from the resin of lacquer trees (for example, Toxicodendron vernicifluum, previously called Rhus verniciflua) that are indigenous in East Asia but rarely grow in other regions. However, we learned from the documentary Masters in the Forbidden City that farmers need to collect resin from high, dangerous mountains through rigorous labor in the hottest days in summer, and the resin is likely to lead to hypersensitive reaction. Hence, we determined make a change of this situation. Raw lacquer consists of four components: urushiol, laccase, water, and gummy substance, the first two of which are the main effective components.
Urushiol
Urushiol is a catechol substituted in the third carbon site with a hydrocarbon chain that has 15- or 17-carbon chain, which can be either saturated or unsaturated. It is urushiol that forms the thin film that protects artifacts. The toluene dioxygenase pathway in Pseudomonas putida provides us a chance to synthesize urushiol using biological method. Four enzymes form a cluster (in the sequence of TodC1C2BAD) in the system and they catalyze the precursor toluene under the presence nicotinamide adenine dinucleotide (NADH). The reaction is initiated by ferredoxin reductase (TodA, coded by BBa_K2950004), which removes two electrons from NADH. Then the electrons are transferred to Rieske-type ferredoxinTOL (TodB, coded by BBa_K2950001), and taken by toluene dioxygenase (TodC, coded by BBa_K2950002) in order to oxidize toluene to form cis-toluene dihydrodiol. The intermeiate is then dehydrogenated by toluene dihydrodiol dehydrogenase (TodD) to form the end product 3-methylcatelchol. In our design, alkylbenzene is chosen to replaced toluene and acts as the precursor in the reaction. Since the length of the carbon chain has an unclear effect on the film formation rate, we selected hexadecylbenzene with a 16 carbon side chain. By utilizing the TodC1C2BAD enzyme cluster to carry oxidative hydroxylation and dehydrogenation on the alkylbenzene, the final product urushiol is obtainable.
Laccase
Laccase, a multi-copper oxidase, catalyzes the oxidation of urushiol to form a polymerized thin film, which provides lacquer-pasted artifacts with its gloss. The laccase produced in lacquer trees cannot be replaced by those in fungi, as their accessory functions are different. Gaining the knowledge above, we plan to use synthetic raw lacquer to substitute the natural ones, as it is made possible to produce raw lacquer in industry, workshops or even household settings instead of going through the demanding process of collecting resin. Besides, we can control the components of artificial raw lacquer and their percentage to produce safer--as there won’t be dangerous unknown components--and more custom lacquer.
The background of carminic acid
During our talk with an artifact collector, we became aware that carminic acid , due to its intense red color, is used as the most favored pigment in colorization for Chinese painting and textile which add a unique, dark shade of red. We also learned from our literature review that carminic acid is still a component of the most widely utilized coloring agents for industrial fields such as food, drinks, and cosmetics. Moreover, around the world, especially in the East Asian region, antiquity conservation needs dyes that contain carminic acid to re-color ancient paintings and frescos. However, the existing way of producing dye that is laborious, as workers need to grind cochineals and purify the carmine by themselves; numerous tons of cochineals are required in industry to yield just proper amount for the market, so this is merciless and inefficient. Besides, raising cochineals can be time-consuming, resulting in the low yield of carminic acid in the world. As a result, we decided to produce carminic acid by microorganisms as a cell factory. .
The biosynthesis pathway of carminid acid
The synthetic pathway of carminic acid, however, is still not revealed in standard production host like E. coli or S. cerevisiae. The only successful heterologous expression was achieved in Aspergillus nidulans[1]. Nevertheless, the wildtype A. nidulans produces other pigments that interfere with the carminic acid synthesis, requiring the gene knockout. Considering that it’s hard for us to engineer A. nidulans (especially the gene knockout of A. nidulans), we aim to engineer S. cerevisiae as the chassis organism for the production of carminic acid. The biosynthesis pathway of carminic acid from carbon source involves at least 4 enzymes: (a) the conversion of acetyl-CoA and malonyl-CoA from simple carbon source through the homologous metabolism; (b) the synthesis of linear non-reduced octaketide, the backbone of carminic acid, from acetyl-CoA and malonyl-CoA through the octaketide synthase from Aloe arborescens; (c) the cyclization or aromatization of linear non-reduced octaketide catalyzed by ZhuI or ZhuJ from the organism Streptomyces sp. R1128, or both of them to convert linear non-reduced octaketide to flavokermesic acid anthrone which will further change to flavokermesic acid (FK), the intermediate between carbon source and CA, spontaneously; (d) the hydroxylation of flavokermesic acid through the enzyme monooxygenase from Aspergillus nidulans to form kermesic acid; (e) the glycosylation of kermesic acid to finish the biosynthesis of carminic acid by the Dactylopius coccus C-glucosyltransferase.