Team:RDFZ-China/Project Description
Background
We see iGEM as a part collecting and creating competition where innovative gene sequences are constructed annually as functional parts of gene circuits designed by different teams. However, we found an inconvenient truth that there exist a tremendous amount of excellent submitted parts which cannot be readily researched and accessed by other teams, resulting in countless parts in existence that fail to be effectively used.
Asides from that, we also noticed that parts for aroma compound generation are becoming more and more popular among iGEM teams, as these parts are being more frequently applied in different gene circuits in recent years. Furthermore, with the rising industry of aromatics production in high demand, aroma compound production from biosynthesis has been recognized as a more prevailing method with a series of applications in market. Therefore, it seems that the iGEM community needs a more specified and organized collection of parts that are capable of producing aroma compounds, a collection that can greatly benefit both researchers who are looking for genes, and industries that are racing to meet societal needs.
Inspirations
It all begun with our team’s initial research for a part that encodes for an enzyme which generates odor compounds from metabolites of hosting cell. We were surprised at how inconvenient our research process was. The search engine has drawbacks where keywords sometimes mismatched, and result that we are expecting scattered in team’s websites. In order to find the desired part, we had to go over the whole description. In addition, the valid information and experimental data of parts are always lacking.
Realizing a protruding obstacle that the team, or the whole iGEM community facing, we were inspired by the grand prize winner Oxford, 2009. This team managed to collect, and express pigments in E.coli, as color generated by manipulating input-sensitive promoter. The novel of the project gave us the ideology of constructing a library of parts, and we found that it is necessary to gather and integrate the series of part for other iGEM teams to design gene circuits. Thus, we focused on collect and improve a specified part library on which encodes enzyme generates odorants.
Project
It is meaningful and somehow requisite to construct a library which focus on fragrance yielding parts, with a stable, and controllable promoter, as we have chosen, ptac promoter; we use IPTG to induce the expression of different coding sequences. With the help of crawlers and constant researching, a part summary is made for us to embody parts, and record information of each part.
For another major subject, we have considered thoughts on applying the project for education and public engagement. As we recognized that a common expression approach, cell free system could be potentially used on a classroom scenario. By doing so, we found an effective molecular cloning demonstration, which gives a better explaining for students.
(See more on public engagement)
It is known to be about 30 parts that are collected from past projects ,eventually exactly 19 parts were successfully synthesized. What’s more, 4 parts are newly introduced by us, which were found from the Ehrlich pathway. These parts generate an amino acid, phenylalanine, and with the present of 4 enzymes,2-phenylethylacetate will generated.
Phenylethylacetate is an important compound used in multiple aspects of manufacture industries, including cosmetics, food, perfume, household products et cetera. As the compound has a strong scent of rose, it quickly acquires fondness among consumers(Kuo et al., 2014)(Etschman et al., 2002). As one of the acetate ester, phenyethyl acetate is traditionally extracted from rose, but that only achieves very low yield, which makes the production costly. What’s more, chemical synthesis gives unwanted byproducts and thus made the production process complicated(Manabe et al., 2002). But with the use of bioengineering, those problems can be solved effectively.
The pathway was introduced in engineered in E. coli for microbial production. In this specific pathway, aminotransferase(ARO8) is responsible for transamination of L-phenylalanine to phenylpyruvate, then phenylacetaldehyde is generated from 2-keto acid decarboxylase(KDC) for the decarboxylation process. After that, Aldehyde reductase(ADH6) will reduce the aldehyde to 2-phenyethanol(2-PE), which is an crucial molecule in industrial production. Ultimately, 2-phenylehylacetate(2-PEAc) is produced by 2-PE and acetyl-CoA via the esterification by alcohol transferase(ATF1).
References
Etschmann et al..(2002). Biotechnological production of 2-phenylethanol. Applied Microbology & Biotechnology, 59(1), 1-8..
Manabe et al. (2002). Dehydration reactions in water. Journal of the American Chemical Society, 124(40), 11971.
Kuo et al. (2014). Kinetics and optimization of lipase-catalyzed synthesis of rose fragrance 2-phenylethyl acetate through transesterification. Process Biochemistry, 49(3),437-444.
Huang, A, Nguyen, P.Q., Stark, J.C., Takahashi, M.K., Donghia, N., Ferrante, T., …Collins, J.J (2018). BioBitsTM Explorer: A modular synthetic biology education kit. Retrieved from https://advances.sciencemag.org/content/4/8/eaat5105
Guo, et, al. "Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine" Microbiology Open, doi: 10.1002/mbo3.486
Dzialo, et, al. "Physiology, ecology and industrial applications of aroma formation in yeast" FEMS microbiology reviews, doi: 10.1093/femsre/fux031d
