Implementation

    2-Phenylethanol (2-PE), an aliphatic alcohol with a pleasant rose aroma is considered to be an important fragrance ingredient used in the food and cosmetic industries due to its mild, warm, and rose-honey like odor(Wang et al., 2019). In nature, 2-PE is mainly extracted from the essential oil of flowers and plant tissues, such as rose, jasmine, tomato, and buckwheat(Wang et al., 2019). However, the extraction process is very complicated and costly, because the 2-PE concentration in these plants is very low(Wang et al., 2018a). Currently, the global market output of 2-PE is in excess of 10,000 tons annually, most of which is chemically synthesized through ethylene oxidation of benzene or reduction of styrene oxide(Wang et al., 2018b). Chemical synthesis processes are generally operated under harsh conditions, such as high temperature, high pressure, and strong acid or alkali environments, causing many undesirable by-products, such as ethylbenzene and styrene, which not only increase the downstream costs, but also seriously debase the grade of 2-PE. The increasing demand for environmental friendly processes and the preference for “natural” products for consumers have considerably stimulated the development of biological production processes for flavors and fragrances(Noda & Kondo, 2017). Therefore, attention has turned to the bioproduction of 2-PE. Specifically, a number of microorganisms synthesize 2-PE naturally at low concentration as a communication molecule. Therefore, we attempted to engineer the nonconventional oleaginous yeast Yarrowia lipolytica as a competitive platform host to produce 2-PE, which can be considered as an economically viable alternative to plant extraction.

    This year, we are committed to the use of microorganisms to synthesize high-quality 2-PE, hoping to use microbial synthesis methods to solve the production and environmental problems caused by the abuse of traditional 2-PE production methods.

    In addition to exploring the practicability and feasibility of 2-PE, we also constructed an engineering strain of Y. lipolytica with high 2-PE production through metabolic engineering and synthetic biology method. As a result, high-purity 2-PE can be obtained by fermentation using phenylalanine as a substrate. We hope our technology can be used in factories, food production and cosmetics.

    1. Fermentation Factory

    One of our target users is manufacturers of 2-PE. The advantage of our project is to construct an engineering Y. lipolytica with effective production of 2-PE. These high-yield host can be directly used in the fermentation production of 2-PE, and after downstream separation, purification, concentration, freeze-drying and other steps, 2-PE preparations can be produced and used in medicine, food and cosmetics industries. The whole flow chart of 2-PE production in factory has been designed (Fig. 2), providing a detailed guide.

    2. Food factory

    Food is indispensable for human survival and its balance is essential for people’s health and wellbeing. With the development of society and technology, people’s concept of food consumption has changed dramatically and the demand for food has switched from basic “guarantee supply ” to “nutrition and health ”. In addition, the increasing environmental pollution and world population, novel processes are required to meet the higher demand while maintaining safety, nutritional value and sustainability. 2-PE can be used as a food additive to impart special flavor to food. For example, adding 2-PE to ice cream can make ice cream have rose flavor, so it has important application prospects in the food industry.

    3. Cosmetics industries

    According to L'Oréal, a leading manufacturer of cosmetic products, the global cosmetic market was valued at US$ 532 Bn in 2018 and is expected to expand at a CAGR of approximately 5.3% by 2020. The global cosmetic market is primarily driven by the rise in urbanization; continuous increase in online beauty spending; expansion of social networks; increase in consumer interest in new, different, and premium products; growth in global population of senior citizens; and surge in the upper middle class population.

    According to a repor published by Infinitus, a China-based company specializing in manufacture of cosmetic products, the cosmetics market in Latin America is projected to reach US$ 68.92 Bn by 2020. Additionally, the personal care & cosmetics industry in the region is expanding at a rapid pace. It is followed by energy drinks, pharmaceuticals, malls, and smartphones industries. Phenethyl alcohol is a primary ingredient in the formulation of cosmetics products. Thus, the global phenethyl alcohol market has been expanding in tandem with the cosmetics industry.

    The leakage of Y. lipolytica from the laboratory may have an impact on natural environmental microorganisms and humans. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). Moreover, the species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions.

    1) Up to now, there is no evidence shows it pose risks to humans.

    2) When the engineered Y. lipolytica finished work, we will sterilize the culture through high temperature and high pressure, and then dispose the waste according to the requirements of relevant departments. So, they will pose no harm to individuals or the environment and does not use any other organism.

    To ensure the productivity and feasibility of our project in the real-world applications as mentioned before, it is essential for us to do a lot of testing and verification. For example, Major considerations should include 2-PE tolerance/toxicity, medium composition, and optimal fermentation processes. In general, mutagenesis coupled with high throughput screening is an efficient and convenient way to evolve tolerance phenotype. In addition, small amounts of organic nitrogen sources, such as peptone and yeast extract, benefit cell growth without inhibiting the Ehrlich pathway. Moreover, it was found that both Ca2+ and Mg2+ salts protect cell and facilitate 2-PE production by increasing membrane stability and integrity. Besides, by adding organic solvents as overlays during fermentation, 2-PE titer could be increased to 12.6 g/L in the concentrated organic layer. These strategies will enable us to build a sustrainable 2-PE platform at low-cost and high-efficiency. (We will follow the safety and responsibility rules of iGEM and will NOT do any in vivo experiments within the regulation scope of iGEM Competition)

    Moreover, potential end users’ insufficient or biased understanding towards genetic engineering and related derivatives should also be considered in our implementation. We should conduct education and promotion activities to enhance the potential end users’ understanding of genetic engineering, and our novel techniques to mitigate their biases or misunderstanding.

    Noda, S., Kondo, A. 2017. Recent Advances in Microbial Production of Aromatic Chemicals and Derivatives. Trends Biotechnol, 35(8), 785-796.

    Wang, J., Shen, X., Rey, J., Yuan, Q., Yan, Y. 2018a. Recent advances in microbial production of aromatic natural products and their derivatives. Appl Microbiol Biotechnol, 102(1), 47-61.

    Wang, Y., Zhang, H., Lu, X., Zong, H., Zhuge, B. 2019. Advances in 2-phenylethanol production from engineered microorganisms. Biotechnol Adv, 37(3), 403-409.

    Wang, Z., Jiang, M., Guo, X., Liu, Z., He, X. 2018b. Reconstruction of metabolic module with improved promoter strength increases the productivity of 2-phenylethanol in Saccharomyces cerevisiae. Microb Cell Fact, 17(1), 60.