Team:NJTech China/Implementation

• Home
• Project
  • Description
  • Design
  • Perspective
  • Modeling
  • Proposed Implementation
• Achievements
  • Contribution
  • Engineering
  • Parts
  • Judging Form
• Team
  • Team
  • Attributions
• HP
  • HumanPractices
  • Collaborations
  • Partnership
  • Communication
  • Excellence in Another Area
• Safety
• Lab
  • Notebook
  • Protocols
  • Experiments
  • Results

Proposed Implementation

Who are your proposed end users? How do you envision others using your project? How would you implement your project in the real world?

One of the application prospects of our project is the large-scale production of natural phenylethanol. Our proposed end users are fragrance companies, air freshener manufacturers or deodorant manufacturers. Our project intends to add our engineered yeasts into their products to improve the quality of their products, because the phenylethanol produced by our engineered yeasts has the characteristics of high-level biosafety and high purity.

Microorganisms have the characteristics of small size, rapid reproduction, high absorption, fast transformation and strive adaptability. The products produced by microorganisms are relatively pure and have features of low cost, short cycle, high efficiency and green environmental protection. Among them, Saccharomyces cerevisiae has clear genetic background, mature genetic manipulation methods, and high-level biosafety. In particular, studies have shown that yeasts have the de novo synthetic pathway of phenylethanol, and phenylalanine can also be directly converted to phenylethanol by amino acid decomposition metabolic pathway. However, the titer of phenylethanol is still low. To improve the biosynthesis capability of phenylethanol, phenylacetaldehyde synthase, a key enzyme in the phenethylamine pathway of plants, was introduced into S. cerevisiae. And the enzyme activity was enhanced through promoter engineering.

At present, there are generally three ways to obtain phenylethanol, namely physical extraction, chemical synthesis and biosynthesis^[1]. Because physical extraction and chemical synthesis have disadvantages such as high cost and serious pollution, biosynthesis is a better choice. Hence phenylethanol biosynthesized by engineered yeasts in our project has a certain market advantage. Our team will also conduct a market research through questionnaires about the phenylethanol produced by our project to fragrance companies, air freshener manufacturers and deodorant manufacturers to explore the market's acceptance to our project, which played a major role in our cooperation with these companies. After the laboratory phase of our project, we will conduct large-scale production in the fermenter with companies based on the data obtained.

What are the safety aspects you would need to consider?

Once the engineered S. cerevisiae leaking into the environment, they may cause some harm to the environment. Therefore, a leak-proof device will be designed to improve the safety of our project before it being put into practical application. Our engineered yeasts may lead to resistance gene transfer, affecting the growth of wild-type yeast. The media carrying antibiotics in the experiment are harmful to the environment and may alter the composition of environmental microorganisms or promote the emergence of super-resistant bacteria. To avoid the environmental and safety hazards of engineered organisms, we strictly abide by all laboratory regulations to prevent accidental leakage of engineered organisms.

What other challenges would you need to consider?

During the fermentation process of phenylethanol production, the composition and proportion of fermentation medium is critical for the production of phenylethanol. In addition, the initial amount of microalgae, pH of medium, stirring speed, fermentation time and other conditions are also vital to the yeast-microalgae microbial consortia system. Therefore, after consulting many literatures and conducting continuous experiments, we will establish a robust, stable, and controllable artificial microbial consortia system, and on this basis, continue to optimize the system, to achieve the green and efficiency phenylethanol biosynthesis.

Identically, the Ehrlich pathway naturally present in the S. cerevisiae to produce phenylethanol may compete with the imported phenylethylamine pathway from plants, which may aggravate cell metabolic burden^[2]. More refined modification plans will be implemented to reduce damage to the normal metabolic process of cells.

In terms of challenges of lab safety. S. cerevisiae are airborne and able to survive in the air for long periods of time. In this way they can contaminate all kinds of other cultures. The escape of engineered yeasts from the laboratory will destroy the ecological balance of the natural yeast community. In addition, allergic people might suffer from allergic reactions after having been exposed to yeasts or its spores. If the transgenic yeast escapes from the laboratory, the vector and the anti-sequence may be potentially harmful, such as the uncontrolled spread of the organism or its genetic material.

E. coli DH5α is a versatile strain used for general cloning. E. coli BL21 and E. coli Rosetta are versatile strains used for protein expression. They are indeed safe strains. However, if the transgenic E. coli escapes from the laboratory, the vectors and the antibiotic resistance genes may be potentially harmful, such as the uncontrolled spread of the organism or its genetic material. We need to make further considerations for environmental safety through the waste classification mechanism and recycling.

References

1. Yin, S.; Zhou, H.; Xiao, X.; Lang, T.; Liang, J.; Wang, C., Improving 2-phenylethanol production via Ehrlich pathway using genetic engineered Saccharomyces cerevisiae strains. Curr Microbiol 2015, 70 (5), 762-7.

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