Team:SCUT-China/Implementation
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Instructions to Stakeholders
In Integrated Human Practices we analyze the project's stakeholders from a variety of perspectives. Because different groups have different requests, we provide recommendations for project-related content here.
For Claimant Stakeholders
The instructions for the subsequent products in the project will be easy to follow. Because we want our mosquito repellent products to be available to as many people as possible, it is likely that the product will be designed to be used in a way that references the common sprays used today. This will only require following the conventional methods of using mosquito repellent sprays, such as spraying evenly and applying to the skin surface and avoiding sensitive areas.
Although there is still a long way to go before the project can be successfully implemented and produced to benefit the public, there are some practical ideas in the project design and subsequent industrialization ideas that may give new inspirations to takeholders.
For Collaborator Stakeholde
Upstream Fermentation Engineering Researchers: Strategy Selection and Regulation of Modified Strains
UP Strategy (UAS Promoter Strategy)
In our project, we realized that the transcriptional intensity and characteristics of UAS would be different after hybrid promoters constructed with different insertion sites in the promoter sequence. The difference in affinity with nucleosomes at different positions of the promoter sequence may also be different, and this may be one of the reasons why UAS inserts at different sites may lead to differences in regulatory strength. The UAS in the tightly bound part of the nucleosome may reduce the probability of recognition and binding to the transcription factors due to, for example, site-block. Therefore, we constructed hybrid promoters by combining different UAS and inserting them into the genome to different positions to obtain more stable production efficiency.
Based on the validation of the production capacity of our engineered strains, we believe that adding or modifying the UAS of promoters is a set of design ideas worth promoting. To better illustrate how the hybrid promoter modification approach of our project should be used, we propose the UP Strategy (UAS Promoter strategy) to guide the use of the stakeholders.
DFig.1 UP strategy flow chart
See more instructions on the UP Strategy in the pdf below!
In realistic implementation, our UP Strategy will likely bring new ideas to stakeholders in these areas.
1. Based on the UP Strategy, you can add or remove such as UAS, URS, and others from the promoter. In this way, you can combine eukaryotic hybrid promoter elements with different characteristics, including but not limited to extending the duration of highly active transcripts, or customizing regulation in response to specific signaling factors. And this can be applied not only in fermentation engineering modification, we believe that UP Strategy can bring new options in other fields as well.
2. obtaining insertion sites that are more likely to be suitable for gene expression. In eukaryotic gene expression, not only the transcription factors will recognize and bind to DNA. Therefore, we believe that the nucleosome affinity analysis in the UP Strategy can be used to screen for suitable insertion sites in eukaryotic exogenous gene introduction studies.
3. further improve the UP Strategy and design a more efficient strategy. Although theoretically genes located at nucleosomal affinity sites are more likely to be expressed, our experimental investigations have shown that some of the results of the actual transformation are not as predicted. This suggests that there is still room for improvement in UPS. We also expect that subsequent researchers will carry out new analysis of the influencing factors or improve the algorithm to build a better UP Strategy.
More and Better Hybrid promoters for S. cerevisiae
In our wet lab, we proposed a new idea to improve the yield of late fermentation according to the carbon source response mechanism of S. cerevisiae promoter. By combining the UAS related to different glucose metabolism pathways, we modified the natural constituent promoters into those with inductivity, which can be activated by yeast “diauxic shift”, so that VS can be expressed at a higher level for a longer period of time without additional inducers. Through our design and engineering, we ended up with a stronger promoter, M8, and a series of promoters with different intensities (Table. 1). With the very exciting results, we got in touch with the technicians at QR Pharmaceuticals. QR Pharmaceuticals is the first company in China to set foot in the biological synthesis of Nootkatone, with rich experience. After effective communication, we have clearly understood the application significance of our idea in industrial production and how factories can learn from our idea in the future:
（1） Compared with wild-type PPDC1, the M8 promoter we finally obtained significantly increased the yield of Vlencene, Nootkatol, Nootkatone, which provided a new idea for companies wanting to obtain higher Nootkatone yield.
（2） Through different combinations of UAS from different promoters, a series of hybrid promoters with different intensities and a wide dynamic range of promoter libraries can be used for fine regulation of metabolic pathways in S. cerevisiae to improve the yield of target products.
（3） The hybrid promoter obtained by our engineering success can be activated by “diauxic Shift” in yeast growth to maintain a good ability of initiation transcription, that is, it has certain "inductivity"., and do not need to add inducers. There are two advantages: one is to reduce unnecessary costs, the other is to reduce the toxicity to cells, both of which are of great significance in the factory application.
（4） Technicians mentioned that in practical production, they often need to balance the relationship between fermentation time and yield. There are many considerations, such as avoiding costs caused by excessively long fermentation time. But if the fermentation time is too short, the yield may not reach the target. And frequent tank changes can also be costly. So we extended the duration of the promoter's high strength, making it easier for technicians to find a balance between input and output.
Table1. Promoters with Different Intensities.
（Promoter strength has been defined as the relative production of nootkatone from a promoter.）
Upstream Fermentation Engineering Researchers: Strategy Selection and Regulation of Modified Strains
Surface-enhanced Raman spectroscopy is an advanced technique for characterizing the reaction process. Fermentation processes commonly require real-time online monitoring to automate the control of some environmental parameters. Commonly, dissolved oxygen, pH, temperature, etc. are measured by electrodes. When the computer monitors excessive fluctuation of a certain index, which exceeds the set optimum range, the machine will automatically control the fermenter for acid-base adjustment and temperature adjustment of hot & cold-water flow to keep it within the set range.
However, during the interview with QR pharmaceuticals, we learned that in fermentation factories nowadays, except for the common pH and temperature parameters which are detected by electrodes, the values of reducing sugars need to be sampled offline and detected manually. By using surface-enhanced Raman spectroscopy, it is possible to measure online in real time and get more realistic and accurate curves, so that special cases can be detected and handled faster.
In the interview with QR pharmaceuticals, the other side also pointed out that there are new instrument technologies for measuring dissolved oxygen and carbon dioxide. However, from the cost and applicability of the spread may not be common enough and the application method may have a relatively large deviation, the engineers will prefer to use the electrode detection equipment that is already popular and widely used.
It means that the new reaction analysis equipment like Surface-enhanced Raman spectroscopy needs to be more utilized and popularized in the market before it can be practically applied in fermentation plants. The small and medium-sized plants or laboratories that are in a position to do so can try to adopt it before the equipment is updated faster.
Project Proposed Implementation
We want to contribute to the solution of nootkatone production challenges. Therefore, our team of microbial fermentation innovators should also consider more. We should extend our focus from the laboratory to the subsequent industrialization process. The conversion rate of university research results is not high, and many projects lack feasibility in production. This problem may be due to the fact that the upstream design does not take into account the subsequent processes.
Therefore, it is necessary to analyze and design the subsequent industrialization process to help us better transform the project into real productivity.
For a more detailed design process, see in Integrated Human Practices
Fig.2 Project Proposed Implementation
Safety points to Consider
1. Industrial production process
Because our product nootkatone has been shown in animal studies to have a low potential for toxic effects in humans. Therefore, it is sufficient to use the usual protective measures in industrial processes. However, there are data that suggest a risk of sensitization of nootkatone at low purity levels, so try to avoid contact of the concentrated crude product with the skin before the isolation and purification process.
2. product design
nootkatone has a certain degree of phytotoxicity, which can cause chlorosis or even necrosis of plant leaves. But because nootkatone has high volatility and is unstable under light and heat [1], its toxicity to the environment though is minimal. However, as a mosquito repellent that may be sprayed directly into the environment, it may form localized areas of high concentration in the environment and lead to poisoning of plants in the environment. The volatility and instability may also limit its effectiveness in the field of mosquito repellents that require long duration of action. Therefore, after searching the literature, we found that these risks and limitations can be greatly improved by encapsulating nootkatone.
The existing studies on nootkatone encapsulation technology are shown in Table.2. The use of lignin for encapsulation not only reduces the phytotoxicity and slows down the volatilization of nootkatone, but even has a more significant killing effect on tick than raw materials.[2] In contrast, encapsulation with cydodexrins greatly affected the physical properties of nootkatone. cydodexrins- encapsulated nootkatone exhibited greater photothermal stability, and the encapsulated cydodexrins-encapsulated nootkatone has better water solubility [3], a property that would facilitate the preparation of safer mosquito repellents.
Table.2 nootkatone encapsulation technology[2,3]
Different encapsulation systems need to be prepared in different ways. We expect that future products will be successfully converted to productivity, so whether the encapsulation method can be implemented in the factory is also a factor we should consider.
In an interview with Prof. Ye, who teaches bioreaction engineering at SCUT, we learned that the ultrasonic treatment used in the preparation of lignin-encapsulated nootkatone is only applicable to fermentation systems in small- or medium-scale plants, and that large-scale fermentation is not feasible. The freezing equipment used for the preparation of cydodexrins-encapsulated nootkatone can be used in any scale. Therefore, in future projects, we can choose the plant size or investigate other more suitable encapsulants and encapsulation methods.
References
[1]National Center for Biotechnology Information (2021). PubChem Annotation Record for Nootkatone, Source: Hazardous Substances Data Bank (HSDB). Retrieved October 17, 2021 from https://pubchem.ncbi.nlm.nih.gov/source/hsdb/8272.
[2]Behle RW, Flor-Weiler LB, Bharadwaj A, Stafford KC 3rd. A formulation to encapsulate nootkatone for tick control. J Med Entomol. 2011;48(6):1120-1127. doi:10.1603/me10282
[3]Kfoury M, Landy D, Ruellan S, Auezova L, Greige-Gerges H, Fourmentin S. Nootkatone encapsulation by cyclodextrins: Effect on water solubility and photostability. Food Chem. 2017;236:41-48. doi:10.1016/j.foodchem.2016.12.086