Team:SCUT-China/Description
Overview
A group of students from SCUT in Guangzhou came up with an idea to alleviate the scourge of mosquitoes. In inspiration, we skillfully used Baidu index big data analysis, investigated the official data of the WORLD Health Organization and EPA, and known a new mosquito repellent ingredient--Nootkatone. In the conception stage of our project, based on the rise of biological manufacturing and synthetic biology, we considered from three aspects: the selection of chassis cells, the identification of synthetic pathways and how to increase yield, so as to build a cell factory with high yield of Nootkatone and to realize our Nootka-Boom!
Inspiration
Regional necessity:
South China University of Technology is located in the humid and rainy Guangzhou, where the warm climate with unclear seasons is not only suitable for human to live but also for mosquito breeding. Whether indoors or outdoors, the mosquito problem has always plagued us, as well as other people living in Guangzhou. According to the data of Baidu index, from 2013 to 2021, the users who search the keyword of mosquito repellent on the Internet are most from Guangdong. And in Guangdong, the most dominant users having searched relevant questions are from Guangzhou. This can show that the mosquito problem is a high concern and urgent problem for people around us.
Image Source:baidu.com
International necessity:
Mosquito bites bring not only itchy skin, but also the risk of contracting infections mosquito-borne diseases. Mosquitoes are vector organisms and diseases that use mosquitoes as vectors include yellow fever, dengue fever, epidemic B encephalitis, malaria, lymphatic filariasis, Chikungunya, West Nile virus disease, and Rift Valley fever. In the case of dengue fever, for example, WHO listed it as one of the top 10 threats to global health in 2019. About 390 million people in 128 countries are at risk of dengue fever, with an average of 96 million people infected each year, including asymptomatic carriers. The mortality rate has remained stable at around 0.12%, which means that thousands of people die each year from dengue infection.
Data Source: World Health Organization
The most direct solution to the problem seems to be the use of insecticide. However, monitoring has found that insecticide resistance is widespread among malaria vectors. According to the World Health Organization, 65 of 89 malaria-endemic countries have reported at least one endemic vector resistant to pyrethroids since 2010. All four classes of insecticides currently used for adult malaria vector control, pyrethroids, organochlorines, carbamates and organophosphates, have spread widely in Africa, the Americas, Southeast Asia, the eastern Mediterranean and the Western Pacific. Moreover, malaria vectors in some parts of Africa are now able to survive in environments with high concentrations of insecticides, suggesting that resistance in some malaria vector organisms is now increasing over time.
Source: WHO Malaria Threats Map.
In the EPA's report on new repellent ingredients, we noted that compared with existing repellent ingredients, Nootkatone can maintain high repellent effect stably for a long time. As proven by EPA's data, the high-purity Nootkatone not only has high safety, but also has no irritation, no skin sensitization, and is a qualified mosquito repellent ingredient. According to the EPA, the creation of a new effective insecticide ingredient,Nootkatone, will help address the growing insecticide resistance to other products currently in use. However, currently there is no popular product related to Nootkatone on the market because its current primary production method is plant-based extraction, which is expensive and difficult to produce in large quantities. In order to further enable the wider application of Nootkatone, to exploit its value and to explore more of its possibilities, we have undertaken our project - Nootka-Boom!
Nootka-Boom！
Source of project
Synthetic biology with engineering design concept, design and transformation of organism to have a goal, subvert the biological technique innovation, is expected to decode the human resources, environment and other fields important challenge to provide a new solution With the rapid development of synthetic biology, through the microbial production of high value-added compounds become attractive In April 2013,Nature published a study on artemisinin, a highly effective semi-synthetic antimalarial drug in Saccharomyces cerevisiae, which greatly promoted the industrialization of microbial semi-synthetic artemisinin. This is the most successful case of heterologous synthesis of high value-added products by microorganisms.
Nootkatone is a natural ingredient found in some Alaskan yellow cedar herbs and citrus fruits that is responsible for grapefruit's distinctive smell. Nootkatone has long been used in many food products that have a citrus flavor or smell. DVBD scientists have found that Nootkatone is an effective mosquito repellent and insecticide that can be used against mosquito ticks and other pests[1].
There are three main production methods of Nootkatone: Plant extraction method, chemical synthesis and microbial synthesis. Terpenoids content in plants is usually very low, the extraction method is easy to cause serious damage to wild plant resources, and the planting and processing of its crops are affected by many factors such as climate, environment and transportation, so the yield, quality and price of Nootkatone have uncontrollable changes, and the economic feasibility is poor. At present, the industry is often through a precursor of the direct oxidation of price is relatively cheap Valencene to obtain Nootkatone. But the reaction usually involves some environmental unfriendly oxidant, such as heavy metal salts. By contrast, microbial synthesis method is not restricted by raw material Green clean production process, has a great advantage[2]. Such as the Nature The focus of the synthetic biology industry is rapidly shifting to the over $20 billion fine chemicals market, which will rapidly advance the heterologous biosynthesis of natural products Therefore, to meet the growing demand for Nootkatone, SCUT-China wants to use synthetic biology methods to create a cell factory with high-yield of Nootkatone.
Selection of chassis cell
In the field of biomanufacturing, the selection of different chassis cells plays a key role in the biosynthesis of terpenoids. IPP and DMAPP, the precursors of terpenoids in E. coli, are mainly used for prenylation of tRNA and synthesis of FPP, and then for synthesis of quinones and cell walls[3]. Therefore, these chassis cells are mainly used for biosynthesis of alcohols and acids rather than terpenoids. In addition, due to the lack of post-translational modification, it is difficult for bacteria to express cytochrome P450, and many terpenoids have antibacterial activity. However, Saccharomyces cerevisiae has been widely used in terpenoid biosynthesis because it is generally regarded as safe (GRAS),10 industrially robust and able to functionally express eukaryotic cytochrome P450 enzymes[4]. Therefore, SCUT-China will also consider using S. cerevisiae to build our Nootkatone cell factory.
Identification of biosynthetic pathways
All terpenes are biosynthesized through mevalonate pathway (MVA) in yeast, originating from acetyl-CoA. And the intermediate product farnesyl diphosphate (FPP) is the direct precursor of valencene, which is catalyzed by valencene synthase (VS) to synthesize valencene. Then, valencene is oxidated to β-Nootkatol and (+)-nootkatone by HPO, AtCPR and ADH[2]. SCUT-China will introduce the Nootkatone synthesis pathway into S. cerevisiae to build our cell factory.
Idea to realize Nootka-Boom!
After constructing a saccharomyces cerevisiae cell factory capable of producing Nootkatone, we found that the construction of metabolic pathways usually involves the expression of multiple genes, whose expression levels span several orders of magnitude, and the regulation of gene expression by endogenous gene regulatory elements alone is not sufficient[5]. In addition, we also found that CnVS is the limiting enzyme in the metabolic pathway of Nootkatone synthesis, and its expression level plays a key role in Nootkatone production[2]. How to effectively improve the expression level of VS to improve Nootkatone precursor (valencene) and Nootkatone production is a big problem we are facing.
To solve the above two problems, we turned our attention to promoters that control gene transcription[6]. As the initiation of gene transcription, promoters are key components of cell factory design and metabolic pathway modification. Due to the limited number of well-characterized promoters in yeast and their small dynamic ranges, it is often difficult to satisfy the fine regulation of genes, optimize metabolic flux, and improve the yield of the target product[7]. Therefore, SCUT-China wants to obtain high-strength promoters to increase the expression of CnVS through engineering modification of S. cerevisiae natural promoters, and to obtain a good dynamic promoter library, which will provide useful and efficient help for the improvement of metabolic flux optimization target product yield and the design of cell factory.
In labs, our promoter engineering will be carried out from Wet Lab and Dry Lab respectively. Wet Lab mainly focuses on UAS combining different promoters to construct a series of hybrid-promoters with different strengths. Dry Lab obtained promoters with different intensities mainly from nucleosome affinity of promoters.
References
1. Clarkson, T.C., et al., Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus. INSECTS, 2021. 12(5).
2. Ouyang, X., et al., Stepwise engineering of Saccharomyces cerevisiae to produce (+)-valencene and its related sesquiterpenes. RSC ADVANCES, 2019. 9(52): p. 30171-30181.
3. Wang, D., Z. Dai, and X. Zhang, [Production of plant-derived natural products in yeast cells - A review]. Wei sheng wu xue bao = Acta microbiologica Sinica, 2016. 56(3): p. 516-29.
4. Dai, Z.B., et al., Yeast synthetic biology for high-value metabolites. Fems Yeast Research, 2015. 15(1): p. 11.
5. Chen, X., et al., DCEO Biotechnology: Tools To Design, Construct, Evaluate, and Optimize the Metabolic Pathway for Biosynthesis of Chemicals. Chemical Reviews, 2018. 118(1): p. 4-72.
6. Young, E. and H. Alper, Synthetic Biology: Tools to Design, Build, and Optimize Cellular Processes. Journal of Biomedicine and Biotechnology, 2010.
7. Maury, J., et al., Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae. Febs Letters, 2008. 582(29): p. 4032-4038