Abstract

Mosquito-borne diseases are a major global health concern, affecting hundreds of millions of people annually. Nearly 700 million people are infected with diseases such as malaria, dengue fever, and Zika virus each year, resulting in over one million deaths. Traditional mosquito control primarily relies on chemical insecticides and repellents (e.g., DEET). While these synthetic chemicals are effective, they also pose challenges: improper use can cause skin irritation or poisoning, and repeated use may disrupt ecosystems or induce insect resistance [1]. As society advances, there is a growing demand for safer and more environmentally friendly repellents.

Plant essential oils offer a natural alternative. Citronella oil, extracted from plants of the Cymbopogon genus, has long been used as a mosquito repellent. Its active ingredients, including citronellal, citronellol, and citral, repel mosquitoes by activating the insects' sensory neurons (TRPA1 channels) and inhibiting their olfactory/gustatory receptors [2].

Plant-based repellents like citronellal also have limitations: the yield of essential oils extracted from plants is relatively low and subject to weather conditions. Relying on large-scale cultivation and extraction is costly and unsustainable, making it unable to meet global demand [3].

If we can biosynthesize citronellal using engineered microorganisms, we can achieve large-scale supply of this biorenewable mosquito repellent, reducing reliance on chemical synthesis or plant cultivation. This will meet the demand for safer mosquito control tools and ensure easy access to these tools for communities at risk of mosquito-borne diseases.

1. Project Inspiration

1.1 Inspiration from the Real World

Every summer, mosquitoes are the most annoying nuisances on campus. We conducted small-scale recordings and interviews across dormitories, classrooms, and study rooms: being bitten and distracted while reviewing with a fan on late at night, swarmed by mosquitoes when pausing near campus green belts during the day, and even encountering the scenario of "mosquitoes arriving before people sit down" during military training and club camping activities. For us, the biggest annoyance is not a single bite, but the decreased sleep quality and impaired learning efficiency caused by repeated harassment. These real, seemingly trivial pain points form the primary motivation for our project—to develop a human-friendly and eco-friendly green mosquito prevention solution.

Zooming out, the problem is more than just a "little itch". With the urban heat island effect and changes in rainfall patterns, the active period of mosquito vectors has lengthened and intensified in some regions, and public health authorities are more frequently reminding people to prevent and control mosquito-borne diseases such as dengue fever and chikungunya fever. Through communications with teachers in disease control and ecology, we realized that the campus is not an isolated island; students' dormitories, community green spaces, and even urban drainage systems form an interconnected ecological network. Solving a "small matter"—reducing mosquito harassment—can simultaneously improve comfort, concentration, and mitigate health risks.

1.1.1 Problem Identification

We first noticed common high-concentration chemical mosquito repellents. Through a small-scale questionnaire survey on campus, most respondents expressed dislike for the pungent smell, residues, skin irritation of chemical insecticides, and potential impacts on pets. Although traditional products work "instantly", they are often unfriendly in enclosed spaces and barely meet the threefold demand for "safety, pleasant smell, and long-lasting effect". Thus, the direction of "green and effective" became clear: Can we use milder active substances combined with smarter release methods to achieve the same mosquito-repelling effect?

1.1.2 Literature Research

After reviewing numerous literatures, we focused on citronellal. Derived from nature, it has a fresh scent, is relatively friendly to the human body, and possesses excellent repellent and behavioral interference potential. However, its current yield is not high, mainly relying on plant extraction or chemical synthesis. This precisely provides a platform for our engineering transformation—using synthetic biology to build a stable, efficient, and low-carbon "cell factory" for citronellal production.

1.1.3 Expert Consultation

We consulted experts in the field and learned that the microbial fermentation route uses simple raw materials, has a low carbon footprint, and can scale smoothly from shake flasks to small fermenters. Their advice provided significant guidance for our project and effectively solved various problems encountered during our experiments.

1.1.4 Team Brainstorming

Equipped with expert guidance and insights, we held a brainstorming session and put forward our team's idea: using an E. coli system to produce citronellal and increasing the yield through the "DBTL" (Design-Build-Test-Learn) cycle to develop a cell factory capable of efficiently producing citronellal. This will increase the yield of green insecticides and realize a "green insect repellent revolution" in society. We hope this project is more than just a paper or a demonstration video, but a replicable path: using synthetic biology to produce green molecules in a stable and accessible manner, transforming them into practical daily products through materials and engineering, and then verifying their value with real-scene experiences and data. We believe that bringing "fewer bites and more peace of mind" back to dormitories and classrooms is the most touching significance of iGEM—starting from the laboratory bench to solve a small, daily problem for people around us that is often overlooked.

1.2 Inspiration from Previous iGEM Projects

In recent years at the iGEM competition, projects focused on "mosquito prevention" have developed several clear technical routes. One route involves intervening in mosquitoes themselves at the source. For example, the 2021 Aix-Marseille team used Asaia, a symbiotic bacterium in the mosquito midgut, to design engineering modules in the gut of Aedes mosquitoes to affect their survival and transmission capabilities. In the same year, the Jilin_China team took the opposite approach: simulating the chemical signals of "human sweat", they used engineered bacteria to produce volatile acids and ammonia to strongly attract and trap mosquitoes, creating an integrated "attraction-killing" device. The SCUT-China team approached from the consumer goods perspective: based on crowd surveys and education, they selected daily chemical solutions such as mosquito repellent sprays and supporting commercialization ideas and materials. These three types of routes represent the thinking frameworks of "parasitic symbiosis-targeted intervention", "behavioral trapping-physical killing", and "low-toxic daily chemicals-scene application" respectively.

The second main route is "detection and early warning". Many teams have focused on identifying pathogens "on mosquitoes or at the early on-site stage". For instance, Tokyo Tech’s 2022 project "Dengnosis" focused on dengue fever typing prediction and detection; CCU_Taiwan's 2020 project "DENDETX" developed a dengue detection reagent using peptide-based recognition. In the 2023 Parts Registry, on-site detection designs for pathogens such as West Nile virus carried by mosquitoes can also be found, which, combined with paper-based/isothermal amplification methods, serve rapid monitoring and community prevention and control. Such "rapid detection" routes work in parallel with source control to jointly reduce transmission risks.

From 2023 to 2024, projects have further evolved towards a "green, safe, and sustainable" combination strategy. In 2024, AIS-China combined the "attractant sugar bait" ATSB with RNAi, using HMBPP to strongly attract blood-feeding mosquitoes and deliver specific shRNA. In the same year, GEMS-Taiwan proposed "Dengue Beeters", which combined recombinant biological larvicide, natural attractants from beet pulp, and sticky egg traps to interrupt the mosquito life cycle. UniMünster's "NOsquito" adopted the route of biosynthesizing mosquito-repellent molecules, exploring sustainable production and evaluating efficacy. Formosa's "BOROHMA" attempted to combine "fragrance + mosquito repellent", building a customizable fragrance-type mosquito repellent product around L-camphorol. Additionally, the iGEM BioInnovation Fair showcased innovations like "Algeavity"—a device using bioluminescent algae to kill mosquitoes—indicating that the community is collectively shifting towards a new paradigm of "ecologically friendly" vector control.

The inspiration for our project is drawn from these efforts. On one hand, we draw on Jilin China’s "behavior-driven" concept and AIS-China's "precision delivery" idea, emphasizing the use of more sensitive chemical signals and safer biological methods to influence mosquitoes' feeding and landing behaviors. On the other hand, like UniMünster and Formosa, we take "biomanufacturing of green active molecules + user-friendly formulations" as the core path to reduce risks and improve accessibility, committing to developing natural-source molecules into product forms that are "long-lasting, low-irritant, and suitable for campuses and communities". Meanwhile, we also continue the experience of teams like Tokyo Tech in public education and health practices, incorporating popular science and standardized usage into the implementation phase of our project's "from laboratory bench to real scenes".

3. Project

3.1 Project Overview

Our team plans to engineer E. coli to enable it to biosynthesize citronellal, thereby creating a living factory for this natural mosquito repellent. The strategy involves reconstructing the monoterpene biosynthetic pathway in E. coli, allowing it to produce citronellal using simple carbon sources. Starting from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)—the universal precursors of isoprenoids—we will generate geranyl pyrophosphate (GPP), convert GPP to geraniol, and then oxidize geraniol to citronellal.

E. coli naturally produces IPP and DMAPP through the methylerythritol 4-phosphate (MEP) pathway during its core metabolism [7]. These C5 structural units are the building blocks of all terpenoids. By introducing several heterologous enzymes, we can convert the natural isoprenoid precursors of E. coli into our target monoterpene (citronellal):

• Geranyl Pyrophosphate Synthase (GPS): An enzyme that condenses IPP and DMAPP into geranyl pyrophosphate (GPP). GPP serves as the 10-carbon skeleton for monoterpenes. We have cloned the GPS gene to ensure E. coli can efficiently convert its C5 precursors into GPP [8].
• Geraniol Synthase (CsTPS1): A terpene synthase that converts GPP to geraniol. This gene is known to catalyze the formation of geraniol from GPP and is a key enzyme in our pathway for producing the intermediate geraniol [9].
Geraniol Dehydrogenase (GeDH): An enzyme that oxidizes geraniol to citronellal. We have identified an enzyme with specificity for geraniol[10]. In our design, this enzyme will perform the final oxidation step to generate citronellal.

By assembling these enzymes in E. coli, our engineered strain can complete the entire citronellal production process. When the engineered bacteria are induced to express this pathway, citronellal accumulates in the culture medium. This biologically produced citronellal can be extracted and used in mosquito repellent formulations, providing a renewable source of mosquito repellent.

This solution leverages synthetic biology technology to sustainably produce natural mosquito repellents. The fermentation-based production of citronellal can be carried out year-round, unaffected by climate or arable land area. It avoids harmful chemicals found in some existing mosquito repellents and produces a product with an identical chemical composition to plant-sourced citronellal. Ultimately, this will make mosquito repellents more affordable and environmentally friendly, thereby supporting public health efforts in mosquito-plagued regions.

3.2 Gene Design and Plasmid Construction

To express these three enzymes (GPS, CsTPS1, GeDH) in E. coli, we have designed a multi-plasmid system to optimize the expression of each enzyme. Each gene has been codon-optimized for *E. coli* and placed under an inducible promoter, allowing us to control the timing of expression (to minimize metabolic burden before the culture is ready):

• pET28a-GPS: Contains the geranyl pyrophosphate synthase gene (GPS) under the T7 promoter (IPTG-inducible). The pET28a vector provides a high-expression T7 system and kanamycin resistance for selection. We designed this plasmid to achieve high-level GPS production.
• pET21a-CsTPS1: Contains the CsTPS1 geraniol synthase gene under the T7 promoter (IPTG-inducible) with ampicillin resistance. This plasmid drives the production of geraniol from GPP. We selected a separate plasmid for CsTPS1 to balance its expression level.
• pBAD33-GeDH: Contains the geraniol dehydrogenase gene under the L-arabinose-inducible promoter (araBAD promoter) in the pBAD33 vector, which confers chloramphenicol resistance. This allows us to independently induce the final oxidation step using arabinose.

Three compatible plasmids with different origins of replication and antibiotic markers can be co-transformed into the same E. coli cell. We verified the plasmid design with Snap Gene prior to synthesis. Figure 1 shows a simplified map of our plasmids and their key features (promoters, antibiotic markers, and inserted genes).

Figure 1: CPlasmid Maps of the Engineered Pathway. (a) pET28a-GPS; (b) pET21a-CsTPS1; (c) pBAD33-GeDH

Each plasmid is equipped with a strong ribosome binding site and a His-tag attached to the enzyme for detection. This combination enables the simultaneous expression of all pathway enzymes in E. coli. (Plasmid maps are for reference only; drawn based on our SnapGene design.)

3.3 Verification of the Physiological Activity of the Engineered Strain

During expert consultations, we emphasized that multiple antibiotics may affect the inherent physiological activity of bacteria. After constructing the engineered strain, we designed experiments to verify that there was no significant change in the growth capacity of the strain (Figure 2).

Figure 2:Comparison of growth ability among different strains
Group 1: Engineered bacteria + LB + antibiotics
Group 2: Wild-type bacteria + LB
Group 3: Empty vector bacteria + LB + antibiotics

Subsequently, we tested the engineered strain at the transcriptional level and used qPCR experiments to verify the increased transcription levels of the target genes (Figure 3).

Figure 3:Relative expression level of GPS, CsTPS1 and GeDH

Finally, we verified the successful expression of the target proteins through Western blot experiments (Figure 4).

Figure 4:The Western blot results of GPS, CsTPS1, and GeDH

3.4 Production of Citronellal

The final step of our project was to demonstrate the production of citronellal. We conducted shake flask fermentation and monitored product formation throughout the process. High-Performance Liquid Chromatography (HPLC) was coupled with ultraviolet (UV) absorption spectroscopy to detect the product concentration, yielding accurate quantitative data.

3.5 Optimization of Citronellal Yield

Under the guidance of experts, we designed a Response Surface Methodology (RSM) experiment with three factors and three levels to optimize the yield. Meanwhile, we leveraged computer-aided tools to perform molecular thermal stability scoring, molecular docking, and molecular dynamics simulations. Based on these analyses, we redesigned the recombinant protein plasmids, ultimately achieving an increase in citronellal yield.