In a continuously modernizing world, insect pests are an increasing nuisance and a significant threat to public health, agriculture, and overall quality of life. This problem is pervasive, affecting both urban dwellers and rural residents alike; while dense urban populations exacerbate spread of disease, the lack of healthcare infrastructure, preventative measures, and education in rural areas makes them equally as vulnerable.

In many tropical and subtropical countries with rapid urbanization and unsustainable waste management, pests like mosquitoes, flies, and roaches breed in abundance. Pest diseases such as dengue fever, malaria, and chikungunya are particularly prevalent, with millions of cases reported annually, leading to substantial mortality. The World Health Organization (WHO) reported that in 2023, Southeast Asia accounted for 60% of global dengue cases, with over 2.8 million cases and nearly 4,000 deaths. Not only so, the rest of the world is also facing this issue due to increased temperatures caused by climate change creating ideal conditions for the proliferation of these pests; this problem is only getting worse.

In our country Taiwan, a pronounced pest problem exists due to its year-long humidity and presence of stagnant water in population-dense residential zones. Both urban and rural communities in Taiwan face considerable challenges related to pest infestations. Our island frequently experiences outbreaks of insect-borne diseases such as dengue fever and, more recently, the Zika virus. In 2023, Taiwan reported over 20,000 cases of dengue fever, marking a significant resurgence of the disease since the 2014/2015 outbreak. Current solutions like repellents, while effective, are unhealthy, unsustainable, and often uncomfortable to use.

In the face of these annoying buzzing creatures that bring about everything from itchy skin to infectious diseases, many have taken to insect repellants as their personal on-the-go solution. No matter the form, whether it be sprays, stickers, creams, or even roll-on applicators, nearly all effective and commercially available repellents make use of the synthetic compound diethyltoluamide (DEET), with most natural alternatives being ineffective.

DEET, also referred to as diethyltoluamide, is the oldest and one of the most effective known active ingredients in insect repellants. It has been estimated that about 30% of the US population use products that contain DEET every year (Agency for Toxic Substances and Disease Registry (US), 2017). However, DEET is also associated with side effects of skin irritation, unpleasant odor, material damage (to synthetic fabrics), greasy feel / stickiness, and general toxicity to human health. In more serious cases, exposure of humans to DEET have shown severe neurological effects including seizures, ataxia, restlessness, uncontrolled limb movements, aggressive behavior, and other similar symptoms (Agency for Toxic Substances and Disease Registry (US), 2017).

Alternatively, many also use spatial solutions like bug-zapping lanterns and mosquito coils to avoid the unfavorable aspects of DEET. Bug-zapping lanterns attract flying pests with ultraviolet (UV) or blue/purple light and zap them with high-voltage electric grids. Mosquito coils repel mosquitoes and other flying pests through the emission of smoke from a burning coil containing insecticides such as pyrethroids or pyrethrins. Although effective, both of these solutions have spatial limitations as they only repel mosquitoes in the general vicinity they are situated in; burning coils can also cause over-inhalation and is a potential fire hazard.

Camphor trees (Camphora officinarum) are a type of evergreen tree native to many East and Southeast Asian countries, including Taiwan, China, and India. They often have a large trunk, live for hundreds of years, and are a great addition to forests as they host many species of butterflies and other wildlife. Camphor wood with its pleasant smell and diverse functionality—insect repellency, medicinal uses, and culinary spice—has been prized by many for centuries. Taiwan was once home to many camphor trees and its export has benefited Taiwan for decades, but camphor populations have since declined due to overharvesting during the Japanese colonization era. Despite this, camphor remains a big part of Taiwanese culture. In fact, many traditional Taiwanese “old streets” (in Mandarin: 老街, Pinyin: laojie) still sell camphor wood and concentrated camphor oil for its aromatic and medicinal properties. However, these compounds are often sticky and uncomfortable, energy-consuming to achieve a high quality, and minimally effective at repelling mosquitoes.

Borneol is a Camphor derivative without its drawbacks (Borneol is called “冰片 or bing pian” in Mandarin, meaning “cool chips” since it has a cooling effect and is typically distilled into crystal/chip form); Borneol is typically used in Traditional Chinese Medicine (TCM) to treat inflammation, but more than that, borneol is also an effective insect repellent that doesn’t exhibit bad odor or high toxicity. Borneol binds to the OR49 receptor in insects, which activates a neuron signal competing with the olfactory receptors involved in animal host location. This acts as a repellent by inhibiting the ability of insects to detect humans.

Typical borneol used in TCM is synthetic borneol, derived through chemical means. However, synthetic borneol is impure since it contains the camphor derivatives D-Isoborneol, L-Isoborneol, D-Borneol, and L-Borneol; D-Isoborneol and L-Isoborneol mildly irritates the skin, eyes, and digestive tract. D-Borneol and L-Borneol are both relatively safe, with L-Borneol being the more effective insect repellent. Currently, producing L-Borneol is extremely expensive with 50 grams of pure (97%+) borneol costing over 30 USD dollars, making it an unpopular option for many consumers. Not only so, L-Borneol refinement often requires a Camphor tree or other plants, making it an environmentally unfriendly and unsustainable practice.

Ever since the 1940s, fragrances have steadily grown in popularity; more now than ever, people are using fragrances in their daily routines. Our project, BOROHMA, aims to utilize fragrances' mass appeal to increase insect repellency in Taiwan and worldwide. By combining effective insect repellency with great aromas, we are creating a customizable insect-repellent fragrance for the everyday commuter. We do so by engineering the L-Borneol biosynthesis pathway into E. coli to produce optically pure L-Borneol that is then encapsulated into a specially designed minicell for controlled release. The L-Borneol-containing minicells are then combined with solvents and other fragrance ingredients to create BOROHMA, our allergy-aware custom insect-repellent fragrance.

Take our fragrance quiz now to customize your very own perfume or cologne, allergen-free!

To achieve large-yield biosynthesis of L-Borneol, we aim to introduce one foreign pathway to E. coli, the mevalonate (MVA) pathway, and increase the efficiency of another native pathway, the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The MVA and MEP pathway have two common products, IPP (Isopentenyl pyrophosphate) and DMAPP (Dimethylallyl pyrophosphate); the balanced interconversion between IPP and DMAPP is controlled by IDI, an enzyme present in both pathways. IPP and DMAPP collectively form the terpene geranyl diphosphate (GPP). By strategically engineering both the MVA and MEP pathways in E. coli, we can effectively increase the yield of GPP, and in turn, increase the yield of BPP and finally optically pure L-Borneol through L-bornyl diphosphate synthase (LBPPS) and alkaline phosphatase (AP).

To produce all the enzymes required for L-borneol biosynthesis, we have designed two constructs: one for overexpression (regulated by T7 promoter in the pACYC vector) and another for controlled expression (regulated by the araBAD promoter in the pBAD vector), hence the distinction between red and green color for protein names; the final engineered plasmids are named pACYC-LBB & pBAD-LBB (LBB = L-Borneol Biosynthesis).

The MEP pathway, although natively present in E. coli, displays low efficiency and metabolite flux; therefore, the overexpression of key enzymes in charge of rate-limiting steps [1-deoxy-d-xylulose-5-phosphate (DXP) synthase (DXS), DXS reductoisomerase (DXR), and isopentenyl diphosphate isomerase (IDI)] is crucial to improve the production efficiency of DMAPP and IPP via the MEP pathway. The rest of the enzymes in the MEP pathway (IspD, IspE, IspF, IspG, and IspH) are subjected to controlled expression.

While both are capable of it, the MVA pathway, completely foreign to E. coli, is a more energy-efficient pathway than the MEP pathway for the production of IPP and DMAPP. The overexpression of the rate-limiting enzymes in the MVA pathway [3-hydroxy-3-methylglutaryl-CoA (HmgR) and IDI] and the controlled regulation of non-rate-limiting enzymes [acetoacetyl-CoA thiolase (ACCT), Hmg-CoA synthase (HmgS), mevalonate kinase (MK), mevalonate-5-phosphate (MVAP) kinase (PMK), mevalonate-5-diphosphate (MVAPP) decarboxylase (MDD)] will be able to effectively engineer the whole MVA pathway into E. coli for the further increased yield of IPP and DMAPP.

The final pathway for high-yield production of L-Borneol requires the conversion of GPP [the combined product of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)] into bornyl diphosphate (BPP) and then into L-Borneol. The overexpression of the enzymes in charge of GPP to BPP to L-Borneol conversion, LBPPS, and AP, respectively, will allow for the proportional high-yield conversion of GPP to L-Borneol, our final desired product.

Overall, by strategically engineering the MEP and MVA pathways to overexpress only crucial enzymes, we can effectively increase DMAPP and IPP production without unnecessary protein buildup that may otherwise decrease overall yield. Additionally, the strong expression of LBPPS and AP in charge of GPP to L-Borneol synthesis highlights the final step of converting high-yield GPP to high-yield L-Borneol.

pACYC-LBB and pBAD-LBB are transformed into E. coli BLR(DE3) to create our L-Borneol-producing bacteria strain; in the future, we aim to perform gene knock-in to ensure L-Borneol production stability and longevity.

Minicells are small, anucleated structures produced from uneven bacterial cell division. These minicells retain most normal bacteria features like protein production (under certain conditions) and functioning cell membrane, but they lack chromosomal DNA. We created E. coli BLR(DE3) minicells by knocking out the minCDRE genes from the bacterial genome to disrupt normal cell division.

Our project leverages the characteristics of minicells to:

1. Simplify the L-Borneol purification process.
   One of the challenges of traditional L-Borneol production—whether it be through chemical synthesis or refining from camphor wood—is that the purification process to separate optically pure L-Borneol from impurities and byproducts is complex, laborious, and energy-intensive. It usually involved a multi-step process of extraction, separation, crystallization, and recrystallization. We bypass this complex process by using minicells as a chassis to continually produce, hold, and release optically pure L-Borneol produced by our engineered E. coli, ridding the need for the complex purification process of traditional methods. Since minicells are significantly smaller and less dense than their parent E. coli cells, separating them is a simple centrifugation process that differentiates based on size and density.


2. Create a controlled L-Borneol release system.
   To enhance the longevity and effectiveness of L-borneol as an insect repellent, we also leverage the unique properties of minicells as a controlled release system. Minicells have a natural membrane that can encapsulate L-Borneol in a water-based solution, effectively trapping the compound within. When this solution is sprayed, the water evaporates, allowing the L-Borneol to be gradually released from the minicell membrane. This controlled release mechanism not only ensures the prolonged effect but also optimizes the efficiency of the repellent by delivering a steady dose of L-Borneol over time.


3. Ensure the biosafety of our final product.
   Since minicells lack the ability to reproduce, they cannot proliferate in uncontrolled environments; this makes them the perfect candidate to ensure the biosafety of BOROHMA. Since our product plans to be released through a spraying mechanism, minicell holds an important role in ensuring that our product is environmentally safe and responsible.

In conclusion, the use of minicells not only enhances biosafety and environmental friendliness but also ensures a consistent and controlled release of L-Borneol, maximizing its effectiveness and longevity. This approach makes BOROHMA a safe, effective, and sustainable solution for L-Borneol delivery and application.

The fragrance industry, currently valued at $67.53 billion USD, continues to grow as more people join the middle and upper classes (Fragrance & Perfume Industry Size & Share Analysis - Growth Trends & Forecasts (2024 - 2029), 2024). This trend is evident in Taiwan, where the fragrance market is expanding at an annual rate of over 2.7%. Alongside the expansion of the market, demand for a more customizable and inclusive product also grows; that’s why we created AroMatch, our specially designed fragrance customization quiz that accounts for allergies. The quiz is designed to filter out user allergies without compromising usability, arranging a combination of top, middle, and base notes to match the needs and preferences of each customer. We hope that by making a quiz like this, we can encourage more people to utilize our product and in turn increase insect repellency in Taiwan and world-wide.

To use AroMatch, click here.

To learn more about AroMatch, visit our software page.

AroFuse is designed to effectively contain and deliver our unique product to make its adoption convenient and appealing. Since our repellent fragrance utilizes minicells as the delivery mechanism, we will incorporate innovative hardware features designed to maximize the longevity of the minicells. This ensures that users won't need to frequently purchase new repellents, providing a more convenient and cost-effective solution. By separating the minicell solution from the fragrance we are preventing the dilution of the minicell broth which allows the minicells to sustain themselves for a longer time. This separation also makes the customization aspect of BOROHMA easy to achieve as we won’t have to consider which fragrances will be damaging to our minicells and automatically sets the ratio of minicell broth to fragrance at 2:8 respectively.

The second aspect of our design ensures our spray bottle is inclusive toward a wide range of consumers. By making our spray nozzle easy to press we are appealing to customers with muscle deficiency who might otherwise be unable to use our product. We also designed our spray bottle to be easy to grip to accommodate people with weaker grip strength, children and the elderly. The high-contrast print makes our product inclusive to people with visual impairments. Overall, our product combines effective hardware design with inclusivity in mind to create a user-friendly experience for everyone.

To learn more about our AroFuse, visit our hardware page.