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.
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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.