1. Demand for the product: people with body odors
Body odor refers to the acrid, unpleasant odor that originates from people’s axillary region. Stimulated by the secretion of sweat, this odor is easily formed, especially during summer or during exercise. This pervasive and easily formed smell can be devastating to a person’s social life. In society, people tend to keep a physical distance away from anyone who has body odor, and even discriminate or wrongfully judge a person based on his/her body odor. These reactions toward people with body odor can be especially severe in situations such as large social events, where people judge one another based on first impressions, or in enclosed spaces, such as the elevator or the subway, where the odor is more distinctive. Unfortunately, the modern population cannot avoid being in these situations; and for people who have body odor, this means that they are frequently faced with social embarrassments or social isolation in life. Furthermore, as the average standard of life increases, society becomes more judgmental towards body odor, enhancing the social dilemma of people with body odor. The social dilemma mentioned above is proven by the results of a survey that we sent out. Overall, it can be concluded that people with body odor are constantly faced with social mistreatment, which negatively impacts their lives.
Visit our results page Results PAGE for more information.
2. Current solutions to body odor
As a result of the social dilemma that they face, people with body odor are in strong need of solutions to mediate their body odor. However, the current strategies of body odor control—deodorants and surgeries—have critical disadvantages that create issues for people with body odor.
The most prevalent solution to body odor–deodorants and antiperspirants—can be toxic and harmful to human skin. The effective substance within most, if not all, chemical deodorants and antiperspirants is aluminum. When applied excessively on skin, aluminum may induce contact dermatitis, and even worse being absorbed into human body. Except chemical deodorants, to solve body malodor, some people might choose to use surgical measures to remove their sweat glandand or inject Botulinum toxin. However, surgery leaves a possibility of the recurrence of body odor. Strict requirement for the instruments and high cost make it replaceable.
Therefore, there is a need in the world for a safer and cheaper product for the effective mediation of body malodor; and our product, AROMATA, was created to fit this need.
3. Staphylococcus spp. & Body odor
Human body odor is produced by bacterial transformation of odorless precursor molecules secreted onto the surface of the skin by apocrine glands (Figure 1). Axillary malodor, specifically, is constituted of volatile organic compounds with volatile fatty acids (VFAs) and thioalcohols – both are primary components of malodor, but thioalcohols are the most stifling and sharp. Staphylococcus, one of the dominant bacteria genera that colonizes human axilla, performs the transformation of 3-methyl-3-sulfanylhexan-1-ol (3M3SH), the most abundant secretion of thioalcohol by the odorless precursor Cys-Gly-3M3SH through biochemical pathways. As Cys-Gly-3M3SH is secreted to the skin surface by the subcutaneous axillary apocrine glands, inside the cell, the terminal glycine is cleaved by dipeptidase (PepA) to release Cys-3M3SH which is metabolized by C-S β-lyase to release volatile 3M3SH. 3M3SH then is diffused or exported extracellularly, starting to produce the malodor. (Rudden et al, 2020)
Figure 1. Overview on the biotransformation of the odorless physiological malodor precursor Cys-Gly-3M3SH into the odor molecule 3M3SH Staphylococcus hominis. (Rudden et al, 2020)
4. Fengycins is a promising body odor control strategy
Fengycin (C72H110N12O20) is a cyclic lipopeptide which has been proven to be broad-spectrum antimicrobial (Sreyoshi Sur et al, 2019). As for Staphylococcus spp., it is reported that fengycin is structurally similar to its autoinducing-peptide (AIP), which enables fengycin to compete with AIPs, then bind to membrane-located AgrC, and thus inhibit the accessory gene regulator quorum sensing (QS) system (Piewngam et al, 2018).
Since fengycin is an optimal molecule as a replacement of AIP in the QS system, fengycin becomes our chosen molecule that could inhibit QS system of Staphylococcus spp., the guilty bacteria to be blamed for the production of 3M3SH. Afterwards fengycins could effectively decrease the sinners’ detachment to skin and realize deodorization (Ruddens et al, 2020).
For the production of such a promising component to prevent body odor, we engineered Bacillus subtilis 168. Previously, the production of fengycins was accomplished through activating its dormant native fengycin synthetase. More works have been built on this approach and make modifications to gene sequence of the condition of fermentation to reach a higher production rate (Tan et al, 2021).
Figure 2. Competitive inhibition of S. aureus AIP activity by fengycins. (Piewngam et al, 2020)
5. Fengycins biosynthesis in Bacillus subtilis
We want to produce fengycin through Bacillus subtilis 168. We started by investigating the native fengycin synthetase pathway. As the Figure 3 shown below, sfp and degQ genes play a critical role in the pathway of fengycins synthesis(Tan et al, 2021).
sfp encodes 4'-phosphopantetheinyl transferase which functions as a primer of nonribosomal peptide synthesis via phosphopantetheinylation of thiotemplates. And degQ gene is pleiotropic regulator gene encoding a 46 amino acid polypeptide, which can regulate the expression of a variety of exocrine enzymes and the production of antibacterial substances. It is reported that increased expression of degQ in B. subtilis 168 results in a 7–10-fold increase in antibiotic production (Chen et al, 2009).
As a model strain for genetic engineering, Bacillus subtilis 168 possesses a natural, invalid sfp gene. Besides, a T base at position -10 in the promoter region of degQ gene in Bacillus subtilis 168 is mutated into a C, which results in the inability to express degQ. Therefore, we knocked in both sfp gene and degQ gene in the region of the natural sfp gene of Bacillus subtilis 168 after being knocking out. Eventually, the modification we implemented would enable Bacillus subtilis 168 to produce fengycin.
Figure 3. A schematic view of the metabolic pathways for the fengycin. (Tan et al, 2021)
6. Santalene biosynthesis in Escherichia coli
AROMATA has decided to produce the sandalwood aroma through genetic engineering technology and techniques. This is because sandalwood not only has a refreshing and clean smell, it’s also considered as a very distinct Eastern smell, presenting our Chinese team to the world.
To discover the chemical composition of sandalwood, previous research has illustrated that the sandalwood oil mainly consists of santalols, α-bergamotol, santalenes and α-bergamotene. Among them, santalenes and α-bergamotene are sesquiterpene and also the precursor of santalol and α-bergamotol. Therefore, the higher yield of santalenes not only provides the more pleasant sandaowood aroma, but also signifies the prospect of more santalols, which makes up more than 70% of sandalwoold oil. (Chonglong et al, 2015)
So far, the pathways and enzymes associated with santalene biosynthesis have been revealed thoroughly (Figure 4). As santalene synthase is a key synthetic enzyme, we heterologously expressed santalene synthase of Clausena lansium (ClSS) for the production of α-santalene in E. coli. Since dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate(IPP) are substrates and FPP is precursor, MVA pathway and ERG20 of Saccharomyces cerevisiae have been introduced into E.coli for the improvement of santalenes’ yield. Afterwards, to probe the possibility of enzyme modification to promote santalene production, we modified ERG20 or ClSS by amino acid mutation, binding to a hydrophillic tag and the construction of fusion protein. Finally, our study elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of santalene by about 20%. (Jia Z. et al, 2022)
Figure 4. Construction and expression of santalene. (a) Enzymes and some of the reaction intermediates necessary for the production of santalene through the MEP pathway and MVA pathway. (b) Schematic representing the structure of pMVA, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL transformed into E.coli DH5α ∆TnaA.
7. Overall design of AROMATA
Our project AROMATA is composed of two basic parts: 1. Producing fengycins from Bacillus subtilis 168 with the aim of reducing people’s body odor. 2. Biosynthesis of santalene from E. coli DH5α for sandalwood aroma.
As mentioned before, we knocked in the sfp and degQ gene to activate the fengycin biosynthesis pathway in Bacillus subtilis 168 (Tan et al, 2021). For the production of santalene, we heterologously expressed MVA pathway and ERG20 of Saccharomyces cerevisiae and santalene synthase of Clausena lansium (ClSS) . Furthermore, to verify our conjecture that AROMATA could inhibit the QS system of Staphylococcus spp., the lipopeptide extract was used for biofilm analysis. Also, to exclude the possibility that extract would induce the formation of drug-resistant bacteria, we studied on the change of growth curve under different concentrations of fengycins.
Figure 5. Bacillus subtilis 168 will produce fengycins to bind with Staphylococcus spp., inhibit their quorum sensing, and thus decrease their detachment to skin. Escherichia coli DH5a will produce santalene to provide sandalwood aroma. By mixing fengycins and santalene, we can produce a series of AROMATA product, such as spray and hydrogel.
8.Future
Fengycins has a broad potential application in many aspects, but its low yields often limit its use on massive industrial production. Therefore, further improvement of Bacillus subtilis using synthetic biology to elevate its yields can not only benefit our product, making it more competitive once it fits into industrial production, but make it possible to use more fengycins in other living aspects such as agriculture, medicine and food industry due to its antifungal effect. In future, optimization of Ppps promoter and over-expression of acs, birA, accACD for strengthening the fatty acid synthesis would be implemented upon Bacillus subtilis 168 (Yazen et al, 2016; Tan et al, 2022). For future development of integrating live bacteria in the applied product, a suicide mechanism of the Bacillus subtilis is needed because the principle of DO NOT RELEASE should be put first in consideration of safety to both human and environment. In a word, our product AROMATA development still needs our further research and verification.
Reference
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