Proof of Concept | KEYSTONE

Introduction

Our project aims to create a bio-body deodorant by producing fengycins through engineering B. subtilis 168 to inhibit the quorum sensing system of Staphylococcus spp., the dominant bacteria genera colonizing the human axilla and producing malodorous molecule 3M3SH. With the delocalization of Staphylococcus spp., our aim of deodorization would be achieved. Meanwhile, we utilized engineered E. coli to produce santalene as a sandalwood aroma addition to optimize consumer experience. We sincerely hope that our product can solve the problem of body odor in a safer, more eco-friendly and more convenient approach to alleviate the social dilemmas faced by people, and then become a new trend in the cosmetic industry.

Figure 1. Bacillus subtilis 168 will produce fengycins to bind with Staphylococcus spp., inhibit their quorum sensing, and thus decrease their detachment to skin. Escherichia coli DH5α 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.

Production of fengycins

The quantification analysis of HPLC demonstrated that we have successfully produced 25.06 mg/L of fengycins through modifying Bacillus subtilis 168, representing the effectiveness of gene editing and laying a foundation for our product development. Visit our results page for more information: https://2022.igem.wiki/keystone/results

Figure 2. Quantification analysis of fengycins production. (a) The sequencing result of engineering Bacillus subtilis 168 strain for production. (b) The standard curve of peak area obtained by HPLC (y) and its corresponding fengycins’ concentration (x). (c) HPLC results of sample and various concentrations of fengycins standard.

Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus hemolyticus

The result of biofilm quantitative detection illustrated that the biofilm content of S. haemolyticus was significantly decreased under the treatment of 1.25 g/L and 2.5 g/L lipopeptide extract. It verified that our extract could inhibit the quorum sensing system of S. haemolyticus. Besides, 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. As it is shown in Figure 3, 1.25 g/L of extract (containing 5.625μM fengycins) did not inhibit S. hemolyticus growth and thus 1.25 g/L of lipopeptide extract would be the optimal inhibitory concentration to be applied in our product. Visit our results page for more information: https://2022.igem.wiki/keystone/results

Figure 3. Fengycins’ inhibitory effect on the quorum sensing system of Staphylococcus haemolyticus. (a) Biofilm quantitative detection of Staphylococcus hemolyticus at different concentrations of lipopeptide extract. (b) Measurement of the growth curve of Staphylococcus hemolyticus at different concentrations of lipopeptide extract.

Production of santalene

With the heterologous expression of MVA pathway and ERG20 from Saccharomyces cerevisiae and santalene synthase from Clausena lansium, we have successfully produced santalene in our fermentation broth. Among the four strains we constructed, our study elucidates the mutation of 96th amino acid into tryptophan (strain CEM) could increase the yield of α-santalene by about 20%, substantiating the prominent performance of ERG20F96W in enhancing the supply of FPP and α-santalene production in E. coli.Visit our results page for more information: https://2022.igem.wiki/keystone/results

Figure 4. Quantification analysis of α-santalene production. (a) Measurement santalene production of different strains by GC/MS results. (b) Quantification of α-santalene is analyzed with 0.475 g/L α-humulene as an internal standard. And the GC/MS results demonstrate that the peaks at the retention time of 26-27 min and 28-29 min respectively were α-santalene and α-humulene. (c) GC/MS results of samples originated from CE, CEM, TCEM and CEM-FL strains.