Project Description

In contrast, our production of 1,8-cineole using synthetic biology methods theoretically enables high purity, high efficiency, stable, and controllable production. This is precisely the key to addressing the pain points of traditional processes and enhancing product quality.

1. Address resource and sustainability challenges

Currently, 1,8-cineole is mainly extracted from plants such as eucalyptus. This leads to several issues: competition for land and water resources (competing with food crops), long growth cycles (eucalyptus requires several years before harvest), and low extraction yield with unstable composition (affected by weather and geographical origin).

2. Change the drawbacks of traditional chemical synthesis

The chemical synthesis of 1,8-cineole may involve complicated steps, the use of toxic catalysts, and the generation of harmful by-products, which does not align with the modern trend of "green cosmetics."

1. Gentle yet effective anti-acne solution

While current mainstream anti-acne ingredients like salicylic acid, benzoyl peroxide, and retinoic acid are effective, they commonly cause significant irritation, leading to skin dryness, peeling, and redness, deterring many users with sensitive skin. Literature has proven that 1,8-cineole possesses confirmed antibacterial, anti-inflammatory, and antioxidant activities.

2. Addressing antibiotic resistance

Due to the misuse of antibiotics, the issue of C. acnes drug resistance is increasingly severe, leading to reduced efficacy of many traditional anti-acne ointments, while 1,8-cineole, as a natural antibacterial agent, has a mechanism of action different from conventional antibiotics and is less likely to induce bacterial resistance. This provides a highly promising alternative solution for addressing antibiotic-resistant acne.

3. Sustainability-conscious consumers

Contemporary consumers, especially Generation Z, are increasingly concerned about whether product ingredients are sourced in an environmentally friendly, humane, and sustainable manner. The synthetic biology approach allows for the production of large quantities of 1,8-cineole without felling a single tree.

1. IUP Pathway to Increase Substrate (IPP/DMAPP) Production

Targeting Substrate Concentration:

In S. cerevisiae, the galactose regulatory network is one of the classic transcription induction systems, activated by galactose and repressed by glucose. This regulation is achieved through two signaling pathways: Gal4p-Gal80p, responsible for galactose utilization, and Snf1, involved in glucose repression. In the galactose regulation system, in the absence of galactose, Gal80p binds to the transcription activator Gal4p, repressing transcription from the Gal1 and Gal10 promoters. Conversely, when galactose is present, the transcription factor Gal3p binds to Gal80p, allowing Gal4p to function normally and activate transcription from the Gal1 and Gal10 promoters. Therefore, disrupting Gal80p makes the Gal promoter independent of galactose, leaving the Snf1 network as the sole controller, thus constructing a glucose-dependent regulatory system.

Consequently, under the control of the Gal promoter, after glucose depletion, the target product synthesis pathway in the Gal80p knockout strain is activated. By knocking out Gal80p and using the Gal1 and Gal10 promoters to control the target pathway, when glucose is depleted, the expression of ScCK and AtIPK is activated. Adding isoprenol or prenol to the medium at this point can avoid substrate toxicity to cell growth. In the Gal80p knockout strain, we overexpress S. cerevisiae-derived ScCK and Arabidopsis thaliana-derived AtIPK, and detect intracellular IP/DMAP and IPP/DMAPP levels via LC-MS/MS.

Based on growth inhibition assays, the optimal supplementation concentration for isoprenol or prenol was determined to be 30 mM.

2. Downregulating ERG20 Activity by Promoter Replacement

Targeting FPP and GPP Relative Yields:

Strategies for downregulating ERG20 activity include replacing its native promoter with an inducible weak promoter and employing N-degron-dependent protein degradation. The strength of the glucose-sensitive promoter P_HXT1 is regulated by the glucose concentration in the medium and is positively correlated with glucose concentration. By using the P_HXT1 promoter to replace the native promoter of the ERG20 gene, the activity of ERG20 can be effectively downregulated by reducing the glucose concentration in the medium.

3. Mutating the MVA Pathway Rate-Limiting Enzyme HMGR

Targeting Substrate Concentration:

S. cerevisiae has two HMGR isozymes: HMG1 and HMG2. Overexpression of HMG1 with the N-terminal membrane binding domain truncated (tHMG1) in S. cerevisiae helps increase terpenoid yield; the mutant HMG2 (K6R) can slow down the degradation of the synthase.

4. Overexpressing IDI

Targeting FPP and GPP Relative Yields:

One molecule of IPP and one molecule of DMAPP synthesize one molecule of GPP (the monoterpene precursor). Sesquiterpene synthesis requires the further addition of IPP to GPP. Therefore, reducing the IPP/DMAPP ratio can increase monoterpene yield. IDI is the enzyme that isomerizes IPP to generate DMAPP. Overexpression of IDI can balance the production of IPP and DMAPP.