Description

Background

Nowadays，a plenty of people are suffering from various sleep-related problems. According to the World Health Organization (WHO) statistics of 2019, 27% of people have insomnia problem worldwide. Additionally, in the China Sleep Quality Report released on World Sleep Day in 2019, more than 80% of respondents are often plagued by sleep problems. In the 2018 self-assessment survey of sleep quality of Chinese netizen, more than half of them said they had sleep quality problems.

To alleviate this symptom, many people used to buy physical products such as eye patches or medicine pillow. However, an increasing number of people tend to buy aromatherapy products now.

It is proved that fragrance has positive effect on improving people’s sleep quality. Through literature review, we learned that limonene has a refreshing function, while linalool can promote peoples sleep. We decide to use E. coli to produce these two fragrance molecules. Furthermore, we use a light-controlled switch to regulate the expression of two substances, allowing E. coli to express specific aroma molecules at specific times.

Biosynthesis of Two Fragrance Molecules Limonene and Linalool

Most of fragrances come from plants. Limonene and linalool are both monoterpenoids, which are commonly synthesized in lemon and lavender by Mevalonate pathway. According to literatures, carbon sources like glucose is first metabolised to acetyl-coA which is then converted into IPP and DMAPP, the two universal precursor of all terpenes. Then IPP and DMAPP will be further converted to geranyl pyrophosphate (GPP) by a geranyl pyrophosphate synthase (GPPS) which is a common precursor of fragrance molecules. The synthesis of limonene or linalool from GPP can be efficiently achieved by limonene synthase (CS) or linalool synthase (LS).

In conclusion, MVA pathway and GPPS enable the GPP synthesis, and limonene synthase and linalool synthase enable the production of limonene and linalool respectively. Thus we plan to construct a precursor circuit for GPP synthesis, and several production circuits to produce limonene and linalool (See Circuits).

Light-responsive System

In the brainstorming stage, we referred to the project of 2018 team UCAS-China, who used several light-responsive systems to control the synthesis of their target molecules. Light-switchable proteins enable unparalleled control of molecular biological processes in live organisms only by physical conditions alternations. In our design, a red/far-red photoreversible two-component signal transduction system (TCS) with transcriptional outputs was employed in E. coli to construct a biodirectional switch so that limonene and linalool could be produced separately in different phases. Furthermore, PCB is an exocellular co-factor essential for the light control system, which can be synthesized by two enzymes -- ho1 and pcyA. As shown in Figure 1, one previously engineered photoreversible Escherichia coli TCSs, Cph8-OmpR. Cph8 has high kinase activity toward the E. coli RR OmpR in the ground state (Pr) and low kinase (high phosphatase) activity in a far-red absorbing activated state (Pfr) OmpR~P binds and activates transcription from the PompF146 promoter.

In our design, this TCS system and PCB generating system are coupled into the light-controlling circuit to yield switchable control of gene expressions. (See circuits)

Inspiration

Can plant fragrances improve sleep quality?

Researches by the University of Southampton in UK has found that lavender essential oils or jasmine aromas can improve sleep quality by 20%. A study published in the Journal of Biochemistry showed that sleep with jasmine flower around can alleviate anxiety and insomnia.^[3]

More relevant studies indicate that the main component in the lavender or jasmine, linalool and limonene respectively, which exhibit the pleasant aroma, have excellent effects on solving sleep-related problems. Linalool is reported to promote sound sleep with peaceful mind, and limonene allows people to feel less sleepy and distraction. Therefore, we chose limonene and linalool as two fragrance molecule, to help solve the sleep-related problems in our project.

What are we doing?

Inspired by the teams Paris Bettencourt and Great-Bay China, we decided to biosynthesize linalool and limonene in engineered E. coli using GPP (geranyl pyrophosphate) as a precursor, as an alternative for high-polluted and less attractive chemical synthesis methods. Based on our design, linalool and limonene need to be produced at different time, which means linalool will be synthesized at night for a nice sleep while limonene will start to be produced in the morning to remove sleepy mind. The 2018-UCAS project which combined science and art using the smart light-control devices, is super attractive for us and give us an clear idea to design our gene circuits. We decided to use the thermosensitive protein and light-controlled switching elements in their project to enable the production of two fragrances separately, that is, red light will enable the expression of limonene synthase and thermosensitive CI protein, and inhibit the expression of linalool synthase at the same time by the regulation of λ promoter. By contrast, the linalool synthase will be expressed without red light. By designing this light-switched gene circuit, selective production of linalool and limonene can be effectively achieved.

All in all, our project focuses on the selectively production of two fragrance molecules to solve the increasingly growing sleep-related problems. Two basic plasmids, precursor-produce plasmid and light-controlled fragrances-produce plasmid are constructed, co-transformed into engineered E. coli, and works together to produce pleasant fragrances alternatively, for a sound sleep and exciting morning!

References

CDC's Division of Population Health, CDC - Data and Statistics, May 2, 2017, "Sleep and Sleep Disorders", https://www.cdc.gov/sleep/data_statistics.html
Xinhua Net, Xinhua Public, March 20, 2019, "On World Sleep Day 'China Sleep Quality Survey Report' Released", http://www.xinhuanet.com/gongyi/2019-03/20/c_137909697.htm
Paul Jordan, Sleep Habits, The Most Effective Herbal Sleep Aids", https://sleephabits.net/herbal-sleep-aids

Team:XJTU-CHINA/Description