Our inspiration: the corrosion of silk

Silk is a traditional textile in China. The invention and large-scale production of silk products by the working people in ancient China opened the first large-scale trade exchange between the East and the West in world history. The route of commodity circulation was called the "Silk Road", and China was also called "Silk Country". Silk is favored by people for its rich colors and comfortable wearing experience. But silk is liable to mildew and rot if it is not carefully cared for. What's more, in modern times, chemicals are inevitably used in the dyeing process of silk, causing a lot of pollution to water and air.

Our solution: FUNCDYES!

Hoping to solve these problems, SCUT-China team came up with the great idea to produce functional dyes for silk, which are environmentally friendly and could slow down the rot of silk. FUNCDYES is mainly composed of two functional terpenoid substances, lycopene and patchoulol.

i. Lycopene

Lycopene is a red pigment belonging to the group of carotenoids, especially found in fruit and vegetables. It is recognized as a functional food additive integrating coloration and nutrition.

Lycopene exerts beneficial effects on human health by protecting lipids, proteins, and DNA from damage by oxidation^[1]. Medical evidence that dietary intake can reduce the incidence of cardiovascular disease and some cancers^[2].

ii. Patchoulol

Patchoulol is a volatile sesquiterpene with aromatic odor, which is an important natural flavor in fragrance industry. Patchoulol has been proven to have numerous health benefits, including immunomodulatory, anti-inflammatory, antitumor, antimicrobial, insecticidal, and sedative activity^[3].

One of the main ingredients of patchouli oil is patchoulol, which can relieve feelings of anger and anxiety and help treat depression. It can also be used as a sedative to relax the body and mind and to treat insomnia. At the same time, patchoulol also has outstanding bacteriostatic and insect repellent effects, which can inhibit wound infection and drive away insects.

iii. Current production methods of terpenes

Lycopene and patchoulol are natural terpenoids abundant in plants. At present, the production of these two substances mainly depends on plant extraction and chemical synthesis. However, terpenoids content in plants is usually very low, the extraction method is easy to cause damage to wild plant resources, and the yield and quality have uncontrollable changes, so the economic feasibility is poor. What's more, the chemical synthesis methods also have low efficiency and high cost. By contrast, microbial synthesis method is not restricted by raw material and the production process is green and clean, which has great advantages.

iv. How to produce our FUNCDYES?

1. Synthesis pathway

Pichia pastoris is widely used as a chassis for biosynthesis factories due to its powerful methanol-induced promoter and safe and operable background. Based on the mevalonate pathway (MVA) derived from P.pastoris, exogenous lycopene synthase genes ( crtI, crtB, crtE ) and patchoulol synthase gene ( PTS ) were transferred to produce lycopene and patchoulol simultaneously in P.pastoris. (Fig.1)

Fig.1 Synthesis pathway of lycopene and patchoulol in Pichia pastoris

2. Promoter selection

As the initiation of gene transcription, promoters are key components of cell factory design and metabolic pathway modification. Due to the limited number of well-characterized promoters in P.pastoris and their small dynamic ranges, it is often difficult to satisfy the fine regulation of genes, optimize metabolic flux, and improve the yield of the target product^[4]. Therefore, SCUT-China hopes to establish a promoter library by screening existing promoters and mutating efficient promoters, which will provide useful and efficient help for the improvement of metabolic flux optimization target product yield and the design of cell factory.

3. PTS mutation

PTS is the key enzyme for the production of patchoulol in cell factory. The specificity and activity of the enzyme are very important for the production of patchoulol. Therefore, SCUT-China mutated PTS gene by Mn²⁺ ion induction, hoping to obtain higher activity of PTS enzyme, so as to improve the yield of patchoulol.

4. Adapting optogenetics systems to adjust the ratio of dye molecules

We hope that our dyes have both excellent antibacterial and repellent effects and bright colors. Therefore, it is important to adjust the proportion of lycopene and patchoulol. Since our two target terpenoids have the same precursor: FPP, during the production process, when the flow of FPP to lycopene increases, the corresponding output of patchoulol decreases. We came up with the idea of dynamically regulating the production ratio of the two substances by adding a light-controlled switch along the route to patchoulol. Optogenetics is based on the use of proteins that change their conformation in the presence of light to become active. It is a physical parameter that can easily be modified, just by switching on and off lights. Compared to chemically regulated systems, such regulations do not require additional molecules. Through the application of blue light regulation system, we can realize the dynamic adjustment of color depth and functional strength of FUNCDYES!