| BIT-China - iGEM 2023

Background

In the 10th century, the Kingdom of Champa presented fifteen bottles of rose dew as tribute to the Central Plains, marking the first perfume introduced in China. Perfume has a history of 2000 years in China. Two natural plant spices, santalol and sclareol are the main components of santal and ambergris. Besides, these two chemicals can also be used in medicine, cosmetics, aromatherapy and so on. Thus, our project “Lights Control iT (intermixing Terpenoid)” has designed a yeast factory, which will manufacture santalol and sclareol. Based on various illumination schemes (such as different wavelength of light, different duration of illumination), the factory will achieve the customization of the fragrance base of different proportions.

Why perfume?

Perfume originated in the 4th century BC in Ancient Greece and Ancient Egypt. After a long period of development, it has become one of the important components in the beauty industry, with the lobal perfume market reaching $46.5 billion in 2022, is expected to grow at a compound annual growth rate (CAGR) of 8.97%.

Why cells?

Nowadays, chemical synthesis is the common method for producing santalol and sclareol. However, this process generates significant industrial waste, which can harm the environment. Additionally, blending the fragrance base requires mixing santalol and sclareol in specific proportions, increasing production costs. Therefore, we employ a cell factory as a more environmentally-friendly approach to controllably produce fragrance bases at the production level.

Why photoinduction?

While chemical inducers are currently the primary substances used for induction, some of these chemicals exhibit multiple effects that can impact the expression of other endogenous genes. Furthermore, these inducers cannot be decomposed within cells, limiting gene expression regulation at the temporal level rather than the spatial level. In contrast, photoinduction offers a cost-effective and flexible dynamic regulation switch. It has advantages such as operational reversibility, high spatiotemporal resolution, non-disruption of native cell function, and high production efficiency. Additionally, using light for control simplifies the mixing of different spices at the production level, thereby reducing the procedures and energy consumption associated with separation and purification.

Figure 1. Sketch maps of two induction systems: (A)Chemical induction; (B) Photoinduction

Overview

We have designed a circuit to ensure that our two target products can not only be synthesized independently but can also be blended in a specific ratio under different illumination schemes. After conducting a series of experiments, the results suggest that our cell factory is potentially capable of producing a fragrant base consisting of a specific proportion of santalol and sclareol.

Figure 2.The whole gene circuit we designed.

Building of ideal chassis cells

We employed S. cerevisiae BY4742 as our starting strain. During our preliminary discussions, we decided to begin by knocking out certain genes native to S. cerevisiae to enhance the precursor production. After modeling, we determined that the spe4 and genes should be knocked out. Additionally, for gene expression regulation, we also knocked out the gal80 and gal4 genes. As a result, we obtained an ideal chassis.

Light-Regulation System

In our circuit, the light-regulation element comprises the photosensitive transcription regulator EL222 and the promoter(P_C120) recognized by EL222. EL222 consists of a N-terminal LOV (Light-Oxygen-Voltage) structural domain and a C-terminal helix-turn-helix DNA binding domain, and is constitutively expressed under control of P_TEF1 promoter. When flavin mononucleotides, acting as colorless molecules, absorb light at 450nm, the LOV domain dimerizes and binds to DNA to initiate transcription of downstream genes.

Synthesis of sclareol

When exposed to light at 450nm, gal4 gene downstream of P_C120 is expressed, which turns on the expression of genes downstream of P_Gal1-S ,leading to the production of the necessary enzymes (labdenediol diphosphate synthase(LPPS) and terpene synthase(TPS)) for sclareol synthesis. Under the catalysis of geranylgeranyl pyrophosphate synthase(GGPPS), Farnyl pyrophosphate(FPP) is converted into geranyl geranyl pyrophosphate(GGPP). Subsequently, LPPS catalyzes the conversion of GGPP into labdenediol diphosphate(LPP). Finally, with the assistance of TPS, LPP is converted into sclareol.

Synthesis of santalol

P_TDH3 is a constitutive promoter with strength equivalent to P_Gal1-S. Under 450nm light, P_Gal1-S and P_TDH3 both initiate transcriptions simultaneously. Subsequently, the mRNAs transcribed downstream of P_Gal1-S and P_TDH3 hybridize through complementarity and prevent the translation of mRNA. However, in the absence of light, P_Gal1-S does not transcribe, allowing mRNA transcribed from P_TDH3 to undergo normal translation. Initially, santalene/bergamotene synthase(STS) catalyzes the conversion of FPP to β-Santalene. Then, with the assistance of the proteins expressed by cyp76 and cpr, β-Santalene is further converted to β-Santalenol.

Envisage

The circuit we have designed is not limited to the production of santalol and sclareol. By ensuring that the products can be synthesized, we can achieve the synthesis and mixing of other products simply by replacing the genes used to produce santalol and sclareol with other functional genes. In essence, we provide templates for the production of various other products.

Reference

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