| BIT-China - iGEM 2023

Overview

The purpose of this project aims to synthesize a green, convenient, and large-scale blend of santalol and sclareol through the construction of brewing yeast cell engineering.This project proposed the use of OptoAMP system for the light-controlled synthesis of santalol and sclareol with different ratios. Its advantages are as follows: first, the synthesis of santalol and sclareol in S. cerevisiae has been documented in the literature and the yield is high and the biosynthetic pathway is clear. Secondly, light-controlled synthesis is non-intrusive, transient, and convenient, and can be used to input different pulse values to the same batch of engineered yeast, thereby producing different ratios of spice blends without the need for re-mixing of single products.

The optical control element enhances the interest, sensitivity, controllability and efficiency of the system. The project has both science popularization advantages and economic benefits. In addition, the practice of controlled synthesis of the two spices will also provide theoretical guidance for the synthesis of more kinds of spices.

**Figure 1.** Gene circuit diagram (general diagram)

Gene Pathways

Generate GAL4 regulatory protein

In normal yeast cells, the constitutive promoter P_TEF1 is self initiated, and the downstream el222 gene is expressed to generate the photosensitive transcription factor EL222.

Under 450 nm light illumination, the photosensitive transcription factor EL222 undergoes homologous dimerization, and then binds to the P_c120 promoter, initiating the transcription and expression of the downstream target gene gal4.

**Figure 2.** Regulation protein GAL4 and its synthesis route

Sclareol biosynthesis

The regulatory protein GAL4, controlled by the P_c120 promoter, can activate the transcription of downstream genes of P_gal1-s.

lpps and tps genes downstream of P_gal1-s will be expressed to produce the required enzymes, to convert FPP to geranylgeranyl pyrophosphate (GGPP) by geranylgeranyl pyrophosphate synthase (GGPPS), and then to LPP through LPPS. Finally, pyrophosphate geranyl glycol (LPP) is converted into sclareol by TPS, thereby achieving the goal of synthesizing sclareol.

**Figure 3.** Synthesis route of Sclareol

Santalol biosynthesis

P_TDH3 is a constitutive promoter. Under dark conditions, the promoter P_TDH3 was activated normally, and downstream genes related to sass, cyp76, and cprs were expressed normally. The obtained mRNA was able to translate into enzymes normally.

And was converted into farnesyl pyrophosphate (FPP) through STS enzyme β-Sandalwood alkene（β- Santalene), and then through CYP76 and CPR enzymes β Sandalwood alkene（β- Santalene) converted to β-santalol（β- santalol） enables us to achieve the goal of converting the precursor substance FPP into santalol.

However, under 450nm light conditions, P_gal1-s binds to the regulatory protein GAL4 and initiates transcription. The mRNA transcribed from P_gal1-s hybridizes with P_TDH3, making it impossible to translate and synthesize santalol.

**Figure 4.** Synthetic route of Santalol

Design Highlights

"Not" Gate Design

In this design, the light control system is a “NOT” gate structure, and the GAL4 regulatory protein triggered by 450nm illumination acts as a switch. Therefore, the composition and proportion of the final products could be changed by changing the illumination conditions to achieve controllable synthesis of santalol and sclareol with different ratios.

"In general, "NOT" gate gene circuits are usually composed of repressors and promoters they act on, that is, by connecting the input promoter with the repressor to turn off the output promoter. However, in this circuit design, we did not use chemical substances to curb the promoter, but transcribed the two promoters together to translate the complementary mRNA. Through antisense transcription, the original mRNA could not be translated, to achieve the blocking of gene expression in this direction.

Knockout of spe4 gene

Through bioinformatics and related model calculations, it was found that if adl1 and dfr1 genes were knocked out in S. cerevisiae, the content of FPP could be significantly increased, which greatly improved the precursors of biosynthetic pathways in this project, and then more target products could be obtained.

However, in the subsequent iterative optimization, we found that knocking out the above genes in S. cerevisiae would have a certain impact on the growth of the strain. Therefore, we further explored the model, and finally identified and knocked out spe4 gene after improving the model.

**Figure 6.** Distal bypass gene knockout based on modeling

Chassis Construction

Knockout of GAL80 and GAL4

The transcription activation of inductive promoter P_gal1-s by GAL4 is repressed by the transcription factor GAL80 under non-inducing condition (absence of galactose) in yeast.

In order to abolishes the indirect repression of GAL80 on P_gal1-s and eliminate the dependence of P_gal1-s on galactose, we knocked out the chassis’ endogenous gal80.

Meanwhile, to make sure that gal4 is expressed under blue light-inducing condition in our strain, the chassis endogenous gal4 was knocked out, too.

**Figure 7.** Knockout of *gal80* and *gal4* genes in the yeast chassis

Method Design

Fluorescent protein detection gene circuit

During the experiment, in order to verify whether the light control system can function as expected, we also added genes encoding fluorescent proteins expression gene in the preliminary circuit design.

Through literature research and information retrieval, we found that there are studies on this system in prokaryotic organisms.Therefore, we first designed a light-controlled system in E. coli, which is easy to operate (Step 1);afterwards, we replaced the product synthesis-related genes with fluorescent protein genes to facilitate the detection of whether the circuit is functioning properly (Step 2);subsequently, we integrated the product synthesis-related genes into the yeast chassis with gal80 and gal4 double knockouts, added a green fluorescent protein gene at the end of the sclareol biosynthesis gene circuit, and added a red fluorescent protein gene to the santalol biosynthesis gene circuit to facilitate the detection of whether the genes are expressed smoothly (Step 3);finally, on this basis, we knocked out the fluorescent protein and further obtained the Saccharomyces cerevisiae strain with spe4 gene knockout (Step 4), thus achieving the goal of light-controlled regulation of Saccharomyces cerevisiae to produce different proportions of mixed spices.

**Figure 8.** The entire process of designing a light control system

Reference

[1] Yang W, Zhou Y, Liu W, Shen H, Zhao ZK. [Engineering Saccharomyces cerevisiae for sclareol production]. Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1185-92. Chinese.
[2] Wang Y, Wen M, Li M, Zhao J, Han X. [Progress in biosynthesis of santalene and santalol]. Sheng Wu Gong Cheng Xue Bao. 2018 Jun 25;34(6):862-875. Chinese. doi: 10.13345/j.cjb.170465.
[3] Zhao, Evan M. , et al. "Optogenetic regulation of engineered cellular metabolism for microbial chemical production." Nature (2018).
[4] Zhao, Evan M , et al. "Optogenetic Amplification Circuits for Light-Induced Metabolic Control." ACS Synthetic Biology (2021).
[5] Jayaraman P, Devarajan K, Chua TK, Zhang H, Gunawan E, Poh CL. Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res. 2016 Aug 19;44(14):6994-7005. doi: 10.1093/nar/gkw548.
[6] Zhao EM, Lalwani MA, Chen JM, Orillac P, Toettcher JE, Avalos JL. Optogenetic Amplification Circuits for Light-Induced Metabolic Control. ACS Synth Biol. 2021 May 21;10(5):1143-1154. doi: 10.1021/acssynbio.0c00642.