In order to synthesis salidroside in vivo. We establishes a complete biosynthetic pathway from glucose in Escherichia coli. The pathway first directs carbon flux from glucose through the native shikimate pathway to synthesize the key intermediate 4-hydroxyphenylpyruvate (4-HPP). We will then introduce a heterologous three-enzyme cascade to convert this precursor into salidroside. Specifically, a decarboxylase (KivD) will convert 4-HPP into 4-hydroxyphenylacetaldehyde (4-HPAAL), which is then reduced to tyrosol by a reductase (PAR). Then, an introduced UDP-glycosyltransferase (UGT) will catalyze the glycosylation of tyrosol with UDP-glucose to yield the final product, salidroside.

Rapid and accurate product detection is critical for the Design-Build-Test-Learn (DBTL) cycle, yet high school teams often lack access to expensive analytical equipment. To overcome this barrier and establish a universal strategy for other high school iGEM teams, we developed transcription factor based colorimetric assay. This method enables both the qualitative identification of high-yielding strains through visual color change and the quantitative measurement of production levels using a common spectrophotometer, facilitating a low-cost and efficient workflow.
Therefore，our project is composed of three primary parts:

1. Salidroside Detection Module: A genetic circuit designed for the rapid detection of salidroside was constructed and tested.

2. Salidroside Producing Module: An efficient salidroside synthesis pathway was established by mining and screening key genes from different organisms.

3. Salidroside Producing Enhancing Module: The supply of precursors, tyrosol and UDP-glucose, was enhanced to increase the product yield.

Our salidroside detection module is engineered around a biosensor circuit designed for rapid and low-cost analysis. The design is inspired by the work of Yang et al. (2024), who successfully modified the transcriptional repressor protein HucR from Deinococcus radiodurans to create a specific response to salidroside. The system comprises three functional modules: a regulatory operator module, a sensing module, and a reporter module, collectively enabling sensitive and visual detection of salidroside.
The regulatory mechanism of our circuit is as follows:

1. "Gene Off" State (Absence of Salidroside): The HucR^SD gene is constitutively expressed, continuously producing the HucR^SD repressor protein. This protein binds specifically to its operator site located within the P23119 promoter, blocking RNA polymerase. This repression prevents the transcription of the downstream reporter genes, GFP and LacZ. Consequently, in the absence of salidroside, the cells exhibit low fluorescence and no color change.

2. "Gene On" State (Presence of Salidroside): When salidroside is produced by the cell or added to the medium, it functions as an inducer. As shown in the Figure 2, salidroside molecules bind directly to the HucR repressor protein, forming a HucR-Salidroside complex. This binding event causes a conformational change in the HucR protein, which loses its affinity for the DNA operator site and detaches from the promoter.

With the repressor removed, the promoter is activated, and the reporter genes GFP and LacZ are transcribed and translated. This results in two measurable outputs: green fluorescence from GFP, which can be quantified with a spectrophotometer, and the production of the LacZ enzyme. LacZ converts the colorless substrate ONPG into the yellow product onitrophenol, providing a simple, visual color change for qualitative screening. This dual-reporter system creates a direct correlation between the intracellular salidroside concentration and the intensity of the fluorescent and colorimetric signals.

Through a comprehensive literature review, we established that E. coli lacks the inherent metabolic competence to biosynthesize salidroside. we engineered a de novo pathway starting with its precursor, hydroxyphenylpyruvate (4-HPP) . Then 4-HPP was converted to tyrosol by introducing two heterologous enzymes: a decarboxylase (KivD) and a reductase (PAR). The final critical step is the glycosylation of tyrosol, which is catalyzed by a UDP-dependent glycosyltransferase (UGT) which dictates the overall yield and purity. To ensure high efficiency, we selected and screened multiple UGT candidates to identify the optimal variant for converting tyrosol into salidroside.


To identify the most effective UGT for salidroside synthesis, we screened several candidates based on phylogenetic analysis and previous research. Our final selections included UGT85A1 from Arabidopsis thaliana for its known high activity, RrUGT33 and UGT72B14 from Rhodiola species, and a site-directed mutant, UGT85A1(A21G), reported to have enhanced efficiency. Each candidate gene was cloned into an expression plasmid under the control of a strong promoter and expressed in E. coli.

For the third cycle of our project, we focused on enhancing the supply of salidroside’s two essential precursors: UDP-glucose (UDPG) and tyrosol. To increase the intracellular concentration of UDPG, we overexpressed the genes pgm and galU, which encode the key rate-limiting enzymes phosphoglucomutase and UTP–glucose-1-phosphate uridylyltransferase. This modification ensures a plentiful supply of the activated glucose donor needed for the final synthesis step.
To improve the tyrosol production, we targeted the shikimate pathway's natural feedback inhibition mechanisms. The native enzymes AroG and TyrA are typically suppressed by the accumulation of aromatic amino acids. We replaced their genes with feedback-resistant (fbr) variants, AroG^fbr and TyrA^fbr, which are insensitive to product levels. This engineering step ensures a sustained and uninterrupted carbon flux towards tyrosol.


By combining these two strategies, we have created a comprehensively optimized metabolic chassis. The engineered strain benefits from both an increased supply of UDPG and a stable, high-flux pathway for tyrosol production. This dual-pronged approach systematically removes key bottlenecks, establishing a robust platform for high-yield salidroside biosynthesis.

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