Our project successfully addresses the unsustainable and inefficient extraction of salidroside from its natural plant source by establishing a sustainable, scalable, and cost-effective production platform in engineered Escherichia coli. We developed a novel, low-cost whole-cell biosensor that enables rapid, high-throughput screening, thereby accelerating the optimization of the biosynthetic pathway without reliance on expensive analytical equipment.

Now, overwork has become a global health epidemic, contributing to a surge in chronic conditions, most notably cardiovascular disease. Nature, however, offers a powerful countermeasure in the form of salidroside, a remarkable compound found in the Rhodiola plant. Hailed as the "Ginseng of the Highlands," salidroside is a potent cardioprotective agent with a rich history in traditional medicine, offering a natural way to combat the physiological damage of stress and fatigue.
Yet, this therapeutic promise is locked away by the limitations of its natural source. The Rhodiola plant is a slow-growing, need high-altitude environments, and its tissues contain only trace amounts of salidroside. This reality makes natural extraction unsustainable, environmentally damaging.
Our project confronts this challenge head-on. Our purpose is to harness the power of synthetic biology to create a sustainable, scalable, and cost-effective production platform for salidroside.We aim to transform this rare botanical treasure into an accessible modern therapeutic. To achieve this, our project is built upon two core objectives:

1. To Engineer an Efficient Biosynthetic Pathway: We will "teach"  E. coli to produce salidroside from a simple, inexpensive sugar—glucose. This involves designing, building, and optimizing a novel metabolic pathway by introducing key enzymes from a variety of organisms.

2. To Develop a Low-Cost Detection Tool: High-yield production requires rapid testing and iteration. To break this barrier, we will engineer a whole-cell biosensor that allows us to "see" salidroside production through a simple color change. This tool is not just for our project; it is a contribution designed to empower future iGEM teams, especially those with limited resources, to accelerate their own research.

By integrating a robust production chassis with an accessible detection system, our project aims to deliver more than just a novel strain. We are creating a complete, open-source toolkit that democratizes the production of salidroside, unlocking its therapeutic potential and providing a powerful example of how synthetic biology can solve real-world health challenges.
Our primary contribution is the development and characterization of a whole-cell biosensor for the rapid detection of salidroside. This tool directly addresses a common challenge for iGEM teams: the lack of access to expensive equipment like HPLC for high-throughput screening.

· What we provide: A new composite part HucR^SD-P119-HucO-GFP-LacZ based on the HucR^SD transcriptional repressor. It features a dual-reporter system (GFP and LacZ), enabling both quantitative analysis via fluorescence and simple, qualitative screening via a colorimetric (yellow) assay.

· Why it's a contribution: We have rigorously documented its performance, demonstrating a linear response to increasing salidroside concentrations and, most importantly, validating its accuracy by comparing its output directly against HPLC measurements. This provides future iGEMers with a reliable, well-characterized, and inexpensive tool to accelerate their DBTL cycles for any project involving salidroside production.

Building a functional pathway requires choosing the right enzymes. We have contributed valuable characterization data that will help future teams make informed decisions, saving them significant time and resources.

· What we provide: Quantitative, comparative data on the in vivo catalytic efficiency of four different UDP-glycosyltransferases (UGTs) for the final step of salidroside synthesis.

· Why it's a contribution: Our results clearly identify UGT72B14 from Rhodiola sachalinensis as the most effective enzyme, showing a 43% higher productivity than the commonly used UGT85A1. This new documentation on the performance of these parts in an E. coli chassis provides a clear starting point for any team looking to build a high-yield salidroside pathway.

Achieving high yields in metabolic engineering often requires overcoming precursor limitations. Fundamentally, biosynthesis is a chemical process that demands an adequate supply of both precursors and cofactors. We have successfully implemented and documented a robust strategy for this common problem.

· What we provide: A validated method for significantly boosting salidroside production by simultaneously enhancing the supply of its two key precursors. We detail the use of feedback-resistant variants (AroG^fbr, TyrA^fbr) to improve the tyrosol pathway and the overexpression of pgm and galU to increase the UDP-glucose pool.

· Why it's a contribution: AroG and TyrA overexpression may be helpful for many kinds of aromtic compound biosynthesis. This serves as a well-documented case study and a practical guide for fellow iGEMers, illustrating an effective troubleshooting and optimization workflow that can be adapted for the production of other glycosylated natural products. This principle is broadly applicable, since a robust strategy for cofactor regeneration is a fundamental requirement for synthesizing any glycoside.

One of the most significant contributions of our project is the establishment of a versatile and generalizable framework for biosensors design. Recognizing that high-throughput screening is a universal bottleneck in metabolic engineering, we designed our detection module not as a one-off solution, but as a standardized architectural model that can be easily adapted by future iGEM teams to detect a wide variety of natural products.
Our framework is built on three distinct and interchangeable modules, utilizing synthetic promoters to ensure predictable and tunable expression:

1. The Regulator Module: The sensory-regulator protein (in our case, HucR) is expressed under the control of a constitutive synthetic promoter. This ensures a consistent and stable intracellular concentration of the protein that will sense the target molecule, forming the core of the biosensor.

2. The Output Module: The reporter genes (e.g., GFP and LacZ) are placed downstream of a different strong synthetic promoter. Crucially, the operator sequence that the regulator protein binds to (e.g., HucO) is positioned within this promoter region. This physically separates the regulator's expression from its site of action, creating a clean and modular regulatory switch.

3. The Reporter Module: Our design incorporates dual reporters for both quantitative (fluorescence) and qualitative (colorimetric) readouts, but this module is inherently "plug-and-play." Future teams can easily swap in other reporters like luciferase or different fluorescent proteins to suit their experimental needs.

Why this is a key contribution for iGEM:

· Versatility and Adaptability: This framework is not limited to salidroside. By simply swapping the Regulator Module (e.g., replacing HucR with a different transcriptional repressor that responds to another molecule, like a flavonoid or alkaloid) and its corresponding operator sequence in the Output Module, teams can rapidly re-purpose this entire architecture to detect their own specific target compound.

· Tunable Performance: The use of synthetic promoters from a standardized library (like the Anderson promoter collection) allows for fine-tuning. Teams can adjust the expression levels of both the regulator and the reporter genes to modify the sensor's sensitivity, dynamic range, and response time, enabling true genetic engineering of the sensor's properties.

· A Clear Design Blueprint: We provide a well-documented and validated "recipe" for biosensor construction. This lowers the barrier to entry, allowing future teams—even those with limited prior experience in sensor design—to confidently build their own custom, high-throughput screening tools.

Our project delivers a comprehensive toolkit for salidroside production and detection. We have engineered a high-yield biosynthetic pathway in E. coli and, to enable rapid optimization, we developed a validated biosensor that correlates directly with HPLC results. More importantly, this sensor is built on a modular design framework that we are contributing to iGEM, providing a versatile blueprint for future teams to create custom biosensors for a wide range of natural products.

1. Yang J, Xia Y, Shen W, Yang H, Chen X. Development of a gene-coded biosensor to establish a high-throughput screening platform for salidroside production. Biochemical and Biophysical Research Communications. 2024;712-713:149942.

2. Lai D, Chen Y, Wang L, Sun H, Chen F, Zhang G. Glycosyltransferases: Mining, engineering and applications in biosynthesis of glycosylated plant natural products. Biotechnology Advances. 2021;52:107817.

3. Li H, Zhang W, Wang J, Sun T. Design and optimization of genetically encoded biosensors for high-throughput screening of chemicals. Biotechnology Advances. 2021;51:107810.