Contribution

Our contributions to the iGEM community and synthetic biology

Project Contribution: A Modular Toolkit for High-Yield Monoterpene Production in Yeast

Our project not only successfully engineered Saccharomyces cerevisiae to produce 1,8-cineole but also developed and refined a suite of genetic tools, optimized protocols, and strategic insights. We are proud to contribute the following resources to the iGEM community and the broader field of synthetic biology, designed to empower future teams working on terpene engineering and complex pathway optimization in yeast.

1. Novel and Enhanced BioBrick Parts

We have created and characterized several new genetic parts, all of which will be thoroughly documented in the iGEM Registry:

High-Efficiency Cineole Synthase (CinS1 from Salvia fruticosa)

We characterized this plant-derived synthase in yeast, confirming its high specificity for producing 1,8-cineole (~72%) from GPP. This provides a reliable, high-performance part for teams targeting this valuable compound.

Engineered ERG20 Promoter System

We constructed a context-adapted part for replacing the native ERG20 promoter with the glucose-sensitive PHXT1 promoter. This part is a significant improvement over using PHXT1 alone, as it includes homologous arms for precise genomic integration in yeast, providing future teams with a standardized tool to dynamically control GPP/FPP flux.

A Modular Plasmid Backbone for GAL-System Based Expression

Our modified pYES2 vector, pre-equipped with GAL1 and GAL10 promoters in a polycistronic arrangement, simplifies the co-expression of multiple pathway genes (like ScCK and AtIPK) under a unified, glucose-dependent regulatory system.

2. A Strategic Framework and Optimized Protocols

We share a comprehensive "Plug-and-Play" metabolic engineering strategy for enhancing monoterpene yields in yeast. This goes beyond individual parts and provides a holistic blueprint:

A Multi-Pronged Engineering Strategy

We demonstrate that simultaneously addressing precursor supply (via tHMG1, IDI), key flux node regulation (via ERG20 promoter swap), and toxic intermediate management (via inducible ScCK/AtIPK) is crucial for success. This integrated approach can be adapted by other teams engineering pathways with similar challenges (e.g., toxicity, intermediate competition).

Optimized Experimental Protocols

We have refined and documented key wet-lab protocols that are critical for yeast metabolic engineering, including:

  • A highly efficient CRISPR-Cas9 protocol for GAL80 knockout and promoter replacement in BY4741.
  • A standardized fermentation protocol for two-stage production (growth on glucose followed by pathway induction and precursor feeding).
  • Troubleshooting guides for common issues in LC-MS/MS and GC-MS analysis of terpenes.

3. Software and Modeling Tool

To help future teams predict the outcome of their engineering efforts, we developed:

A Simple Kinetic Model of the MVA Pathway

This model, built on our experimental data, allows users to simulate the effect of modulating enzyme levels (e.g., IDI, ERG20) on the IPP/DMAPP and GPP/FPP ratios. This tool is invaluable for in silico testing of design strategies before moving to the lab.

4. Knowledge and Design Principles

Our most valuable contribution may be the "Learn" phase insights we generated:

  • Validation of the "ERG20-weakening" strategy for monoterpene production and its limitations, guiding the next iteration.
  • Data on the performance of heterologous enzymes (CinS1, ScCK, AtIPK) in a unified yeast chassis, saving others the time of initial screening.
  • Clear documentation of "what worked" and "what didn't," including the challenges of balancing growth and production, which provides a realistic roadmap for future iGEMers.

Why is this a Valuable Contribution?

Lowers the Barrier to Entry

Teams new to yeast or terpene engineering can adopt our well-characterized parts and proven strategies, avoiding time-consuming trial-and-error.

Saves Time and Resources

Our optimized protocols and pre-validated designs help other teams accelerate their project cycles.

Promotes Standardization

By contributing standardized, Registry-compliant parts for promoter replacement and modular vectors, we enhance reproducibility and collaboration across the iGEM community.

Inspires Future Projects

Our work on a high-value compound like 1,8-cineole demonstrates the power of synthetic biology and provides a foundational toolkit that can be expanded to produce other monoterpenes or more complex terpenoids.

We believe that by generously sharing these tools, data, and hard-earned knowledge, we are strengthening the collaborative spirit of iGEM and contributing to a more efficient and innovative global synthetic biology community.