Engineering

Our engineering approach and design principles

Engineering Yeast for 1,8-Cineole Biosynthesis: An Engineering Cycle Practice

This project aims to construct an engineered strain of Saccharomyces cerevisiae capable of efficiently synthesizing 1,8-cineole (eucalyptol), a monoterpenoid widely used in pharmaceuticals, fragrances, and insecticides. We selected BY4741 as the host strain due to its well-defined auxotrophic background (His, Leu, Met, Ura), which facilitates multiple rounds of screening and plasmid co-transformation.

Design → Build → Test → Learn

1. Design

Key Design Strategies Included:

A. Introduction of a Cineole Synthase

The high-efficiency cineole synthase CinS1 (UniProt: A6XH05) from Salvia fruticosa was selected. It utilizes GPP as a substrate and achieves a cineole yield of up to 72.4%.

B. Enhancement of Precursor Supply

  • Overexpression of truncated HMG1 (tHMG1) to strengthen the MVA pathway.
  • Overexpression of IDI to balance the IPP/DMAPP ratio.
  • Replacement of the native ERG20 promoter with the glucose-sensitive PHXT1 promoter to regulate the GPP/FPP ratio.
  • Introduction of ScCK and AtIPK to enhance isoprenol utilization.

C. Construction of a Regulatory System

Utilization of GAL1 and GAL10 promoters, activated upon glucose depletion, to express heterologous pathways and avoid substrate toxicity. We chose the pYES2 plasmid as the expression vector, featuring the URA3 selection marker and AmpR resistance, suitable for manipulation in both yeast and E. coli.

2. Build

A. Plasmid Construction

  • pYES2 was double-digested with BamHI and EcoRI, and the synthesized CinS1 gene fragment was inserted.
  • Expression cassettes containing GAL1-ScCK and GAL10-AtIPK were constructed.
  • The CRISPR-Cas9 system was used to knock out GAL80 and replace the native ERG20 promoter with PHXT1.
  • All constructs were verified by colony PCR and Western blot.

B. Yeast Transformation

  • Competent cells were prepared using the LiAc method.
  • Electroporation was performed, followed by plating on SD/-Ura plates containing G418.
  • Positive clones were selected via dual screening (auxotrophy and antibiotic resistance).
Engineering Workflow

3. Test

A. Phenotypic Validation

  • Positive transformants showing robust growth were selected on SD/-Ura plates.
  • Colony PCR confirmed the integration of target genes.
  • Western blot verified the expression of the cineole synthase and other key enzymes.

B. Metabolite Analysis

  • LC-MS/MS was used to detect IPP/DMAPP and GPP levels.
  • GC-MS was used to analyze 1,8-cineole production.
  • OD600 and substrate consumption were monitored during fermentation.

C. Preliminary Results

  • The engineered strain successfully activated the GAL promoters after glucose depletion.
  • 1,8-Cineole production was significantly higher than in the control strain.
  • Replacement of the ERG20 promoter led to increased GPP accumulation, favoring monoterpene synthesis.

4. Learn and Next Iteration Design

A. Key Findings

  • The cineole synthase CinS1 was expressed well in yeast, but production might be limited by endogenous GPP supply.
  • The ERG20 promoter replacement was effective but incomplete; further attenuation of its activity might be necessary.
  • The initial design did not fully account for the impact of byproduct accumulation on cell growth.

B. Design Improvements for the Next Cycle

  • Construct an erg20 mutant plasmid: To further reduce ERG20 activity and enhance GPP availability.
  • Introduce a Dynamic Regulation System: Implement a two-stage fermentation strategy (glucose/galactose) for more precise pathway control.
  • Optimize Promoter Combinations: Experiment with stronger or more sensitive promoters to drive IDI and tHMG1.
  • Build a Multi-Gene Co-expression Vector: Modularize the cineole synthesis pathway to facilitate future debugging and standardization.

Conclusion

We successfully completed a full engineering cycle: starting from rational design, we constructed an engineered yeast strain capable of synthesizing 1,8-cineole and experimentally verified its feasibility. The data obtained provides clear directions for the next round of engineering optimization.

All constructed plasmids and strains have been documented in the iGEM Registry for reference and use by future teams.