We designed our Super Safety Strain of S. cerevisiae yeast with industrial scale-up in mind. For engineered strains whose purpose is to synthesize small molecules, such as biofuels and drugs, adequate safeguards are necessary to prevent accidental release and potential production of contaminating small molecules in ecosystems. With that in mind, we had several criteria in mind for our strain:

1. The strain should be incapable of dividing outside of the production facility
2. During production, the strain should be capable of good growth to ensure economic yields of small molecules
3. The strain should be incapable of reverting to a wildtype phenotype capable of growth outside of the production facility
4. The phenotype of the Super Safety Strain should be readily identifiable when the safety system is active

To these ends, we have set out to address these challenges by designing our genetic platform around strains that can switch off expression of telomerase, an enzyme that is critical for maintaining the ends of chromosomes (telomeres), along with a battery of additional mutations to shorten or lengthen the lifespan of the cells.






There are several subunits of yeast telomerase that, when deleted, result in a non-functional telomerase. We chose to utilize the well characterized gene EST2 (Ever Shortening Telomeres 2) which codes for the catalytic subunit of yeast telomerase. The EST2 gene's main function is as a telomerase reverse transcriptase, and EST2 plays an essential role in telomerase activity. The EST2 gene causes telomere extension, and in addition, EST stands for Even Shorter Telomeres. A mutation, such as a deletion, in the EST2 gene will cause the telomeres to shorten and eventually die off.



However, yeast cells also possess a backup system called ALT (Alternative Lengthening of Telomeres) based on telomere extension via homologous recombination. Several genes are required for this pathway to function, There have been many strain backgrounds generated containing EST2 and RAD52 knockouts, with no strains exhibiting "rescue" from senescence.

The RAD52 gene is a DNA recombinase that allows for cells with shortened telomeres to use homologous recombination to re-extend their telomeres. It is involved in repairing DNA double strand breaks by working with RAD51. In cells with a single EST2 deletion, the RAD52 gene codes for an alternate pathway of recovery. In cells with a double deletion for both EST2 and RAD52, they will reach senescence and not recover.

There are certain genes within S. cerevisiae yeast that have been tested with genome-wide screens for deletion mutants that affect telomere length. Two such genes are MAK31 and VPS75. MAK31, generally categorized as a gene involved in protein modification, specifically with N-acetyltransferase, was found by Askree et al. to lengthen the yeast telomeres by 50-150 base pairs. VPS75, a gene that normally deals with vesicular traffic and vacuolar sorting proteins, was found in the same study to shorten yeast telomeres by 50-150 base pairs.

In cells with either a double or triple deletion of MAK31 or VPS75 in conjunction with EST2 and/or RAD52, growth curves should show shifts in when senescence occurs.

-SIR1 Gene Knockout: For Generating Non-Mating Sterile Strains
-Galactose Activated Cre Recombinase Switch: for Controllable EST2 deletion and ADE2 Reporter Gene Activation

References

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