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Updated: Jul 10, 2026
Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
Chaperone overexpression boosts heterologous small molecule production in Saccharomyces cerevisiae
Andreas M Vestergaard1, Wasti Nurani1, Paul Cachera2
1Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens lyngby, Denmark.
Background:
Chaperones play an important role in maintaining cellular proteostasis by mediating protein folding. As a result, chaperone overexpression has been widely used as a tool for enhancing folding and improving production of heterologous proteins in host organisms such as Saccharomyces cerevisiae. In contrast, this strategy has been much less explored for small molecule (SM) production. This is surprising, as SM pathways typically depend on multiple enzymes including large multi-domain synthases or synthetases, which may all benefit from folding assistance to enhance the catalytic power of the pathway.
Results:
We have established an S. cerevisiae strain library of 68 strains overexpressing endogenous cytosolic chaperones and a mating-based method that allows the chaperone library to be combined with a query strain that contains the pathway of a desirable SM. Using the small molecule aspulvinone E from Aspergillus terreus as a model compound, we screened the chaperone library for chaperones that improve production of aspulvinone E. Screening of the library identified several chaperones and chaperone combinations that improved aspulvinone E production. Specifically, the combined overexpression of YDJ1 and SSA1 was identified as the best hit in our screen. Subsequently, we demonstrated that overexpression of YDJ1 and SSA1 improved aspulvinone E production by 84% in 1.5 mL scale batch fermentations. The observed increase is likely due to higher levels of the MelA synthetase responsible for aspulvinone E synthesis, as overexpression of YDJ1 and SSA1 increases the amounts of fluorescent MelA-mRFP in cells producing this fusion protein.
Conclusion:
The endogenous cytosolic chaperone overexpression library and mating based screening method presented in this report constitute a tool allowing for fast and efficient identification of specific chaperones and chaperone combinations that benefit production of a given SM in S. cerevisiae-based cell factories.
