Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron
Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AI...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2023-05-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1188119/full |
| _version_ | 1797815534934294528 |
|---|---|
| author | Zhenlin Han Jessica Maruwan Yinjie Tang Wei Wen Su |
| author_facet | Zhenlin Han Jessica Maruwan Yinjie Tang Wei Wen Su |
| author_sort | Zhenlin Han |
| collection | DOAJ |
| description | Conditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag. |
| first_indexed | 2024-03-13T08:24:16Z |
| format | Article |
| id | doaj.art-d97ce06ae38f4b779b5143db6ed2d6d9 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-13T08:24:16Z |
| publishDate | 2023-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-d97ce06ae38f4b779b5143db6ed2d6d92023-05-31T04:53:37ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-05-011110.3389/fbioe.2023.11881191188119Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degronZhenlin Han0Jessica Maruwan1Yinjie Tang2Wei Wen Su3Department of Molecular Biosciences and Bioengineering, University of Hawai’i at Manoa, Honolulu, HI, United StatesDepartment of Molecular Biosciences and Bioengineering, University of Hawai’i at Manoa, Honolulu, HI, United StatesDepartment of Energy, Environmental and Chemical Engineering, Washington University, Saint Louis, MO, United StatesDepartment of Molecular Biosciences and Bioengineering, University of Hawai’i at Manoa, Honolulu, HI, United StatesConditional protein degradation is a powerful tool for controlled protein knockdown. The auxin-inducible degron (AID) technology uses a plant auxin to induce depletion of degron-tagged proteins, and it has been shown to be functional in several non-plant eukaryotes. In this study, we demonstrated AID-based protein knockdown in an industrially important oleaginous yeast Yarrowia lipolytica. Using the mini-IAA7 (mIAA7) degron derived from Arabidopsis IAA7, coupled with an Oryza sativa TIR1 (OsTIR1) plant auxin receptor F-box protein (expressed from the copper-inducible MT2 promoter), C-terminal degron-tagged superfolder GFP could be degraded in Yarrowia lipolytica upon addition of copper and the synthetic auxin 1-Naphthaleneacetic acid (NAA). However, leaky degradation of the degron-tagged GFP in the absence of NAA was also noted. This NAA-independent degradation was largely eliminated by replacing the wild-type OsTIR1 and NAA with the OsTIR1F74A variant and the auxin derivative 5-Ad-IAA, respectively. Degradation of the degron-tagged GFP was rapid and efficient. However, Western blot analysis revealed cellular proteolytic cleavage within the mIAA7 degron sequence, leading to the production of a GFP sub-population lacking an intact degron. The utility of the mIAA7/OsTIR1F74A system was further explored in controlled degradation of a metabolic enzyme, β-carotene ketolase, which converts β-carotene to canthaxanthin via echinenone. This enzyme was tagged with the mIAA7 degron and expressed in a β-carotene producing Y. lipolytica strain that also expressed OsTIR1F74A controlled by the MT2 promoter. By adding copper and 5-Ad-IAA at the time of culture inoculation, canthaxanthin production was found to be reduced by about 50% on day five compared to the control culture without adding 5-Ad-IAA. This is the first report that demonstrates the efficacy of the AID system in Y. lipolytica. Further improvement of AID-based protein knockdown in Y. lipolytica may be achieved by preventing proteolytic removal of the mIAA7 degron tag.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1188119/fullconditional protein degradationdegronmetabolic engineeringsynthetic biologyYarrowia lipolytica |
| spellingShingle | Zhenlin Han Jessica Maruwan Yinjie Tang Wei Wen Su Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron Frontiers in Bioengineering and Biotechnology conditional protein degradation degron metabolic engineering synthetic biology Yarrowia lipolytica |
| title | Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron |
| title_full | Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron |
| title_fullStr | Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron |
| title_full_unstemmed | Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron |
| title_short | Conditional protein degradation in Yarrowia lipolytica using the auxin-inducible degron |
| title_sort | conditional protein degradation in yarrowia lipolytica using the auxin inducible degron |
| topic | conditional protein degradation degron metabolic engineering synthetic biology Yarrowia lipolytica |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1188119/full |
| work_keys_str_mv | AT zhenlinhan conditionalproteindegradationinyarrowialipolyticausingtheauxininducibledegron AT jessicamaruwan conditionalproteindegradationinyarrowialipolyticausingtheauxininducibledegron AT yinjietang conditionalproteindegradationinyarrowialipolyticausingtheauxininducibledegron AT weiwensu conditionalproteindegradationinyarrowialipolyticausingtheauxininducibledegron |