Dataset Information

Light-inducible protein degradation in E. coli with the LOVdeg tag.

ABSTRACT: Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in Escherichia coli using blue light. We demonstrate the modularity of LOVdeg by using it to tag a range of proteins, including the LacI repressor, CRISPRa activator, and the AcrB efflux pump. Additionally, we demonstrate the utility of pairing the LOVdeg tag with existing optogenetic tools to enhance performance by developing a combined EL222 and LOVdeg system. Finally, we use the LOVdeg tag in a metabolic engineering application to demonstrate post-translational control of metabolism. Together, our results highlight the modularity and functionality of the LOVdeg tag system and introduce a powerful new tool for bacterial optogenetics.

SUBMITTER: Tague N

PROVIDER: S-EPMC10945698 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Publications

Light-inducible protein degradation in <i>E. coli</i> with the LOVdeg tag.

Tague Nathan N Coriano-Ortiz Cristian C Sheets Michael B MB Dunlop Mary J MJ

eLife 20240125

Molecular tools for optogenetic control allow for spatial and temporal regulation of cell behavior. In particular, light-controlled protein degradation is a valuable mechanism of regulation because it can be highly modular, used in tandem with other control mechanisms, and maintain functionality throughout growth phases. Here, we engineered LOVdeg, a tag that can be appended to a protein of interest for inducible degradation in <i>Escherichia coli</i> using blue light. We demonstrate the modular ...[more]

PMID: 38270583

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Project 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.

Dataset Information

Light-inducible protein degradation in E. coli with the LOVdeg tag.

Publications

Light-inducible protein degradation in <i>E. coli</i> with the LOVdeg tag.

Similar Datasets

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