Abstract
The auxin-inducible degron (AID) system enables targeted protein degradation in vivo. Conventionally, the AID tag is fused directly to the terminus of the target protein; however, its intrinsically disordered nature and destabilizing motifs can render it susceptible to premature proteolytic removal from its tagged protein, resulting in incomplete degradation upon auxin induction. In addition, direct terminal fusion of the conventional AID tag may compromise protein stability. To address these limitations, we introduce a new degron design in which the conventional AID tag, mIAA7, is inserted into an exposed loop of green fluorescent protein (GFP). The resulting engineered GFP variant, termed the “constrained AID” (cAID) tag, was validated in the industrially important oleaginous yeast Yarrowia lipolytica. Inserting mIAA7 into GFP, rather than fusing it directly at the GFP terminus, elevated expression of the GFP fusion protein and enabled more complete degradation upon auxin induction. The utility of the novel cAID tag was further validated by tagging a soluble cytosolic anti-mCherry nanobody and by targeting β-carotene ketolase, squalene synthase, and squalene epoxidase to modulate carotenoid biosynthesis. Compared to direct fusion with the conventional AID tag, cAID tag enabled more complete protein degradation and tighter temporal control of metabolism, while minimizing perturbation to the tagged protein.