Engineering a Transcription Activation Toolkit by Rewiring Prokaryotic Transcription Factors and Synthetic Yeast Promoters in Yarrowia lipolytica

Abstract Yarrowia lipolytica is a promising industrial host, yet its metabolic engineering potential remains limited by insufficient genetic tools. Here, we engineered a synthetic Transcription Activation Toolkit (TAT) based on LacI–VPRH, a chimeric protein fusing the prokaryotic DNA-binding domain LacI with the eukaryotic activation domain VPRH. Systematic optimization of LacO copy numbers and core promoter composition achieved up to 205-fold gene activation. The TAT platform was further expanded to construct bidirectional expression systems and enable multiplexed gene control. Applied to resveratrol biosynthesis via a “push-pull” strategy, CRISPR/Cas9-mediated integration of TAT-controlled synthetic promoters upregulated the shikimate pathway genes aroM10 and aroC alongside the rate-limiting enzyme ST1, achieving a shake-flask titer of 2.715 g/L─the highest reported to date. Additionally, an IPTG-inducible “turn-on” system (TAT-2.0) incorporating the antiLacI9 mutant was developed for small-molecule-responsive transcriptional control. Collectively, the modular TAT system provides a versatile strategy for precise metabolic pathway optimization in Y. lipolytica.