Development of a β-Arbutin Hyperproducer in <i>Escherichia coli</i> by Rational Modular and Transcription Factor Engineering

β-Arbutin is a valuable aromatic compound with multiple biological activities. The exploitation of efficient engineering microbial platforms for β-arbutin production has been an appealing research avenue in recent years due to l-tyrosine auxotrophy and the limited distribution of metabolic flux. Herein, an inducer-free and nonauxotrophic cell factory is successfully constructed in Escherichia coli toward high-titer β-arbutin production by combining metabolic engineering strategies. First, the β-arbutin de novo synthesis pathway was constructed, and the expression level of the heterologous enzyme was optimized by promoter engineering. Next, the biosynthetic pathway was modified by rational modular engineering strategies to further enhance β-arbutin production. Subsequently, the transcription factor TorI was identified to negatively regulate β-arbutin biosynthesis based on transcriptomic data. Finally, a dynamic toggle switch controlling the expression of the tyrA gene was designed to reconstruct a nonauxotrophic pathway for β-arbutin production. Without additional supplements of l-tyrosine, the final engineering strain AT25 generated the highest production level ever reported among inducer-free, nonauxotrophic strains, producing 30.52 g/L β-arbutin with a productivity of 0.51 g/L/h. The systematic strategies demonstrated in this study can provide a reference for establishing efficient microbial cell factories for the production of β-arbutin together with other aromatic compounds.