Constructing an Adenine Base Editor in

Adenine base editors (ABEs) can be used to fine-tune metabolic flux by editing gene promoters. However, their targeting scope is constrained by the PAM requirement of Cas9, making it difficult to target promoter regions where NGG motifs are rare. To overcome this limitation, we fused the SpRY variant, a near-PAMless Cas9, with the highly active adenine deaminase TadA8e to construct a near-PAMless ABE. After multi-round optimization, the editor achieved A → G edit at the T7 promoter, along with a bystander C → A edit at neighboring position. The ABE was used to edit the T7 promoter in the 1,4-butanediamine-producing strain, thereby altering the promoter strength and reallocating metabolic flux into 1,4-butanediamine biosynthesis. After shake-flask fermentation, the engineered strain produced 478.8 mg/L of 1,4-butanediamine within 48 h, representing a 106% increase compared to the control. This study provides a feasible strategy for constructing near-PAMless base editors and lays a foundation for modulating metabolic pathways. Moreover, the engineered strain achieved a marked increase in 1,4-butanediamine production, demonstrating that promoter editing via ABE can effectively regulate metabolic flux and enhance the production of the target product, as well as serving as a valuable reference for the biosynthesis of other high-value-added chemicals.