Tunable T7 Promoter Orthogonality on T7RNAP for cis-Aconitate Decarboxylase Evolution via Base Editor and Screening from Itaconic Acid Biosensor

The deaminase-fused T7 RNA polymerase (T7RNAP) presents a promising toolkit for in vivo target-specific enzyme evolution, offering the unique advantage of simultaneous DNA modification and screening. Previous studies have reported the mutation efficiency of base editors relying on different resources. In contrast, the mechanism underlying the T7RNAP/T7 system is well-understood. Therefore, this study aimed to establish a new platform, termed dT7-Muta, by tuning the binding efficiency between T7RNAP and the T7 promoter for gene mutagenesis. The strategy for proof-of-concept involves alterations in the fluorescence distribution through dT7-Muta and screening of the mutants via flow cytometry. The cis-aconitate decarboxylase from Aspergillus terreus (AtCadA) was evolved and screened via an itaconate-induced biosensor as proof-of-function of enzyme evolution. First, the degenerated codons were designed within the binding and initial region of T7 promoters (dT7s), including upstream (U), central (C), and downstream (D) regions. Three strength variants of dT7 promoter from each design, i.e., strong (S), medium (M), and weak (W), were used for evaluation. Mutation using dT7s of varying strength resulted in a broader fluorescence distribution in sfGFP mutants from the promoters CW and DS. On the other hand, broader fluorescence distribution was observed in the AtCadA mutants from the original promoter T7, UW, and DS, with the highest fluorescence and itaconic acid titer at 860 a.u. and 0.51 g/L, respectively. The present platform introduces a novel aspect of the deaminase-based mutagenesis, emphasizing the potential of altering the binding efficiency between T7RNAP and the T7 promoter for further efforts in enzyme evolution.