Fig. 1: Design, development, optimization, and application of the orthogonal transcription mutation system.

a Design and mechanism of the orthogonal transcription mutation system. The host RNAP recognizes the endogenous promoter and transcribes to produce mRNA with a low background mutation efficiency. Orthogonal transcription mutators are based on the fusion of deaminase PmCDA1 and TadA variants with phage RNAPs. The phage RNAP domain specifically recognizes the corresponding phage promoter and opens the double-stranded target gene. The deaminase domain generates random mutations on single-stranded DNA with a high mutation efficiency. b Systematic optimization of the orthogonal transcription mutation system. Optimization of single type mutators (C:G to T:A or A:T to G:C type mutators) by combining PmCDA1 and TadA variants with different phage RNAPs, regulating the expression of UGI, and controlling the inducer concentration to increase the mutation rate. Three dual type mutators were designed and tested to generate C:G to T:A and A:T to G:C mutations. Then, a dual promoter strategy was implemented to distribute mutations across the target gene evenly. Finally, orthogonality tests among three phage RNAP-based mutators demonstrated high specificity. c Applications of the orthogonal transcription mutation system. Orthogonal transcription mutators were used to mutate fluorescent proteins and chromoproteins to produce colorful cells, mutate cytoskeleton and cell division-related proteins to achieve morphological diversity, and mutate sigma 70 factor RpoD and LysE exporter to achieve higher tolerance to L-arginine.