T7 RNA polymerase-guided base editor for accelerated continuous evolution in Bacillus subtilis

Abstract

Targeted in vivo hypermutation mediated by base deaminase-T7 RNA polymerase (T7 RNAP) fusions promotes genetic diversification and accelerates continuous directed evolution. Due to the lack of a T7RNAP expression regulation system and functionally compatible linker for fusion protein expression, T7RNAP-guided continuous evolution has not been established in Bacillus subtilis, which limited long gene fragment continuous evolution targeted on genome. Here, we developed BS-MutaT7 system, which introduced mutations into specific genomic regions by leveraging chimeric fusions of base deaminases with T7RNAP in B. subtilis. We selected seven different sources of adenosine and cytosine deaminases, 14 fusion protein linkers to be fused with T7RNAP, constructing four libraries with the size of 5000, where deaminases were fused at either the N- or C-terminus of T7RNAP. Based on the efficiency of binding to T7 promoter and high mutagenesis activity, two optimal chimeric mutators, BS-MutaT7^A (TadA8e-Linker0-T7RNAP) and BS-MutaT7^C (PmCDA1-(GGGGS)₃-T7RNAP co-expressed with UGI) were identified. The target mutation rates reached 1.2 × 10^-5 per base per generation (s.p.b.) and 5.8 × 10^-5 s.p.b., representing 7000-fold and 37,000-fold increases over the genomic mutation rate, respectively. Both exhibited high processivity, maintaining mutation rates of 5.8 × 10^-6 s.p.b. and 2.9 × 10^-5 s.p.b. within a 5 kb DNA region. Notably, BS-MutaT7^C exhibited superior mutagenic activity, making it well-suited for applications requiring intensive and sustained genomic diversification. Application of BS-MutaT7 enabled a 16-fold increase in tigecycline resistance and enhanced β-lactoglobulin (β-Lg) expression by evolving the global transcriptional regulator codY, achieving a β-Lg titer of 3.92 g/L. These results highlight BS-MutaT7 as a powerful and versatile tool for genome-scale continuous evolution in B. subtilis.

Keywords: BS-MutaT7 system; Bacillus subtilis; Continuous directed evolution; T7 RNA polymerase.

Fig. 1

Graphic summary of the BS-MutaT7 system in B. subtilis. (a) Schematic of the T7 RNA polymerase-guided base editor system. Temperature-sensitive plasmids were constructed to express the fusion protein comprising the base deaminase and T7RNAP. The fusion protein specifically binds to the T7 promoter inserted downstream of the target gene on the genome. As the T7 RNAP transcribes along the target gene, the deaminase introduces point mutations. Upon encountering the T7 terminator array, the fusion protein detaches from the DNA, terminating transcription and mutagenesis. The mutagenesis plasmid is lost by increasing the cultivation temperature. BD-T7RNAP: chimeric fusion of base deaminase with T7RNAP. (b) Representation of the reporter Ery^R expression cassettes and P_T7-gfp expression cassette. The expression cassettes were integrated into the genome to assess mutagenic and transcriptional activities of the BS-MutaT7 system. In BS-MutaT7^A, the distance from the T7 transcription start site (TSS) to the “TAA” codon was 616 bp, while in BS-MutaT7^C, the distance to “GGA” codon was 571 bp. The green circle represented the ugi gene. The bold bases represented the target sites for mutagenesis. The RFI was used to assess the transcriptional activity. (c) Verification of Ery^R gene mutation inactivation. The B. subtilis strains carrying Ery^R_L43∗ (“TAA” stop codon) or Ery^R_E58G (inactive mutation) cassettes were erythromycin sensitive and unable to grow on selective LB agar plates containing 0.75 μg/mL erythromycin.

Fig. 2

Construction and screening of BS-MutaT7^A with transcriptional and mutagenic activity in B. subtilis. (a) Schematic of the BD-T7RNAP mutagenesis library construction. The plasmid vector pWL-P_tet was used to express BD-T7RNAP fusion proteins, induced by 0.5 μM aTC. (b) Transcriptional and mutagenic activity analysis of 30 single colonies from ALib1 during preliminary screening. The host strain E43G contained two cassettes: the reporter Ery^R_L43∗ expression cassette and the P_T7-gfp expression cassette. An asterisk indicated strains with relatively high mutation rates selected for rescreening. (c) Validation of the inactivated promoter KOT7. The FI assay was used to assess the KOT7 activity and the KOT7G strain exhibited no measurable fluorescence. (d) Analysis of the target mutation rate and untargeted mutation rate of the screened strains. Plasmids were transformed into E43 to evaluate the mutation rates, identifying EA1-C10 as the optimal BS-MutaT7^A with the highest target mutation efficiency. The T7 promoters in the C-terminus of Ery^R_L43∗ expression cassette of the strains were replaced with KOT7 to assess the untargeted mutation rates. The numbers indicated the fold increase in target mutation rates compared to the genomic spontaneous mutation rate. (e) Evaluation of the genomic off-target mutations in BS-MutaT7^A. The rifampicin resistance assay was used to test the off-target mutation rate. Induced cultures were plated on LB agar plates containing 50 μg/mL rifampicin for colony counting and fluctuation analysis. Error bar represents data from biological triplicate.

Fig. 3

Construction and optimization of BS-MutaT7^C with transcriptional and mutagenic activity in B. subtilis. (a) Transcriptional and mutagenic activity of 40 single colonies from CLib1 during preliminary screening. The host strain E58G contained two cassettes: the reporter Ery^R_E58G expression cassette and P_T7-gfp expression cassette. An asterisk indicated strains with relatively high mutation rates selected for rescreening. (b) Analysis of the target mutation rate and untargeted mutation rate of the screened strains. Plasmids were transformed into E58 to evaluate the mutation rates, with EC6-F8 achieving the highest target mutation efficiency. The numbers indicated the fold increase in target mutation rates compared to the genomic spontaneous mutation rate. (c) Engineering the ugi expression to enhance the target mutation efficiency. (d) Evaluation of the genomic off-target mutations in BS-MutaT7^C. The plasmid pWL-T7RNAP-P_tet-ugi was transformed into E58 to evaluate the effect of ugi expression on off-target mutation rate. Error bar represents data from biological triplicate.

Fig. 4

Testing the sustained mutagenic activity of BS-MutaT7^A and BS-MutaT7^C in B. subtilis. (a) Schematic of the expanded editing window. By repositioning the T7 promoter on the genomes of E43 and E58, the editing window was extended to 5000 bp for BS-MutaT7^A and 4955 bp for BS-MutaT7^C. The values in the box represented the corresponding editing window lengths for BS-MutaT7^C. (b) The sustained mutagenic activity of BS-MutaT7^A. (c) The sustained mutagenic activity of BS-MutaT7^C. Error bar represents data from biological triplicate.

Fig. 5

Continuous evolution of tigecycline resistance using the BS-MutaT7 system. (a) Schematic of the continuous evolution process for the tetK gene. Using the BS-MutaT7^A and BS-MutaT7^C systems, three transformants were cultured separately in centrifuge tubes. (b) Validation of the evolved tetK gene using the BS-MutaT7^A system. Sanger sequencing of the key mutations introduced by BS-MutaT7^A revealed T to C transitions on the coding strand of tetK gene and the P₄₃ promoter. Several mutants also exhibited additional A to G transitions. The evolved tetK gene conferred a 16-fold increase in tigecycline resistance. (c) Validation of the evolved tetK gene using the BS-MutaT7^C system. Sanger sequencing analysis of the key mutations introduced by BS-MutaT7^C identified G to A transitions on the coding strand of tetK gene and the P₄₃ promoter. The evolved tetK gene conferred a 12-fold increase in tigecycline resistance. (d) Most enriched tetK mutants identified through continuous evolution with BS-MutaT7 system.

Fig. 6

Continuous evolution of global transcriptional regulator CodY using the BS-MutaT7 system. (a) Expression of β-Lg in the B. subtilis chassis cells visualized by SDS-PAGE analysis. Lane 1, the total intracellular protein of BS-P₅₆₆-Lg. Lane 2, the intracellular soluble supernatant fraction. Lane 3, the intracellular insoluble fraction. Lane M represents the molecular weights of pre-stained protein marker. (b) Flow cytometric analysis and rescreening of high fluorescence intensity mutants. The left panel represents the screening results in BS-MutaT7^A system, while the right panel represents the screening results in BS-MutaT7^C system. (c) β-Lg titers of codY mutants. in shake flask fermentation. (d) Fermentation of C-codY-Lg in a 5-L fermenter.