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 regulat...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-MutaT7A(TadA8e-Linker0-T7RNAP)and BS-MutaT7C(PmCDA1-(GGGGS)3-T7RNAP co-expressed with UGI)were identified.The target mutation rates reached 1.2×105 per base per generation(s.p.b.)and 5.8×105 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 5.8×106 s.p.b.and 2.9×105 s.p.b.within a 5 kb DNA region.Notably,BS-MutaT7C 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 enhancedβ-lactoglobulin(β-Lg)expression by evolving the global transcriptional regulator codY,achieving aβ-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.展开更多
The continuous evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)presents ongoing challenges and risks to public health,as it renders most reported monoclonal antibodies(mAbs)ineffective due to i...The continuous evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)presents ongoing challenges and risks to public health,as it renders most reported monoclonal antibodies(mAbs)ineffective due to immune escape[1,2].Unlike conventional strategies that rely on conserved epitopes across known viral subtypes,the extraordinary pace and unpredictability of SARS-CoV-2 mutations have progressively eroded these epitopes,thereby destabilizing the foundations of traditional broadly neutralizing antibody(bnAb)development[3].展开更多
基金supported by the National Key Research and Development Program(2024YFF1106800)National Natural Sci-ence Foundation of China(32172349)+2 种基金the Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)the Jiangsu Basic Research Center for Synthetic Biology(BK20233003)the Natural Science Foundation of Jiangsu Province(BK20202002).
文摘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-MutaT7A(TadA8e-Linker0-T7RNAP)and BS-MutaT7C(PmCDA1-(GGGGS)3-T7RNAP co-expressed with UGI)were identified.The target mutation rates reached 1.2×105 per base per generation(s.p.b.)and 5.8×105 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 5.8×106 s.p.b.and 2.9×105 s.p.b.within a 5 kb DNA region.Notably,BS-MutaT7C 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 enhancedβ-lactoglobulin(β-Lg)expression by evolving the global transcriptional regulator codY,achieving aβ-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.
基金funded by the External Cooperation Program of CAS(grant number 180GJHZ2023017MI)to G.F.G.
文摘The continuous evolution of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)presents ongoing challenges and risks to public health,as it renders most reported monoclonal antibodies(mAbs)ineffective due to immune escape[1,2].Unlike conventional strategies that rely on conserved epitopes across known viral subtypes,the extraordinary pace and unpredictability of SARS-CoV-2 mutations have progressively eroded these epitopes,thereby destabilizing the foundations of traditional broadly neutralizing antibody(bnAb)development[3].
