ABSTRACT Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently

mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for

continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in _Bacillus thuringiensis_ with an engineered error-prone orthogonal DNA polymerase.

BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host

Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Access Nature and 54

other Nature Portfolio journals Get Nature+, our best-value online-access subscription $29.99 / 30 days cancel any time Learn more Subscribe to this journal Receive 12 print issues and

online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes

which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY

OTHERS SYNTHETIC CROSS-PHYLA GENE REPLACEMENT AND EVOLUTIONARY ASSIMILATION OF MAJOR ENZYMES Article 10 August 2020 ADAPTIVE LABORATORY EVOLUTION RECRUITS THE PROMISCUITY OF SUCCINATE

SEMIALDEHYDE DEHYDROGENASE TO REPAIR DIFFERENT METABOLIC DEFICIENCIES Article Open access 15 October 2024 IN VITRO GENERATION OF GENETIC DIVERSITY FOR DIRECTED EVOLUTION BY ERROR-PRONE

ARTIFICIAL DNA SYNTHESIS Article Open access 24 May 2024 DATA AVAILABILITY All data discussed in this study can be found in the Supplementary Information. The NGS raw data were deposited in

the National Center of Biotechnology Information Sequence Read Archive (BioProject: PRJNA941059). Source data are provided with this paper. REFERENCES * Arnold, F. H. Design by directed

evolution. _Acc. Chem. Res._ 31, 125–131 (1998). Article  CAS  Google Scholar  * Davis, A. M., Plowright, A. T. & Valeur, E. Directing evolution. The next revolution in drug discovery?

_Nat. Rev. Drug Discov._ 16, 681–698 (2017). Article  CAS  PubMed  Google Scholar  * Packer, M. S. & Liu, D. R. Methods for the directed evolution of proteins. _Nat. Rev. Genet._ 16,

379–394 (2015). Article  CAS  PubMed  Google Scholar  * Arnold, F. H. Directed evolution. Bringing new chemistry to life. _Angew. Chem._ 57, 4143–4148 (2018). Article  CAS  Google Scholar  *

Rix, G. & Liu, C. C. Systems for in vivo hypermutation. A quest for scale and depth in directed evolution. _Curr. Opin. Chem. Biol._ 64, 20–26 (2021). Article  CAS  PubMed  PubMed

Central  Google Scholar  * Morrison, M. S., Podracky, C. J. & Liu, D. R. The developing toolkit of continuous directed evolution. _Nat. Chem. Biol._ 16, 610–619 (2020). Article  CAS 

PubMed  Google Scholar  * Meyer, A. J. & Ellington, A. D. Molecular evolution picks up the PACE. _Nat. Biotechnol._ 29, 502–503 (2011). Article  CAS  PubMed  Google Scholar  * Simon, A.

J., d’Oelsnitz, S. & Ellington, A. D. Synthetic evolution. _Nat. Biotechnol._ 37, 730–743 (2019). Article  CAS  PubMed  Google Scholar  * Komor, A. C., Kim, Y. B., Packer, M. S., Zuris,

J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. _Nature_ 533, 420–424 (2016). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Chen, H. et al. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. _Nat. Biotechnol._ 38, 165–168 (2020). Article  CAS  PubMed  Google Scholar  *

Cravens, A., Jamil, O. K., Kong, D., Sockolosky, J. T. & Smolke, C. D. Polymerase-guided base editing enables in vivo mutagenesis and rapid protein engineering. _Nat. Commun._ 12, 1579

(2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hao, W. et al. Development of a base editor for protein evolution via in situ mutation in vivo. _Nucleic Acids Res._ 49,

9594–9605 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Schubert, M. G. et al. High-throughput functional variant screens via in vivo production of single-stranded DNA.

_Proc. Natl Acad. Sci. USA_ 118, e2018181118 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Jensen, E. D. et al. A synthetic RNA-mediated evolution system in yeast. _Nucleic

Acids Res._ 49, e88 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Crook, N. et al. In vivo continuous evolution of genes and pathways in yeast. _Nat. Commun._ 7, 13051

(2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. _Nature_

560, 248–252 (2018). Article  CAS  PubMed  Google Scholar  * Yi, X., Khey, J., Kazlauskas, R. J. & Travisano, M. Plasmid hypermutation using a targeted artificial DNA replisome. _Sci.

Adv._ 7, eabg8712 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Blum, T. R. et al. Phage-assisted evolution of botulinum neurotoxin proteases with reprogrammed specificity.

_Science_ 371, 803–810 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Esvelt, K. M., Carlson, J. C. & Liu, D. R. A system for the continuous directed evolution of

biomolecules. _Nature_ 472, 499–503 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ravikumar, A., Arzumanyan, G. A., Obadi, M. K., Javanpour, A. A. & Liu, C. C.

Scalable, continuous evolution of genes at mutation rates above genomic error thresholds. _Cell_ 175, 1946–1957 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhong, Z.

& Liu, C. C. Probing pathways of adaptation with continuous evolution. _Curr. Opin. Syst. Biol._ 14, 18–24 (2019). Article  PubMed  PubMed Central  Google Scholar  * Ravikumar, A.,

Arrieta, A. & Liu, C. C. An orthogonal DNA replication system in yeast. _Nat. Chem. Biol._ 10, 175–177 (2014). Article  CAS  PubMed  Google Scholar  * Wellner, A. et al. Rapid generation

of potent antibodies by autonomous hypermutation in yeast. _Nat. Chem. Biol_. 17, 1057–1064 (2021). * Javanpour, A. A. & Liu, C. C. Evolving small-molecule biosensors with improved

performance and reprogrammed ligand preference using OrthoRep. _ACS Synth. Biol._ 10, 2705–2714 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Rix, G. et al. Scalable

continuous evolution for the generation of diverse enzyme variants encompassing promiscuous activities. _Nat. Commun._ 11, 5644 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar 

* Muñoz-Espín, D., Holguera, I., Ballesteros-Plaza, D., Carballido-López, R. & Salas, M. Viral terminal protein directs early organization of phage DNA replication at the bacterial

nucleoid. _Proc. Natl Acad. Sci. USA_ 107, 16548–16553 (2010). Article  PubMed  PubMed Central  Google Scholar  * van Nies, P. et al. Self-replication of DNA by its encoded proteins in

liposome-based synthetic cells. _Nat. Commun._ 9, 1583 (2018). Article  PubMed  PubMed Central  Google Scholar  * Gillis, A. & Mahillon, J. Phages preying on _Bacillus anthracis_,

_Bacillus cereus_, and _Bacillus thuringiensis_. Past, present and future. _Viruses_ 6, 2623–2672 (2014). Article  PubMed  PubMed Central  Google Scholar  * Meijer, W. J., Horcajadas, J. A.

& Salas, M. Phi29 family of phages. _Microbiol. Mol. Biol. Rev._ 65, 261–287 (2001). Article  CAS  PubMed  PubMed Central  Google Scholar  * Gillis, A. & Mahillon, J. Influence of

lysogeny of _Tectiviruses_ GIL01 and GIL16 on _Bacillus thuringiensis_ growth, biofilm formation, and swarming motility. _Appl. Environ. Microbiol._ 80, 7620–7630 (2014). Article  PubMed 

PubMed Central  Google Scholar  * Biggel, M. et al. Whole genome sequencing reveals biopesticidal origin of _Bacillus thuringiensis_ in foods. _Front. Microbiol._ 12, 775669 (2022). Article

  PubMed  PubMed Central  Google Scholar  * Verheust, C., Fornelos, N. & Mahillon, J. GIL16, a new gram-positive tectiviral phage related to the _Bacillus thuringiensis_ GIL01 and the

_Bacillus cereus_ pBClin15 elements. _J. Bacteriol._ 187, 1966–1973 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  * Doron, S. et al. Systematic discovery of antiphage defense

systems in the microbial pangenome. _Science_ 359, eaar4120 (2018). Article  PubMed  PubMed Central  Google Scholar  * Wannier, T. M. et al. Improved bacterial recombineering by

parallelized protein discovery. _Proc. Natl Acad. Sci. USA_ 117, 13689–13698 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wu, Y. et al. Design of a programmable

biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in _Bacillus subtilis_. _Nucleic Acids Res._ 48, 996–1009 (2020). Article  CAS  PubMed  Google

Scholar  * Soengas, M. S. et al. Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3′–5′ exonuclease and strand-displacement

activities. _EMBO J._ 11, 4227–4237 (1992). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vega, M., de, Lazaro, J. M., Salas, M. & Blanco, L. Primer-terminus stabilization at

the 3′–5′ exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. _EMBO J._ 15, 1182–1192 (1996). Article 

PubMed  PubMed Central  Google Scholar  * Truniger, V., Lázaro, J. M., Salas, M. & Blanco, L. A DNA binding motif coordinating synthesis and degradation in proofreading DNA polymerases.

_EMBO J._ 15, 3430–3441 (1996). Article  CAS  PubMed  PubMed Central  Google Scholar  * Vega, M., de, Lázaro, J. M. & Salas, M. Phage ø29 DNA polymerase residues involved in the proper

stabilisation of the primer-terminus at the 3′–5′ exonuclease active site. _J. Mol. Biol._ 304, 1–9 (2000). Article  PubMed  Google Scholar  * Pérez-Arnaiz, P., Lázaro, J. M., Salas, M.

& de Vega, M. Functional importance of bacteriophage ϕ29 DNA polymerase residue tyr148 in primer-terminus stabilisation at the 3'-5' exonuclease active site. _J. Mol. Biol._

391, 797–807 (2009). Article  PubMed  Google Scholar  * Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. _Nature_ 596, 583–589 (2021). Article  CAS  PubMed 

PubMed Central  Google Scholar  * Liu, Y., Liu, L., Li, J., Du, G. & Chen, J. Synthetic biology toolbox and chassis development in _Bacillus subtilis_. _Trends Biotechnol._ 37, 548–562

(2019). Article  CAS  PubMed  Google Scholar  * Lu, Z. et al. CRISPR-assisted multi-dimensional regulation for fine-tuning gene expression in _Bacillus subtilis_. _Nucleic Acids Res._ 47,

e40 (2019). Article  PubMed  PubMed Central  Google Scholar  * Tian, R. et al. Titrating bacterial growth and chemical biosynthesis for efficient _N_-acetylglucosamine and

_N_-acetylneuraminic acid bioproduction. _Nat. Commun._ 11, 5078 (2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Cai et al. Cell-free chemoenzymatic starch synthesis from

carbon dioxide. _Science_ 373, 1523–1527 (2021). Article  CAS  PubMed  Google Scholar  * Gao, B. et al. Constructing a methanol-dependent _Bacillus subtilis_ by engineering the methanol

metabolism. _J. Biotechnol._ 343, 128–137 (2022). Article  CAS  PubMed  Google Scholar  * Li, C., Zou, Y., Jiang, T., Zhang, J. & Yan, Y. Harnessing plasmid replication mechanism to

enable dynamic control of gene copy in bacteria. _Metab. Eng._ 70, 67–78 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fornelos, N., Bamford, J. K. H. & Mahillon, J.

Phage-borne factors and host LexA regulate the lytic switch in phage GIL01. _J. Bacteriol._ 193, 6008–6019 (2011). Article  CAS  PubMed  PubMed Central  Google Scholar  * Fornelos, N. et al.

Lytic gene expression in the temperate bacteriophage GIL01 is activated by a phage-encoded LexA homologue. _Nucleic Acids Res._ 46, 9432–9443 (2018). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Fornelos, N. et al. Bacteriophage GIL01 gp7 interacts with host LexA repressor to enhance DNA binding and inhibit RecA-mediated auto-cleavage. _Nucleic Acids Res._ 43,

7315–7329 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa, M. & Díaz-Orejas, R. Replication and control of

circular bacterial plasmids. _Microbiol. Mol. Biol. Rev._ 62, 434–464 (1998). Article  PubMed  PubMed Central  Google Scholar  * Salis, H. M., Mirsky, E. A. & Voigt, C. A. Automated

design of synthetic ribosome binding sites to control protein expression. _Nat. Biotechnol._ 27, 946–950 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Espah Borujeni, A. et

al. Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences. _Nucleic Acids Res._ 45, 5437–5448 (2017).

Article  PubMed  PubMed Central  Google Scholar  * Majidian, P. et al. _Bacillus subtilis_ GntR regulation modified to devise artificial transient induction systems. _J. Gen. Appl.

Microbiol._ 62, 277–285 (2016). Article  CAS  PubMed  Google Scholar  * Brantl, S. Antisense-RNA mediated control of plasmid replication—pIP501 revisited. _Plasmid_ 78, 4–16 (2015). Article

  CAS  PubMed  Google Scholar  * Wang, Y. et al. Synthetic promoter design in _Escherichia coli_ based on a deep generative network. _Nucleic Acids Res._ 48, 6403–6412 (2020). Article  CAS 

PubMed  PubMed Central  Google Scholar  * Yang, H. et al. Efficient extracellular production of recombinant proteins in _E. coli_ via enhancing expression of _dacA_ on the genome. _J. Ind.

Microbiol. Biotechnol._ 49, kuac016 (2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Snapyan, M. et al. Cell-free protein synthesis by diversifying bacterial transcription

machinery. _BioTech_ 10, 24 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Wang, Y. et al. _Bacillus subtilis_ genome editing using ssDNA with short homology regions.

_Nucleic Acids Res._ 40, e91 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Mencía, M., Gella, P., Camacho, A., de Vega, M. & Salas, M. Terminal protein-primed

amplification of heterologous DNA with a minimal replication system based on phage ϕ29. _Proc. Natl Acad. Sci. USA_ 108, 18655–18660 (2011). Article  PubMed  PubMed Central  Google Scholar 

* González-Huici, V., Alcorlo, M., Salas, M. & Hermoso, J. M. Phage φ29 proteins p1 and p17 are required for efficient binding of architectural protein p6 to viral DNA in vivo. _J.

Bacteriol._ 186, 8401–8406 (2004). Article  PubMed  PubMed Central  Google Scholar  * Serna-Rico, A., Muñoz-Espín, D., Villar, L., Salas, M. & Meijer, W. J. J. The integral membrane

protein p16.7 organizes in vivo φ29 DNA replication through interaction with both the terminal protein and ssDNA. _EMBO J._ 22, 2297–2306 (2003). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Verheust, C., Jensen, G. & Mahillon, J. pGIL01, a linear tectiviral plasmid prophage originating from _Bacillus thuringiensis_ serovar israelensis. _Microbiology_ 149,

2083–2092 (2003). Article  CAS  PubMed  Google Scholar  * Peng, D. et al. Elaboration of an electroporation protocol for large plasmids and wild-type strains of _Bacillus thuringiensis_. _J.

Appl. Microbiol._ 106, 1849–1858 (2009). Article  CAS  PubMed  Google Scholar  * He, J. et al. Complete genome sequence of _Bacillus thuringiensis_ mutant strain BMB171. _J. Bacteriol._

192, 4074–4075 (2010). Article  CAS  PubMed  PubMed Central  Google Scholar  * Filsinger, G. T. et al. Characterizing the portability of phage-encoded homologous recombination proteins.

_Nat. Chem. Biol._ 17, 394–402 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Butala, M., Žgur-Bertok, D. & Busby, S. J. W. The bacterial LexA transcriptional repressor.

_Cell. Mol. Life Sci._ 66, 82–93 (2008). Article  Google Scholar  * Fabret, C., Dusko Ehrlich, S. & Noirot, P. A new mutation delivery system for genome-scale approaches in _Bacillus

subtilis_. _Mol. Microbiol._ 46, 25–36 (2002). Article  CAS  PubMed  Google Scholar  * Chang, S. & Cohen, S. N. High frequency transformation of _Bacillus subtilis_ protoplasts by

plasmid DNA. _Mol. Gen. Genet._ 168, 111–115 (1979). Article  CAS  PubMed  Google Scholar  * Foster, P. L. Methods for determining spontaneous mutation rates. _Methods Enzymol._ 409, 195–213

(2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Hall, B. M., Ma, C.-X., Liang, P. & Singh, K. K. Fluctuation analysis calculator. A web tool for the determination of

mutation rate using Luria–Delbrück fluctuation analysis. _Bioinformatics_ 25, 1564–1565 (2009). Article  CAS  PubMed  PubMed Central  Google Scholar  * Alper, H., Fischer, C., Nevoigt, E.

& Stephanopoulos, G. Tuning genetic control through promoter engineering. _Proc. Natl Acad. Sci. USA_ 102, 12678–12683 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Tian, R. et al. Synthetic N-terminal coding sequences for fine-tuning gene expression and metabolic engineering in _Bacillus subtilis_. _Metab. Eng._ 55, 131–141 (2019). Article  CAS  PubMed

  Google Scholar  * Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L. & Wold, B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. _Nat. Methods_ 5, 621–628 (2008).

Article  CAS  PubMed  Google Scholar  * Tjaden, B. A computational system for identifying operons based on RNA-seq data. _Methods_ 176, 62–70 (2020). Article  CAS  PubMed  Google Scholar  *

Wang, Y. et al. Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered _Corynebacterium glutamicum_. _Nat. Commun._ 11, 3120 (2020). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Badran, A. H. & Liu, D. R. Development of potent in vivo mutagenesis plasmids with broad mutational spectra. _Nat. Commun._ 6, 8425 (2015).

Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to M. Sun from Huazhong Agricultural University for providing us with strain _B. thuringiensis_

HD-1 and _B. thuringiensis_ BMB171. We are also grateful to L. Ma from Jiangsu Academy of Agricultural Sciences for providing us with strain _B. thuringiensis_ JW-1. We also thank W. Chu

from the Science Center for Future Foods, Jiangnan University, for preparing all the NGS samples. In addition, we thank L. Zhang from the School of Biotechnology, Jiangnan University, for

doing all the flow cytometry. This study is financially supported by the National Key Research and Development Program of China (2018YFA0900300), the National Science Fund for Excellent

Young Scholars (32222069), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (32021005), the National Natural Science Foundation of China

(32172349), the Natural Science Foundation of Jiangsu Province (BK20202002 and BK20200085) and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX20_1824).

AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China

Rongzhen Tian, Runzhi Zhao, Haoyu Guo, Kun Yan, Chenyun Wang, Cheng Lu, Xueqin Lv, Jianghua Li, Long Liu, Guocheng Du & Yanfeng Liu * Science Center for Future Foods, Jiangnan

University, Wuxi, China Rongzhen Tian, Runzhi Zhao, Haoyu Guo, Kun Yan, Chenyun Wang, Xueqin Lv, Jianghua Li, Long Liu, Guocheng Du, Jian Chen & Yanfeng Liu * Engineering Research Center

of Ministry of Education on Food Synthetic Biotechnology, Jiangnan University, Wuxi, China Rongzhen Tian, Runzhi Zhao, Haoyu Guo, Kun Yan, Chenyun Wang, Xueqin Lv, Jian Chen & Yanfeng

Liu * Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology, Jiangnan University, Wuxi, China Rongzhen Tian, Runzhi Zhao, Haoyu Guo, Kun Yan, Chenyun Wang, Xueqin Lv, 

Jian Chen & Yanfeng Liu Authors * Rongzhen Tian View author publications You can also search for this author inPubMed Google Scholar * Runzhi Zhao View author publications You can also

search for this author inPubMed Google Scholar * Haoyu Guo View author publications You can also search for this author inPubMed Google Scholar * Kun Yan View author publications You can

also search for this author inPubMed Google Scholar * Chenyun Wang View author publications You can also search for this author inPubMed Google Scholar * Cheng Lu View author publications

You can also search for this author inPubMed Google Scholar * Xueqin Lv View author publications You can also search for this author inPubMed Google Scholar * Jianghua Li View author

publications You can also search for this author inPubMed Google Scholar * Long Liu View author publications You can also search for this author inPubMed Google Scholar * Guocheng Du View

author publications You can also search for this author inPubMed Google Scholar * Jian Chen View author publications You can also search for this author inPubMed Google Scholar * Yanfeng Liu

View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.L. and R.T. designed the experiments. R.T., R.Z., K.Y., H.G. and C.W. performed the

biochemical experiments and analyzed the data. R.T. and C.L. performed protein structure modeling and analysis. Y.L., X.L., J.L., L.L., G.D. and J.C. conceived the project and supervised the

research. R.T., Y.L., X.L., J.L., L.L., G.D. and J.C. wrote the paper. CORRESPONDING AUTHOR Correspondence to Yanfeng Liu. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no

competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Chemical Biology_ thanks Mattheos Koffas, Jumi Shin and the other, anonymous, reviewer(s) for their contribution to the peer

review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED

DATA EXTENDED DATA FIG. 1 THE FIRST TWO APPROACHES TO CONSTRUCT LINEAR PLASMIDS BASED ON LYTIC PHAGE AND TEMPERATE PHAGE IN _B. SUBTILIS_. (A) Linear plasmid system construction using lytic

phage φ29 replication system. During the phage φ29 lytic cycle, phage φ29 first injects its linear double-stranded DNA (dsDNA) genome into the cell. Then, it replicates the phage genome and

synthesizes phage capsid and tail proteins. In addition, cells are lysed, and progeny phages are released after packaging into progeny phage particles. Developing linear plasmids in _B.

subtilis_ was tested by expressing linear dsDNA replication machinery proteins and rationally designing linear plasmids. TP, terminal proteins. DNAP, DNA polymerase. (B) Transferring the

dormant state phage to _B. subtilis_ by protoplast fusion of _B. thuringiensis_-harboring temperate phage GIL01 and _B. subtilis_. Linear plasmid system construction using temperate phage

GIL01 replication system. _B. thuringiensis_ temperate phage GIL01/GIL16 are capable of persistent intracellular dormancy unless DNA damage occurs. Therefore, it potentially can be used as a

linear plasmid in _B. subtilis_. EXTENDED DATA FIG. 2 LINEAR PLASMID CONSTRUCTION WORKFLOW BASED ON PHAGE Φ29 DNA REPLICATION SYSTEM. (A) Genome map of lytic phage φ29. The genome is mainly

composed of two early operons on both sides (mainly expressed in the early stage of phage infection) and a late operon in the middle (mainly expressed in the late stage of phage infection).

A2b, A2c and C2 are promoters of corresponding operons. TP, terminal proteins. DNAP, DNA polymerase. (B) Map of designed linear plasmid (LP). _cm_, chloromycetin resistance gene. _gfp_,

green fluorescent protein gene. MoriL, minimally replicated region on the left (191 bp). MoriR, minimally replicated region on the right (MoriR, 194 bp). GOI, gene of interest. 5’-phosphate

modification of linear dsDNA is one of the necessary conditions for TP to covalently bind to it. (C) Electroporation protocol optimization. Data are the mean ± SD from four (n = 4)

biologically independent replicates. (D) Replication machinery expression optimization. 18 promoters with gradient strength were selected, and 36 new strains expressing the φ29 replication

machinery via plasmid expression and genome-integrated expression were constructed, respectively. Expression levels of 18 promoters were characterized using GFP. (E) Orthogonal DNAP strict

expression regulation. Expression of φ29 DNAP using the tightly self-regulated promoter P-PIP501 and 5 RBSs. Expression levels of strong promoter P224 and promoter P-PIP501 were

characterized using GFP. For D and E, data are the mean ± SD from five (n = 5) biologically independent replicates. (F) Right early operon expression. Expression of the right early operon

using a gluconic acid-inducible promoter. Expression levels of genes under different gluconic acid concentrations were characterized using GFP. Data are the mean ± SD from four (n = 4)

biologically independent replicates. Source data EXTENDED DATA FIG. 3 LYSOGENIC CONTROL MECHANISM VERIFICATION AND LINEAR PLASMID CONSTRUCTION BASED ON TEMPERATE PHAGE GIL01 REPLICATION

SYSTEM. (A) Genome map and lysogenic control mechanism of temperate phage GIL01/GIL16. The genome of phage GIL01/GIL16 consists of two operons with clear functions. Phage GIL01 leads to

turbid plaques typical of temperate phages. The complex of gp7 with bacterial SOS transcription factor LexA achieves tight control of GIL01 gene expression. P1P2 and P3 are

_dinBox_-containing promoters. (B) The theoretical functional mechanism of the GIL01 lysogeny control system in _B. subtilis_. When mitomycin C (MMC) is not added, the complex composed of

LexA and gp7 binds to the promoter containing the _dinBox_ sequence and inhibits its expression; when MMC is added, the ssDNA generated by genomic DNA damage activates RecA, which further

enables LexA to undergo self-cleavage thereby releasing repression of the promoter. (C) Colony images when transforming different plasmids. (D) _dinBox_ sequences of three promoters. (E)

Design for inducible expression of gp1 and gp7. gp1 and gp7 were expressed under the control of IPTG-inducible promoter P_graC_. (F) GFP expression levels under the control of three

_dinBox_-containing promoters. All the data are expressed as the mean ± SD from three (n = 3) biologically independent replicates. (G) Design of the protoplast fusion process. Kmr, kanamycin

resistance gene expression cassette, Spcr, spectinomycin resistance gene expression cassette. Successfully fused strains are capable of growing on plates supplemented with both kanamycin

(Km) and spectinomycin (Spc). Scale bar, 2 μm. Source data EXTENDED DATA FIG. 4 CHARACTERIZING THE GROWTH RATES OF _B. THURINGIENSIS_ AND OPTIMIZING THE ELECTROPORATION PROTOCOL FOR _B.

THURINGIENSIS_ HD-1. (A) Growth curves and maximum specific growth rates (_µ_) of different strains at 30 °C and 37 °C, respectively. Strains include _B. thuringiensis_ JW-1 containing

lysogenic prophage GIL01, _B. thuringiensis_ HD-1 containing lysogenic prophage GIL16, _B. thuringiensis_ mutant strain BMB171, gram-negative model bacterium _E. coli_, and gram-positive

model bacterium _B. subtilis_. Data are the mean ± SD from six biologically independent replicates. (B) Electroporation protocol optimization. Data are the mean ± SD from four biologically

independent replicates (C) Tolerance concentration of _B. thuringiensis_ HD-1 to different antibiotics. Source data EXTENDED DATA FIG. 5 OPTIMIZATION OF LINEAR PLASMID EDITING PROTOCOLS. (A)

Illustration of linear plasmid editing. (B) Linear plasmid editing efficiency when additionally expressing different DNA annealing-assistance proteins. Exo, 5′ to 3′ double-stranded DNA

exonuclease in the λ-Red system. CspRecT, _Collinsella stercoris_ phage single-stranded DNA-annealing proteins. EcoSSB, _E. coli_ single-stranded DNA-binding protein. BtComK, _B.

thuringiensis_ ComK protein. BsComK, _B. subtilis_ ComK protein. (C) Illustration of linear plasmid structure. _spc_, spectinomycin resistance gene. _em_, erythromycin resistance gene. TP,

terminal protein. (D) Validation of successfully edited linear plasmids by PCR. Three times experiments were repeated independently with similar results. Source data EXTENDED DATA FIG. 6

DEVELOP A CRISPRI REPRESSION TOOL TO DEMONSTRATE THE ORTHOGONALITY OF GIL16 DNAP AND LINEAR PLASMIDS. (A) CRISPRi repression tool design and test. Data are expressed as the mean ± SD from

six (n = 6) biologically independent replicates. (B) sgRNA design to repress the expression of the entire linear plasmid (LP) replication and regulation gene cluster or the expression of

GIL16 DNAP. (C) Using CRISPRi repression tool to demonstrate the orthogonality of the GIL16 DNAP and the LP. (D) Measurement of cell growth curve. Data are expressed as the mean ± SD from

three (n = 3) biologically independent replicates. Source data EXTENDED DATA FIG. 7 RATIONAL SEARCH FOR TARGET MUTATION SITES THROUGH SEQUENCE ALIGNMENT. Functional domains common to

B-family DNAPs are shown above. All red and yellow shaded areas indicate regions that have been reported to affect φ29 DNAP fidelity. For example, mutations corresponding to the error-prone

synthetic DNA of φ29 DNAP (shown in grey) through sequence alignment and homology analysis were found in GIL16 DNAP (shown in green). EXTENDED DATA FIG. 8 SEQUENCES OF PROMOTER VARIANTS

OBTAINED BY CONTINUOUS EVOLUTION. Red letters represent known functional sequences. Blue represents mutations. Black lines represent sequence insertions. ‘(xxx)n’ represents the number of

sequence repeats. EXTENDED DATA FIG. 9 COMPARE PM4 TO OTHER REPORTED STRONG PROMOTERS IN THREE _E. COLI_ STRAINS. To test the universality of the PM4 promoter among different _E. coli_

strains, it was compared with several strong _E. coli_ promoters selected from recent publications. All promoters were tested under the same conditions, including the same plasmid vector

(pUC plasmid) and the same RBS. _E. coli_ strains including _E. coli_ MG1655, _E. coli_ BL21, and _E. coli_ Nissle1917. Data are the mean ± SD from six (n = 6) biologically independent

replicates. Source data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Fig. 1, Tables 1–6, References and Note REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 2 Statistical

source data. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 2 Statistical

source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data and

an unprocessed gel. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 9 Statistical source data. RIGHTS AND PERMISSIONS Springer Nature or its

licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the

accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Tian, R., Zhao, R., Guo, H. _et al._ Engineered bacterial orthogonal DNA replication system for continuous evolution. _Nat Chem Biol_ 19, 1504–1512 (2023).

https://doi.org/10.1038/s41589-023-01387-2 Download citation * Received: 09 July 2022 * Accepted: 16 June 2023 * Published: 13 July 2023 * Issue Date: December 2023 * DOI:

https://doi.org/10.1038/s41589-023-01387-2 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative