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Engineered bacterial orthogonal DNA replication system for continuous evolution

Nature Chemical Biology volume 19, pages 1504–1512 (2023)Cite this article

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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 genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.

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Fig. 1: Design and construction of BacORep.
Fig. 2: Developing a linear plasmid system by hijacking the DNA replication system of the temperate phage GIL16 in B. thuringiensis.
Fig. 3: Rational design and characterization of error-prone ODNAPs.
Fig. 4: Continuous evolution for obtaining generally applicable strong promoters in different bacteria and strains.
Fig. 5: Continuous evolution for obtaining efficient methanol-assimilating B. thuringiensis.

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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.

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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).

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Authors and Affiliations

  1. 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

  2. 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

  3. 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

  4. 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

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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.

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Correspondence to Yanfeng Liu.

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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 PgraC. (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.

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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.

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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.

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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.

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Supplementary information

Supplementary Information

Supplementary Fig. 1, Tables 1–6, References and Note

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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

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