Abstract
Phage recombinase function unit (PRFU) plays a key role in the life cycle of phage. Repurposing this system such as lambda-Redαβ or Rac-RecET for recombineering has gained success in Escherichia coli. Previous studies have showed that most PRFUs only worked well in its native hosts but poorly in the distant species. Thus, identification of new PRFUs in specific species is necessary for the development of its corresponding genetic engineering tools. Here, we present a thorough study of PRFUs in the genomes of genus Corynebacterium. We first used a database to database searching method to facilitate accurate prediction of novel PRFUs in 423 genomes. A total number of 60 sets of unique PRFUs were identified and divided into 8 types based on evolution affinities. Recombineering ability of the 8 representative PRFUs was experimentally verified in the Corynebacterium glutamicum ATCC 13032 strain. In particular, PRFU from C. aurimucosum achieved highest efficiency in both ssDNA and dsDNA mediated recombineering, which is expected to greatly facilitate genome engineering in genus Corynebacterium. These results will provide new insights for the study and application of PRFUs.
Key points
• First report of bioinformatic mining and systematic analysis of Phage recombinase function unit (PRFU) in Corynebacterium genomes.
• Recombineering ability of the representative PRFUs was experimentally verified in Corynebacterium glutamicum ATCC 13032 strain.
• PRFU with the highest recombineering efficiency at 10-2 magnitude was identified from Corynebacterium aurimucosum.
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Data Availability
1. Genomes: ftp://ftp.ncbi.nlm.nih.gov/genomes/all
2. Conserved domain: https://www.ncbi.nlm.nih.gov/cdd/
3. Genome information: https://www.ncbi.nlm.nih.gov/genome/, the Genome Assembly and Annotation report section
4. Protein structure: https://www.rcsb.org/
References
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402. https://doi.org/10.1093/nar/25.17.3389
Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, Wishart DS (2016) PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 44(W1):W16–W21. https://doi.org/10.1093/nar/gkw387
Binder S, Siedler S, Marienhagen J, Bott M, Eggeling L (2013) Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation. Nucleic Acids Res 41(12):6360–6369. https://doi.org/10.1093/nar/gkt312
Caldwell BJ, Zakharova E, Filsinger GT, Wannier TM, Hempfling JP, Chun-Der L, Pei D, Church GM, Bell CE (2019) Crystal structure of the Redbeta C-terminal domain in complex with lambda Exonuclease reveals an unexpected homology with lambda Orf and an interaction with Escherichia coli single stranded DNA binding protein. Nucleic Acids Res 47(4):1950–1963. https://doi.org/10.1093/nar/gky1309
Cho JS, Choi KR, Prabowo CPS, Shin JH, Yang D, Jang J, Lee SY (2017) CRISPR/Cas9-coupled recombineering for metabolic engineering of Corynebacterium glutamicum. Metab Eng 42:157–167. https://doi.org/10.1016/j.ymben.2017.06.010
Datta S, Costantino N, Zhou X, Court DL (2008) Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages. Proc Natl Acad Sci U S A 105(5):1626–1631. https://doi.org/10.1073/pnas.0709089105
Dong H, Tao W, Gong F, Li Y, Zhang Y (2014) A functional recT gene for recombineering of Clostridium. J Biotechnol 173:65–77. https://doi.org/10.1016/j.jbiotec.2013.12.011
Drozdetskiy A, Cole C, Procter J, Barton GJ (2015) JPred4: a protein secondary structure prediction server. Nucleic Acids Res 43(W1):W389–W394. https://doi.org/10.1093/nar/gkv332
Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797. https://doi.org/10.1093/nar/gkh340
Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Muller R, Stewart AF, Zhang Y (2012) Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat Biotechnol 30(5):440–446. https://doi.org/10.1038/nbt.2183
Garriss G, Poulin-Laprade D, Burrus V (2013) DNA-damaging agents induce the RecA-independent homologous recombination functions of integrating conjugative elements of the SXT/R391 family. J Bacteriol 195(9):1991–2003. https://doi.org/10.1128/JB.02090-12
Huang Y, Li L, Xie S, Zhao N, Han S, Lin Y, Zheng S (2017) Recombineering using RecET in Corynebacterium glutamicum ATCC14067 via a self-excisable cassette. Sci Rep 7(1):7916. https://doi.org/10.1038/s41598-017-08352-9
Iyer LM, Koonin EV, Aravind L (2002) Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52. BMC Genomics 3:8. https://doi.org/10.1186/1471-2164-3-8
Jiang Y, Qian F, Yang J, Liu Y, Dong F, Xu C, Sun B, Chen B, Xu X, Li Y, Wang R, Yang S (2017) CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 8:15179. https://doi.org/10.1038/ncomms15179
Kagawa W, Kurumizaka H, Ishitani R, Fukai S, Nureki O, Shibata T, Yokoyama S (2002) Crystal structure of the homologous-pairing domain from the human Rad52 recombinase in the undecameric form. Mol Cell 10(2):359–371. https://doi.org/10.1016/S1097-2765(02)00587-7
Li C, Swofford CA, Ruckert C, Sinskey AJ (2021) Optimizing recombineering in Corynebacterium glutamicum. Biotechnol Bioeng 118:2255–2264. https://doi.org/10.1002/bit.27737
Liu J, Wang Y, Lu Y, Zheng P, Sun J, Ma Y (2017) Development of a CRISPR/Cas9 genome editing toolbox for Corynebacterium glutamicum. Microb Cell Factories 16(1):205. https://doi.org/10.1186/s12934-017-0815-5
Lloyd JA, McGrew DA, Knight KL (2005) Identification of residues important for DNA binding in the full-length human Rad52 protein. J Mol Biol 345(2):239–249. https://doi.org/10.1016/j.jmb.2004.10.065
Lopes A, Amarir-Bouhram J, Faure G, Petit MA, Guerois R (2010) Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res 38(12):3952–3962. https://doi.org/10.1093/nar/gkq096
Murphy KC (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180(8):2063–2071
Murphy KC (2012) Phage recombinases and their applications. Adv Virus Res 83:367–414. https://doi.org/10.1016/b978-0-12-394438-2.00008-6
Muyrers JP, Zhang Y, Buchholz F, Stewart AF (2000) RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev 14(15):1971–1982. https://doi.org/10.1101/gad.14.15.1971
Nakamura Y, Nishio Y, Ikeo K, Gojobori T (2003) The genome stability in Corynebacterium species due to lack of the recombinational repair system. Gene 317(1-2):149–155. https://doi.org/10.1016/S0378-1119(03)00653-X
Oliveira A, Oliveira LC, Aburjaile F, Benevides L, Tiwari S, Jamal SB, Silva A, Figueiredo HCP, Ghosh P, Portela RW, Azevedo VAD, Wattam AR (2017) Insight of Genus Corynebacterium: Ascertaining the Role of Pathogenic and Non-pathogenic Species. Front Microbiol 8. https://doi.org/10.3389/fmicb.2017.01937
Ploquin M, Bransi A, Paquet ER, Stasiak AZ, Stasiak A, Yu X, Cieslinska AM, Egelman EH, Moineau S, Masson JY (2008) Functional and structural basis for a bacteriophage homolog of human RAD52. Curr Biol 18(15):1142–1156. https://doi.org/10.1016/j.cub.2008.06.071
Resende BC, Rebelato AB, D'Afonseca V, Santos AR, Stutzman T, Azevedo VA, Santos LL, Miyoshi A, Lopes DO (2011) DNA repair in Corynebacterium model. Gene 482(1-2):1–7. https://doi.org/10.1016/j.gene.2011.03.008
Smith CE, Bell CE (2016) Domain Structure of the Redbeta Single-Strand Annealing Protein: the C-terminal Domain is Required for Fine-Tuning DNA-binding Properties, Interaction with the Exonuclease Partner, and Recombination in vivo. J Mol Biol 428(3):561–578. https://doi.org/10.1016/j.jmb.2016.01.008
Su TY, Jin HY, Zheng Y, Zhao Q, Chang YZ, Wang Q, Qi QS (2018) Improved ssDNA recombineering for rapid and efficient pathway engineering in Corynebacterium glutamicum. J Chem Technol Biotechnol 93(12):3535–3542. https://doi.org/10.1002/jctb.5726
Swingle B, Bao Z, Markel E, Chambers A, Cartinhour S (2010) Recombineering using RecTE from Pseudomonas syringae. Appl Environ Microbiol 76(15):4960–4968. https://doi.org/10.1128/aem.00911-10
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat Methods 4(2):147–152. https://doi.org/10.1038/nmeth996
van Kessel JC, Marinelli LJ, Hatfull GF (2008) Recombineering mycobacteria and their phages. Nat Rev Microbiol 6(11):851–857. https://doi.org/10.1038/nrmicro2014
van Pijkeren JP, Britton RA (2012) High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res 40(10):e76. https://doi.org/10.1093/nar/gks147
Vellani TS, Myers RS (2003) Bacteriophage SPP1 Chu is an alkaline exonuclease in the SynExo family of viral two-component recombinases. J Bacteriol 185(8):2465–2474
Wang B, Hu Q, Zhang Y, Shi R, Chai X, Liu Z, Shang X, Zhang Y, Wen T (2018) A RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum. Microb Cell Factories 17(1):63. https://doi.org/10.1186/s12934-018-0910-2
Yang P, Wang J, Qi Q (2015) Prophage recombinases-mediated genome engineering in Lactobacillus plantarum. Microb Cell Factories 14:154. https://doi.org/10.1186/s12934-015-0344-z
Yin J, Zhu H, Xia L, Ding X, Hoffmann T, Hoffmann M, Bian X, Muller R, Fu J, Stewart AF, Zhang Y (2015) A new recombineering system for Photorhabdus and Xenorhabdus. Nucleic Acids Res 43(6):e36. https://doi.org/10.1093/nar/gku1336
Yu X, Jin H, Liu W, Wang Q, Qi Q (2015) Engineering Corynebacterium glutamicum to produce 5-aminolevulinic acid from glucose. Microb Cell Factories 14:183. https://doi.org/10.1186/s12934-015-0364-8
Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20(2):123–128. https://doi.org/10.1038/2417
Zhao N, Li L, Luo G, Xie S, Lin Y, Han S, Huang Y, Zheng S (2020) Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum. J Ind Microbiol Biotechnol 47(8):599–608. https://doi.org/10.1007/s10295-020-02304-5
Acknowledgements
We thank Haiying Jin for her help in the recombineering with ssDNA experiment. We also thank Qilong Qin for reviewing the manuscript.
Author and contributors
YZ participated in the conceptualization, data curation, formal analysis, investigation, methodology, project administration, software, validation, visualization, and writing process. QS participated in the conceptualization, funding acquisition, project administration, resources, supervision, writing review and editing process. QW participate in the funding acquisition and writing-review and editing process. TY participated in the conceptualization of oligo-mediated recombineering and methodology of oligo-mediated recombineering, resources and writing-review and editing process.
Funding
This work was financially supported by the grant from the National Natural Science Foundation of China (31730003), National Key Research and Development Program of China (2019YFA0904004), Key R&D Program of Shandong Province (2020CXGC010602), and the China Postdoctoral Science Foundation funded project (2019M662341).
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Chang, Y., Wang, Q., Su, T. et al. Identification of phage recombinase function unit in genus Corynebacterium. Appl Microbiol Biotechnol 105, 5067–5075 (2021). https://doi.org/10.1007/s00253-021-11384-x
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DOI: https://doi.org/10.1007/s00253-021-11384-x