Abstract
Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon–based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon–based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aparicio T, Jensen SI, Nielsen AT, de Lorenzo V, Martínez-García E (2016) The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42. Biotechnol J 11:1309–1319. https://doi.org/10.1002/biot.201600317
Aparicio T, de Lorenzo V, Martínez-García E (2018) CRISPR/Cas9-based counterselection boosts recombineering efficiency in Pseudomonas putida. Biotechnol J 13:e1700161. https://doi.org/10.1002/biot.201700161
Aranda-Olmedo I, Ramos JL, Marques S (2005) Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0. Appl Environ Microbiol 71:4191–4198. https://doi.org/10.1128/AEM.71.8.4191-4198.2005
Barrangou R, van Pijkeren JP (2016) Exploiting CRISPR-Cas immune systems for genome editing in bacteria. Curr Opin Biotechnol 37:61–68. https://doi.org/10.1016/j.copbio.2015.10.003
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 41:6360–6369. https://doi.org/10.1093/nar/gkt312
Blatny JM, Brautaset T, Winther-Larsen HC, Karunakaran P, Valla S (1997) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid 38:35–51
Chen J, Qin J, Zhu YG, de Lorenzo V, Rosen BP (2013) Engineering the soil bacterium Pseudomonas putida for arsenic methylation. Appl Environ Microbiol 79:4493–4495. https://doi.org/10.1128/AEM.01133-13
Chen Z, Ling W, Shang G (2016) Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion. FEMS Microbiol Lett 363:fnw231. https://doi.org/10.1093/femsle/fnw231
Choi KR, Cho JS, Cho IJ, Park D, Lee SY (2018) Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida. Metab Eng 47:463–474. https://doi.org/10.1016/j.ymben.2018.05.003
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143
Cook TB, Rand JM, Nurani W, Courtney DK, Liu SA, Pfleger BF (2018) Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. J Ind Microbiol Biotechnol 45:517–527. https://doi.org/10.1007/s10295-017-2001-5
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645
de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis of Pseudomonas gene products using lacI q/P trp -lac plasmids and transposons that confer conditional phenotypes. Gene 123:17–24
Ellis HM, Yu D, DiTizio T, Court DL (2001) High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci U S A 98:6742–6746
Galvao TC, de Lorenzo V (2005) Adaptation of the yeast URA3 selection system to gram-negative bacteria and generation of a ΔbetCDE Pseudomonas putida strain. Appl Environ Microbiol 71:883–892
Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–444. https://doi.org/10.1126/science.aaq0179
Graf N, Altenbuchner J (2011) Development of a method for markerless gene deletion in Pseudomonas putida. Appl Environ Microbiol 77:5549–5552. https://doi.org/10.1128/AEM.05055-11
Graf N, Altenbuchner J (2013) Functional characterization and application of a tightly regulated MekR/P mekA expression system in Escherichia coli and Pseudomonas putida. Appl Microbiol Biotechnol 97:8239–8251. https://doi.org/10.1007/s00253-013-5030-7
Hoffmann J, Altenbuchner J (2015) Functional characterization of the mannitol promoter of Pseudomonas fluorescens DSM 50106 and its application for a mannitol-inducible expression system for Pseudomonas putida KT2440. PLoS One 10:e0133248. https://doi.org/10.1371/journal.pone.0133248
Jakočiūnas T, Jensen MK, Keasling JD (2016) CRISPR/Cas9 advances engineering of microbial cell factories. Metab Eng 34:44–59. https://doi.org/10.1016/j.ymben.2015.12.003
Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31:233–239. https://doi.org/10.1038/nbt.2508
Kang Y, Norris MH, Wilcox BA, Tuanyok A, Keim PS, Hoang TT (2011) Knockout and pullout recombineering for naturally transformable Burkholderia thailandensis and Burkholderia pseudomallei. Nat Protoc 6:1085–1104. https://doi.org/10.1038/nprot.2011.346
Kast P (1994) pKSS–a second-generation general purpose cloning vector for efficient positive selection of recombinant clones. Gene 138:109–114
Ko KS, Kong IC (2017) Application of the freeze-dried bioluminescent bioreporter Pseudomonas putida mt-2 KG1206 to the biomonitoring of groundwater samples from monitoring wells near gasoline leakage sites. Appl Microbiol Biotechnol 101:1709–1716. https://doi.org/10.1007/s00253-016-7974-x
Li D, Lv L, Chen JC, Chen GQ (2017) Controlling microbial PHB synthesis via CRISPRi. Appl Microbiol Biotechnol 101:5861–5867. https://doi.org/10.1007/s00253-017-8374-6
Libis V, Delepine B, Faulon JL (2016) Expanding biosensing abilities through computer-aided design of metabolic pathways. ACS Synth Biol 5:1076–1085. https://doi.org/10.1021/acssynbio.5b00225
Luo X, Yang Y, Ling W, Zhuang H, Li Q, Shang G (2016) Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination. FEMS Microbiol Lett 363:fnw014. https://doi.org/10.1093/femsle/fnw014
Martínez-García E, de Lorenzo V (2011) Engineering multiple genomic deletions in gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ Microbiol 13:2702–2716. https://doi.org/10.1111/j.1462-2920.2011.02538.x
Martínez-García E, Nikel PI, Aparicio T, de Lorenzo V (2014) Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression. Microb Cell Factories 13:159. https://doi.org/10.1186/PREACCEPT-1816907831139858
Mosberg JA, Lajoie MJ, Church GM (2010) Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate. Genetics 186:791–799. https://doi.org/10.1534/genetics.110.120782
Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der Oost J (2017) Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat Commun 8:1647. https://doi.org/10.1038/s41467-017-01591-4
Murphy KC (1998) Use of bacteriophage λ recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063–2071
Murphy KC (2016) λ Recombination and Recombineering. EcoSal Plus. https://doi.org/10.1128/ecosalplus.ESP-0011-2015
Nelson KE, Weinel C, Paulsen IT, Dodson RJ, Hilbert H, Martins dos Santos VA, Fouts DE, Gill SR, Pop M, Holmes M, Brinkac L, Beanan M, DeBoy RT, Daugherty S, Kolonay J, Madupu R, Nelson W, White O, Peterson J, Khouri H, Hance I, Chris Lee P, Holtzapple E, Scanlan D, Tran K, Moazzez A, Utterback T, Rizzo M, Lee K, Kosack D, Moestl D, Wedler H, Lauber J, Stjepandic D, Hoheisel J, Straetz M, Heim S, Kiewitz C, Eisen JA, Timmis KN, Dusterhoft A, Tummler B, Fraser CM (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808
Nikel PI, de Lorenzo V (2013) Engineering an anaerobic metabolic regime in Pseudomonas putida KT2440 for the anoxic biodegradation of 1,3-dichloroprop-1-ene. Metab Eng 15:98–112. https://doi.org/10.1016/j.ymben.2012.09.006
Nikel PI, Martinez-Garcia E, de Lorenzo V (2014) Biotechnological domestication of pseudomonads using synthetic biology. Nat Rev Microbiol 12:368–379. https://doi.org/10.1038/nrmicro3253
Noirot P, Kolodner RD (1998) DNA strand invasion promoted by Escherichia coli RecT protein. J Biol Chem 273:12274–12280
Nyerges A, Csörgő B, Nagy I, Bálint B, Bihari P, Lázár V, Apjok G, Umenhoffer K, Bogos B, Pósfai G, Pál C (2016) A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. Proc Natl Acad Sci U S A 113:2502–2507. https://doi.org/10.1073/pnas.1520040113
Obeng EM, Brossette T, Ongkudon CM, Budiman C, Maas R, Jose J (2018) The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison. Appl Microbiol Biotechnol 102:4829–4841. https://doi.org/10.1007/s00253-018-8987-4
Oh JH, van Pijkeren JP (2014) CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res 42:e131. https://doi.org/10.1093/nar/gku623
Poblete-Castro I, Becker J, Dohnt K, dos Santos VM, Wittmann C (2012) Industrial biotechnology of Pseudomonas putida and related species. Appl Microbiol Biotechnol 93:2279–2290. https://doi.org/10.1007/s00253-012-3928-0
Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183. https://doi.org/10.1016/j.cell.2013.02.022
Ravi K, Garcia-Hidalgo J, Gorwa-Grauslund MF, Liden G (2017) Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost. Appl Microbiol Biotechnol 101:5059–5070. https://doi.org/10.1007/s00253-017-8211-y
Rybalchenko N, Golub EI, Bi B, Radding CM (2004) Strand invasion promoted by recombination protein β of coliphage λ. Proc Natl Acad Sci U S A 101:17056–17060. https://doi.org/10.1073/pnas.0408046101
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Sasnow SS, Wei H, Aristilde L (2016) Bypasses in intracellular glucose metabolism in iron-limited Pseudomonas putida. Microbiologyopen 5:3–20. https://doi.org/10.1002/mbo3.287
Sawitzke JA, Costantino N, Li XT, Thomason LC, Bubunenko M, Court C, Court DL (2011) Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. J Mol Biol 407:45–59. https://doi.org/10.1016/j.jmb.2011.01.030
Shapiro RS, Chavez A, Collins JJ (2018) CRISPR-based genomic tools for the manipulation of genetically intractable microorganisms. Nat Rev Microbiol 16:333–339. https://doi.org/10.1038/s41579-018-0002-7
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223
Sun J, Wang Q, Jiang Y, Wen Z, Yang L, Wu J, Yang S (2018) Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system. Microb Cell Factories 17:41. https://doi.org/10.1186/s12934-018-0887-x
Tang W, Liu DR (2018) Rewritable multi-event analog recording in bacterial and mammalian cells. Science 360:eaap8992. https://doi.org/10.1126/science.aap899
Wang HH, Zhou XR, Liu Q, Chen GQ (2011) Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida. Appl Microbiol Biotechnol 89:1497–1507. https://doi.org/10.1007/s00253-010-2964-x
Wang H, Li Z, Jia R, Hou Y, Yin J, Bian X, Li A, Muller R, Stewart AF, Fu J, Zhang Y (2016) RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat Protoc 11:1175–1190. https://doi.org/10.1038/nprot.2016.054
Acknowledgements
We thank Dr. Josef Altenbuchner, Dr. Svein Valla, and Dr. Barry Wanner for providing the plasmids used in this study.
Funding
Funding was provided by National Natural Science Foundation of China (NSFC 81273412) and National High Technology Research and Development Program of China (2012AA02A702).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the author.
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 859 kb)
Rights and permissions
About this article
Cite this article
Wu, Z., Chen, Z., Gao, X. et al. Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing. Appl Microbiol Biotechnol 103, 2783–2795 (2019). https://doi.org/10.1007/s00253-019-09654-w
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-09654-w