Abstract
Bacteria are versatile living systems that enhance our understanding of nature and enable biosynthesis of valuable chemicals. Long fragment editing techniques are of great importance for accelerating bacterial genome engineering to obtain desirable and genetically stable strains. However, the existing genome editing methods cannot meet the needs of engineers. We herein report an efficient long fragment editing method for large-scale and scarless genome engineering in Escherichia coli. The method enabled us to insert DNA fragments up to 12 kb into the genome and to delete DNA fragments up to 186.7 kb from the genome, with positive rates over 95%. We applied this method for E. coli genome simplification, resulting in 12 individual deletion mutants and four cumulative deletion mutants. The simplest genome lost a total of 370.6 kb of DNA sequence containing 364 open reading frames. Additionally, we applied this technique to metabolic engineering and obtained a genetically stable plasmid-independent isobutanol production strain that produced 1.3 g/L isobutanol via shake-flask fermentation. These results suggest that the method is a powerful genome engineering tool, highlighting its potential to be applied in synthetic biology and metabolic engineering.
Key points
• This article reports an efficient genome engineering tool for E. coli.
• The tool is advantageous for the manipulations of long DNA fragments.
• The tool has been successfully applied for genome simplification.
• The tool has been successfully applied for metabolic engineering.
Similar content being viewed by others
References
Atsumi S, Hanai T, Liao JC (2008) Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 451:86–89. https://doi.org/10.1038/nature06450
Atsumi S, Li Z, Liao JC (2009) Acetolactate synthase from Bacillus subtilis serves as a 2-ketoisovalerate decarboxylase for isobutanol biosynthesis in Escherichia coli. Appl Environ Microbiol 75:6306–6311. https://doi.org/10.1128/AEM.01160-09
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:8 https://doi.org/10.1038/msb4100050
Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1462. https://doi.org/10.1126/science.277.5331.1453
Bujard H, Gentz R, Lanzer M, Stueber D, Mueller M, Ibrahimi I, Haeuptle MT, Dobberstein B (1987) A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol 155:416–433. https://doi.org/10.1016/0076-6879(87)55028-5
Chayot R, Montagne B, Mazel D, Ricchetti M (2010) An end-joining repair mechanism in Escherichia coli. Proc Natl Acad Sci U S A 107:2141–2146. https://doi.org/10.1073/pnas.0906355107
Chen CT, Liao JC (2016) Frontiers in microbial 1-butanol and isobutanol production. FEMS Microbiol Lett 363:fnw020. https://doi.org/10.1093/femsle/fnw020
Chung ME, Yeh IH, Sung LY, Wu MY, Chao YP, Ng IS, Hu YC (2017) Enhanced integration of large DNA into E. coli chromosome by CRISPR/Cas9. Biotechnol Bioeng 114:172–183. https://doi.org/10.1002/bit.26056
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. https://doi.org/10.1073/pnas.120163297
Elowitz MB, Leibler S (2000) A synthetic oscillatory network of transcriptional regulators. Nature 403:335–338. https://doi.org/10.1038/35002125
Fang H, Li D, Kang J, Jiang P, Sun J, Zhang D (2018) Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12. Nat Commun 9:4917. https://doi.org/10.1038/s41467-018-07412-6
Feng X, Zhao D, Zhang X, Ding X, Bi C (2018) CRISPR/Cas9 assisted multiplex genome editing technique in Escherichia coli. Biotechnol J 13:1700604. https://doi.org/10.1002/biot.201700604
Garst AD, Bassalo MC, Pines G, Lynch SA, Halweg-Edwards AL, Liu R, Liang L, Wang Z, Zeitoun R, Alexander WG, Gill RT (2017) Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering. Nat Biotechnol 35:48–55. https://doi.org/10.1038/nbt.3718
Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109:e2579–e2586. https://doi.org/10.1073/pnas.1208507109
Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318
Hashimoto M, Ichimura T, Mizoguchi H, Tanaka K, Fujimitsu K, Keyamura K, Ote T, Yamakawa T, Yamazaki Y, Mori H, Katayama T, Kato J (2005) Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome. Mol Microbiol 55:137–149. https://doi.org/10.1111/j.1365-2958.2004.04386.x
Huang C, Ding T, Wang J, Wang X, Guo L, Wang J, Zhu L, Bi C, Zhang X, Ma X, Huo YX (2019) CRISPR-Cas9-assisted native end-joining editing offers a simple strategy for efficient genetic engineering in Escherichia coli. Appl Microbiol Biotechnol 103:8497–8509. https://doi.org/10.1007/s00253-019-10104-w
Huo YX, Cho KM, Rivera JG, Monte E, Shen CR, Yan Y, Liao JC (2011) Conversion of proteins into biofuels by engineering nitrogen flux. Nat Biotechnol 29:346–351. https://doi.org/10.1038/nbt.1789
Huo YX, Ren H, Yu H, Zhao L, Yu S, Yan Y, Chen Z (2018) CipA-mediating enzyme self-assembly to enhance the biosynthesis of pyrogallol in Escherichia coli. Appl Microbiol Biotechnol 102:10005–10015. https://doi.org/10.1007/s00253-018-9365-y
Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM (2011) Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333:348–353. https://doi.org/10.1126/science.1205822
Jeong J, Cho N, Jung D, Bang D (2013) Genome-scale genetic engineering in Escherichia coli. Biotechnol Adv 31:804–810. https://doi.org/10.1016/j.biotechadv.2013.04.003
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
Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 81:2506–2514. https://doi.org/10.1128/AEM.04023-14
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Kang Y, Durfee T, Glasner JD, Qiu Y, Frisch D, Winterberg KM, Blattner FR (2004) Systematic mutagenesis of the Escherichia coli genome. J Bacteriol 186:4921–4930. https://doi.org/10.1128/JB.186.15.4921-4930.2004
Kato J, Hashimoto M (2007) Construction of consecutive deletions of the Escherichia coli chromosome. Mol Syst Biol 3:132. https://doi.org/10.1038/msb4100174
Kato J, Hashimoto M (2008) Construction of long chromosomal deletion mutants of Escherichia coli and minimization of the genome. Methods Mol Biol 416:279–293. https://doi.org/10.1007/978-1-59745-321-9_18
Keseler IM, Mackie A, Peralta-Gil M, Santos-Zavaleta A, Gama-Castro S, Bonavides-Martínez C, Fulcher C, Huerta AM, Kothari A, Krummenacker M, Latendresse M, Muñiz-Rascado L, Ong Q, Paley S, Schröder I, Shearer AG, Subhraveti P, Travers M, Weerasinghe D, Weiss V, Collado-Vides J, Gunsalus RP, Paulsen I, Karp PD (2013) EcoCyc: fusing model organism databases with systems biology. Nucleic Acids Res 41:D605–D612. https://doi.org/10.1093/nar/gks1027
Keseler IM, Mackie A, Santos-Zavaleta A, Billington R, Bonavides-Martínez C, Caspi R, Fulcher C, Gama-Castro S, Kothari A, Krummenacker M, Latendresse M, Muñiz-Rascado L, Ong Q, Paley S, Peralta-Gil M, Subhraveti P, Velázquez-Ramírez DA, Weaver D, Collado-Vides J, Paulsen I, Karp PD (2017) The EcoCyc database: reflecting new knowledge about Escherichia coli K-12. Nucleic Acids Res 45:D513–D550. https://doi.org/10.1093/nar/gkw1003
Koob MD, Shaw AJ, Cameron DC (1994) Minimizing the genome of Escherichia coli. Ann N Y Acad Sci 745:1–3. https://doi.org/10.1111/j.1749-6632.1994.tb44359.x
Lan EI, Liao JC (2013) Microbial synthesis of n-butanol, isobutanol, and other higher alcohols from diverse resources. Bioresour Technol 135:339–349. https://doi.org/10.1016/j.biortech.2012.09.104
Lee SK, Chou HH, Pfleger BF, Newman JD, Yoshikuni Y, Keasling JD (2007) Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters. Appl Environ Microbiol 73:5711–5715. https://doi.org/10.1128/AEM.00791-07
Li Y, Lin Z, Huang C, Zhang Y, Wang Z, Tang Y, Chen T, Zhao H (2015) Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing. Metab Eng 31:13–21. https://doi.org/10.1016/j.ymben.2015.06.006
Li L, Liu X, Wei K, Lu Y, Jiang W (2019a) Synthetic biology approaches for chromosomal integration of genes and pathways in industrial microbial systems. Biotechnol Adv 37:730–745. https://doi.org/10.1016/j.biotechadv.2019.04.002
Li Y, Yan F, Wu H, Li G, Han Y, Ma Q, Fan X, Zhang C, Xu Q, Xie X, Chen N (2019b) Multiple-step chromosomal integration of divided segments from a large DNA fragment via CRISPR/Cas9 in Escherichia coli. J Ind Microbiol Biotechnol 46:81–90. https://doi.org/10.1007/s10295-018-2114-5
Liang S, Chen H, Liu J, Wen J (2018) Rational design of a synthetic Entner-Doudoroff pathway for enhancing glucose transformation to isobutanol in Escherichia coli. J Ind Microbiol Biotechnol 45:187–199. https://doi.org/10.1007/s10295-018-2017-5
Mignon C, Sodoyer R, Werle B (2015) Antibiotic-free selection in biotherapeutics: now and forever. Pathogens 4:157–181. https://doi.org/10.3390/pathogens4020157
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
Paddon CJ, Keasling JD (2014) Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development. Nat Rev Microbiol 12:355–367. https://doi.org/10.1038/nrmicro3240
Pines G, Freed EF, Winkler JD, Gill RT (2015) Bacterial recombineering: genome engineering via phage-based homologous recombination. ACS Synth Biol 4:1176–1185. https://doi.org/10.1021/acssynbio.5b00009
Rong M, He B, McAllister WT, Durbin RK (1998) Promoter specificity determinants of T7 RNA polymerase. Proc Natl Acad Sci U S A 95:515–519. https://doi.org/10.1073/pnas.95.2.515
Saini M, Wang ZW, Chiang CJ, Chao YP (2017) Metabolic engineering of Escherichia coli for production of n-butanol from crude glycerol. Biotechnol Biofuels 10:173. https://doi.org/10.1186/s13068-017-0857-2
Sharan SK, Thomason LC, Kuznetsov SG, Court DL (2009) Recombineering: a homologous recombination-based method of genetic engineering. Nat Protoc 4:206–223. https://doi.org/10.1038/nprot.2008.227
Shipman SL, Nivala J, Macklis JD, Chruch GM (2017) CRISPR-Cas encoding of a digital movie into the genomes of a population of living bacteria. Nature 547:345–349. https://doi.org/10.1038/nature23017
Su T, Liu F, Gu P, Jin H, Chang Y, Wang Q, Liang Q, Qi Q (2016) A CRISPR-Cas9 assisted non-homologous end-joining strategy for one-step engineering of bacterial genome. Sci Rep 6:37895. https://doi.org/10.1038/srep37895
Su T, Liu F, Chang Y, Guo Q, Wang J, Wang Q, Qi Q (2019) The phage T4 DNA ligase mediates bacterial chromosome DSBs repair as single component non-homologous end joining. Synth Syst Biotechnol 4:107–112. https://doi.org/10.1016/j.synbio.2019.04.001
Tao H, Bausch C, Richmond C, Blattner FR, Conway T (1999) Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J Bacteriol 181:6425–6440. https://doi.org/10.1128/JB.181.20.6425-6440.1999.
Thompson MG, Sedaghatian N, Barajas JF, Wehrs M, Bailey CB, Kaplan N, Hillson NJ, Mukhopadhyay A, Keasling JD (2018) Isolation and characterization of novel mutations in the pSC101 origin that increase copy number. Sci Rep 8:1590. https://doi.org/10.1038/s41598-018-20016-w
Tischer BK, von Einem J, Kaufer B, Osterrieder N (2006) Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli. Biotechniques 40:191–197. https://doi.org/10.2144/000112096
Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460:894–898. https://doi.org/10.1038/nature08187
Warner JR, Reeder PJ, Karimpour-Fard A, Woodruff LB, Gill RT (2010) Rapid profiling of a microbial genome using mixtures of barcoded oligonucleotides. Nat Biotechnol 28:856–862. https://doi.org/10.1038/nbt.1653
Wu Z, Wang Y, Zhang Y, Chen W, Wang Y, Ji Q (2019) Strategies for developing CRISPR-based gene editing methods in bacteria small methods 4:https://doi.org/10.1002/smtd.201900560
Yamamoto N, Nakahigashi K, Nakamichi T, Yoshino M, Takai Y, Touda Y, Furubayashi A, Kinjyo S, Dose H, Hasegawa M, Datsenko KA, Nakayashiki T, Tomita M, Wanner BL, Mori H (2009) Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5:335. https://doi.org/10.1038/msb.2009.92
Yang J, Sun B, Huang H, Jiang Y, Diao L, Chen B, Xu C, Wang X, Liu J, Jiang W, Yang S (2014) High-efficiency scarless genetic modification in Escherichia coli by using lambda red recombination and I-SceI cleavage. Appl Environ Microbiol 80:3826–3834. https://doi.org/10.1128/AEM.00313-14
Yu BJ, Sung BH, Koob MD, Lee CH, Lee JH, Lee WS, Kim MS, Kim SC (2002) Minimization of the Escherichia coli genome using a Tn5-targeted Cre/loxP excision system. Nat Biotechnol 20:1018–1023. https://doi.org/10.1038/nbt740
Yu BJ, Kang KH, Lee JH, Sung BH, Kim MS, Kim SC (2008) Rapid and efficient construction of markerless deletions in the Escherichia coli genome. Nucleic Acids Res 36:e84. https://doi.org/10.1093/nar/gkn359
Yu Y, Zhu X, Xu H, Zhang X (2019) Construction of an energy-conserving glycerol utilization pathways for improving anaerobic succinate production in Escherichia coli. Metab Eng 56:181–189. https://doi.org/10.1016/j.ymben.2019.10.002
Zhang H, Cheng QX, Liu AM, Zhao GP, Wang J (2017) A novel and efficient method for bacteria genome editing employing both CRISPR/Cas9 and an antibiotic resistance cassette. Front Microbiol 8:812. https://doi.org/10.3389/fmicb.2017.00812
Zhao D, Yuan S, Xiong B, Sun H, Ye L, Li J, Zhang X, Bi C (2016) Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Factories 15:205. https://doi.org/10.1186/s12934-016-0605-5
Zhao D, Feng X, Zhu X, Wu T, Zhang X, Bi C (2017) CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency. Sci Rep 7:16624. https://doi.org/10.1038/s41598-017-16998-8
Zheng X, Li SY, Zhao GP, Wang J (2017) An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis. Biochem Biophys Res 485:768–774. https://doi.org/10.1016/j.bbrc.2017.02.129
Acknowledgments
This work was jointly supported by The National Key Research and Development Program of China (No. 2019YFA0904100, to Yi-Xin Huo) and The National Natural Science Foundation of China (No. 31961133014, to Yi-Xin Huo).
Author information
Authors and Affiliations
Contributions
YXH, CH, and LG conceived and designed research. CH, LG, JW, and NW conducted experiments. YXH contributed new reagents and analytical tools. YXH, CH, and LG analyzed data. YXH, CH, and LG wrote the manuscript. All authors read and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants performed by any of the authors.
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 26951 kb)
Rights and permissions
About this article
Cite this article
Huang, C., Guo, L., Wang, J. et al. Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering. Appl Microbiol Biotechnol 104, 7943–7956 (2020). https://doi.org/10.1007/s00253-020-10819-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10819-1