Abstract
Numerous expression systems, engineered strains, and cultivation systems have been developed globally but producing recombinant proteins in the soluble form continues to remain a challenge. Escherichia coli, a preferred host for the recombinant production of biopharmaceuticals and other proteins. Up to 75% of human proteins expressed in E. coli have only 25% in an active soluble form. The proteolytic activity of Lon encoded protease triggers the inclusion bodies leading to heterogenous secreted proteins thereby hampering downstream processing and isolation. Putrescine monooxygenases are versatile with applications in iron acquisition, pathogen control, biotransformation, bio-remediation and redox reaction are still isolated from plant and microbial sources at low yields. As a prerequisite to developing protease knockout E. coli strains, using the Cre-loxP recombination strategy we have built a full-length Lon disruption cassette (5′lon-lox66-cre-KanR-lox71-3′lon) (3368 bp) consisting of upstream and downstream regions of Lon, loxP sites, and Cre gene driven by T7 promoter to the expression of Cre recombinase and a selectable kanamycin resistance gene. Here, after the integration of the knock-out cassette into the host genome, we show the production of homogeneous protein species of recombinant Putrescine monooxygenase by using an E. coli platform strain in which Lon gene is deleted. This Lon knock-out strain secreted more homogeneous protein at a volumetric yield of 60% of the wild-type strain.
Similar content being viewed by others
References
Demain AL, Preeti V (2009) Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv 27(3):297–306. https://doi.org/10.1016/j.biotechadv.2009.01.008
Gupta SK, Shukla P (2016) Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 36(6):1089–1098. https://doi.org/10.3109/07388551.2015.1084264
Bird LE (2011) High throughput construction and small scale expression screening of multi-tag vectors in Escherichia coli. Methods 55:29–37. https://doi.org/10.1016/j.ymeth.2011.08.002
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbial Biotechnol 60:523–553. https://doi.org/10.1007/s00253-002-1158-6
Jana S, Deb JK (2005) Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol 67:289–298. https://doi.org/10.1007/s00253-004-1814-0
Rozkov A, Schweda T, Veide A (2000) Dynamics of proteolysis band and it s influence and on the accumulation of intracellular recombination proteins. Enzyme Microb Technol 27(10):743–748. https://doi.org/10.1016/s0141-0229(00)00294-5
Pacheco B, Crombet L, Loppnau P, Cossar D (2012) A Screening strategy for heterologous protein expression in Escherichia coli with the highest return of investment. Protein Expr Purif 81:33–41. https://doi.org/10.1016/j.pep.2011.08.030
Studier FW, Daegelen P, Lenski RE, Maslov S, Kim JF (2009) Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E coli B and K-12 genomes. J Mol Biol 394(4):653–80. https://doi.org/10.1016/j.jmb.2009.09.021
Hui CY, Guo Y, He QS, Peng L, Wu SC, Cao H, Huang SH (2010) Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides. Microbiol Immunol 54(8):452–459. https://doi.org/10.1111/j.1348-0421.2010.00238.x
Sreenivas S, Krishnaiah SM, Anil HS, Mallikarjun N, Govindappa N, Chatterjee A, Kedarnath NS (2016) Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris. Protein Expr Purif 118:1–9. https://doi.org/10.1016/j.pep.2015.10.002
Yan X, Yu H, Hong Q, Li S (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol 74(17):5556–5562. https://doi.org/10.1128/AEM.01156-08
Qian W, Song H, Liu Y, Zhang C, Niu Z, Wang H, Qiu B (2009) Improved gene disruption method and Cre-loxP mutant system for multiple gene disruptions in Hansenula polymorpha. J Microbiol Methods 79:253–259. https://doi.org/10.1016/j.mimet.2009.09.004
Dutra BE, Sutera VA, Lovett ST (2007) RecA-independent recombination is efficient but limited by exonucleases. PNAS 104(1):216–221.
Werten MW, Van den Bosch TJ, Wind RD, Mooibroek H, DeWolf FA (1999) High yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1077–1096. https://doi.org/10.1002/(SICI)1097-0061(199908)15:11
Serra-Moreno R, Acosta S, Hernalsteens JP (2006) Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes. BMC Molecular Biol 7:31–37. https://doi.org/10.1186/1471-2199-7-31
Santos LDF, Caraty-Philippe L, Darbon E, Pernodet J-L (2022) Marker-free genome engineering in amycolatopsis using the pSAM2 site-specific recombination system. Microorganisms 10:828. https://doi.org/10.3390/microorganisms10040828
Sharma MS, Mukherjee AK (2014) Genome engineering for improved recombinant protein expression in Escherichia coli. Microb Cell Fact 19(13):177. https://doi.org/10.1186/s12934-014-0177-1
Srividya D, Anil HS, Saroja NR (2020) Expression and purification of codon-optimized Cre recombinase in E. coli. Protein Expr Purif 167:105546. https://doi.org/10.1016/j.pe.2019.105546
Sambrook, J, Russell, DW (2001) Molecular cloning: a laboratory manual; cold spring harbor laboratory press: cold spring harbor, NY, USA, 2001; ISBN 0879695773
Nikolai A, Shevchuk AV, Bryksin YA, Nusinovich F, Cabello C, Margaret S, Stephan L (2004) Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucl Acids Res 32(2):16–19. https://doi.org/10.1093/nar/gnh014
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Reeder R, Sobrado P (2011) Substrate binding modulates the activity of Mycobacterium smegmatis G, a flavin-dependent monooxygenase involved in the biosynthesis of hydroxamate-containing siderophores. Biochemistry 508:489–8496. https://doi.org/10.1021/bi200933h
Visser MB, Majumdar S, Hani E, Sokol PA (2004) Importance of the ornibactin and pyochelin siderophore transport systems in Burkholderia cenocepacia lung infections. Infect Immun 72(5):2850–2857. https://doi.org/10.1128/iai.72.5.2850-2857.2004
Saroja NR, Anil HS, Srividya D, Supreetha K (2019) Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95 Protein Expr Purif, 157:9–16. doi: https://doi.org/10.1016/j.pep.2019.01.006.
Khushoo A, Pal Y, Mukherjee KJ (2005) Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor. Appl Microbiol Biotechnol 68(2):189–197. https://doi.org/10.1007/s00253-004-1867-0
Ohta T, Sutton MD, Guzzo A, Cole S, Ferentz AE, Walker GC (1999) Mutations affecting the ability of the Escherichia coli UmuD′ protein to participate in SOS mutagenesis. J Bacteriol 181(1):177–185. https://doi.org/10.1128/jb.181.1.177-185.1999
Sharma AK, Shukla E, Janoti DS, Mukherjee KJ, Shiloach J (2020) A novel knock out strategy to enhance recombinant protein expression in Escherichia coli. Microb Cell Fact 19:148. https://doi.org/10.1186/s12934-020-01407-z
Yan X, Yu H, Hong Q, Li S (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol 74:5556–5562. https://doi.org/10.1128/AEM.01156-08
Acknowledgements
Funding by Vision Group for Science and Technology, Govt. of Karnataka, (GRD No.869)
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
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.
About this article
Cite this article
Rao, S.N., Kumari, G.M., Srividya, D. et al. Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase. Indian J Microbiol 63, 56–64 (2023). https://doi.org/10.1007/s12088-023-01056-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12088-023-01056-x