Part:BBa_K5246039
Rosetta(DE3)pLysS ΔWecB
Introduction
Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - Caulobacter crescentus and Hirschia baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used Escherichia coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.
Strain description
Escherichia coli Rosetta(DE3)pLysS protein expression strain derived from Escherichia coli BL21(DE3) designed to enhance the expression of proteins that contain codons rarely used in E. coli and have chloramphenicol-resistant plasmid. It produces recombinant proteins under the control of T7 RNA polymerase [1]. pLysS strains express T7 lysozyme, which further suppresses basal expression of T7 RNA polymerase before induction, thus stabilizing pET recombinants encoding target proteins that affect cell growth and viability [2], [3].
| Strain | Genotype |
|---|---|
| Rosetta(DE3)pLysS | F- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSRARE2 (CamR) |
Usage and Biology
Biology
The enterobacterial common antigen (ECA) is an outer membrane glycolipid shared by all members of the Enterobacteriaceae and is restricted to this family. It is a glycophospholipid located in the outer leaflet of the outer membrane, composed of an L-glycerophosphatidyl residue linked to an aminosugar heteropolymer [4]. The carbohydrate consists of trisaccharide repeat units of N-acetyl-α-D-glucosamine, N-acetyl-β-D-mannosaminuronate, and N-acetylthomosamine [5]. The function of this molecule has remained largely unknown, partly because the biosynthesis pathways for ECA, O-antigen, and peptidoglycan overlap and partly because there are three forms of ECA that cannot currently be genetically separated [6]. Current research suggests that its function is to maintain outer membrane stability and immunogenicity, and it is involved in biofilm formation [7], [8],[9].
WecB is UDP-N-acetylglucosamine 2-epimerase that synthesizes UDP-ManNAc - a precursor substrate for the biosynthesis of ECA on an outer membrane. The enzyme catalyzes the first step in UDP-ManNAc biosynthesis, the reversible epimerization at the C-2 position between UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc) [11].
Eliminating the wecB gene results in ECA and lipopolysaccharide 08-side chain inactivation without causing high-stress levels [12].
Usage
Escherichia coli BL21(DE3) protein expression strain is used to produce recombinant proteins under the control of T7 RNA polymerase [1]. BL21(DE3) is widely used in both research and industrial settings. It is particularly popular due to its high efficiency in producing recombinant proteins. More specifically, it is widely used as medical diagnostic reagents in human healthcare, including vaccines, drugs, or antibodies, and in biochemical analysis[13].
Knocking out the wecA gene and inactivating the ECA pathway loads off the metabolic burden of synthesizing polysaccharides. It prevents ECA biosynthesis and increases precursor availability for peptidoglycan biosynthesis [14]. BL21(DE3)∆wecA strain could be used to investigate the ECA pathway further and the cell interactions with other cells or bacteriophages. This strain could be used in biotechnology as cells with altered surface properties. Biofilms are increasingly recognized as a critical global issue in many industries and come out with economic costs. Biofilms contaminate manufacturing equipment and pharmaceuticals, compromising product quality and safety. Biofilms can foul heat exchangers, pipelines, and other industrial equipment, leading to decreased performance and increased energy consumption. A strain with reduced biofilm formation could save power consumption and finances [15]. On top of that, exposed glycan structures often serve as initial receptors for host - N4 bacteriophage - recognition. Subsequent binding to a terminal or secondary receptor directly on the cell surface triggers irreversible adsorption and injection of the phage genome. [16] N4 bacteriophage infects industrially grown bacteria. Eliminating recognition antigens on these bacteria could provide them additional resistance to N4 phage infection. [17].
In our project, Synhesion, we aimed to produce an efficient polysaccharide in E. coli by introducing a new synthesis pathway. This pathway originated from the bacteria C. crescentus</> and <i>H. baltica, which inhabit aquatic environments. Although polysaccharide synthesis pathways in E. coli and marine bacteria are analogous, we aimed to engineer an E. coli strain that could produce desired polysaccharides more efficiently by reducing its metabolic burden. For this reason, we chose to eliminate the polysaccharide-producing ECA pathway from E. coli by knocking out the wecA gene.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1520
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1520
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1520
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1520
Illegal AgeI site found at 385 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
| genotype | F- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSRARE (CamR)(KanR) ΔwecB |
Experimental characterization
To create an ECA pathway deficient E. coli BL21(DE3)∆wecA strain, we used a homology recombineering Red/ET recombination system. Our edited colonies had kanamycin-resistance cassettes as markers in the edited region, and we tested them by colony PCR (Fig 1.). We could distinguish edited and non-edited colonies by size: PCR’ed non-edited region 1544 bp and with edited region 1840 bp.
References
1.Langenscheid, J., Killmann, H., & Braun, V. (2004). A FhuA mutant of Escherichia coli is infected by phage T1 independent of TonB. FEMS Microbiology Letters, 234(1), 133–137. https://doi.org/10.1016/j.femsle.2004.03.019
2.Tegel, H., Tourle, S., Ottosson, J., & Persson, A. (2010). Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3). Protein Expression and Purification, 69(2), 159–167. https://doi.org/10.1016/j.pep.2009.08.017
3.Fu, W., Lin, J., & Cen, P. (2007). 5-Aminolevulinate production with recombinant Escherichia coli using a rare codon optimizer host strain. Applied Microbiology and Biotechnology, 75(4), 777–782. https://doi.org/10.1007/s00253-007-0887-y
4.Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
5.Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
6.Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
7.Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z
8.Jorgenson, M. A., Kannan, S., Laubacher, M. E., & Young, K. D. (2016). Dead-end intermediates in the enterobacterial common antigen pathway induce morphological defects in Escherichia coli by competing for undecaprenyl phosphate. Molecular Microbiology, 100(1), 1–14. https://doi.org/10.1111/mmi.13304
9.Zeidan, A. A., Poulsen, V. K., Janzen, T., Buldo, P., Derkx, P. M. F., Øregaard, G., & Neves, A. R. (2017). Polysaccharide production by lactic acid bacteria: From genes to industrial applications. FEMS Microbiology Reviews, 41(Suppl_1), S168–S200. https://doi.org/10.1093/femsre/fux017
10.Rai, A. K., Carr, J. F., Bautista, D. E., Wang, W., & Mitchell, A. M. (2021). ElyC and cyclic enterobacterial common antigen regulate synthesis of phosphoglyceride-linked enterobacterial common antigen. mBio, 12(5), e02846-21. https://doi.org/10.1128/mBio.02846-21
11.Highmore, C. J., Melaugh, G., Morris, R. J., & Rice, S. A. (2022). Translational challenges and opportunities in biofilm science: A BRIEF for the future. npj Biofilms and Microbiomes, 8(1), 68. https://doi.org/10.1038/s41522-022-00327-7
12.Nobrega, F. L., Vlot, M., de Jonge, P. A., Drost, M., & Brouns, S. J. J. (2018). Targeting mechanisms of tailed bacteriophages. Nature Reviews Microbiology, 16(12), 760–773. https://doi.org/10.1038/s41579-018-0070-8
13.McPartland, J., & Rothman-Denes, L. B. (2009). The tail sheath of bacteriophage N4 interacts with the Escherichia coli receptor. Journal of Bacteriology, 191(2), 525–532. https://doi.org/10.1128/JB.01423-08
14.Rick, P. D., Hubbard, G. L., & Barr, K. (1994). Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12. Journal of Bacteriology, 176(9), 2877–2884. https://doi.org/10.1128/jb.176.9.2877-2884.1994
15.Klena, J. D., & Schnaitman, C. A. (1993). Function of the rfb gene cluster and the rfe gene in the synthesis of O antigen by Shigella dysenteriae 1. Molecular Microbiology, 9(2), 393–402. https://doi.org/10.1111/j.1365-2958.1993.tb01705.x
16.Alexander, D. C., & Valvano, M. A. (1994). Role of the rfe gene in the biosynthesis of the Escherichia coli O7-specific lipopolysaccharide and other O-specific polysaccharides containing N-acetylglucosamine. Journal of Bacteriology, 176(22), 7079–7084. https://doi.org/10.1128/jb.176.22.7079-7084.1994
17.Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z
| genotype | F- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSRARE (CamR)(KanR) ΔwecB |