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HMS147(DE3) ΔWecA

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 - C. crescentus and H. 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 E. 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 HMS174(DE3) protein expression strain alternative to BL21(DE3) and is derived from E. coli K-12[1]. It produces recombinant proteins under the control of T7 RNA polymerase. Unlike BL21(DE3), the HMS174(DE3) strain can metabolize galactose [2],[3].

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].

WecA is Undecaprenyl-phosphate α-N-acetylglucosaminyl transferase, which initiates the biosynthesis of enterobacterial common antigen (ECA) and O-antigen by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc [10]. The O antigen is located on the cell surface and could be recognized by the host immune system and bacteriophages [11].

Eliminating the wecA gene results in ECA and lipopolysaccharide 08-side chain inactivation without causing high-stress levels [12].

Usage

Escherichia coli HMS174(DE3) protein expression strain is used to produce recombinant proteins under the control of T7 RNA polymerase [1]. HMS174(DE3) is 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]. HMS174(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].

We, Vilnius-Lithuania iGEM 2024 team, in our project Synhesion 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. For this strain usage in our project for CB2 system see parts BBa_K5246043 and BBa_K5246046.

Sequence and Features

This sequence is only part of genome with introduced deletion.

Functional Parameters

Experimental characterization

Knocking out the wecA gene by homology recombineering

To create an ECA pathway deficient E. coli HMS174(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.

CB2 system expression and polysaccharide production in HMS174(DE3)∆wecA

CB2 holdfast system proteins are expressed, and polysaccharides are produced in HMS174(DE3)ΔwecA. We discovered that wecA gene deletion did not interfere with CB2 system protein production (for CB2 system see parts BBa_K5246043 and BBa_K5246046). Additionally, we compared it to the not-edited HMS174(DE3) strain (Fig 2.).

We analyzed polysaccharide and ring production after overnight incubation with 1% glucose in an ECA-deficient HMS174(DE3)ΔwecA strain (Fig. 3). Although cells with the CB2 system grew slower than an empty control, they still produced rings with the polysaccharides.

References

1.Hausjell, J., Weissensteiner, J., Molitor, C., & Spadiut, O. (2018). E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3). Microbial Cell Factories, 17(1), 169. https://doi.org/10.1186/s12934-018-1016-6
2.Shiloach, J., Kaufman, J., Guillard, A. S., & Fass, R. (1996). Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (λDE3) and Escherichia coli JM109. Biotechnology and Bioengineering, 49(4), 421–428. https://doi.org/10.1002/(SICI)1097-0290(19960220)49:4<421::AID-BIT5>3.0.CO;2-W
3.Mairhofer, J., Krempl, P. M., Thallinger, G. G., & Striedner, G. (2014). Finished genome sequence of Escherichia coli K-12 strain HMS174 (ATCC 47011). Genome Announcements, 2(6), e00975-14. https://doi.org/10.1128/genomeA.00975-14
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