Part:BBa_K5246040

HMS147(DE3) 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 - 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].

Table 1. Genotype of HMS174(DE3) E. coli Strain

Genotype	F- recA1 hsdR(rK12- mK12+) (DE3) (RifR)

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. Additionally, the exact composition of this polysaccharide was not known. By disabling the wecB gene in E.coli and thereby inhibiting the production of N-acetylmannosaminuronic acid, we could investigate whether this monosaccharide is a component of the C. crescentus holdfast.

Sequence and Features

This sequence is only part of genome with introduced deletion.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 245
  Illegal XbaI site found at 1467
  Illegal PstI site found at 223
  Illegal PstI site found at 810
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 223
  Illegal PstI site found at 810
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 597
  Illegal BamHI site found at 211
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 245
  Illegal XbaI site found at 1467
  Illegal PstI site found at 223
  Illegal PstI site found at 810
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 245
  Illegal XbaI site found at 1467
  Illegal PstI site found at 223
  Illegal PstI site found at 810
  Illegal NgoMIV site found at 1261
  Illegal AgeI site found at 37
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

genotype

F- recA1 hsdR(rK12- mK12+) (DE3) (Rif R)(KanR) ΔwecB

Experimental characterization

Knocking out the wecB gene by homology recombineering

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.

Fig. 1. Knocked out wecB gene in E.coli strain HMS174(DE3). C- is the wecB gene and its region from an unedited strain (~1.5 kb), and C+ is the kanamycin cassette, indicating knockout (~1.8 kb). 1-10 is the cPCR of knocked-out colonies. Colonies the same size as C+ indicate a knockout. M - molecular weight ladder, GeneRuler Mix DNA Ladder (Thermo Scientific). 1% agarose gel in TAE buffer with EtBr.

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

CB2 holdfast system proteins are expressed, but polysaccharides are not produced in HMS174(DE3)ΔwecB. We discovered that wecB gene deletion did not interfere with CB2 system protein production (Fig. 2).

Fig. 2. SDS-PAGE analysis of CB2 system expression in HMS174(DE3)ΔwecB at 0.5mM IPTG for 3h at 37°C and overnight at 30°C. M - molecular weight ladder in kDa, Pageruler Unstained Protein Ladder, 26614 (Thermo Scientific).

We analyzed polysaccharide and ring production after overnight incubation with 1% glucose in the HMS174(DE3)ΔwecB strain. Cells with the CB2 system grew faster than unedited HMS174(DE3) and did not produce rings with polysaccharides. The flask with the E. coli containing the CB2 system looked the same as the E. coli without (Fig 3).

Fig. 3. Appearance of rings in expression flask after the addition of 1% glucose and incubation O/N at 30ºC in HMS174(DE3)ΔwecB. The empty system flask contains no target genes, CB2 system flask contains the required proteins for polysaccharide production, but in both, no thick rings are present.

Additionally, we compared HMS174(DE3)ΔwecA with HMS174(DE3)ΔwecB containing CB2 system to see the difference of polysaccharide and ring production (Fig. 4).

Fig. 4. Difference in ring formation between knocked out HMS174(DE3)ΔwecA and HMS174(DE3)ΔwecB, both containing CB2 system proteins after the addition of 1% glucose and incubation O/N at 30ºC. HMS174(DE3)ΔwecA flask features visible rings, and HMS174(DE3)ΔwecB flask does not.

For CB2 system for holdfast synthesis see parts BBa_K5246043 and BBa_K5246046.

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.Sala, R. F., Morgan, P. M., & Tanner, M. E. (1996). Enzyme-catalyzed formation of stable enolates: Mechanism of the reaction catalyzed by GDP-mannose 3,5-epimerase. Journal of the American Chemical Society, 118(12), 3033–3034. https://doi.org/10.1021/ja960266z
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