Part:BBa_K5246037

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].

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].

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].

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

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1171
  Illegal BamHI site found at 1557
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  Illegal NgoMIV site found at 507
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

genotype

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