Part:BBa_K5246030

HB HfsG Glycosyltransferase, 6xHis tag for purification

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.

This protein is part of the Tetrad assembly system BBa_K5246043 and operon responsible for the addition of N-acetyl-D-glucosamine and deacetylation of the former molecule BBa_K5246042.

Part was used in Vilnius-Lithuania iGEM 2024 project "Synhesion" https://2024.igem.wiki/vilnius-lithuania/.

This part also has a non 6xhis-tagged variant BBa_K5246024.

Usage and Biology

TBA

This part also has a non his-tagged variant BBa_K5246019.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 852
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 747
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental characterization

Bioinformatic analysis

CDD and protein BLAST analysis suggest that HfsG is a glucosyltransferase family 2 protein. Proteins of this family are involved in cell wall biosynthesis. HfsG is similar to the WecA protein in E.Coli that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate. In the case of HfsG, it catalyzes the transfer of UDP-GlcNAc to the sugar acceptor made earlier in the holdfast synthesis pathway.

Protein topology analysis using DeepTMHMM suggests that HfsG is a globular protein located in the cytoplasm. AlphaFold 3 structures further confirm it. A pTM score above 0.5 suggests that the predicted overall structure may closely resemble the true protein fold, while ipTM indicates the accuracy of the subunit positioning within the complex. Values higher than 0.8 represent confident, high-quality predictions.

HfsG is family 2 glycosyltransferase similar to WecA of E.Coli. This globular protein transfers UDP-GlcNAc to the acceptor molecule, our conclusions are in agreement with existing research. [1][2][3]

References

1. Toh, E., Kurtz, Harry D. and Brun, Y.V. (2008) ‘Characterization of the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant redundancy in the initiating glycosyltransferase and polymerase steps’, Journal of Bacteriology, 190(21), pp. 7219–7231. doi:10.1128/jb.01003-08.
2. Hardy, G.G. et al. (2018) ‘Mutations in sugar-nucleotide synthesis genes restore holdfast polysaccharide anchoring to Caulobacter crescentus holdfast anchor mutants’, Journal of Bacteriology, 200(3). doi:10.1128/jb.00597-17.
3. Sulkowski, N.I. et al. (2019) ‘A multiprotein complex anchors adhesive holdfast at the outer membrane of Caulobacter crescentus’, Journal of Bacteriology, 201(18). doi:10.1128/jb.00112-19.