Part:BBa_K1686046

Curdlan synthase gene with codon optimisation for E. coli

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 678
  Illegal XhoI site found at 400
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 801
  Illegal AgeI site found at 1192
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

Curdlan is a bacterial polysaccharide (linear (1->3)-β-glucan) that has been of significant recent interest due to its valuable properties and its bioactivity. In fact, Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses. For example, it can have antitumor, anti-infective, anti-inflammatory, and anticoagulant activities. In particular, this (1->3)-β-glucan can stimulate the plant's immune system.
Our project is focused on the production of Curdlan to stimulate the plant's defense system against Downy Mildew.

The molecular genetics of curdlan production have been investigated in Agrobacterium sp. ATCC31749. Studies have identified three genes (crdA, crdS and crdC) that are essential for Curdlan production The crdASC genes occupy a contiguous 4,948 bp region of the genome ( the crdS gene is flanked by the crdA and crdC genes).

The crdS gene (1965 bp) encodes the Curdlan synthase (CrdS, 73 kDa) protein. In Agrobacterium, CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a N_out–C_in disposition. The UDP-Glucose substrate-binding and the catalytic motif for curdlan polymerisation is observed between TM3 and TM4. The process of Curdlan polymerisation mediated by CrdS occurs on the cytoplasmic face of the inner membrane.

For our project, we wanted to produce Curdlan with E.coli. To do this, our part has been made with a codon optimization of the natural crdS sequence for E.coli.

References

Stasinopoulos, S. J., Fisher, P. R., Stone, B. A., & Stanisich, V. A. (1999). Detection of two loci involved in (1-->3)-beta-glucan (curdlan) biosynthesis by Agrobacterium sp. ATCC31749, and comparative sequence analysis of the putative curdlan synthase gene. Glycobiology, 9(1), 31–41. https://doi.org/10.1093/glycob/9.1.31