Part:BBa_K4130013

Dextransucrase (L. mesenteroides), DexYG

This part encodes the enzyme DexYG from Leuconostoc mesenteroides NRRL B-512F.

Biology

Dextransucrases are extracellular Class II enzymes expressed in lactic acid bacteria strains. They are united in function, catalyzing a reaction that transfers glucosyl units from sucrose to a growing dextran chain [1]:

n Sucrose → n Fructose + Dextran ((glucose)n)

Dextran is a homo-exopolysaccharide with 1:6 glucoside links, consisting primarily of 𝛼-1→6 chain and 𝛼-1→3 branching linkages between glucose monomers [3]. The long chain structure allows the molecule to effectively crosslink with itself and other small molecules. Its colloidal, hydrophilic, biocompatible, and nontoxic properties make it suitable for a variety of applications [6]. Dextrans were initially used as colloids for fluid resuscitation [5] and since then have been identified as a practical and versatile product in molecular, medical, and agricultural research. Some common uses of dextran include treating hemorrhage and burns, drug delivery, and radiological imaging [4]. The molecule is also used as a stabilizing and texturizing agent in food processing [1]. DexYG is a 4,584 bp gene encoding for a dextran sucrase found in L. mesenteroides NRRL B-512F. The resulting protein is 1,527 aa and has a molecular weight of 170 kDa [2]. The protein contains a hydrophobic N-terminus region displaying a host secretion signal peptide sequence for secretion of the protein out of the cell [2]. When expressed in Escherichia coli, this DexYG dextransucrase was found to successfully convert extracellular sucrose into fructose and dextran, the latter of which was found to have a molecular weight of 68 kDa [2].

Usage

Although well-utilized in many industrial and medical fields, dextran is not viewed favorably in the maple syrup industry. Upon contamination with lactic acid bacteria, maple syrup becomes thick and stringy due to the production of dextran. This texturally defective syrup, called “ropy syrup”, cannot be sold for human consumption and is often discarded or sold for industrial purposes at an extremely low price. In our project, we propose a solution for leftover and low-value ropy syrup: sucrose-rich ropy syrup can be incubated with genetically modified E. coli to increase production of valuable dextrans. Dextran can then be isolated and purified and sold for a higher price than the original defective syrup. To accomplish this goal, BioBrick BBa_K130013 was designed. This genetic part contains the coding sequence for the protein DexYG from L. mesenteroides NRRL B-512F. It is accompanied by a strong T7 promoter (BBa_I712074), strong ribosomal binding site (BBa_B0034), and double terminator (BBa_B0015) for efficient protein expression.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2354
  Illegal AgeI site found at 3668
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

Dols, M.; Remaud-Simeon, M. et al. Characterization of the Different Dextransucrase Activities Excreted in Glucose, Fructose, or Sucrose Medium by Leuconostoc mesenteroides. Appl Environ Microbiol 1998, 64(4), 1298-1302. https://doi.org/10.1128/AEM.64.4.1298-1302.1998

Zhang, H. et al. Cloning, sequencing and expression of a dextransucrase gene (dexYG) from Leuconostoc mesenteroides. Biotechnol Lett 2008, 30, 1441–1446. https://doi.org/10.1007/s10529-008-9711-8

Díaz-Montes, E. Dextran: Sources, Structures, and Properties. Polysaccharides 2021, 2, 554–565. https://doi.org/10.3390/ polysaccharides2030033

Miao, K.H.; Guthmiller, K.B. Dextran. National Library of Medicine via StatPearls, 2022. PMID: 32491563

Shnayder, Y. et al. Free Tissue Transfer. Cummings Otolaryngology: Head and Neck Surgery, Seventh Edition 2021, 1098-1118.

Sun, G., Shen, Y.-I., Ho, C.C., Kusuma, S. and Gerecht, S. (2010), Functional groups affect physical and biological properties of dextran-based hydrogels. J. Biomed. Mater. Res., 93A: 1080-1090. https://doi.org/10.1002/jbm.a.32604