Part:BBa_K4621000

EctOperon is a cluster of genes encoding four enzymes that synthesize 5-Hydroxyectoine

Usage, Biology and Characterization：

EctOperon contains ectA, ectB, ectC, and ectD.

ectB is the coding sequence of L-2, 4-diaminobutyrate --2-oxoglutarate aminotransferase, L-Aspartate 4-semialdehyde can be converted to L-2,4-diaminobutanoate. ectA is the coding sequence for L-2,4-diaminobutyric acid acetyltransferase, L-2, 4-diaminobutanoate can be converted to N4-acetyl-L-2,4-diaminobutyrate. ectC is the coding sequence of L-ectoine synthase, which can convert N4-acetyl-L-2,4-diaminobutyrate to L-Ectoine. These three genes encode enzymes Ectoine: (4S)-2-methyl-1,4,5, 6-tetrahydropyrim idine-4-carboxylic acid, located on the same operon, forming the ectABC gene cluster. The Ectoine is then activated by ectoine hydroxylase, which is encoded by the ectD gene, to generate 5-Hydroxyectoine. The synthesis of 5-Hydroxyectoine from L-Aspartate occurs naturally in some moderately halophilic microorganisms, as shown in Figure 1.

Figure 1:Pathways for 5-hydroxyectoine synthesis

Streptomyces SCUT-3 was modified to overexpressed EctOperon. They were then seeded in a 40 mL LB medium system. After fermentation at 40 °C and 220 rpm for 72h, the product 5-Hydroxyectoine was determined as follows.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 3227
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 3227
  Illegal NotI site found at 571
  Illegal NotI site found at 1456
  Illegal NotI site found at 1774
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 753
  Illegal XhoI site found at 927
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 3227
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 3227
  Illegal NgoMIV site found at 788
  Illegal NgoMIV site found at 1194
  Illegal NgoMIV site found at 1200
  Illegal NgoMIV site found at 1491
  Illegal NgoMIV site found at 2540
  Illegal NgoMIV site found at 2732
  Illegal NgoMIV site found at 3242
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 376
  Illegal BsaI.rc site found at 556
  Illegal BsaI.rc site found at 1338
  Illegal BsaI.rc site found at 1866
  Illegal BsaI.rc site found at 2121
  Illegal BsaI.rc site found at 2211
  Illegal BsaI.rc site found at 2286
  Illegal SapI site found at 414