Part:BBa_K4711028

alkB+alkG+alkT

Usage and Biology

In the ω-hydroxylation enzyme system, we have chosen the alkBGT system, which consists of three enzymes: AlkB, AlkG, and AlkT, derived from the plasmid of Pseudomonas putida GPo1. AlkB is a membrane-bound monooxygenase and a key component of the alkane hydroxylation enzyme system. AlkG and AlkT are electron transfer proteins involved in the oxidation of NADH and electron transfer within the system. The operating mechanism of the alkane hydroxylation system in vivo is as follows: AlkT consumes NADH and generates electrons, AlkG transfers the electrons to AlkB, and finally, AlkB introduces a single oxygen atom from molecular oxygen at the terminal position to achieve hydroxylation reaction.

This system exhibits highly specific terminal hydroxylation activity towards fatty acids and has a broad substrate specificity. It can oxidize various substrates including C5-C16 alkanes, cycloalkanes, alkenes, thioether fatty acids, and fatty acids. It shows the highest catalytic activity towards medium-chain fatty acids. Since many hydroxylation enzymes have a preference for longer carbon chain fatty acid substrates, the alkane hydroxylation enzyme system holds great promise in the production of medium-chain hydroxy fatty acids.

Fig 1 Hydroxylase reaction principle

Source

Pseudomonas oleovorans

References

[1]Qiaofei He, George N. Bennett, Ka-Yiu San, Hui Wu*. Biosynthesis of medium-chain ω-hydroxy fatty acids by AlkBGT of Pseudomonas putida GPo1 with native FadL in engineered Escherichia coli. Frontiers in Bioengineering and Biotechnology. 2019. 7:273.

[2] Staijen I E , Beilen J B V , Witholt B .Expression, stability and performance of the three-component alkane mono-oxygenase of Pseudomonas oleovorans in Escherichia coli[J].European journal of biochemistry, 2000, 267(7):1957-65.DOI:10.1046/j.1432-1327.2000.01196.x.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 1509
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 84
  Illegal NgoMIV site found at 996
  Illegal NgoMIV site found at 2276
  
• 1000
  COMPATIBLE WITH RFC[1000]