Part:BBa_K4083004

nadE gene of Pseudomonas aeruginosa

nadE from Pseudomonas aeruginosa is coding for NAD synthetase which synthesizes NAD+

Usage and Biology

Our team extracted nadE gene from P. aeruginosa to add it into novel plasmid called pRGPDuo2 for P. putida. Apart from nadE gene, planned to add rhlA and rhlB genes that are responsible for rhamnolipid synthesis. Thus, we predicted that dual expression of NAD synthetase and Rhamnosyltrnasferse can allow P. putida to express rhamnolipids in higher rates.

Pseudaminas aeuriginosa - is gram negative bacilus and opportunistic pathogen. NH(3) dependent NAD-synthetase converts deamido-NAD+ to NAD+ by ATP-dependent amidation [1]. ATP + deamido-NAD+ + NH4+ -> AMP + diphosphate + H+ + NAD+

The NAD+ is important for metabolism in organisms. NAD+ can reduce into NADH during cell digestion like glucolysis or Krebs cycle. Thus, more available NAD+ can lead to faster substrate catabolism. Moreover, NADH interacts with the electron transport chain where it releases one electron and one proton. As an electron moves, more protons exit the bacterial membrane which increases the proton gradient. Finally, to reach equilibrium, protons enter the cell by ATP synthase, and one proton can generate up to 3 ATP molecules this way. Thus, one NADH that releases one proton can generate 3 ATP molecules. Finally, NAD+ can interact with NAD kinase to convert into NADP+ which plays a direct role in biosynthesis of rhamnolipids. [2] Reference:

[1] UniProt. (n.d.). nadE - NH(3)-dependent NAD(+) synthetase - Pseudomonas aeruginosa (strain PA7) - nadE gene & protein. https://www.uniprot.org/uniprot/A6VD32 [2] Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. New York: Garland Science. Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 208
  Illegal XhoI site found at 475
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 400
  Illegal NgoMIV site found at 470
  Illegal NgoMIV site found at 691
  
• 1000
  COMPATIBLE WITH RFC[1000]