Part:BBa_K4194019

fadD

The gene codes for long chain acyl-CoA synthase(CoA ligase).

CoA ligase

CoA ligase catalyze the reaction of hydroxyl fatty acids with CoA

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 195
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 504
  Illegal XhoI site found at 1006
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 408
  Illegal AgeI site found at 1033
  
• 1000
  COMPATIBLE WITH RFC[1000]

Gas chromatographic analysis of hydrolyzed products

The BUCT-China team attempted to synthesize PHFA (polyhydroxyfatty acid, a biocompatible polymer material) using an enzyme system containing CoA Ligase.The obtained product was hydrolyzed and then analyzed by gas chromatography, and the result analysis showed that the monomer of the polymer was hydroxy hexadecanoic acid. Therefore, it can be seen that the system carried out hydroxylation reaction and subsequent polymerization reaction on the fatty acid of the substrate 16C as expected, and the system basically met our requirements.

Results of gas chromatographic analysis of product hydrolysis.

NMR hydrogen spectrogram analysis of polymers

The fig. shows the NMR of the simulated dimer product

The characteristic peaks determines that the product has dimerization products.

NMR of hydroxyhexadecanoic acid.

NMR of dimeric compounds.

NMR of trimeric compounds.

Integration comparison of the peaks of 2/16/20 C to determine the degree of aggregation.

(No. 16 C: No. 2 C) = 1.12: 0.13 = 8.61 (polymerization degree of 7-8)

(No. 20 C: No. 2 C) = 0.94: 0.19 = 7.23 (polymerization degree of 7-8)

(No. 16 C: No. 20 C) = 1.12: 0.94 = 1.19 (approximately equal to 6/5 = 1.2, polymerization degree of 5-6)
It could be judged that the degree of polymerization degree of the products should be between 5 and 8. Therefore, we determine that the system we designed is able to produce polyhydroxy fatty acids with a certain degree of polymerization, further confirming the better activity of the new FadD enzyme.

Gel Permeation Chromatography（GPC）

Gel Permeation Chromatography（GPC）

We performed gel chromatography on the products, and the results showed that the number average molecular weight (Mn) was the highest at 198588, and the peak molecular weight (Mp) was 191,439. The molecules with molecular weights around this value accounted for the largest proportion of the overall material, which then indicates that the system has a higher proportion of large molecules with a higher degree of polymerization. This further indicates that the efficiency and quality of the system to produce PHFA were improved after replacing FadD enzyme. Meanwhile, the polydispersity coefficient of this gel chromatography is about 1.258 which is closed to 1, which indicates that the substances are more uniformly dispersed during this assay and the results are highly reliable.