Part:BBa_K1129001

Feruloyl-CoA-Synthetase (FCS) from Pseudomonas putida KT2440

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 43
  Illegal XhoI site found at 1153
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 30
  Illegal NgoMIV site found at 294
  Illegal NgoMIV site found at 772
  Illegal NgoMIV site found at 1027
  Illegal NgoMIV site found at 1090
  Illegal NgoMIV site found at 1107
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 802
  

Background

Ferulic acid is best utilized as a precursor for synthesis of aromatic compounds such as vanillin, which involves 2 enzymes: the conversion of ferulic acid to feruloyl-CoA by feruloyl-CoA synthetase (Fcs) enzymes and the conversion of feruloyl-CoA into vanillin by enoyl-CoA hydratase/aldolase (Ech) enzymes BBa_K1129002[1]. BBa_K1129001 is a feruloyl-CoA-synthetase (fcs) from Pseudomonas putida KT2440 which converts ferrulic acid to feruloyl CoA.[2]

Experimental Data

Ech and fcs genes( BBa_K1129022 and BBa_K1129024), both of which are under constitutive promoters, were ligated into one pSB1C3 plasmid. An overnight culture of E.coli 10G cells harboring this plasmid were inoculated into a fresh culture of 5mL minimal media containing ferulic acid. Cells were then harvested for GC-MS after 7 hours of growth.

Figure 1 Compound generation identification by Gas Chromatography Mass Spectometry . Chromatograms(left) and mass spectra(Right) for select peaks are shown. Structures represent predictions based on library matching or comparison to standards. Controls represent plasmids missing the gene of interest. A) Internal control using vanillin(10.486). B) Conversion of Ferulic acid to vanillin acid by constitutive expressed Fcs and Ech through the Feuroyl CoA intermediate, not shown. C) Conversion of Ferulic acid to vanillin acid by Arabinose inducible expressed Fcs and Ech through the Feuroyl CoA intermediate, not shown.

Cross referencing with standards and a compound library

Gas Chromatography Mass Spectrum of Alpha-pinene Synthase Products: We expressed the alpha-pinene synthase (BBa_K517002) in C41 DE3 E. coli and lysed the cell culture to obtain protein extract which was run through a nickel column to purify the HIS-tagged alpha-pinene synthase. The purified synthase was used in an in vitro enzymatic assay to produce alpha-pinene from geranyl diphosphate (GPP) substrate. (A) Monoterpene product spectrum of our alpha-pinene synthase. Through gas chromatography mass spectrometry, we analysed the products of the alpha-pinene synthase and found alpha-pinene, beta-pinene and small quantities of other monoterpenes. This product spectrum agrees with prior findings (Keeling et al. 2011 BMC Plant Biology). (B) Identification of alpha-pinene as a product. Cross referencing with standards and a compound library revealed that the alpha-pinene product had the characteristic base peaks and retention time of 3.6 minutes. Top panel displays peak spectrum for our sample. Bottom panel displays peak spectrum for library standard. (C) Identification of beta-pinene as a product. Cross referencing with standards and a compound library revealed that the beta-pinene product had the characteristic base peaks and retention time of 5.6 minutes. Top panel displays peak spectrum for our sample. Bottom panel displays peak spectrum for library standard.