Part:BBa_K1129024
Feruloyl-CoA-Synthetase (FCS) under pTET constitutive promoter
Const+rbs+fcs+term
Feruoyl-CoA synthase is an enzyme of the vanillin pathway. It adds acetyl-CoA to ferulic acid to make ferruryl CoA the immediate precursor for vanillin.Feruoyl-CoA synthase is under a constitutive promoter Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 104
Illegal XhoI site found at 1214 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 91
Illegal NgoMIV site found at 355
Illegal NgoMIV site found at 833
Illegal NgoMIV site found at 1088
Illegal NgoMIV site found at 1151
Illegal NgoMIV site found at 1168 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 863
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 BBa_K1129001 and the conversion of feruloyl-CoA into vanillin by enoyl-CoA hydratase/aldolase (ech) enzymes [1]. BBa_K1129024 is a feruloyl-CoA-synthetase (fcs) from Pseudomonas putida KT2440 which converts ferrulic acid to feruloyl CoA [2].
BBa_K1129024 expression is under the control of a constitutive promoter.
Experimental Data
Ech and fcs genes( BBa_K1129022 and BBa_K1129024), both of which are under constitutive promoters (BBa_J23118), were ligated into one pSB1C3 plasmid. An overnight culture of E.coli 10G cells harboring this plasmid was 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.
References
[1] Priefert,H., Rabenhorst, J., Steinbiichel, A. (2001). Biotechnological production of vanillin. Appl Microbiol Biotechnol, 56, 296-314.
[2] Plaggenborg, R., Overhage, J., Steinbuchel, A. (2003). Functional analyses of genes involved in the metabolism of ferulic acid in Pseudomonas putida KT2440. Appl Microbiol Biotechnol, 61,528-535.
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