Part:BBa_K1129023
Feruloyl-CoA-Synthetase (FCS) under arabinose promoter
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal XhoI site found at 199
Illegal XhoI site found at 1309 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 186
Illegal NgoMIV site found at 450
Illegal NgoMIV site found at 928
Illegal NgoMIV site found at 1183
Illegal NgoMIV site found at 1246
Illegal NgoMIV site found at 1263 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 958
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_K1129023 is a enoyl-CoA hydratase/aldolase (ech) from Pseudomonas putida KT2440 which converts feruloyl CoA to vanillin [2]. BBa_K1129023 expression is under the control of an arabinose promoter.[2].
Experimental Data
Ech and fcs genes (BBa_K1129021 and BBa_K1129023) under arabinose inducible promoters (BBa_I13453) were ligated together 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 and arabinose. 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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