Part:BBa_K1129021
Enoyl-CoA hydratase/aldolase (Ech) under arabinose promoter
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
Illegal NotI site found at 911 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 340
Illegal NgoMIV site found at 358
Illegal NgoMIV site found at 397
Illegal AgeI site found at 334 - 1000COMPATIBLE WITH RFC[1000]
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_K1129021 is a enoyl-CoA hydratase/aldolase (ech) from Pseudomonas putida KT2440 which converts feruloyl CoA to vanillin [2]. BBa_K1129021 expression is under the control of an arabinose promoter.
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.
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