Difference between revisions of "Part:BBa K1129002"

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Enoyl-CoA hydratase/aldolase (ECH) from Pseudomonas putida

This is a enoyl-CoA hydratase/aldolase from Pseudomonas putida KT2440 which converts feruloyl-CoA to vanillin

Sequence and Features

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 (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.

Similarly, 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.