Difference between revisions of "Part:BBa K1129023"
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<partinfo>BBa_K1129023 short</partinfo>
 
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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 an arabinose inducable promoter. -<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
  
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<partinfo>BBa_K1129023 parameters</partinfo>
 
<partinfo>BBa_K1129023 parameters</partinfo>
 
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<h2>Background</h2>
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<p> 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 <html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129002">BBa_K1129002</b></a></html> [1].
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<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129023">BBa_K1129023</b></a></html> a feruloyl-CoA-synthetase (fcs) from ''Pseudomonas putida'' KT2440 which converts ferrulic acid to feruloyl CoA  [2].
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<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129023">BBa_K1129023</b></a></html> expression is under the control of an arabinose promoter.[2].</p>
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<html><center><IMG id="emp" SRC="https://static.igem.org/mediawiki/2013/b/bf/UBC-Vanillin-structure.png" USEMAP="#vin">
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<map id="vin" name="vin"><area shape="rect" alt="" title="" coords="104,101,242,184" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129000" target="" /><area shape="rect" alt="" title="" coords="353,103,491,189" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129046" target="" /><area shape="poly" alt="" title="" coords="595,76,750,77,750,150,696,151,699,263,647,263,644,164,595,164" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129041" target="" /><area shape="rect" alt="" title="" coords="489,387,630,487" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129024" target="" /><area shape="rect" alt="" title="" coords="205,384,351,487" href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129022" target="" /><!-- Created by Online Image Map Editor (http://www.maschek.hu/imagemap/index) --></map>
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</center></html>
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<h2>Experimental Data</h2>
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Ech and fcs genes (<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129021">BBa_K1129021</b></a></html> and <html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1129023">BBa_K1129023</b></a></html>) under arabinose inducible promoters (<html><b><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_I13453">BBa_I13453</b></a></html>) 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.   
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<p align=center>https://static.igem.org/mediawiki/2013/a/ac/Ech900.jpg</p>
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'''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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<h2>References</h2>
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[1] Priefert,H., Rabenhorst, J., Steinbiichel, A. (2001). Biotechnological production of vanillin. Appl Microbiol Biotechnol, 56, 296-314.
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[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.

Latest revision as of 01:29, 29 October 2013

Feruloyl-CoA-Synthetase (FCS) under arabinose promoter

Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
    Illegal XhoI site found at 199
    Illegal XhoI site found at 1309
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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 a feruloyl-CoA-synthetase (fcs) from Pseudomonas putida KT2440 which converts ferrulic acid to feruloyl CoA [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.



Ech900.jpg


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.