Part:BBa_K323135

VioA and VioB enzymes fused with zinc fingers under pBAD promoter

The part contains two of the enzymes of the violacein biosynthetic pathway, fused to zinc finger proteins. VioA enzyme is linked to the Blues zinc finger (Part:BBa_K323085) and VioB enzyme to the Zif268 zinc finger (Part:BBa_K323134). When translated to protein, two functional chimeric proteins which bind to a specific DNA sequence are produced. We designed a DNA program (Part:BBa_K323066) with these sequences to bind the chimeric enzymes to a closer proximity.

Figure: The DNA-guided violacein biosynthetic pathway. Genes for violacein biosynthesis are arranged in an operon consisting of vioA, vioB, vioC, vioD and vioE. VioA generates an IPA imine from L-tryptophan. VioB converts the IPA imine into a dimer and VioE then acts by converting the dimer into protodexyviolaceinic acid (PVA), which can be spontaneously converted into a green pigment called deoxychromoviridans. VioD and VioC hydroxylate (PVA) to form violacein. Our iGEM team improved the rather poor yield of violacein production in E.coli and reduced the formation of deoxychromoviridans by introducing the DNA-guided biosynthetic pathway based on chimeric enzymes bound in correct order to DNA program.

In combination with Part:BBa_K323132, we have bound all of the enzymes, necessary for synthesis of violacein, on the DNA program. This enabled a faster and increased synthesis of the desired product, as the enzymes were fixed close together and the intermediates were immediately available to the next enzyme. The latter fact also resulted in a decreased formation of an unwanted side product deoxychromoviridans, which most likely also contributed to the overall increased production of violacein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1204
  Illegal NheI site found at 2568
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 6132
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5657
  Illegal AgeI site found at 979
  Illegal AgeI site found at 5853
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961