Part:BBa_M36584

Making acetoacetate from acetic acid and acetoacetyl-CoA

This part is comprised of a promoter, the ctfA coding sequence (BBa_M36581), a strong RBS, the ctfB coding sequence (BBa_M36582, and a termination sequence from T1. This composite part aims to produce acetoacetate, acetyl-CoA, and butyryl-CoA using the ctfA and ctfB genes from Clostridium acetobutylicum. Acetoacetyl-COA acetate CoA-transferase subunit A, and acetoacetyl-COA butyrate CoA-transferase subunit B are important enzymes in the acetone production mechanism because they create the acetoacetate necessary for making acetone. This can be used in projects that are aiming to create the acetoacetate intermediate taken up by the adc gene (BBa_M36587) to produce acetone as in C. acetobutylicum.

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USAGE AND BIOLOGY

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. The operon responsible for this acetone production (thl, ctfA, ctfB, adc) begins by converting acetyl-CoA to acetoaceyl-CoA, acetic acid, and butyric acid through a thiolase promoter. The acetic acid is converted to acetoacetate and acetyl-CoA by ctfA, and the butyric acid is converted to acetoacetate and butyryl-CoA by ctfB. Finally, the acetoacetate is converted to acetone and carbon dioxide by acetoacetate decarboxylase (adc).

Since acetone cannot be used as a biofuel, its production is usually minimized or suppressed in cell or bioreactor engineering experiments. We, however, hope to make use this production of acetone for the degradation of polystyrene. Polystyrene (molecular formula [−CH(C6H5)CH2−]n), commonly produced as Styrofoam (expanded polystyrene foam), is classified as a durable, non-biodegradable plastic, yet it is often used in products designed for a short or one-time use due its low manufacturing costs. Unfortunately, this has led to a rapidly-increasing accumulation of polystyrene waste in the environment. Although styrene monomers and oligomers are susceptible to biodegradation, polystyrene is generally considered non-biodegradable as a result of its high molecular weight and highly-stable structure. Acetone has long been known to swell and depolymerize polystyrene, leaving behind a viscous liquid of styrene monomers and oligomers as the Styrofoam breaks down. This viscous styrene can then be reused as styrene glue, commonly used in industrial, craft, and construction applications.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 91
  Illegal XbaI site found at 772
  Illegal PstI site found at 841
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 91
  Illegal PstI site found at 841
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 91
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 91
  Illegal XbaI site found at 772
  Illegal PstI site found at 841
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 91
  Illegal XbaI site found at 772
  Illegal PstI site found at 841
  
• 1000
  COMPATIBLE WITH RFC[1000]