Part:BBa_K3846360

Alpha-humulen production casette

Mevalonate pathway

The synthesis of sesquiterpenes, our main focus of this project, relies on the availability of common building blocks like farnesyl diphosphate (FPP). It has been shown for different organisms that the amount of FPP can be increased by overexpressing proteins of the mevalonat (MevT) and the MBIS pathway which we organised to be expressed into operon structures for our project. Both operons are IPTG-inducible and use lacUV5 promoter and rrnB T1 and T2 terminator sequence.

MevT operon: This operon contains the acetoacetyl-CoA synthase (ackA), HMG-CoA synthases (HMG), and an N-terminally truncated version of HMG-CoA reductase (tHMGR).

MBIS operon: This operon contains mevalonate kinase (MK), phosphomevalonate kinase (PMK), phosphomevalonate decarboxylase (PMD), isopentenyl diphosphate isomerase (idi), and farnesyl diphosphate synthase (ispA).

By combining both of these operons in a single composite part and further adding synthases, it is possible to synthesize different bioactive compounds.

Humulene

Humulene is the precursor of the sesquiterpene ketone zerumbone, which has immunoregulatory and anticancer activities. The biosynthesis of cyclic sesquiterpenes is catalysed by FPP. In the case of humulene biosynthesis, this cyclase is alpha humulene synthase.

BBa:K3846360

This part combines the MevT and MBIS operons and the alpha humulene synthase expression cassette to produce alpha-humulene.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 9575
  Illegal NheI site found at 9579
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]