Part:BBa_K3846354

Artemisinic acid 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.

BBa_K3846354

This phyto brick additionally to the MBIS and MevT operon features expression cassettes of the amorpha-4,11-diene synthase and CYP71AV1-ATR2 fusion protein under the control of a T7 promoter. Fermentation in presence of IPTG will first lead to the formation of Amorpha-4,11-diene, which is then converted by CYP71AV1-ATR2 to artemisinic acid. Artemisinic acid can be extracted and chemically oxidised to obtain the antimalarial drug artemisinin.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 12957
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

iGEM Hamburg 2021 part collection

Terpenoids are an important group of natural products used as biofuels, drugs or fragrances. Naturally occuring in plants it has been shown that microbial terpene production in microorganisms like yeast, E. coli or cyanobacteria is possible. Nevertheless iGEM projects seem to rarely focus on this interesting class of natural products which is correlated with a lack of useful parts inside the iGEM registry.

Fortunately we were able to change that and designed a novel golden gate based toolbox which allows.

1. production of terpenoid precursors and simple terpenoids
2. creation of CYP P450-reductase fusion enzymes to optimise processing of terpenoid precursors and production of bioactive target products
3. modularity of the system to enable exchange of linker sequences/promoters/etc. (MoClo-compatible toolbox)

MoClo-based Part Design 2.0

To improve the usefulness of our parts, we then aimed to make our parts compatible with the MoClo standard of goten gate based IIS restriction enzyme assembly. Thereby we expanded the Common Genetic Syntax for fusion sites to allow the creation of a) fusion proteins connected by linker sequences and b) multiple CDS expressed in an operon. More useful information and an overview of all our parts can be found on our wiki.

Figure 1: MoClo syntax of the part collection.