Part:BBa_K4179007

Umbelliferone dimethylallyltransferase (UDT)

UDT, umbelliferone dimethylallyltransferase, is an enzyme that belongs to the prenyl transferase family of enzymes, which catalyzes the prenytlation reaction. Its gene was mined based on the local BLAST alignment with homologous sequences as described by Song et al [1].

Prenylation

Prenylation is a structural modification in which an isoprenoid moiety, prenyl being the most common, is transferred from a donor molecule to an acceptor. Prenylation of aromatic substrates causes the enhancement of their bioactivity which is a crucial step in the biosynthesis of biologically active secondary metabolites, which are important in the survival and disease resistance of many plant species. To perform prenylation, plants use membrane-bound aromatic prenyltransferases (PTs) that transfer isoprenoid moieties from pyrophosphate donor substrates to aromatic acceptor substrates [2].

Usage and biology

The team of Technion 2022 used this part in a construct (BBa_K4179003) designed for the purpose of introducing decursin’s biosynthetic pathway into E. coli. The team’s starting point was umbelliferone, which had to be prenylated to yield DMS. and thus, this enzyme was needed to perform the first reaction in the pathway.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1195
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

This part includes the gene of UDT, in addition to two restriction sites, one at every end of the gene. NdeI restriction site was added to the N-terminus side of the sequence, directly before the methionine codon of the UDT gene. NheI restriction site was added to the C-terminus side of the sequence, directly after the end of the gene.

A stop codon is not present at the end of the UDT gene, due to the gene being cloned upstream to a P2A (BBa_K4179005)-mCherry (BBa_K106005) sequence. In this genetic system (BBa_K4179003) , the mCherry expression is tied directly to the start codon of UDT, while not being covalently bound to it. In such a design the mCherry signal corresponds to a 1:1 ratio, at the very least, which allows for a lower estimate of UDT’s expression.

References

1. Song J, Luo H, Xu Z et al. Mining genes associated with furanocoumarin biosynthesis in an endangered medicinal plant, Glehnia littoralis. J Genet 2041;

2. de Bruijn WJC, Levisson M, Beekwilder J, van Berkel WJH, Vincken J-P. Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories. 2020;