Part:BBa_K4179000
Petroselinum crispum prenyl transferase (PcPT)
This sequence codes for a prenyl transferase. An enzyme which prenylates umbelliferone in the presence of dimethylallyl diphosphate (DMAPP) at the 6th carbon position to produce demethylsuberosin (DMS) as the main product, in addition to a minor amount of osthenol corresponding to umbelliferone prenylated at the 8th carbon position (figure 2) [1,2].
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 gene of PcPT, Petroselinum crispum prenyl transferase, was first isolated from the genome of parsley leaves by Karamat et al and codon optimized for E. coli. It is a transmembrane protein with 8 predicted transmembrane helices. Its subcellular localization was found to be plastids. Functional characterization of PcPT done by Karamat et al showed that PcPT has a strong substrate specificity for DMAPP as a prenyl donor, with a dominant prenylation activity of umbelliferone at the 6th position, yielding DMS as the major product. The enzyme contains a cleavage site for a signal peptide sequence (48 aa) at the N-terminus.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 127
Illegal EcoRI site found at 559
Illegal EcoRI site found at 922
Illegal PstI site found at 82
Illegal PstI site found at 445 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 127
Illegal EcoRI site found at 559
Illegal EcoRI site found at 922
Illegal NheI site found at 1207
Illegal PstI site found at 82
Illegal PstI site found at 445 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 127
Illegal EcoRI site found at 559
Illegal EcoRI site found at 922 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 127
Illegal EcoRI site found at 559
Illegal EcoRI site found at 922
Illegal PstI site found at 82
Illegal PstI site found at 445 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 127
Illegal EcoRI site found at 559
Illegal EcoRI site found at 922
Illegal PstI site found at 82
Illegal PstI site found at 445 - 1000COMPATIBLE WITH RFC[1000]
This part includes the gene of PcPT, 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 PcPT 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 PcPT 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 PcPT, 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 PcPT’s expression.
References
1. Karamat F, Olry A, Munakata R et al. A coumarin-specific prenyltransferase catalyzes the crucial biosynthetic reaction for furanocoumarin formation in parsley. The Plant Journal 2014; 77: 627–638.
2. de Bruijn WJC, Levisson M, Beekwilder J, van Berkel WJH, Vincken J-P. Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories. 2020;
3. Bateman A, Martin MJ, Orchard S et al. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 2021; 49: D480–D489.
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