Part:BBa_K5120003
2-Hydroxyisoflavanone dehydratase (HID) in Pueraria Mirifica Var. Lobata
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Figure 1: Isoflavonoid Biosynthetic Pathway
Hydroxyisoflavanone dehydratase (HID) in the pEAQ DEST 1 plasmid is a genetic construct designed for the expression of the HID enzyme from Pueraria Mirifica Var. Lobata in plants like Nicotiana benthamiana. HID catalyzes the conversion of flavanones into isoflavones, which are key compounds in the biosynthesis of isoflavonoids. This construct, which is 1566 base pairs long, is optimized for efficient expression in plant systems, using the pEAQ-HT-DEST1 backbone for high-level transient expression. With its assembly compatibility and regulatory elements, it serves as a powerful tool for exploring isoflavonoid biosynthesis and metabolic engineering in synthetic biology. .
The enzyme 2-Hydroxyisoflavanone dehydratase (HID) from Pueraria Mirifica Var. Lobata is involved in the biosynthesis of isoflavonoids. HID catalyzes the dehydration of 2-hydroxyisoflavonones, facilitating the formation of isoflavonoid compounds. This enzyme part is designed to be compatible with assembly standards for incorporation into various systems for further research or synthetic biology applications. It is 972 base pairs long and is compatible with several assembly standards, allowing it to be integrated into constructs for expression in plants like Nicotiana benthamiana.Proof of Function
This part was used in a composite part along with other key enzymes in the isoflavonoid biosynthetic pathway and was agroinfiltrated into N. benthamiana using Agrobacterium tumefaciens.
Figure 2: PCR results for composite parts with genes for selected enzymes from the Isoflavonoid biosynthetic pathway
The enzyme 2-Hydroxyisoflavanone dehydratase (HID) from Pueraria Mirifica Var. Lobata is involved in the biosynthesis of isoflavonoids. HID catalyzes the dehydration of 2-hydroxyisoflavonones, facilitating the formation of isoflavonoid compounds. This enzyme part is designed to be compatible with assembly standards for incorporation into various systems for further research or synthetic biology applications. It is 972 base pairs long and is compatible with several assembly standards, allowing it to be integrated into constructs for expression in plants like Nicotiana benthamiana.
Figure 3: HPLC Chromatogram showing the detection of puerarin, daidzin, genistin, iso-vitexin, daidzien and genistein in transformed Nicotiana benthamiana samples
After transformation, the modified plants were tested for isoflavonoid production using High-Performance Liquid Chromatography (HPLC). The chromatogram shows the amounts of each target isoflavonoid: puerarin, daidzein, and genistein with the first peak, observed at around 16.0 minutes, representing puerarin, followed by a peak at approximately 17.0 minutes, which corresponds to daidzin. Further along, a peak at 22.0 minutes is attributed to genistin. Traces of all three compounds were detected in N. benthamiana, a plant that does not naturally produce any of these because it lacks the enzymes needed to do so. This shows that the sequence for HID did function as intended because if it hadn't then the pathway wouldn't have progressed further and produced these isoflavonoids.
References
- Sohn, Soo In, et al. “Metabolic Engineering of Isoflavones: An Updated Overview.” Frontiers in Plant Science, U.S. National Library of Medicine, 7 June 2021, www.ncbi.nlm.nih.gov/pmc/articles/PMC8216759/.
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