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Characterization of P450 enzyme gene responsible for Cheilanthifoline alkaloid in greater celandine (Chelidonium majus L.) by transgenic yeast Pichia pastoris | ||
| تحقیقات گیاهان دارویی و معطر ایران | ||
| Article 8, Volume 37, Issue 6 - Serial Number 110, January and February 2022, Pages 989-1000 PDF (881 K) | ||
| Document Type: Research Paper | ||
| DOI: 10.22092/ijmapr.2021.356257.3071 | ||
| Authors | ||
| M. Yahyazadeh* 1; N. Hadi1; Z. Shirazi1; K. Jaimand1; Kh. Karimzadeh Asl1; M. Makizadeh Tafti1; S. Fekri Qomi1; M. Rahimifard1; M. Gorji1; F. Askari1; Z. Behrad1; D. Selmar2 | ||
| 1Research Institute of Forests and Rangelands, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran | ||
| 2Institute of Plant Biology, TU Braunschweig, Mendelssohnstr, Braunschweig, Germany | ||
| Abstract | ||
| Plants are the main sources of secondary metabolites with high medical value. The most important member of these valuable compounds are alkaloids with the different drug purposes. Concerning the limited production of some of these metabolites in the plants, these medicinal compounds can be produced naturally and commercially with the identification and transfer of alkaloids-producing enzymes corresponding plant genes to the microorganisms as an alternative method. In this way, the characterization of the corresponding genes is the first step. Among the different enzymes involved in the alkaloid biosynthesis, the cytochrome P450 enzymes play an important role. Due to the endoplasmic reticulum (ER) localization of these enzymes and their glycoprotein characters, they cannot be expressed functionally in the standard bacterial systems. Consequently, the heterologous expression aimed to verify the enzymatic activity can favorably be performed using the eukaryotic systems, like yeast or insect cells. Herein, in this study, with employing a phylogenic comparison of cheilanthifoline synthase sequence of Eschscholzia californica Cham. and comparing the sequence with the homolog amino acid sequences of Chelidonium majus L. achieved from bioinformatics databases, six cytochrome P450 enzymes responsible for cheilanthifoline synthase in Ch. majus were identified. To prove the efficacy of these enzymes practically, their genes were cloned into the pPIC3.5 vector. Then, these recombinant vectors were transferred to the yeast cell (Pichia pastoris) and the scoulerine alkaloid was given to its media. Finally, the cheilanthifoline alkaloid microbial production by P. pastoris containing the recombinant plasmids was evaluated by LC-MS. The results of the present study indicated that among the enzymes genes cloned and introduced to the yeast host, only the Contig8931 enzyme had the cheilanthifoline synthase activity. | ||
| Keywords | ||
| P450 enzymes; isoquinoline alkaloids; microbial production; yeast | ||
| References | ||
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