Document Type : Research Paper

Authors

• M. Yahyazadeh ¹
• N. Hadi ¹
• Z. Shirazi ¹
• K. Jaimand ¹
• Kh. Karimzadeh Asl ¹
• M. Makizadeh Tafti ¹
• S. Fekri Qomi ¹
• M. Rahimifard ¹
• M. Gorji ¹
• F. Askari ¹
• Z. Behrad ¹
• D. Selmar ²

¹ Research Institute of Forests and Rangelands, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran

² Institute 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

20.1001.1.17350905.1400.37.6.7.8

Main Subjects

• Biotechnology