title: Transcriptome-wide identification and characterization of CAD isoforms specific for podophyllotoxin biosynthesis from Podophyllum hexandrum.
abstract: Podophyllotoxin (ptox) is a therapeutically important lignan derived from Podophyllum hexandrum and is used as a precursor for the synthesis of anticancer drugs etoposide, teniposide and etopophose. In spite of its enormous economic significance, genomic information on this endangered medicinal herb is scarce. We have performed de novo transcriptome analysis of methyl jasmonate (MeJA)-treated P. hexandrum cell cultures exhibiting enhanced ptox accumulation. The results revealed the maximum up-regulation of several isoforms of cinnamyl alcohol dehydrogenase (CAD). CAD catalyzes the synthesis of coniferyl alcohol and sinapyl alcohol from coniferaldehyde (CAld) and sinapaldehyde respectively. Coniferyl alcohol can produce both lignin and lignan while sinapyl alcohol produces only lignin. To isolate the CAD isoforms favoring ptox, we deduced full length cDNA sequences of four CAD isoforms: PhCAD1, PhCAD2, PhCAD3 and PhCAD4 from the contigs of the transcriptome data. In vitro enzyme assays indicated a higher affinity for CAld over sinapaldehyde for each isoform. In silico molecular docking analyses also suggested that PhCAD3 has a higher binding preference with CAld over sinapaldehyde, followed by PhCAD4, PhCAD2, and PhCAD1, respectively. The transgenic cell cultures overexpressing these isoforms independently revealed that PhCAD3 favored the maximum accumulation of ptox as compared to lignin followed by PhCAD4 and PhCAD2, whereas, PhCAD1 favored both equally. Together, our study reveals transcriptome-wide identification and characterization of ptox specific CAD isoforms from P. hexandrum. It provides a useful resource for future research not only on the ptox biosynthetic pathway but on overall P. hexandrum, an endangered medicinal herb with immense therapeutic importance.
authors.affiliation: Division of Biotechnology, Chonbuk National University, 79 Gobong-ro, Iksan-si, Jeollabuk-do, 570-752, Republic of Korea.
authors.forename: Dipto
authors.initials: D
authors.lastname: Bhattacharyya
authors.affiliation: Plant Biology Lab., Organic and Medicinal Chemistry Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Saptarshi
authors.initials: S
authors.lastname: Hazra
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Anindyajit
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Plant Biology Lab., Organic and Medicinal Chemistry Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Riddhi
authors.initials: R
authors.lastname: Datta
authors.affiliation: Plant Biology Lab., Organic and Medicinal Chemistry Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kumar
authors.affiliation: Structural Biology and Bioinformatics Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India.
authors.forename: Saikat
authors.initials: S
authors.lastname: Chakrabarti
authors.affiliation: Plant Biology Lab., Organic and Medicinal Chemistry Division, CSIR-Indian Institute Chemical Biology, 4, Raja S. C. Mullick Road, Kolkata, 700 032, India. sharmila@iicb.res.in.
authors.forename: Sharmila
authors.initials: S
authors.lastname: Chattopadhyay
mesh_terms: D000085:Acetates / Q000494:pharmacology; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics; D054883:Oxylipins / Q000494:pharmacology; D011034:Podophyllotoxin / Q000096:biosynthesis*; D011035:Podophyllum / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D054883:Oxylipins; D020033:Protein Isoforms; C072239:methyl jasmonate; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase; D011034:Podophyllotoxin
keywords: Cinnamyl alcohol dehydrogenase; Molecular docking; Podophyllotoxin; Podophyllum hexandrum; Transcriptome; Transgenic