Investigation and Expression of the Secoisolariciresinol Dehydrogenase Gene Involved in Podophyllotoxin Biosynthesis.
Podophyllotoxin (PPT) is a plant natural product that serves as a precursor for the synthesis of many well-known chemotherapeutic drugs. The limited availability and high demand for the source plants of PPT have led to the exploration of alternative sources for this compound. In this study, we utilized the endophytic fungus Phialocephala podophylli (strain PPE7) that we isolated from the rhizomes of Podophyllum peltatum and is known to produce detectable amounts of PPT in broth culture. To date, the complete PPT biosynthetic pathway has yet to be determined in any species. Since fungi are well known for clustering genes that belong to secondary metabolite pathways, use of a fungal system for investigation of the PPT biosynthesis genes may ultimately lead to elucidation of the entire pathway. In this study, we investigated the secoisolariciresinol dehydrogenase (SD) gene that facilitates the dehydrogenation of secoisolariciresinol to form matairesinol, a mid-pathway intermediate product in PPT biosynthesis. We utilized PCR amplification to acquire the complete SD gene sequence in PPE7 and opted to synthesize the P. peltatum SD sequence for expression. Through western blotting, we confirmed the expression of the recombinant SD (PpSD) and verified protein functionality with a bioconversion assay followed by HPLC and LC-MS analyses. Here, we report the identification of the SD gene in PPE7; this is the first report of the SD gene in an endophytic fungus. Additionally, we established the groundwork for the future expression of the complete PPT biosynthetic pathway in the heterologous host Pichia pastoris.
Department of Biological Sciences, University of the Sciences, 600 South 43rd Street, Philadelphia, PA, 19104, USA. sarneaud@mail.usciences.edu.
Sonja L B
SL
Arneaud
Department of Biological Sciences, University of the Sciences, 600 South 43rd Street, Philadelphia, PA, 19104, USA. j.porter@usciences.edu.
John R
http://orcid.org/0000-0001-8332-7313
JR
Porter
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D053898:Biosynthetic Pathways; D003001:Cloning, Molecular; D003470:Culture Media; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011034:Podophyllotoxin / Q000096:biosynthesis*; D028144:Podophyllum peltatum / Q000382:microbiology; D011487:Protein Conformation; D017422:Sequence Analysis, DNA
D003470:Culture Media; D004271:DNA, Fungal; D005656:Fungal Proteins; D064430:NAD (+) and NADP (+) Dependent Alcohol Oxidoreductases; C426673:secoisolariciresinol dehydrogenase; D011034:Podophyllotoxin
Heterologous expression; Phialocephala podophylli; Pichia pastoris; Podophyllotoxin biosynthesis; Podophyllum peltatum; Secoisolariciresinol dehydrogenase; Secondary metabolite