Molecular Cloning and Identification of NADPH Cytochrome P450 Reductase from Panax ginseng

Abstract

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.

Transcriptome-Wide Identification and Integrated Analysis of a UGT Gene Involved in Ginsenoside Ro Biosynthesis in Panax ginseng

“…In order to improve the ginsenoside content of P. ginseng , several genes from different gene families capable of affecting the synthesis of ginsenosides were identified and analyzed, such as, bHLHs [ 36 ], bZIPs [ 37 ], P450s [ 38 ], GRASs [ 39 ], and MYBs [ 40 ]. With the development of synthetic biology, the heterologous synthesis of ginsenosides in microbial cell factories offers a sustainable solution [ 41 , 42 , 43 , 44 ].…”

Class I and II NADPH-cytochrome P450 reductases exhibit different roles in triterpenoid biosynthesis in Lotus japonicus

“…Both CPR classes were reported to be able to support the in vitro activities of CYPs involved in specialized metabolism ( Rana et al., 2013 ). However, based on numerous functional analyses in planta , CPR class I was believed to be responsible for basal or constitutive metabolisms, while CPR class II was more responsible for adaptation and defense mechanisms, involving numerous specialized metabolisms ( Rana et al., 2013 ; Parage et al., 2016 ; Huang et al., 2021 ; Zou et al., 2021 ). This study showed that LjCPR1 is closely involved with CYP716A51, a C-28 oxidase, which is involved in triterpenoid biosynthesis, one of the specialized metabolisms in L. japonicus .…”

“…CPR class I is reported to be constitutively expressed and plays a role in primary or basal constitutive metabolism, while CPR class II is inducible by environmental stimuli and is involved in defense mechanisms through plant secondary metabolism ( Parage et al., 2016 ). Different tissue expression profiles of CPR classes I and II were reported in Withania somnifera ( Rana et al., 2013 ), Panax ginseng ( Zou et al., 2021 ), Camellia sinensis ( Huang et al., 2021 ), and Catharanthus roseus ( Parage et al., 2016 ). The differences in the protein sequences and expression levels of CPR classes I and II suggest that they have different roles in plants.…”

“…Transcript and metabolite profiling of stress/elicitor-treated plants or cell cultures can be used to determine gene function in secondary metabolism ( Misra et al., 2014 ). CPR class II genes from W. somnifera , P. ginseng , C. sinensis , and C. roseus were highly induced by methyl jasmonate (MeJA) treatment, whereas their CPR class I genes showed less or no induction ( Rana et al., 2013 ; Parage et al., 2016 ; Huang et al., 2021 ; Zou et al., 2021 ). Interestingly, this different effect of phytohormone treatment was also observed in OSC s and CYP s. RT-PCR analysis of MeJA-treated Ocimum basilicum showed that ObbAS1 and ObCYP2 were significantly and continually induced until 12 h of treatment, while the phytohormone effect on ObbAS2 , ObCYP1 , and ObCYP3 was not that apparent ( Misra et al., 2014 ).…”

A Trifolium repens flavodoxin‐like quinone reductase 1 (TrFQR1) improves plant adaptability to high temperature associated with oxidative homeostasis and lipids remodeling

“…Many enzymes including nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)‐cytochrome P450, NAD(P)H:quinone reductase (NQR) and flavodoxin‐like quinone reductase (FQR) are responsible for reduction of quinones in plants. NADH dehydrogenase and NADPH‐cytochrome P450 catalyze reduction of quinones by one‐electron reaction leading to the accumulation of reactive intermediates such as semiquinone (SQ), which passes a single electron to O 2 to form O 2 − and subsequent other ROS (Møller et al., 2021; Zou et al., 2021). Somewhat differently, NAD(P)H‐dependent quinone reductase family such as NQR and FQR catalyzes the transformation of quinone directly to dihydroquinones (DHQs) by carrying out two electrons, thereby decreasing the formation of SQ (Laskowski et al., 2002; Sparla et al., 1999; Wrobel et al., 2002).…”