Comparative Analysis of NADPH-Cytochrome P450 Reductases From Legumes for Heterologous Production of Triterpenoids in Transgenic Saccharomyces cerevisiae

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Because of their low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. In this study, we first analyzed the CPR expression level of three legumes species, Medicago truncatula, Lotus japonicus, and Glycyrrhiza uralensis, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. We then co-expressed different combinations of CYP716As and CYP72As with different CPR classes from these three legumes in transgenic yeast. We found that CYP716As worked better with CPR-I from the same species, while CYP72As worked better with any CPR-IIs. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest level of 11-oxo-β-amyrin, the important precursor of high-value metabolites glycyrrhizin. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.

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

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.

Contribution of NADPH-cytochrome P450 Reductase to Azole Resistance in Fusarium oxysporum

Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens-mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments.

Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults

Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.