P450 Enzyme Genes Research Articles

Systematic metabolic engineering for improved synthesis of perillic acid in Candida tropicalis

Perillic acid has been studied as an anticancer and antimicrobial drug. Production of perillic acid has attracted considerable attention. Meanwhile, Candida tropicalis is an unconventional diploid yeast, most significantly characterized by its ability to metabolize alkanes or fatty acids for growth and proliferation. Therefore, perillic acid’s precursor (L-limonene) in C. tropicalis was firstly synthesized by expressing a Mentha spicata L-limonene synthase gene, LS_Ms in this work. Expression of a gene which encoded for a truncated version of tLS_Ms increased the production of L-limonene with a 2.78-fold increase in the titer over C. tropicalis GJR-LS-01. Compartmentalized expression of the gene tLS_Ms inhibited the production of L-limonene in C. tropicalis compared to cytoplasmic expression. Cytoplasmic overexpression of seven precursor synthesis genes significantly enhanced the production of L-limonene in C. tropicalis compared to their compartmentalized expression (mitochondria or peroxisomes), which increased by 31.7-fold in C. tropicalis GJR-tLS-01. The L-limonene titer in C. tropicalis GJR-EW-tLS-04 overexpressing the mutant gene ERG20WW in the cytoplasm was significantly increased, 11.33-fold higher than the control. The titer of L-limonene for 60 g/L glucose was increased by 1.40-fold compared to the control. Finally, a Salvia miltiorrhiza cytochrome P450 enzyme gene CYP7176 and an Arabidopsis thaliana NADPH cytochrome P450 reductase gene CPR were heterologously expressed in C. tropicalis GJR-EW-tLS-04C for the synthesis of perillic acid, which reached a titer of 106.69 mg/L in a 5-L fermenter. This is the first report of de novo synthesis of perillic acid in engineered microorganisms. The results also showed that other chemicals may be efficiently produced in C. tropicalis.Key points• Key genes cytoplasmic expression was conducive to L-limonene production in C. tropicalis.• Perillic acid was first synthesized de novo in engineered microorganisms.• The titer of perillic acid reached 106.69 mg/L in a 5-L fermenter.

Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

Exploration of Candidate Genes Involved in the Biosynthesis, Regulation and Recognition of the Male-Produced Aggregation Pheromone of Halyomorpha halys

The aggregation pheromone of the brown marmorated stink bug, Halyomorpha halys (Stål), is produced by adult males, and plays an important role in the behavioral regulation of H. halys. However, information on the molecular mechanisms underlying this pheromone's biosynthesis is limited. In this study, HhTPS1, a key candidate synthase gene in the aggregation pheromone biosynthesis pathway of H. halys, was identified. Then, through weighted gene co-expression network analysis, the candidate P450 enzyme genes in the biosynthetic downstream of this pheromone and the related candidate transcription factor in this pathway were also identified. In addition, two olfactory-related genes, HhCSP5 and HhOr85b, involved in the recognition of the aggregation pheromone of H. halys, were detected. We further identified the key amino acid sites of HhTPS1 and HhCSP5 that interact with substrates by using molecular docking analysis. This study provides basic information for further investigations into the biosynthesis pathways and recognition mechanisms of aggregation pheromones in H. halys. It also provides key candidate genes for bioengineering bioactive aggregation pheromones necessary for the development of technologies for the monitoring and control of H. halys.

Toxicological effects of trace amounts of pyriproxyfen on the midgut of non-target insect silkworm

Pyriproxyfen is an insect growth regulator that is widely used in public health and pest control in agriculture. Our previous studies have shown that trace amounts of pyriproxyfen in the environment can cause serious toxic effects in the non-target insect silkworm, including failing to pupate, metamorphose and spin cocoons. However, it is unknown why pyriproxyfen not only has no lethal effects on fifth instar larvae but also tend to increase their body weight. The midgut is the main digestive organs of the silkworm, our results showed that the residual of pyriproxyfen in the silkworm at 24 h after 1 × 10−4 mg/L pyriproxyfen treatment caused severe damage to the midgut microvilli, goblet cells, and nuclei of the silkworm, but body weight and digestibility of the larval were both increased. In addition, pyriproxyfen significantly (p < 0.05) increased the activities of digestive enzymes (α-amylase, trehalase, trypsin and lipase) in the midgut of silkworm. However, it caused down-regulation of ecdysone synthesis-related genes at the end of the fifth instar silkworm, decreased ecdysone titer, and prolonged larval instar. At the same time, pyriproxyfen also activated transcription of detoxification enzymes-related genes such as the cytochrome P450 enzyme genes Cyp9a22 and Cyp15C1, the carboxylesterase genes CarE-8 and CarE-11, and the glutathione S-transferase gene GSTo2. This study elucidated a novel toxicological effect of pyriproxyfen to insects, which not only expands the understanding of the effects of juvenile hormone pesticides on lepidopteran insects but also provides a reference for exploring the ecological security of non-target organisms.

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation.

The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment.

BackgroundIndigo-containing plant tissues change blue after a freezing treatment, which is accompanied by changes in indigo and its related compounds. Phaius flavus is one of the few monocot plants containing indigo. The change to blue after freezing was described to explore the biosynthesis of indigo in P. flavus.MethodsIn this study, we surveyed the dynamic change of P. flavus flower metabolomics and transcriptomics.ResultsThe non-targeted metabolomics and targeted metabolomics results revealed a total of 98 different metabolites, the contents of indole, indican, indigo, and indirubin were significantly different after the change to blue from the freezing treatment. A transcriptome analysis screened ten different genes related to indigo upstream biosynthesis, including three anthranilate synthase genes, two phosphoribosyl-anthranilate isomerase genes, one indole-3-glycerolphosphate synthase gene, five tryptophan synthase genes. In addition, we further candidate 37 cytochrome P450 enzyme genes, one uridine diphosphate glucosyltransferase gene, and 24 β-D-glucosidase genes were screened that may have participated in the downstream biosynthesis of indigo. This study explained the changes of indigo-related compounds at the metabolic level and gene expression level during the process of P. flavus under freezing and provided new insights for increasing the production of indigo-related compounds in P. flavus. In addition, transcriptome sequencing provides the basis for functional verification of the indigo biosynthesis key genes in P. flavus.

Nuclear receptor HR96 up‐regulates cytochrome P450 for insecticide detoxification in Tribolium castaneum

Red flour beetle, Tribolium castaneum (T. castaneum), is a major agricultural pest that causes significant damage to stored grains and products. Although hormone receptor 96 (HR96) is known to be the single ortholog corresponding to mammalian constitutive androstane receptor and pregnane X receptor, the structural features of Tribolium HR96 (TcHR96) and its role in insecticide-mediated transcription control of cytochrome P450 enzyme genes in T. castaneum have not been elucidated yet. We cloned full-length complementary DNA encoding TcHR96 and revealed the role of TcHR96 in transcriptional control of cytochrome P450 enzyme genes. Interestingly, genome-wide transcriptome analysis of HR96-deficient beetles using RNA sequencing showed a positive correlation between TcHR96 and gene transcription of metabolizing enzymes involved in phase I detoxification processes. Moreover, TcHR96 overexpression significantly increased the promoter activity of genes encoding phase I P450 enzymes such as CYP4Q4, CYP4G7, CYP4BR3, and CYP345A1. Chromatin immunoprecipitation analysis showed that TcHR96 could directly bind to the promoter of gene encoding CYP345A1, an enzyme for metabolizing insecticides in T. castaneum. Furthermore, imidacloprid, a neonicotinoid insecticide, significantly increased gene expression of phase I P450 enzymes in old larvae of T. castaneum, which were reversed by TcHR96 knockdown. Finally, TcHR96 knockdown significantly decreased the resistance of old larvae to imidacloprid concomitant with reduction of imidacloprid-mediated phase I P450 enzyme gene expression. TcHR96 plays a major role in transcriptional control of P450 enzyme for imidacloprid detoxification. Controlling TcHR96 might facilitate the regulation of insecticide tolerance in T. castaneum, thus providing a promising new strategy to manage pest beetle populations. © 2021 Society of Chemical Industry.

Hepatic Dysregulation of Bile Acid Homeostasis in Hyperandrogenemic Female Mouse Model of Polycystic Ovary Syndrome

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is recognized as the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities compared with healthy individuals. Bile acids are endocrine signaling molecules that modulate hepatic lipid, glucose, and energy metabolism by aiding in absorption of lipids. Alteration of bile acid homeostasis affects overall metabolic homeostasis and contributes to pathogenesis of an array of metabolic diseases, although the molecular mechanisms of this have not been studied in PCOS. Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Weekly body weight, food intake, and body composition was assessed. Fasting serum was obtained and the oral glucose tolerance test (OGTT) was performed in the last week of treatment. Animals were euthanized on treatment day 90 and livers were harvested. Expression levels of mRNA were assessed using RT-qPCR. Results: DHT treated females had significantly higher liver mass (1,387 ± 51 vs 1,197 ± 29 g, p<0.05), increased lean mass (21.25 ± 0.27 vs 19.58 ± 0.23 g, p<0.05) and increased fat mass (4.83 ± 0.47 vs 3.59 ± 0.36 g, p<0.05) compared to the vehicle counterparts. These hyperandrogenemic females additionally showed altered glucose homeostasis, having increased fasting glucose (201.10 ± 11.11 vs 152.80 ± 9.23 mg/dL, p<0.05) and an increased area under the curve (209.2± 11.0 vs 160.8± 3.5 mg.min/dL, p<0.05) following OGTT. Hepatic expression of both classic (Cyp8b1, 1.4 ± 0.1-fold, p<0.05) and alternative (Cyp7b1, 2.0 ± 0.3-fold, p<0.05) bile acid synthesis cytochrome P450 enzyme genes were significantly upregulated in DHT treated animals. Additionally, expression of sulfotransferase Sult2a2 was completely abolished in DHT treated animals compared with vehicle animals, indicating the possibility of androgen regulation of the sulfonation of bile acids marked for elimination. Liver expression of both the bile acid receptor G-protein coupled bile acid receptor 1 and the androgen receptor were both significantly downregulated (Gpbar1: 0.68 ± 0.08-fold, AR: 0.46 ± 0.04-fold, p<0.05) in DHT treated animals. Conclusions: Bile acid synthesis, transport, and elimination are tightly controlled processes in the liver to maintain a constant bile acid pool and limit reabsorption. Together, our results highlight the potential role of androgens in DHT-treated female mice in the dysregulation of bile acid homeostasis and its potential contribution to influence metabolic dysfunction. (Supported by NIH grants NIGMS P20GM-121334 to LLYC and DGR, and NIH NIDDK R21DK-113500 to DGR and the Mississippi Center of Excellence in Perinatal Research.)

Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa.

Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3,4-dihydroxyphenylalanine 4,5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research.

Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua.

The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.

Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence

In this study, based on the transcriptome database of suspension cells of Arnebia euchroma, we explored two candidate cytochrome P450 enzyme genes that might relate to the shikonin biosynthesis downstream pathway when CYP76B74 sequence was referenced. We constructed interference-type hairy roots of candidate genes and cultured them. We measured the fresh weight, dry weight, total naphthoquinone content, shikonin and its derivatives content and expression levels of key enzyme genes involved in shikonin biosynthesis pathway. The effects of candidate genes on the growth and shikonin production of A. euchroma hairy roots were discussed, and the possible regulatory mechanisms that candidate genes affected shikonin synthesis were discussed. Through local Blast and phylogenetic analysis, two candidate CYP450 genes(CYP76B75 and CYP76B100) with high homology to CYP76B74 in A. euchroma were screened, and corresponding interference hairy roots were constructed. Compared with the control(RNAi-control), the fresh weight of CYP76B75 interfered hairy root(RNAi-CYP76B75) and CYP76B100 interfered hairy root(RNAi-CYP76B100) were significantly reduced, while dry weight were not affected, so the dry rate increased significantly. Except for β-acetoxyisovalerylalkannin, which is high in three groups of hairy roots, the contents of shikonin, deoxyshikonin, acetylshikonin, β,β'-dimethacrylicalkannin, β-hydroxyisovalerylshikonin,β-hydroxyisovalerylshikonin, isobutyrylshikonin and total naphthoquinones showed a consistent pattern: RNAi-CYP76B75&gt;RNAi-CYP76B100&gt;RNAi-control. Among them, the synthesis of β-hydroxyisovalerylshikonin was most significantly promoted by interfering with the expression of CYP76B75. The content of β-hydroxyisovalerylshikonin in RNAi-CYP76B75 was 11.7 times that of RNAi-control. RESULTS:: of real-time qPCR analysis showed that compared to RNAi-control, the expression levels of AePGT gene in RNAi-CYP76B75 and RNAi-CYP76B100 were not changed significantly, and the expression levels of CYP76B74 and AeHMGR were up-regulated. In addition, the expression level of CYP76B100 in RNAi-CYP76B75 was down-regulated, whereas in RNAi-CYP76B100, the expression of CYP76B75 was significantly up-regulated. Therefore, this study confirmed that when the expression of CYP76B75 and CYP76B100 were interrupted, the growth of hairy roots were suppressed, but the synthesis of shikonin were promoted. They might increase the shikonin biosynthesis by up-regulating the expression of CYP76B74 in the hairy roots of A. euchroma.

Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae)

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Long‐term outcomes of elderly patients with CYP2C9 and VKORC1 variants treated with vitamin K antagonists

Background The long-term effect of polymorphisms of the vitamin K-epoxide reductase (VKORC1) and the cytochrome P450 enzyme gene (CYP2C9) on clinical outcomes remains unclear. Objectives We examined the association between CYP2C9/VKORC1 variants and long-term clinical outcomes in a prospective cohort study of elderly patients treated with vitamin K antagonists for venous thromboembolism (VTE). Methods We followed 774 consecutive patients aged ≥ 65 years with acute VTE from nine Swiss hospitals for a median duration of 30 months. The median duration of initial anticoagulant treatment was 9.4 months. The primary outcome was the time to any clinical event (i.e. the composite endpoint of overall mortality, major and non-major bleeding, and recurrent VTE. Results Overall, 604 (78%) patients had a CYP2C9 or VKORC1 variant. Three hundred and thirty-four patients (43.2%) had any clinical event, 119 (15.4%) died, 100 (12.9%) had major and 167 (21.6%) non-major bleeding, and 100 had (12.9%) recurrent VTE. After adjustment, CYP2C9 (but not VKORC1) variants were associated with any clinical event (hazard ratio [HR], 1.34; 95% confidence interval [CI], 1.08-1.66), death (HR, 1.74; 95% CI, 1.19-2.52) and clinically relevant non-major bleeding (sub-hazard ratio [SHR], 1.39; 95% CI, 1.02-1.89), but not with major bleeding (SHR, 1.03; 95% CI, 0.69-1.55) or recurrent VTE (SHR, 0.95; 95% CI, 0.62-1.44). Patients with genetic variants had a slightly lower anticoagulation quality. Conclusions CYP2C9 was associated with long-term overall mortality and non-major bleeding. Although genetic variants were associated with a slightly lower anticoagulation quality, there was no relationship between genetic variants and major bleeding or VTE recurrence.

Cyclophosphamide treatment-induced leukopenia rates in ANCA-associated vasculitis are influenced by variant CYP450 2C9 genotypes.

Correlation of outcomes of cyclophosphamide (CP) therapy in antineutrophil cytoplasmic antibody-associated vasculitis with genotype polymorphisms in prodrug activating cytochrome P450 enzyme genes CYP2C9 and CYP2C19. One hundred and ninety six patients with antineutrophil cytoplasmic antibody-associated vasculitis treated with CP, either as intravenous pulse or as daily oral medication, were included. Genotypes of CYP2C9 and CYP2C19 were correlated with clinical outcomes (leukopenia, infection, urotoxicity and treatment response). Sixty five (33.2%) patients had variant CYP2C9 and 55 (28.1%) had variant CYP2C19 genotype. In patients bearing variant CYP2C9, leukopenia was documented significantly more frequent than in carriers of wild-type CYP2C9 (55.4 vs 37.4%; odds ratio: 2.08; 95% CI: 1.14-3.80; p = 0.017). The impact of the CYP2C9 genotype was stronger in patients treated with oral CP (69.6 vs 45.6%; odds ratio: 2.73; 95% CI: 1.27-5.89; p = 0.009), but was not present in patients treated with intravenous pulsed CP. We observed less refractory disease courses in patients with variant CYP2C9, not reaching statistical significance. Patients with variant CYP2C9 are at increased risk for cyclophosphamide-induced leukopenia but may have a better chance to respond to treatment.

Structure and biosynthesis of fumosorinone, a new protein tyrosine phosphatase 1B inhibitor firstly isolated from the entomogenous fungus Isaria fumosorosea

Fumosorinone, isolated from the entomogenous fungus Isaria fumosorosea, is a new 2-pyridone alkaloid which is elucidated by HRESIMS 1D and 2DNMR. Fumosorinone is structurally similar to tenellin and desmethylbassianin but it differs in chain length and degree of methylation. It is characterized by a classic noncompetitive inhibitor of protein tyrosine phosphatase 1B (IC50 14.04μM) which was implicated as a negative regulator of insulin receptor signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. For further study, we identified the biosynthetic gene cluster of fumosorinone from ongoing genome sequencing project, and it was verified by a direct knock-out strategy, reported for the first time in I. fumosorosea, using the Agrobacterium-mediated transformation in conjunction with linear deletion cassettes. The biosynthetic gene cluster includes a hybrid polyketide synthase–nonribosomal peptide synthetase gene, two cytochrome P450 enzyme genes, a trans-enoyl reductase gene, and other two transcription regulatory genes. Comparison of fumosorinone biosynthetic cluster with known gene clusters gives further insight into biosynthesis of pyridone alkaloids and provides the foundation for combinatorial biosynthesis for new fumosorinone derivatives.

Diversity of platelet function and genetic polymorphism in clopidogrel-treated Chinese patients.

We investigated the correlation between genetic polymorphisms of cytochrome P450 enzyme genes and the outcome of clopidogrel treatment in 118 coronary disease patients after percutaneous coronary intervention at the Chinese PLA General Hospital. Patients were divided into an ischemia event relapse group (IERG) and a non-IERG group (NIERG) based on relapse of ischemia events within 6 months after percutaneous coronary intervention. Ischemia occurred in 26.27% of patients. Thromboelastogram platelet mapping results showed that compared with the NIERG, the ADP-induced platelet inhibition ratio in the IERG was significantly lower (31.33 ± 24.91% vs 54.68 ± 26.63%, P < 0.05). The platelet inhibition ratio of patients carrying mutant alleles CYP3A5*3 (41.98 ± 29.33% vs 52.89 ± 26.49%), CYP2C19*2 (43.15 ± 27.97% vs 55.89 ± 26.71%), and P2Y12*1 (38.74 ± 24.36% vs 52.19 ± 28.58%) was lower than patients with the wild-type alleles. The frequency of ischemia event relapse in patients with the mutant alleles CYP3A5*3 and CYP2C19*2 was significantly higher than patients carrying the G/G genotype; however, there was no significant difference between patients carrying the T/T genotype and C allele of P2Y12*1. Thus, coexisting polymorphisms of CYP3A5*3 and 2C19*2, but not P2Y12*1, play an important role in the variability of clopidogrel's curative effect.

Expression of P450 and nuclear receptors in normal and end-stage Chinese livers.

To investigate the expression of P450 enzyme genes by using end-stage liver disease samples and trimmed normal Chinese donor livers. The end-stage liver disease samples [n = 93, including hepatocellular carcinoma (HCC), peri-HCC tissue, hepatitis B virus cirrhosis, alcoholic cirrhosis, and severe cirrhosis] and trimmed normal Chinese donor livers (n = 35) from The Institute of Organ Transplantation in Beijing, China. Total RNA was extracted, purified, and subjected to real-time RT-PCR analysis. For cytochrome P450 enzymes 1 (CYP1) family, the expression of CYP1A2 was decreased 90% in HCC, 80% in alcoholic cirrhosis, and 65% in severe cirrhosis. For CYP2 family, the expression of CAR was decreased 50% in HCC, but increased 50% in peri-HCC tissues. Similar decreases (about 50%) of CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 were observed in HCC, as compared to peri-HCC tissues and normal livers. CYP2C19 were decreased in all end-stage liver diseases and CYP2E1 also decreased in alcoholic cirrhosis and severe cirrhosis. For CYP3 family, the expression of PXR was decreased 60% in HCC, together with decreases in CYP3A4, CYP3A5, and CYP3A7. In contrast, the expression of CYP3A7 was slightly increased in HBV cirrhosis. The expression of CYP4A11 was decreased 85% in HCC, 7% in alcoholic cirrhosis and severe liver cirrhosis, along with decreases in PPARα. The 93 end-stage livers had much higher inter-individual variations in gene expression than 35 normal livers. The expression of CYP enzyme genes and corresponding nuclear receptors was generally decreased in end-stage liver diseases, and significant differences in gene expression were evident between peri-HCC and HCC.

Rutaecarpine effects on expression of hepatic phase-1, phase-2 metabolism and transporter genes as a basis of herb–drug interactions

Ethnopharmacological relevanceRutaecarpine is an alkaloid of Evodia rutaecarpa which is traditionally used to treat human diseases. Rutaecarpine has been used in combination with other drugs in the treatment of disorders and found to produce herb–drug interactions. The basis of these herb–drug interactions is not completely understood. Aim of studyTo examine the effects of rutaecarpine on the expression of drug processing genes, including Phase-1 (P450 enzyme genes), Phase-2 (glucuronidation and sulfation genes) and Phase-3 (drug transporters) in liver of mice. Materials and methodsMice were orally administered rutaecarpine at the doses of 10, 20, and 30mg/kg for consecutive 7 days. Twenty-four hours after the last dose, blood and liver were collected. Total RNA was isolated, purified, and subjected to real-time RT-PCR analysis of genes of interest. ResultsRutaecarpine administration induced Cyp1a2, 2b10 and 2e1 as previously reported. Cyp3a11 and Cyp4a10 were also induced. For phase-2 enzyme genes, rutaecarpine increased glucuronyltransferases (Ugt1a1 and Ugt1a6), but had no effects on sulfotransferase (Sult1a1 and Sult1b1). Most interestingly, rutaecarpine increased hepatic uptake of organic anion transporting peptides (Oatp1a1, Oayp1a4, Oatp1b2, and Oatp2b1) and induced efflux transporter such as multidrug resistance-associated proteins (Mrp1, Mrp2, Mrp3, and Mrp4), especially at the doses of 20mg/kg and above. ConclusionThe interactions of rutaecarpine with drugs involve not only the induction of cytochrome P450 enzyme genes, but also the induction of hepatic transporters and phase-2 enzyme genes. The effects of rutaecarpine on these drug processing genes could play integrated roles in producing herb–drug interactions.

An oligonucleotide microarray for detection of cytochrome P450 enzyme gene polymorphisms related to paclitaxel metabolism

Objective To develop an accurate,rapid,high throughout genotyping method based on oligonucleotide microarray for cytochrome P450 gene polymorphisms related to paclitaxel metabolism.Methods The mutant points of 2C8 * 3,3A4 * 18 and 3A5 * 3C from cytochrome P450 gene were regarded as targets.Based on the sequences in the GenBank,the wild-type and mutant-type probes were specially designed for each mutant point.PCR primers were located in the both sides of mutant point,and furthermore the fragments of PCR products were less than 200 bp.Each type of standard plasmids was constructed.Thus,all the olignucleotide probes were modified with 3'amino-group,and the reverse primers were labeled with fluorescein (Cy3).The probes were immobilized onto certain glass slides.The specific fragments of three genes were amplified and then hybridized with oligonucleotide microarray.The results were analyzed by using certain software.Finally this assay was applied to detect 50 clinical blood specimens.Results When PCR products from standard plasmids were hybridized with DNA microarray,the corresponding probes produced positive signals.Meanwhile,the non-specific hybridization signals did not appear.The results of clinical specimens showed that the mutant rate of CYP2C8 * 3 was 2%.The point of CYP3A4 * 18 for all the clinical specimens was wild-type and the mutant rate of CYP3A5 * 3C was 62%. Meanwhile,the results from detecting 50 clinical blood specimens using oligonucleotide microarray were the same as sequencing analysis.Conclusions Oligonucleotide microarray is a reliable and accurate genotyping assay for cytochrome P450 2C8 * 3.3A4 * 18 and 3A5 * 3C polymorphisms related to paclitaxel simultaneously.This genotyping assay is a high-throughout method for guiding personalized therapy and analyzing metabolism of paclitaxel in vivo. Key words: Cytochrome P-450 enzyme system; Paclitaxel; Polymorphism,genetic; Oligonucleotide array sequence analysis

Cytochrome P450 enzyme polymorphisms and adverse drug reactions.

Adverse drug reactions (ADR) are common and many are thought to have a genetic predisposition. There has been a great deal of interest in the role of P450 enzyme gene polymorphisms in the pathogenesis of adverse reactions. The major impact to date of polymorphic P450 expression has been on pre-clinical drug development. However, the direct clinical impact of P450 polymorphisms on prediction of ADRs has been limited, largely because studies have been small and retrospective, and the literature shows an abundance of contradictory data. Furthermore, the clinical- and cost-effectiveness of pre-prescription genotyping for P450 polymorphisms has not been convincingly demonstrated. Further studies that address these deficiencies are urgently needed--only then will prospective P450 genotyping become routine in clinical practice.