Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae.

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L-1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

Probing the functions of friedelane-type triterpene cyclases from four celastrol-producing plants.

Triterpenes are among the most diverse plant natural products, and their diversity is closely related to various triterpene skeletons catalyzed by different 2,3-oxidosqualene cyclases (OSCs). Celastrol, a friedelane-type triterpene with significant bioactivities, is specifically distributed in higher plants, such as Celastraceae species. Friedelin is an important precursor for the biosynthesis of celastrol, and it is synthesized through the cyclization of 2,3-oxidosqualene, with the highest number of rearrangements being catalyzed by friedelane-type triterpene cyclases. However, the molecular mechanisms underlying the catalysis of friedelin production by friedelane-type triterpene cyclases have not yet been fully elucidated. In this study, transcriptome data of four celastrol-producing plants from Celastraceae were used to identify a total of 21 putative OSCs. Through functional characterization, the friedelane-type triterpene cyclases were separately verified in the four plants. Analysis of the selection pressure showed that purifying selection acted on these OSCs, and the friedelane-type triterpene cyclases may undergo weaker selective restriction during evolution. Molecular docking and site-directed mutagenesis revealed that changes in some amino acids that are unique to friedelane-type triterpene cyclases may lead to variations in catalytic specificity or efficiency, thereby affecting the synthesis of friedelin. Our research explored the functional diversity of triterpene synthases from a multispecies perspective. It also provides some references for further research on the relative mechanisms of friedelin biosynthesis.

[Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius].

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 µmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

A cytochrome P450 CYP81AM1 from Tripterygium wilfordii catalyses the C-15 hydroxylation of dehydroabietic acid.

MAIN CONCLUSION: A novel cytochrome P450 from Tripterygium wilfordii, CYP81AM1, specifically catalyses the C-15 hydroxylation of dehydroabietic acid. This is the first CYP450 to be found in plants with this function. Cytochrome P450 oxygenases (CYPs) play an important role in the post-modification in biosynthesis of plant bioactive terpenoids. Here, we found that CYP81AM1 can catalyze the formation of 15-hydroxydehydroabietic acid by in vitro enzymatic reactions and in vivo yeast feeding assays. This is the first study to show that CYP81 family enzymes are involved in the hydroxylation of abietane diterpenoids. At the same time, we found that CYP81AM1 could not catalyse abietatriene and dehydroabietinol, suggesting that the occurrence of the reaction may be related to the carboxyl group. Through molecular docking and site mutations, it was found that some amino acid sites (F104, K107) near the carboxyl group had an important effect on enzyme activity, also suggesting that the carboxyl group played an important role in the occurrence of the reaction.

Engineering chimeric diterpene synthases and isoprenoid biosynthetic pathways enables high-level production of miltiradiene in yeast.

Miltiradiene is a key intermediate in the biosynthesis of many important natural diterpene compounds with significant pharmacological activity, including triptolide, tanshinones, carnosic acid and carnosol. Sufficient accumulation of miltiradiene is vital for the production of these medicinal compounds. In this study, comprehensive engineering strategies were applied to construct a high-yielding miltiradiene producing yeast strain. First, a chassis strain that can accumulate 2.1 g L-1 geranylgeraniol was constructed. Then, diterpene synthases from various species were evaluated for their ability to produce miltiradiene, and a chimeric miltiradiene synthase, consisting of class II diterpene synthase (di-TPS) CfTPS1 from Coleus forskohlii (Plectranthus barbatus) and class I di-TPS SmKSL1 from Salvia miltiorrhiza showed the highest efficiency in the conversion of GGPP to miltiradiene in yeast. Moreover, the miltiradiene yield was further improved by protein modification, which resulted in a final yield of 550.7 mg L-1 in shake flasks and 3.5 g L-1 in a 5-L bioreactor. This work offers an efficient and green process for the production of the important intermediate miltiradiene, and lays a foundation for further pathway reconstruction and the biotechnological production of valuable natural diterpenes.

The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the first enzyme in the plant 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoid synthesis. TwDXS is a prominent protein in the Tripterygium wilfordii proteome, with especially high expression in the root periderm. It is significantly regulated by methyl jasmonate. Here, we studied the influence of TwDXS expression on bioactive terpenoids in T. wilfordii. Specific fragments of TwDXS (GenBank: AKP20998.1) with lengths of 2148 and 437 bp were amplified to construct the overexpression (OE) and RNA-interference (RNAi) vectors, respectively. After transformation of suspension cells, the expression of TwDXS and genes related to the terpenoid biosynthetic pathway was measured using qRT-PCR. TwDXS mRNA level was 153 and 43% of the control in the OE and RNAi lines. Related genes in the 2-C-methyl-d-erythritol 4-phosphate (MEP), mevalonic acid (MVA) and downstream pathways showed similar trends to the changes of TwDXS expression. Ultra Performance Liquid Chromatography (UPLC) was employed to measure the accumulation of terpenoids. Importantly, the triptolide content showed significant differences in both the TwDXS OE (222.35% of the control) and RNAi (34.86% of the control). However, there were no obvious changes in the celastrol content. In this study, we verified that the expression of TwDXS affects triptolide but not celastrol in T. wilfordii via both TwDXS OE and RNAi experiments.

Overexpression and RNA interference of TwDXR regulate the accumulation of terpenoid active ingredients in Tripterygium wilfordii.

OBJECTIVE: To examine the putative regulatory role of TwDXR in terpenoid biosynthesis and terpenoid biosynthetic pathway-related gene expression, through overexpression and RNA interference with TwDXR. RESULTS: We obtained 1410 and 454 bp TwDXR-specific fragments to construct overexpression and RNAi vectors. qRT-PCR was used to detect the expression of TwDXR and terpenoid biosynthesis pathway-related genes. The overexpression of TwDXR led to a 285% upregulation and the TwDXR RNAi led to a reduction to 26% of the control (empty vector-transformed cells) levels. However, pathway-related genes displayed different trends. When TwDXR was overexpressed, TwDXS expression decreased by 31% but increased to 198% when TwDXR expression was inhibited. The accumulation of terpenoids was also assayed. In the overexpression group, differences were not significant whereas the contents of triptolide and celastrol in the TwDXR RNAi samples were diminished by 27.3 and 24.0%, respectively. CONCLUSION: The feedback regulation of gene transcription and the accumulation of terpenoids in terpenoid biosynthesis in Tripterygium wilfordii were verified by TwDXR overexpression and RNAi experiments.

Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae.

Triptolide is a valuable multipotent antitumor diterpenoid in Tripterygium wilfordii, and its C-14 hydroxyl group is often selected for modification to enhance both the bioavailability and antitumor efficacy. However, the mechanism for 14-hydroxylation formation remains unknown. Here, we discover 133 kb of tandem duplicated CYP82Ds encoding 11 genes on chromosome 12 and characterize CYP82D274 and CYP82D263 as 14-hydroxylases that catalyze the metabolic grid in triptolide biosynthesis. The two CYP82Ds catalyze the aromatization of miltiradiene, which has been repeatedly reported to be a spontaneous process. In vivo assays and evaluations of the kinetic parameters of CYP82Ds indicate the most significant affinity to dehydroabietic acid among multiple intermediates. The precursor 14-hydroxy-dehydroabietic acid is successfully produced by engineered Saccharomyces cerevisiae. Our study provides genetic elements for further elucidation of the downstream biosynthetic pathways and heterologous production of triptolide and of the currently intractable biosynthesis of other 14-hydroxyl labdane-type secondary metabolites.

Electro-enhanced sulfamethoxazole degradation efficiency via carbon embedding iron growing on nickel foam cathode activating peroxymonosulfate: Mechanism and degradation pathway.

The combination of peroxymonosulfate (PMS) activation by hetero-catalysis and electrolysis (EC) attracted incremental concerns as an efficient antibiotics degradation method. In this work, carbon embedding iron (C@Fe) catalysts growing on nickel foam (NF) composite cathode (C@Fe/NF) was prepared via in-situsolvothermal growth and carbonization method and used to activate PMS toward sulfamethoxazole (SMX) degradation. The EC-[C@Fe/NF(II)]-PMS system exhibited an excellent PMS activation, with 100% SMX removal efficiency achieving within 30 min. Reactive oxygen species (ROS) generation and their roles in SMX degradation were confirmed by quenching experiments and electron paramagnetic resonance. It was found that singlet oxygen (1O2) and surface-bound radicals were responsible for SMX degradation, and 1O2 contributed the most. Furthermore, the possible SMX degradation pathways were proposed on the base of the detected degradation intermediates and density functional theory (DFT) calculation. Toxicity changes were also assessed by the Ecological Structure Activity Relationships (ESAR). This work provides a practicable strategy for synergistically enhancing PMS activation efficiency and promoting antibiotics removal.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.