Discovery of unique CYP716C oxidase involved in pentacyclic triterpene biosynthesis from Camptotheca acuminata.

Dozens of triterpenes have been isolated from Camptotheca acuminata, however, triterpene metabolism in this plant remains poorly understood. The common C28 carboxy located in the oleanane-type and ursane-type triterpenes indicates the existence of a functionally active triterpene, C28 oxidase, in this plant. Thorough mining and screening of the CYP716 genes were initiated using the multi-omics database for C. acuminata. Two CYP716A (CYP716A394 and CYP716A395) and three CYP716C (CYP716C80-CYP716C82) were identified based on conserved domain analyses and hierarchical cluster analyses. CYP716 microsomal proteins were prepared and their enzymatic activities were evaluated in vitro. The CYP716 classified into the CYP716C subfamily displays ß-amyrin oxidation activity, and CYP716A displays α-amyrin and lupeol oxidation activity, based on gas chromatography-mass spectrometry analyses. The oxidation products were determined based on their mass and nuclear magnetic resonance spectrums. The optimum reaction conditions and kinetic parameters for CYP716C were determined, and functions were verified in Nicotiana benthaminana. Relative quantitative analyses revealed that these CYP716C genes were enriched in the leaves of C. acuminata plantlets after 60 d. These results indicate that CYP716C plays a dominant role in oleanane-type triterpene metabolism in the leaves of C. acuminata via a substrate-specific manner, and CYP716A is responsible for ursane- and lupane-type triterpene metabolism in fruit. This study provides valuable insights into the unique CYP716C-mediated oxidation step of pentacyclic triterpene biosynthesis in C. acuminata.

Proteomics-Guided Mining and Characterization of Epoxidase Involved in Camptothecin Biosynthesis from Camptotheca acuminata.

The detailed metabolic map for camptothecin (CPT) biosynthesis in Camptotheca acuminata has been proposed according to our combined omics results. However, the CYP450-mediated epoxidation step in CPT biosynthesis remains unexplored. A proteomics-guided approach was used to identify and annotate the proteins enriched during the vigorous CPT metabolism period in mature C. acuminata and seedlings. Comparative analyses revealed that the CPT and flavonoid biosyntheses were vigorous in stems and all of the samples except the leaves, respectively. The CYP71BE genes were screened based on their enrichment patterns at the transcriptomic-proteomic level and biochemically characterized in Saccharomyces cerevisiae WAT11. Four CYP71BE proteins exhibited in vitro isoliquiritigenin epoxidase activity. Additionally, CYP71BE206 showed epoxidase activity toward strictosamide, the critical precursor for CPT biosynthesis, both in vitro and in Nicotiana benthamiana. In planta functional verification suggested that CYP71BE206 is involved in CPT biosynthesis. Their catalytic conditions were optimized, and the enzymatic parameters were determined. This study provides valuable insight into the CYP71BE-mediated epoxidation step for CPT biosynthesis and offers evidence to verify that the newly characterized epoxidase (CYP71BE206) is simultaneously responsible for the biosynthesis of CPT and the flavonoid in this plant. An evolution event probably happened on ancestral CYP71BE, resulting in the neofunctionalization of CYP71BE206.

Biosynthesis-inspired mining and identification of untapped alkaloids in Camptotheca acuminate for enzyme discovery using ultra-high performance liquid chromatography coupled with quadrupole-time of flight-mass spectrometry.

Leaves, flowers, fruits and stems (44 sample groups) were collected from mature Camptotheca acuminate during 2017.3-2018.3 and classified by ultra-high performance liquid chromatography coupled with quadrupole-time of flight-mass spectrometry based metabolomics. One hundred metabolites including forty-seven alkaloids, fifteen terpenes, thirty-two polyphenols and six other metabolites were rapidly identified through the in-house database alignment at first glance. Thirty-three alkaloids classified into five groups including camptothecin group (CG1-13), pumiloside group (PG1-5), strictosidinic acid group (SG1-3), vincosamide group (VG1-7), and a new hybrid group, vincosamide-camptothecin group (VC1-5) were mined and further characterized by MS/MS analyses. The identification of two untapped biosynthetic precursors, 2-hydroxypumiloside (PG2) and 16­hydroxy­15, 16-dihydrocamptothecoside (CG3), along with sixteen new alkaloids enables us for a better understanding of camptothecin biogenetic reasoning. The underlying enzymes involved in camptothecin biosynthesis were also proposed according to the guiding metabolic map, thus purposefully mining of enzymes involved in the downstream biosynthetic pathway of camptothecin could be initiated with the help of this map.

Highly Efficient Metal-Free Two-Dimensional Luminescent Melem Nanosheets for Bioimaging.

Two-dimensional (2D) luminescent materials have received tremendous attention for their intrinsic properties and promising practical applications. Achieving 2D luminescent materials with high photoluminescence (PL) efficiency is still a great challenge. Here, ultrathin metal-free 2D luminescent nanosheets of 2,5,8-triamino-tri-s-triazine (melem) are synthesized through a facile liquid exfoliation process assisted by ultrasound. The as-obtained melem nanosheets distribute in the size range from a few nanometers to around 150 nm with a thickness of about 5 to 6 atomic layers. Melem nanosheets exhibit efficient blue emission with a PL efficiency as high as 77.09%, much higher than the heavily explored 2D luminescent g-C3N4 nanosheets. The high efficiency of melem nanosheets comes from the absence of atom vacancies and the low carrier mobility. Benefiting from the easy synthesis, good stability, low cell toxicity, and high efficiency, melem nanosheets are successfully applied as bioimaging materials on human breast cancer cells, requiring no extra treatments such as surface coating or functionalization. These metal-free 2D luminescent melem nanosheets hold great potential for various applications including bioimaging and other biorelated applications.

Possible clues for camptothecin biosynthesis from the metabolites in camptothecin-producing plants.

The plant derived camptothecin (CPT) is a pentacyclic pyrroloquinoline alkaloid with unique antitumor activity. Successive discoveries of new CPT-producing plants occurred in recent years due to market demands. The scattered distribution among angiosperms drew researchers' attention. The aim of this review is to appraise the literature available to date for CPT distribution and the phytochemistry of these CPT-producing plants. Metabolite comparative analyses between the plants were also conducted for tracking of possible clues for CPT biosynthesis. Forty-three plant species in total were reported to possess CPT-producing capability, and one hundred twenty-five alkaloids classified into three major categories are summarized herein. Metabolite comparative analysis between these plants suggests the probability that the formation of the central intermediate for CPT biosynthesis has multiple origins. A more complete biogenetic reasoning for CPT and its structural homolog was delineated based on this fragmentary phytochemical evidence from a chemical point of view. Furthermore, an in-house compound database was constructed for further metabolomic analysis.

[A neuritogenic compound from Tremella fuciformis].

OBJECTIVE: To search for low-molecular-weight neuritogenic compounds from the traditional Chinese medicine (TCM). METHOD: An extract library of TCM was prepared. Targeted isolation guided by biological screening led to the discovery of compound 1, and its structure was elucidated by analysis of spectroscopic methods and comparison of spectroscopic data with these reported from the literature. RESULT: A neuritogenic compound 1, 3-O-beta-D-glucopyranosyl-22E, 24R-5alpha, 8alpha-epidioxyergosta-6, 22-diene, was isolated and identified from the methanol extract of T. fuciformis. This compound showed a significant neuritogenic activity against PC12 cells at 3 micromol x L(-1)). CONCLUSION: Methonal extract of T. fuciformis and targeted compound 1 both showed significant neuritogenic activity against PC12 cells. These results suggested that the extract and compound 1 might be used to prevent and treat neurodegenerative disease such as Alzheimer's disease.

[Induce of laccase from Trametes gallica and its degradation on neutral dyes and organophosphorus pesticides].

The characteristics of the induction of laccase in Trametes gallica under different initial cultural pH, incubation time by different inducers were discussed, as well as the effects of temperature, pH and time on laccase degradation of six dyes and four organophosphors. The results showed that RB-bright blue, ABTS and o-toluidine affected the production of laccase at different levels, and ABTS was the best inductive agent in our test conditions, whose optimal initial pH and incubation time were 4.0 and 13 days, respectively. The appropriate reaction temperature of the laccase produced was 38 degrees C, and it got a good stability, for it could retain 78.6% of the enzyme activity after 20 min holding at 40 degrees C. Mediated by ABTS, the optimal temperature for laccase to degrade the six types of neutral dyes could be divided into two cases, that was 30 degrees C (neutral black, neutral bordeaux, neutral pink, methyl orange) and 60 degrees C (neutral dark yellow, cresol red), the optimal pH were 6.0 (neutral black), 2.0 (neutral bordeaux, neutral pink) and 4.0 (methyl orange, neutral dark yellow, cresol red), respectively, while the optimal times separately were 6 h (methyl orange, neutral dark yellow, cresol red), 12 h (neutral pink) and 24 h (neutral bordeaux). And using the same inductive agent, the best temperature for laccase to degrade dimethoate, chlorpyrifos, trichlorfon and parathion-pyridazine was 25 degrees C, the suitable time was 9 h, and the optimal pH was 10.0 for dimethoate, chlorpyrifos and parathion-pyridazine, and 8.0 for trichlorfon.