LC-MS based untargeted metabolomics studies of the metabolic response of Ginkgo biloba extract on arsenism patients.

Arsenic is an environmentally ubiquitous toxic metalloid. Chronic exposure to arsenic may lead to arsenicosis, while no specific therapeutic strategies are available for the arsenism patients. And Ginkgo biloba extract (GBE) exhibited protective effect in our previous study. However, the mechanisms by which GBE protects the arsenism patients remain poorly understood. A liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics analysis was used to study metabolic response in arsenism patients upon GBE intervention. In total, 39 coal-burning type of arsenism patients and 50 healthy residents were enrolled from Guizhou province of China. The intervention group (n = 39) were arsenism patients orally administered with GBE (three times per day) for continuous 90 days. Plasma samples from 50 healthy controls (HC) and 39 arsenism patients before and after GBE intervention were collected and analyzed by established LC-MS method. Statistical analysis was performed by MetaboAnalyst 5.0 to identify differential metabolites. Multivariate analysis revealed a separation in arsenism patients between before (BG) and after GBE intervention (AG) group. It was observed that 35 differential metabolites were identified between BG and AG group, and 30 of them were completely or partially reversed by GBE intervention, with 14 differential metabolites significantly up-regulated and 16 differential metabolites considerably down-regulated. These metabolites were involved in promoting immune response and anti-inflammatory functions, and alleviating oxidative stress. Taken together, these findings indicate that the GBE intervention could probably exert its protective effects by reversing disordered metabolites modulating these functions in arsenism patients, and provide insights into further exploration of mechanistic studies.

Comprehensive analysis of JAZ family members in Ginkgo biloba reveals the regulatory role of the GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis.

Ginkgo biloba L., an ancient relict plant known as a 'living fossil', has a high medicinal and nutritional value in its kernels and leaves. Ginkgolides are unique diterpene lactone compounds in G. biloba, with favorable therapeutic effects on cardiovascular and cerebrovascular diseases. Thus, it is essential to study the biosynthesis and regulatory mechanism of ginkgolide, which will contribute to quality improvement and medication requirements. In this study, the regulatory roles of the JAZ gene family and GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis were explored based on genome and methyl jasmonate-induced transcriptome. Firstly, 18 JAZ proteins were identified from G. biloba, and the gene characteristics and expansion patterns along with evolutionary relationships of these GbJAZs were analyzed systematically. Expression patterns analysis indicated that most GbJAZs expressed highly in the fibrous root and were induced significantly by methyl jasmonate. Mechanistically, yeast two-hybrid assays suggested that GbJAZ3/11 interacted with both GbMYC2 and GbCOI1, and several GbJAZ proteins could form homodimers or heterodimers between the GbJAZ family. Moreover, GbMYC2 is directly bound to the G-box element in the promoter of GbLPS, to regulate the biosynthesis of ginkgolide. Collectively, these results systematically characterized the JAZ gene family in G. biloba and demonstrated that the GbCOI1/GbJAZs/GbMYC2 module could regulate ginkgolides biosynthesis, which provides a novel insight for studying the mechanism of JA regulating ginkgolide biosynthesis.

Inferring the Regulatory Network of miRNAs on Terpene Trilactone Biosynthesis Affected by Environmental Conditions.

As a medicinal tree species, ginkgo (Ginkgo biloba L.) and terpene trilactones (TTLs) extracted from its leaves are the main pharmacologic activity constituents and important economic indicators of its value. The accumulation of TTLs is known to be affected by environmental stress, while the regulatory mechanism of environmental response mediated by microRNAs (miRNAs) at the post-transcriptional levels remains unclear. Here, we focused on grafted ginkgo grown in northwestern, southwestern, and eastern-central China and integrally analyzed RNA-seq and small RNA-seq high-throughput sequencing data as well as metabolomics data from leaf samples of ginkgo clones grown in natural environments. The content of bilobalide was highest among detected TTLs, and there was more than a twofold variation in the accumulation of bilobalide between growth conditions. Meanwhile, transcriptome analysis found significant differences in the expression of 19 TTL-related genes among ginkgo leaves from different environments. Small RNA sequencing and analysis showed that 62 of the 521 miRNAs identified were differentially expressed among different samples, especially the expression of miRN50, miR169h/i, and miR169e was susceptible to environmental changes. Further, we found that transcription factors (ERF, MYB, C3H, HD-ZIP, HSF, and NAC) and miRNAs (miR319e/f, miRN2, miRN54, miR157, miR185, and miRN188) could activate or inhibit the expression of TTL-related genes to participate in the regulation of terpene trilactones biosynthesis in ginkgo leaves by weighted gene co-regulatory network analysis. Our findings provide new insights into the understanding of the regulatory mechanism of TTL biosynthesis but also lay the foundation for ginkgo leaves' medicinal value improvement under global change.

Expression profiling of the phenylalanine ammonia-lyase (PAL) gene family in ginkgo biloba L.

The PAL gene family plays an important role in plant growth, development, and response to abiotic stresses and has been identified in a variety of plants. However, a systematic characterization is still lacking in Ginkgo biloba. Using a bioinformatics approach, 11 GbPAL members of the PAL gene family identified in ginkgo were identified in this study. The protein structure and physicochemical properties indicated that the GbPAL genes were highly similar. Based on their exon-intron structures, they can be classified into three groups. A total of 62 cis-elements for hormone, light, and abiotic stress responses were identified in the promoters of GbPAL genes, indicating that PAL is a multifunctional gene family. GbPAL genes were specifically expressed in different tissues and ploidy of ginkgo. These results provide a theoretical basis for further studies on the functional expression of the GbPAL genes.

Ginkgo biloba GbbZIP08 transcription factor is involved in the regulation of flavonoid biosynthesis.

Ginkgo biloba is the oldest relict plant on Earth and an economic plant resource derived from China. Flavonoids extracted from G. biloba are beneficial to the prevention and treatment of cardiovascular and cerebrovascular diseases. Basic leucine zipper (bZIP) transcription factors (TFs) have been recognized to play important roles in plant secondary metabolism. In this study, GbbZIP08 was isolated and characterized. It encodes a protein containing 154 amino acids, which belongs to hypocotyl 5 in group H of the bZIP family. Tobacco transient expression assay indicated that GbbZIP08 was localized in the plant nucleus. GbbZIP08 overexpression showed that the contents of total flavonoids, kaempferol, and anthocyanin in transgenic tobacco were significantly higher than those in the wild type. Transcriptome sequencing analysis revealed significant upregulation of structural genes in the flavonoid biosynthesis pathway. In addition, phytohormone signal transduction pathways, such as the abscisic acid, salicylic acid, auxin, and jasmonic acid pathways, were enriched with a large number of differentially expressed genes. TFs such as MYB, AP2, WRKY, NAC, bZIP, and bHLH, were also differentially expressed. The above results indicated that GbbZIP08 overexpression promoted flavonoid accumulation and increased the transcription levels of flavonoid-synthesis-related genes in plants.

Screening and Evaluation of Novel α-Glucosidase Inhibitory Peptides from Ginkgo biloba Seed Cake Based on Molecular Docking Combined with Molecular Dynamics Simulation.

Food-derived α-glucosidase inhibitory peptides have gained significant interest in treating type 2 diabetes mellitus (T2DM) owing to their favorable safety profiles. Molecular docking combined with molecular dynamics simulation was performed to screen α-glucosidase inhibitory peptides from Ginkgo biloba seed cake (GBSC), and two novel peptides (Met-Pro-Gly-Pro-Pro (MPGPP) and Phe-Ala-Pro-Ser-Trp (FAPSW)) were acquired. The results of molecular docking and molecular dynamics simulation suggested that FAPSW and MPGPP could generate stable complexes with 3wy1, and the electrostatic and van der Waals forces played contributory roles in FAPSW and MPGPP binding to 3wy1. The α-glucosidase inhibition assay corroborated that FAPSW and MPGPP had good α-glucosidase inhibition capacity, with IC50 values of 445.34 ± 49.48 and 1025.68 ± 140.78 µM, respectively. In vitro simulated digestion results demonstrated that FAPSW and MPGPP strongly resisted digestion. These findings lay a theoretical foundation for FAPSW and MPGPP in treating T2DM.

Ginkgo biloba supplement abates lead-induced endothelial and testicular dysfunction in Wistar rats via up-regulation of Bcl-2 protein expression, pituitary-testicular hormones and down-regulation of oxido-inflammatory reactions.

BACKGROUND: Apoptotic and oxido-inflammatory pathways have been found to be up-regulated in lead acetate poisoning which has been associated to endothelial and testicular dysfunctions. It is yet uncertain, nevertheless, if treatment with Ginkgo biloba supplements (GBS), a flavonoid-rich natural product can lessen the adverse effects of lead on endothelial and testicular functions. This study investigated the impact of Ginkgo biloba supplementation on lead-induced endothelial and testicular dysfunctions. METHODS: The animals were treated with GBS (50 mg/kg and 100 mg/kg orally) for 14 days following oral exposure to lead acetate (25 mg/kg) for 14 days. After euthanasia, blood samples, epididymal sperm, testes, and aorta were collected. The quantities of the hormones (testosterone, follicle stimulating hormone (FSH) and luteinizing hormone (LH), as well as the anti-apoptotic, oxidative, nitrergic, inflammatory markers, were then determined using immunohistochemistry, ELISA, and conventional biochemical methods. RESULTS: GBS reduced lead-induced oxidative stress by increasing the levels of the antioxidant enzymes catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD), while lowering malondialdehyde (MDA) in endothelium and testicular cells. Normal testicular weight was restored by GBS which also decreased endothelial endothelin-I and increased nitrite levels. TNF-α and IL-6 were decreased while Bcl-2 protein expression was enhanced. Lead-induced alterations in reproductive hormones (FSH, LH, and testosterone) were also restored to normal. CONCLUSION: According to our result, using Ginkgo biloba supplement prevented lead from causing endothelial and testicular dysfunction by raising pituitary-testicular hormone levels, boosting Bcl-2 protein expression and lowering oxidative and inflammatory stress in the endothelium and testes.

Molecular Docking and Network Pharmacology Interaction Analysis of Gingko Biloba (EGB761) Extract with Dual Target Inhibitory Mechanism in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is the most common type of neurodegenerative dementia affecting people in their later years of life. The AD prevalence rate has significantly increased due to a lack of early detection technology and low therapeutic efficacy. Despite recent scientific advances, some aspects of AD pathological targets still require special attention. Certain traditionally consumed phytocompounds have been used for thousands of years to treat such pathologies. The standard extract of Gingko biloba (EGB761) is a combination of 13 macro phyto-compounds and various other micro phytocompounds that have shown greater therapeutic potential against the pathology of AD. OBJECTIVE: Strong physiological evidence of cognitive health preservation has been observed in elderly people who keep an active lifestyle. According to some theories, consuming certain medicinal extracts helps build cognitive reserve. We outline the research employing EGB761 as a dual target for AD. METHODS: This study investigates various inhibitory targets against AD using computational approaches such as molecular docking, network pharmacology, ADMET (full form), and bioactivity prediction of the selected compounds. RESULTS: After interaction studies were done for all the phytoconstituents of EGB761, it was concluded that all four of the phytocompounds (kaempferol, isorhamnetin, quercetin, and ginkgotoxin) showed the maximum inhibitory activity against acetylcholinesterase (AChE) and GSK3ß. CONCLUSION: The highly active phytocompounds of EGB761, especially quercetin, kaempferol, and isorhamnetin, have better activity against AChE and GSK3ß than its reported synthetic drug, according to molecular docking and network pharmacology research. These compounds may act on multiple targets in the protein network of AD. The AChE theory was primarily responsible for EGB761's therapeutic efficacy in treating AD.

Genome-Wide Identification of Expansin Gene Family and Their Response under Hormone Exposure in Ginkgo biloba L.

Expansins are pH-dependent enzymatic proteins that irreversibly and continuously facilitate cell-wall loosening and extension. The identification and comprehensive analysis of Ginkgo biloba expansins (GbEXPs) are still lacking. Here, we identified and investigated 46 GbEXPs in Ginkgo biloba. All GbEXPs were grouped into four subgroups based on phylogeny. GbEXPA31 was cloned and subjected to a subcellular localization assay to verify our identification. The conserved motifs, gene organization, cis-elements, and Gene Ontology (GO) annotation were predicted to better understand the functional characteristics of GbEXPs. The collinearity test indicated segmental duplication dominated the expansion of the GbEXPA subgroup, and seven paralogous pairs underwent strong positive selection during expansion. A majority of GbEXPAs were mainly expressed in developing Ginkgo kernels or fruits in transcriptome and real-time quantitative PCR (qRT-PCR). Furthermore, GbEXLA4, GbEXLA5, GbEXPA5, GbEXPA6, GbEXPA8, and GbEXPA24 were inhibited under the exposure of abiotic stresses (UV-B and drought) and plant hormones (ABA, SA, and BR). In general, this study expanded our understanding for expansins in Ginkgo tissues' growth and development and provided a new basis for studying GbEXPs in response to exogenous phytohormones.

Genome-Wide Identification, Evolutionary and Functional Analyses of WRKY Family Members in Ginkgo biloba.

WRKY transcription factors (TFs) are one of the largest families in plants which play essential roles in plant growth and stress response. Ginkgo biloba is a living fossil that has remained essentially unchanged for more than 200 million years, and now has become widespread worldwide due to the medicinal active ingredients in its leaves. Here, 37 WRKY genes were identified, which were distributed randomly in nine chromosomes of G. biloba. Results of the phylogenetic analysis indicated that the GbWRKY could be divided into three groups. Furthermore, the expression patterns of GbWRKY genes were analyzed. Gene expression profiling and qRT-PCR revealed that different members of GbWRKY have different spatiotemporal expression patterns in different abiotic stresses. Most of the GbWRKY genes can respond to UV-B radiation, drought, high temperature and salt treatment. Meanwhile, all GbWRKY members performed phylogenetic tree analyses with the WRKY proteins of other species which were known to be associated with abiotic stress. The result suggested that GbWRKY may play a crucial role in regulating multiple stress tolerances. Additionally, GbWRKY13 and GbWRKY37 were all located in the nucleus, while GbWRKY15 was located in the nucleus and cytomembrane.

Inhibitory effect of Ginkgo biloba seeds peptides on methylglyoxal-induced glycations.

The aim of this study was to investigate the antiglycation activity and mechanism of two identified peptides, Valine-Valine-Phenylalanine-Proline-Glycine-Cysteine-Proline-Glutamic acid (VVFPGCPE) and Serine-Valine-Aspartic acid-Aspartic acid-Proline-Arginine-Threonine-Lysine (SVDDPRTL), from Ginkgo biloba seeds protein hydrolysates. Both VVFPGCPE and SVDDPRTL were efficient in bovine serum albumin (BSA)-methylglyoxal (MGO) model to inhibit BSA glycation, while VVFPGCPE showed higher antiglycation activity than SVDDPRTL. In antioxidant assays, VVFPGCPE scavenged more hydroxyl and super anion radicals, and chelated more Fe2+. Moreover, VVFPGCPE was more efficient in alleviating glycoxidation since it retained higher content of tryptophan and reduced dityrosine and kynurenine generation. Compared with SVDDPRTL, VVFPGCPE showed better performance in inhibiting protein aggregation and amyloid-like fibrillation formation. Therefore, VVFPGCPE was selected for further mechanism study. The circular dichroism analysis suggested VVFPGCPE could preserve α-helix structure and stabilize protein structure. The MGO trapping assay indicated VVFPGCPE (5 mg/mL) could capture 66.25% MGO within 24 h, and the mass spectrometry revealed VVFPGCPE could trap MGO by forming VVFPGCPE-mono-MGO adducts. Besides, molecular simulations suggested VVFPGCPE could interact with key glycation residues, arginine and lysine residues, of BSA mainly through van der Waals and hydrogen bonds. This study might supply a theoretical basis for the development of VVFPGCPE as an effective antiglycation agent.

Effects of dietary Ginkgo biloba leaf extract on growth performance, immunity and environmental stress tolerance of Penaeus vannamei.

Ginkgo biloba leaf extract (GBE) has been extensively used in the treatment of diseases due to its anti-inflammatory, antioxidant, and immunomodulatory effects. In aquaculture, GBE is widely used as a feed additive, which is important to enhance the immunity of aquatic animals. The current study evaluated the effects of adding GBE to the diet of Penaeus vannamei (P. vannamei) under intensive aquaculture. The GBE0 (control group), GBE1, GBE2, and GBE4 groups were fed a commercial feed supplemented with 0.0, 1.0, 2.0, and 4.0 g/kg GBE for 21 days, respectively. The results showed that dietary GBE could alleviate hepatopancreas tissue damage and improve the survival rate of shrimp, and dietary 2 g/kg GBE could significantly increase the total hemocyte count (THC), the hemocyanin content, the antioxidant gene's expression, and the activity of their encoded enzymes in P. vannamei. Furthermore, transcriptome data revealed that immunity-related genes were upregulated in the GBE2 group compared with the GBE0 group after 21 days of culture. Drug metabolism-cytochrome P450, sphingolipid metabolism, linoleic acid metabolism, glycerolipid metabolism, fat digestion and protein digestion and absorption pathways were significantly enriched, according to KEGG results. Surprisingly, all of the above KEGG-enriched pathways were significantly upregulated. These findings demonstrated that supplementing P. vannamei with 2 g/kg GBE improved its environmental adaptability by improving immunity, lipid metabolism, and detoxification. In this study, a comprehensive evaluation of the effects of dietary GBE on the intensive aquaculture of P. vannamei was conducted to provide a reference for the healthy culture of P. vannamei.

Genome-wide identification and expression analysis of NAC family genes in Ginkgo biloba L.

NAC (NAM, ATAF, CUC2) transcription factors constitute one of the largest families of plant-specific transcription factors with important roles in plant growth and development and in biotic and abiotic stresses. The physicochemical properties, gene structure, cis-acting elements and expression patterns of NAC transcription factors in Ginkgo biloba were analysed using bioinformatics, and expression of this gene family was analysed via quantitative reverse transcription PCR. The family of G. biloba NAC transcription factors had 50 members, distributed on 12 chromosomes and divided into 11 groups. Members in the same group share a similar gene structure and motif distribution. Transcriptome data analysis of G. biloba showed that 35 genes were expressed in eight tissues. Correlation analysis suggested that GbNAC007 and GNAC008 might be involved in flavonoid biosynthesis. Expression levels of 12 GbNACs under cold, het, and salt stresses were analysed. Results indicate that NAC transcription factors play an important role in response to abiotic stresses. This study provides a reference for the functional analysis of the G. biloba family of NAC transcription factors, as well as a resource for studies on the involvement of this family in responses to abiotic stresses and flavonoid biosynthesis.

Site-Directed Mutation of Salicylate Decarboxylase Gene and Mechanism of Ginkgo Acid Decarboxylation.

Ginkgo seed is an important Chinese medicine and food resource in China, but the toxicity of ginkgo acid in it limits its application. Previous studies have found that salicylic acid decarboxylase (Sdc) has a decarboxylation degradation effect on ginkgo acid. In order to improve the decarboxylation ability of Sdc to Ginkgo acid, 11 residues of the Sdc around the substrate (salicylic acid) were determined as mutation targets according to the analysis of crystal structure of Sdc (PDB ID:6JQX), from Trichosporon moniliiforme WU-0401, and a total of 30 single point mutant enzymes and one compound mutant enzyme were obtained. With Ginkgo acid C15:1 as the substrate, it was found from activity assay that Sdc-Y64T and Sdc-P191A had higher decarboxylation activity, which increased by 105.18% and 116.74% compared with that of wild type Sdc, respectively. The optimal pH for Sdc Y64T and Sdc-P191A to decarboxylate Ginkgo acid C15:1 was 5.5, which is the same as the wild type Sdc. The optimal temperature of Sdc-P191A was 50 °C, which was consistent with that of the wild type Sdc, but the optimal temperature of the mutant Sdc-Y64T was 40 °C, which was 10 °C lower than that of wild type Sdc.

Time-Series-Dependent Global Data Filtering Strategy for Mining and Profiling of Xenobiotic Metabolites in a Dynamic Complex Matrix: Application to Biotransformation of Flavonoids in the Extract of Ginkgo biloba by Gut Microbiota.

Efficient characterization of xenobiotic metabolites and their dynamics in a changing complex matrix remains difficult. Herein, we proposed a time-series-dependent global data filtering strategy for the rapid and comprehensive characterization of xenobiotic metabolites and their dynamic variation based on metabolome data. A set of data preprocessing methods was used to screen potential xenobiotic metabolites, considering the differences between the treated and control groups and the fluctuations over time. To further identify metabolites of the target, an in-house accurate mass database was constructed by potential metabolic pathways and applied. Taking the extract of Ginkgo biloba (EGB) co-incubated with gut microbiota as an example, 107 compounds were identified as flavonoid-derived metabolites (including 67 original from EGB and 40 new) from 7468 ions. Their temporal metabolic profiles and regularities were also investigated. This study provided a systematic and feasible method to elucidate and profile xenobiotic metabolism.

Antiskin Aging Effects of Indole Alkaloid N-Glycoside from Ginkgo Fruit (Ginkgo biloba fruit) on TNF-α-Exposed Human Dermal Fibroblasts.

Human skin aging has internal and external factors, both of which are characterized by TNF-α overproduction. Therefore, we aimed to identify a natural product that suppresses the damage that occurs in cutaneous dermal fibroblasts exposed to TNF-α. The protective effects of the indole alkaloid N-glycoside, ginkgoside B dimethyl ester (GBDE), isolated from ginkgo fruit (Ginkgo biloba fruit) were evaluated in TNF-α stimulated human dermal fibroblasts (HDFs). GBDE inhibited TNF-α-induced MMP-1 expression to 2.2 ± 0.1-fold (p < 0.01) and reversed the decrease in collagen levels to 0.4 ± 0.00-fold (p < 0.01) at 50 µM. The effect of GBDE was due to the suppression of the phospolylaton of MAPKs (ERK, 0.47 ± 0.05; JNK, 1.21 ± 0.07; p38, 0.77 ± 0.07-folds, p < 0.001) and Akt (0.14 ± 0.03-fold, p < 0.001) compared to the TNF-α group. GBDE also reduced the expression of COX-2 to 2.06 ± 0.12-fold (p < 0.001) and increased the expression of HO-1 to 10.64 ± 0.2-fold (p < 0.001). In addition, GBDE inhibited the expression of the pro-inflammatory cytokines (IL-8, 2.2 ± 0.0; IL-1ß, 1.6 ± 0.0; IL-6, 2.0 ± 0.10-folds, p < 0.05). These results provide experimental evidence that GBDE can protect against skin damage, including aging.

Dynamic transcriptome and network-based analysis of yellow leaf mutant Ginkgo biloba.

BACKGROUND: Golden leaf in autumn is a prominent feature of deciduous tree species like Ginkgo biloba L., a landscape tree widely cultivated worldwide. However, little was known about the molecular mechanisms of leaf yellowing, especially its dynamic regulatory network. Here, we performed a suite of comparative physiological and dynamic transcriptional analyses on the golden-leaf cultivar and the wild type (WT) ginkgo to investigate the underlying mechanisms of leaf yellowing across different seasons. RESULTS: In the present study, we used the natural bud mutant cultivar with yellow leaves "Wannianjin" (YL) as materials. Physiological analysis revealed that higher ratios of chlorophyll a to chlorophyll b and carotenoid to chlorophyll b caused the leaf yellowing of YL. On the other hand, dynamic transcriptome analyses showed that genes related to chlorophyll metabolism played key a role in leaf coloration. Genes encoding non-yellow coloring 1 (NYC1), NYC1-like (NOL), and chlorophyllase (CLH) involved in the degradation of chlorophyll were up-regulated in spring. At the summer stage, down-regulated HEMA encoding glutamyl-tRNA reductase functioned in chlorophyll biosynthesis, while CLH involved in chlorophyll degradation was up-regulated, causing a lower chlorophyll accumulation. In carotenoid metabolism, genes encoding zeaxanthin epoxidase (ZEP) and 9-cis-epoxy carotenoid dioxygenase (NCED) showed significantly different expression levels in the WT and YL. Moreover, the weighted gene co-expression network analysis (WGCNA) suggested that the most associated transcriptional factor, which belongs to the AP2/ERF-ERF family, was engaged in regulating pigment metabolism. Furthermore, quantitative experiments validated the above results. CONCLUSIONS: By comparing the golden-leaf cultivar and the wide type of ginkgo across three seasons, this study not only confirm the vital role of chlorophyll in leaf coloration of YL but also provided new insights into the seasonal transcriptome landscape and co-expression network. Our novel results pinpoint candidate genes for further wet-bench experiments in tree species.

A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis.

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year's history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C-C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

An LC-MS-based metabolomic approach provides insights into the metabolite profiles of Ginkgo biloba L. at different developmental stages and in various organs.

Ginkgo (Ginkgo biloba L.) is a tree valued for the high medicinal and nutritional value of its leaves and seeds. However, the metabolite profiles of ginkgo leaves and seeds and their changes during development have not been comprehensively analyzed, which hinders improvements in the utilization of ginkgo. A comprehensive and systematic untargeted LC-MS metabolomics analysis of the metabolites in ginkgo leaves (male and female) and seeds at two developmental stages identified 8146 known metabolites, which mainly included lipids and lipid-like molecules, phenylpropanoids and polyketides, organoheterocyclic compounds, organic acids and derivatives, organic oxygen compounds, and benzenoids. Some of the identified metabolites have known healthcare and food value, and some of the others are newly discovered metabolites with potential for new drug development. The small number of differential Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between leaves of male and female gingko trees indicated that the developmental stage affected the metabolic pathways more significantly than sex. Among the flavonoid constituents of ginkgo, 653 flavonoid metabolites were identified, and these included some new flavonoid components, which confirmed that the developmental stage is a critical factor in secondary metabolite variations. This study illuminated the metabolites and medicinal and edible values of ginkgo leaves and seeds at different developmental stages and thus supports further effective utilization of ginkgo leaves and seeds.

Production of medium-chain fatty acids by co-fermentation of antibiotic fermentation residue with fallen Ginkgo leaves.

The co-fermentation of antibiotic fermentation residues (AFRs) and fallen Ginkgo leaves at C/N ratios of 10-60 was conducted for medium-chain fatty acids (MCFA) production. It was found that a proper C/N ratio could largely promote the MCFA accumulation. Group with C/N ratio of 50 exhibited highest MCFA production of 133.14 mmol C/L, which was 42 %-121 % higher than the other groups. Through the co-fermentation, substrate condition was optimized with rich micro-nutrients in AFRs and abundant polysaccharides in Ginkgo leaves, the hydrolysis of leaves was promoted by the active microbes in AFRs, and the predominance of CE microbes was also stimulated with the dilution of AFRs. The increased C/N ratio significantly affected the SCFA producers like genus Escherichia Shigella and Proteiniphilum, and enriched CE microbes like genus Romboutsia, Eubacterium and Clostridium_sensu_stricto_12. Functional enzymes analysis showed that both reverse ß oxidation and fatty acid biosynthesis pathways were strengthened with the increased C/N ratio.