Methylerythritol Phosphate Research Articles

Monodopsis and Vischeria Genomes Shed New Light on the Biology of Eustigmatophyte Algae.

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis, have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis, very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis, long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria, which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans-splicing in Monodopsis and Vischeria at 36–61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae.

Mechanism of Action of N-Acyl and N-Alkoxy Fosmidomycin Analogs: Mono- and Bisubstrate Inhibition of IspC from Plasmodium falciparum, a Causative Agent of Malaria.

Malaria is a global health threat that requires immediate attention. Malaria is caused by the protozoan parasite Plasmodium, the most severe form of which is Plasmodium falciparum. The methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential to the survival of many human pathogens, including P. falciparum, but is absent in humans, and thus shows promise as a new antimalarial drug target. The enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the MEP pathway. In addition to a divalent cation (Mg2+), the enzyme requires the substrates 1-deoxy-D-xylulose 5-phosphate (DXP) and NADPH to catalyze its reaction. We designed N-alkoxy and N-acyl fosmidomycin analogs to inhibit the activity of P. falciparum IspC in a bisubstrate manner. Enzyme assays reveal that the N-alkoxy fosmidomycin analogs have a competitive mode of inhibition relative to both the DXP- and NADPH-binding sites, confirming a bisubstrate mode of inhibition. In contrast, the N-acyl fosmidomycin analogs demonstrate competitive inhibition with respect to DXP but uncompetitive inhibition with respect to NADPH, indicating monosubstrate inhibitory activity. Our results will have a positive impact on the discovery of novel antimalarial drugs.

Metabolomic Variability of Different Soybean Genotypes: β-Carotene-Enhanced (Glycine max), Wild (Glycine soja), and Hybrid (Glycine max × Glycine soja) Soybeans.

We obtained a new hybrid soybean (Hybrid) by hybridizing β-carotene-enhanced soybean (BCE; Glycine max L.) containing the phytoene synthase-2A-carotene desaturase gene and wild-type soybean (Wild; Glycine soja). To investigate metabolic changes between variants, we performed metabolic profiling of leaves (three growth stages) and seeds. Multivariate analyses revealed significant metabolic differences between genotypes in seeds and leaves, with seeds showing accumulation of phytosterols, tocopherols, and carotenoids (BCE only), indicating co-induction of the methylerythritol 4-phosphate and mevalonic acid pathways. Additionally, Hybrid produced intermediate levels of carotenoids and high levels of amino acids. Principal component analysis revealed metabolic discrimination between growth stages of soybean leaves and identified differences in leaf groups according to different genotypes at 8, 12, and 16 weeks, with Wild showing higher levels of environmental stress-related compounds relative to BCE and Hybrid leaves. The metabolic profiling approach could be a useful tool to identify metabolic links in various soybean cultivars.

Sclareol and linalyl acetate are produced by glandular trichomes through the MEP pathway

Sclareol, an antifungal specialized metabolite produced by clary sage, Salvia sclarea, is the starting plant natural molecule used for the hemisynthesis of the perfume ingredient ambroxide. Sclareol is mainly produced in clary sage flower calyces; however, the cellular localization of the sclareol biosynthesis remains unknown. To elucidate the site of sclareol biosynthesis, we analyzed its spatial distribution in the clary sage calyx epidermis using laser desorption/ionization mass spectrometry imaging (LDI–FTICR-MSI) and investigated the expression profile of sclareol biosynthesis genes in isolated glandular trichomes (GTs). We showed that sclareol specifically accumulates in GTs’ gland cells in which sclareol biosynthesis genes are strongly expressed. We next isolated a glabrous beardless mutant and demonstrate that more than 90% of the sclareol is produced by the large capitate GTs. Feeding experiments, using 1-13C-glucose, and specific enzyme inhibitors further revealed that the methylerythritol-phosphate (MEP) biosynthetic pathway is the main source of isopentenyl diphosphate (IPP) precursor used for the biosynthesis of sclareol. Our findings demonstrate that sclareol is an MEP-derived diterpene produced by large capitate GTs in clary sage emphasing the role of GTs as biofactories dedicated to the production of specialized metabolites.

Retraction Note: IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance.

Isoprenoids are vital for all organisms, in which they maintain membrane stability and support core functions such as respiration1. IspH, an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, is essential for Gram-negative bacteria, mycobacteria and apicomplexans2,3. Its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), is not produced in metazoans, and in humans and other primates it activates cytotoxic Vγ9Vδ2 T cells at extremely low concentrations4–6. Here we describe a class of IspH inhibitors and refine their potency to nanomolar levels through structure-guided analogue design. After modification of these compounds into prodrugs for delivery into bacteria, we show that they kill clinical isolates of several multidrug-resistant bacteria—including those from the genera Acinetobacter, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Shigella, Salmonella, Yersinia, Mycobacterium and Bacillus—yet are relatively non-toxic to mammalian cells. Proteomic analysis reveals that bacteria treated with these prodrugs resemble those after conditional IspH knockdown. Notably, these prodrugs also induce the expansion and activation of human Vγ9Vδ2 T cells in a humanized mouse model of bacterial infection. The prodrugs we describe here synergize the direct killing of bacteria with a simultaneous rapid immune response by cytotoxic γδ T cells, which may limit the increase of antibiotic-resistant bacterial populations. A class of compounds with a dual mechanism of action—direct targeting of IspH and stimulation of cytotoxic γδ T cells to enhance pathogen clearance—are active against multidrug-resistant bacteria.

Growth condition controls on G-93 parameters of isoprene emission from tropical trees

Despite its major role in global isoprene emission, information on the environmental control of isoprene emission from tropical trees has remained scarce. Thus, in this study, we examined the relationship between parameters of G-93 isoprene emission formula (CT1, CT2, and α), growth temperature and light intensity, photosynthesis (ɸ, Pmax), isoprene synthase (IspS) level, and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway metabolites using sunlit and shaded leaves of four tropical trees. The results showed that the temperature dependence of isoprene emission from shaded leaves did not differ significantly from sunlit leaves. In contrast, there was a lower saturation irradiance in shaded leaves than in sunlit leaves, the same as temperate plants. The photosynthesis rate of shaded leaves showed lower saturation irradiance, similar to the light dependence of isoprene emission. In most cases, the concentration of MEP pathway metabolites was of lower tendency in shaded leaves versus in sunlit leaves, whereas no significant difference was noted in IspS level between sunlit and shaded leaves. Correlation analysis between these parameters found that CT1 of the G-93 parameter was positively correlated with the concentration of DXP and DMADP, whereas CT2 correlated with the concentration of MEP and the average air temperature for the past 48h. Similarly, α closely associated with the initial slope (ɸ) of photosynthesis rate, and the basal emission factor is also linked to the photon flux of past days. These results suggest that growth conditions may control the temperature dependence of isoprene emission from tropical trees via the changes in the profiles of MEP pathway metabolites, causing alteration in the parameters of the isoprene emission formula.

Description of Microbacterium luteum sp. nov., Microbacterium cremeum sp. nov., and Microbacterium atlanticum sp. nov., three novel C50 carotenoid producing bacteria.

We have identified three Microbacterium strains, A18JL200T, NY27T, and WY121T, that produce C50 carotenoids. Taxonomy shows they represent three novel species. These strains shared < 98.5% 16S rRNA gene sequence identity with each other and were closely related to Microbacterium aquimaris JCM 15625T, Microbacterium yannicii JCM 18959T, Microbacterium ureisolvens CFH S00084T, and Microbacterium hibisci CCTCC AB 2016180T. Digital DNA-DNA hybridization (dDDH) values and average nucleotide identity (ANI) showed differences among the three strains and from their closest relatives, with values ranging from 20.4% to 34.6% and 75.5% to 87.6%, respectively. These values are below the threshold for species discrimination. Both morphology and physiology also differed from those of phylogenetically related Microbacterium species, supporting that they are indeed novel species. These strains produce C50 carotenoids (mainly decaprenoxanthin). Among the three novel species, A18JL200T had the highest total yield in carotenoids (6.1 mg/L or 1.2 mg/g dry cell weight). Unusual dual isoprenoid biosynthetic pathways (methylerythritol phosphate and mevalonate pathways) were annotated for strain A18JL200T. In summary, we found strains of the genus Microbacterium that are potential producers of C50 carotenoids, but their genome has to be investigated further.

From Magic Bullet to Magic Bomb: Reductive Bioactivation of Antiparasitic Agents.

Paul Ehrlich coined the term "magic bullet" to describe how a drug kills the parasite inside its human host without harming the host itself. Ehrlich concluded that the drug must have a greater affinity to the parasite than to human cells. Today, the specificity of drug action is understood in terms of the drug target. An ideal target is a protein that is essential for the proliferation of the pathogen but absent in human cells. Examples are the enzymes of folate synthesis or of the nonmevalonate pathway in the malaria parasites. However, there are other ways how a drug can kill selectively. Of particular relevance is the specific activation of a prodrug inside the pathogen but not in the host, as this is how the current frontrunners of parasite chemotherapy work. Artemisinins for malaria, fexinidazole for human African trypanosomiasis, benznidazole for Chagas' disease, metronidazole for intestinal protozoa: these molecules are "magic bombs" that are triggered selectively. They are prodrugs that need to be activated by chemical reduction, i.e., the acquisition of an electron, which occurs in the parasite. Such a mode of action is shared by the novel antimalarial peroxides arterolane and artefenomel, which are activated by reduction of the endoperoxide bond with ferrous heme as the likely electron donor, a metabolic end-product of Plasmodium falciparum. Here we provide an overview on the molecular basis of selectivity of antiparasitic drug action with particular reference to the ozonides, the new generation of antimalarial peroxides designed by Jonathan Vennerstrom.

Listeria monocytogenes-infected human monocytic derived dendritic cells activate V\u03b39V\u03b42 T cells independently of HMBPP production

Gamma-delta (γδ) T cells express T cell receptors (TCR) that are preconfigured to recognize signs of pathogen infection. In primates, γδ T cells expressing the Vγ9Vδ2 TCR innately recognize (E)-4-hydroxy-3-methyl-but- 2-enyl pyrophosphate (HMBPP), a product of the 2-C-methyl-D-erythritol 4- phosphate (MEP) pathway in bacteria that is presented in infected cells via interaction with members of the B7 family of costimulatory molecules butyrophilin (BTN) 3A1 and BTN2A1. In humans, Listeria monocytogenes (Lm) vaccine platforms have the potential to generate potent Vγ9Vδ2 T cell recognition. To evaluate the activation of Vγ9Vδ2 T cells by Lm-infected human monocyte-derived dendritic cells (Mo-DC) we engineered Lm strains that lack components of the MEP pathway. Direct infection of Mo-DC with these bacteria were unchanged in their ability to activate CD107a expression in Vγ9Vδ2 T cells despite an inability to synthesize HMBPP. Importantly, functional BTN3A1 was essential for this activation. Unexpectedly, we found that cytoplasmic entry of Lm into human dendritic cells resulted in upregulation of cholesterol metabolism in these cells, and the effect of pathway regulatory drugs suggest this occurs via increased synthesis of the alternative endogenous Vγ9Vδ2 ligand isoprenyl pyrophosphate (IPP) and/or its isomer dimethylallyl pyrophosphate (DMAPP). Thus, following direct infection, host pathways regulated by cytoplasmic entry of Lm can trigger Vγ9Vδ2 T cell recognition of infected cells without production of the unique bacterial ligand HMBPP.

Overexpression of geranyl diphosphate synthase (PmGPPS1) boosts monoterpene and diterpene production involved in the response to pine wood nematode invasion.

Outbreaks of pine wood nematode (PWN; Bursaphelenchus xylophilus) represent a severe biotic epidemic for the Pinus massoniana in China. When invaded by the PWN, the resistant P. massoniana might secret abundant oleoresin terpenoid to form certain defensive fronts for survival. However, the regulatory mechanisms of this process remain unclear. Here, the geranyl diphosphate synthase (PmGPPS1) gene was identified from resistant P. massoniana. Tissue-specific expression patterns of PmGPPS1 at transcript and protein level in resistant P. massoniana were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. Functional characteristics analysis of PmGPPS1 was performed on transgenic Nicotiana benthamiana by overexpression, as genetic transformation of P. massoniana is, so far, not possible. In summary, we identified and functionally characterized PmGPPS1 from the resistant P. massoniana following PWN inoculation. Tissue-specific expression patterns and localization of PmGPPS1 indicated that it may play a positive role involved in the metabolic and defensive processes of oleoresin terpenes production in response to PWN attack. Furthermore, overexpression of PmGPPS1 may enhance the production of monoterpene, among which limonene reduced the survival of PWN in vitro. In addition, PmGPPS1 upregulated the expression level of key genes involved in mevalonic acid (MVA) pathway, the methylerythritol phosphate (MEP) pathway and gibberellins (GAs) biosynthesis to boost the growth and development of tobacco through a feedback regulation mechanism. Our results offered new insights into the pivotal role of the PmGPPS1 involved in terpene-based defense mechanisms responding to the PWN invasion in resistant P. massoniana and provided a new metabolic engineering scenario to improve monoterpene production in tobacco.

The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway.

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 (th3) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 (DXS1), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

Identification of Key Genes Controlling Carotenoid Metabolism during Apricot Fruit Development by Integrating Metabolic Phenotypes and Gene Expression Profiles.

To explore the metabolic basis of carotenoid accumulation in different developmental periods of apricot fruits, targeted metabonomic and transcriptomic analyses were conducted in four developmental periods (S1-S4) in two cultivars (Prunus armeniaca cv. "Kuchebaixing" with white flesh and P. armeniaca cv. "Shushangganxing" with orange flesh) with different carotenoid contents. 14 types of carotenes and 27 types of carotene lipids were identified in apricot flesh in different developmental periods. In S3 and S4, the carotenoid contents of the two cultivars were significantly different, and β-carotene and (E/Z)-phytoene were the key metabolites that caused the difference in the total carotenoid content between the examined cultivars. Twenty-five structural genes (including genes in the methylerythritol 4-phosphate and carotenoid biosynthesis pathways) related to carotenoid biosynthesis were identified among the differentially expressed genes in different developmental periods of the two cultivars, and a carotenoid metabolic pathway map of apricot fruits was drawn according to the KEGG pathway map. The combined analysis of carotenoid metabolism data and transcriptome data showed that PSY, NCED1, and CCD4 were the key genes leading to the great differences in the total carotenoid content. The results provide a new approach to study the synthesis and accumulation of carotenoids in apricot fruits.

Channeling of Carbon Flux Towards Carotenogenesis in Botryococcus braunii: A Media Engineering Perspective

Microalgae, due to their unique properties, gained attention for producing promising feedstocks having high contents of proteins, antioxidants, carotenoids, and terpenoids for applications in nutraceutical and pharmaceutical industries. Optimizing production of the high-value renewables (HVRs) in microalgae requires an in-depth understanding of their functional relationship of the genes involved in these metabolic pathways. In the present study, bioinformatic tools were employed for characterization of the protein-encoding genes of methyl erythritol phosphate (MEP) pathway involved in carotenoid and squalene biosynthesis based upon their conserved motif/domain organization. Our analysis demonstrates nearly 200 putative genes showing a conservation pattern within divergent microalgal lineages. Furthermore, phylogenomic studies confirm the close evolutionary proximity among these microalgal strains in the carotenoid and squalene biosynthetic pathways. Further analysis employing STRING predicts interactions among two rate-limiting genes, i.e., phytoene synthase (PSY) and farnesyl diphosphate farnesyl synthase (FPPS), which are specifically involved in the synthesis of carotenoids and squalene. Experimentally, to understand the carbon flux of these rate-limiting genes involved in carotenogenesis, an industrial potential strain, namely, Botryococcus braunii, was selected in this study for improved biomass productivity (i.e., 100 mg L–1 D–1) along with enhanced carotenoid content [0.18% dry cell weight (DCW)] when subjected to carbon supplementation. In conclusion, our approach of media engineering demonstrates that the channeling of carbon flux favors carotenogenesis rather than squalene synthesis. Henceforth, employing omics perspectives will further provide us with new insights for engineering regulatory networks for enhanced production of high-value carbon biorenewables without compromising growth.

Comparative transcriptome analysis reveals sesquiterpenoid biosynthesis among 1-, 2- and 3-year old Atractylodes chinensis

BackgroundAtractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood.ResultsIn this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis.ConclusionThe enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

Identification of the diterpenoid biosynthesis genes and their expression status in relation to oleoresin yield of masson pine

Conifer oleoresin is a highly precious raw material for industrial sectors and a precursor for biofuels. Diterpenoid, particularly diterpenoid resin acids (DRAs), is one of the most abundant oleoresin components. Masson pine (Pinus massoniana L.) is one of the most important native species for oleoresin yield in China. To better understand the biosynthesis of diterpenoid in high oleoresin-yield masson pine and develop methods for its production in needle and trunk xylem, we used the combination of SMRT full-length transcriptome and Illumina RNA sequencing technologies to establish a transcriptome dataset. Approximately 20,560 and 17,950 full-length non-redundant transcripts were assembled in trunk xylem and needle, respectively. Of those, 29 and 20 DEGs were identified involving in the Terpenoid Backbone Biosynthesis pathway (TBB) (KO 00900) and the Diterpenoid Biosynthesis pathway (DB) (KO 00904) in trunk xylem, as well as 35 and 12 DEGs in the TBB and the DB in the needle, respectively. Compared with the low oleoresin-yield germplasm, 13 up-regulated DEGs with complete open reading frame (ORF) sequence were obtained by Venn analysis in trunk xylem and needle of a high oleoresin-yield clone. Subsequently, the expression patterns of the 16 diterpenoid biosynthesis-related genes (including three types of DXS genes) were validated by quantitative real-time PCR in both 12-year-old and 1-year-old high and low oleoresin-yield masson pine clones. The results showed that the 13 genes in the higher yield oleoresin trees were proved to be higher expression in comparison with the lower ones irrespectively of the tree age; as well as MCS (2-C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase), and IDI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway demonstrated a higher expression compared to that of other TBB genes. Both cytosol-localized MVA and plastid-localized MEP pathways gave significant contributions to diterpenoid accumulation, and the latter played a more dominant role. To clarify the potential relationships between oleoresin yield and the expression patterns of 16 genes from the TBB and DB pathways, the Pearson correlation analysis was performed using three tissues (young needle, old needle, and trunk xylem) from the 60-year-old tree. Remarkably, HMGR (Hydroxy methylglutaryl-CoA reductase) and ent-CDS (Ent-copalyl diphosphate synthase) were positive correlation with oleoresin yield in the new needle, whereas FPPS (Farnesyl pyrophosphate synthase) was a negative correlation with that in the old needle; furthermore, HMGS (Hydroxy methylglutaryl-CoA synthase), IDI and CDS (Copalyl diphosphate synthase) were positively correlated with oleoresin synthesis in the trunk xylem. Moreover, the Pearson correlation analysis was also carried out to determine the potential relationships between the 13 genes of the TBB pathways and ent-CDS, CDS, and LDS ((13E)-labda-7 13-dien-15-ol synthase) from DB pathways within three tissues of the 60-year-old masson pine. Furthermore, the diterpenoid and diterpene resin acids (DRAs) demonstrated highly positive correlations with oleoresin yield, and DRAs were detected as the most abundant diterpenoids in oleoresin. Promisingly, HMGR, MCS, HDS, IDI, ent-CDS, and CDS may be used as the key target genes of the molecular-assisted selection for high-oleoresin-yielding germplasm in this species.

The Coordinated Upregulated Expression of Genes Involved in MEP, Chlorophyll, Carotenoid and Tocopherol Pathways, Mirrored the Corresponding Metabolite Contents in Rice Leaves during De-Etiolation.

Light is an essential regulator of many developmental processes in higher plants. We investigated the effect of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1/2 genes (OsHDR1/2) and isopentenyl diphosphate isomerase 1/2 genes (OsIPPI1/2) on the biosynthesis of chlorophylls, carotenoids, and phytosterols in 14-day-old etiolated rice (Oyza sativa L.) leaves during de-etiolation. However, little is known about the effect of isoprenoid biosynthesis genes on the corresponding metabolites during the de-etiolation of etiolated rice leaves. The results showed that the levels of α-tocopherol were significantly increased in de-etiolated rice leaves. Similar to 1-deoxy-D-xylulose-5-phosphate synthase 3 gene (OsDXS3), both OsDXS1 and OsDXS2 genes encode functional 1-deoxy-D-xylulose-5-phosphate synthase (DXS) activities. Their expression patterns and the synthesis of chlorophyll, carotenoid, and tocopherol metabolites suggested that OsDXS1 is responsible for the biosynthesis of plastidial isoprenoids in de-etiolated rice leaves. The expression analysis of isoprenoid biosynthesis genes revealed that the coordinated expression of the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway, chlorophyll, carotenoid, and tocopherol pathway genes mirrored the changes in the levels of the corresponding metabolites during de-etiolation. The underpinning mechanistic basis of coordinated light-upregulated gene expression was elucidated during the de-etiolation process, specifically the role of light-responsive cis-regulatory motifs in the promoter region of these genes. In silico promoter analysis showed that the light-responsive cis-regulatory elements presented in all the promoter regions of each light-upregulated gene, providing an important link between observed phenotype during de-etiolation and the molecular machinery controlling expression of these genes.

Optimization of Protein Isolation and Label-Free Quantitative Proteomic Analysis in Four Different Tissues of Korean Ginseng

Korean ginseng is one of the most valuable medicinal plants worldwide. However, our understanding of ginseng proteomics is largely limited due to difficulties in the extraction and resolution of ginseng proteins because of the presence of natural contaminants such as polysaccharides, phenols, and glycosides. Here, we compared four different protein extraction methods, namely, TCA/acetone, TCA/acetone–MeOH/chloroform, phenol–TCA/acetone, and phenol–MeOH/chloroform methods. The TCA/acetone–MeOH/chloroform method displayed the highest extraction efficiency, and thus it was used for the comparative proteome profiling of leaf, root, shoot, and fruit by a label-free quantitative proteomics approach. This approach led to the identification of 2604 significantly modulated proteins among four tissues. We could pinpoint differential pathways and proteins associated with ginsenoside biosynthesis, including the methylerythritol 4–phosphate (MEP) pathway, the mevalonate (MVA) pathway, UDP-glycosyltransferases (UGTs), and oxidoreductases (CYP450s). The current study reports an efficient and reproducible method for the isolation of proteins from a wide range of ginseng tissues and provides a detailed organ-based proteome map and a more comprehensive view of enzymatic alterations in ginsenoside biosynthesis.

Root Endophytes and Ginkgo biloba Are Likely to Share and Compensate Secondary Metabolic Processes, and Potentially Exchange Genetic Information by LTR-RTs.

Ginkgo biloba is a pharmaceutical resource for terpenes and flavonoids. However, few insights discussed endophytes’ role in Ginkgo, and whether genetic exchange happens between Ginkgo and endophytes remains unclear. Herein, functional gene profiles and repetitive sequences were analyzed to focus on these issues. A total of 25 endophyte strains were isolated from the Ginkgo root and distributed in 16 genera of 6 phyla. Significant morphological diversities lead to the diversity in the COG functional classification. KEGG mapping revealed that endophytic bacteria and fungi potentially synthesize chalcone, while endophytic fungi might also promote flavonoid derivatization. Both bacteria and fungi may facilitate the lignin synthesis. Aspergillus sp. Gbtc_1 exhibited the feasibility of regulating alcohols to lignans. Although Ginkgo and the endophytes have not observed the critical levopimaradiene synthase in ginkgolides synthesis, the upstream pathways of terpenoid precursors are likely intact. The MVK genes in Ginkgo may have alternative non-homologous copies or be compensated by endophytes in long-term symbiosis. Cellulomonas sp. Gbtc_1 became the only bacteria to harbor both MEP and MVA pathways. Endophytes may perform the mutual transformation of IPP and DMAPP in the root. Ginkgo and bacteria may lead to the synthesis and derivatization of the carotenoid pathway. The isoquinoline alkaloid biosynthesis seemed lost in the Ginkgo root community, but L-dopa is more probably converted into dopamine as an essential signal-transduction substance. So, endophytes may participate in the secondary metabolism of the Ginkgo in a shared or complementary manner. Moreover, a few endophytic sequences predicted as Ty3/Gypsy and Ty1/Copia superfamilies exhibited extremely high similarity to those of Ginkgo. CDSs in such endophytic LTR-RT sequences were also highly homologous to one Ginkgo CDS. Therefore, LTR-RTs may be a rare unit flowing between the Ginkgo host and endophytes to exchange genetic information. Collectively, this research effectively expanded the insight on the symbiotic relationship between the Ginkgo host and the endophytes in the root.

Introduction of the Carotenoid Biosynthesis α-Branch Into Synechocystis sp. PCC 6803 for Lutein Production.

Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol–1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.

Manipulation of DXP pathway for andrographolide production in callus cultures of Andrographis paniculata.

Greening of in vitro callus cultures and andrographolide over-accumulation was achieved by manipulating light exposure and media composition, when the biosynthetic cascade was channeled through the DXP pathway. Andrographolide, the primary biologically active compound of Andrographis paniculata, is produced through coordinated action of two pathways, the classical cytosolic mevalonate pathway and the alternative plastidial non-mevalonate pathway (Deoxy-xylulose Phosphate pathway). In vitro callus cultures of A. paniculata are useful sources of production, as well as, manipulation of andrographolide, and the present study was designed to explore the strategy of pathway inhibition for its overproduction. When the cytosolic mevalonate pathway blocker, lovastatin, was applied to callus cultures of A. paniculata, andrographolide production was enhanced in comparison to untreated control. In contrast, treatment of the callus tissue with the DXP-pathway blocker, fosmidomycin, led to depletion in andrographolide production. The present study also showed that silver nitrate, a potent elicitor of andrographolide production in in vitro callus culture, when added in combination with the pathway inhibitors resulted in alterations in andrographolide production. The highest andrographolide production was obtained in callus treated with a combination of silver nitrate and lovastatin, indicating a predominant role of the plastidial DXP pathway in andrographolide biosynthesis. A positive co-relation with chlorophyll content and andrographolide production in in vitro callus cultures (untreated and treated) observed also supported the above assumption. It could be inferred from this study that greening of callus tissue through organellar organization was a potent strategy for enhancing andrographolide accumulation in callus tissues of A. paniculata.