Methylerythritol Phosphate Research Articles

Combinatorial transient gene expression strategies to enhance terpenoid production in plants.

The monoterpenoid linalool and sesquiterpenoid costunolide are ubiquitous plant components that have been economically exploited for their respective essential oils and pharmaceutical benefits. In general, monoterpenes and sesquiterpenes are produced by the plastid 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytosolic mevalonate (MVA) pathways, respectively. Herein, we investigated the individual and combinatorial potential of MEP and MVA pathway genes in increasing linalool and costunolide production in Nicotiana benthamiana. First, six genes from the MEP (1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, geranyl pyrophosphate synthase, and linalool synthase) and MVA (acetoacetyl-CoA-thiolase, hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, germacrene A synthase, germacrene A oxidase, and costunolide synthase) pathways were separately cloned into the modular cloning (MoClo) golden gateway cassette. Second, the cassettes were transformed individually or in combination into the leaves of N. benthamiana by agroinfiltration. Five days post infiltration (DPI), all selected genes were transiently 5- to 94-fold overexpressed. Quantification using gas chromatography-Q-orbitrap-mass spectrometry (GC-Q-Orbitrap-MS) determined that the individual and combinatorial expression of MEP genes increased linalool production up to 50-90ng.mg-1 fresh leaf weight. Likewise, MVA genes increased costunolide production up to 70-90ng.mg-1 fresh leaf weight. Our findings highlight that the transient expression of MEP and MVA pathway genes (individually or in combination) enhances linalool and costunolide production in plants.

Transcriptional profiling of the terpenoid biosynthesis pathway and in vitro tests reveal putative roles of linalool and farnesal in nectarine resistance against brown rot

The most devastating fungal disease of peaches and nectarines is brown rot, caused by Monilinia spp. Among the many plant responses against biotic stress, plant terpenoids play essential protective functions, including antioxidant activities and inhibition of pathogen growth. Herein, we aimed to characterize the expression of terpenoid biosynthetic genes in fruit tissues that presented different susceptibility to brown rot. For that, we performed artificial inoculations with Monilinia laxa at two developmental stages (immature and mature fruit) of two nectarine cultivars (‘Venus’ –mid-early season cultivar - and ‘Albared’ –late season cultivar-) and in vitro tests of the key compounds observed in the transcriptional results. All fruit were susceptible to M. laxa except for immature ‘Venus’ nectarines. In response to the pathogen, the mevalonic acid (MVA) pathway of the ‘Venus’ cultivar was highly induced in both stages rather than the methylerythritol phosphate (MEP) pathway, being the expression of some MEP-related biosynthetic genes [e.g., PROTEIN FARNESYLTRANSFERASE (PpPFT), and 3S-LINALOOL SYNTHASE (PpLIS)] different between stages. In ‘Albared’, both stages presented similar responses to M. laxa for both pathways. Comparisons between cultivars showed that HYDROXYMETHYLGLUTARYL-CoA REDUCTASE (PpHMGR1) expression levels were common in susceptible tissues. Within all the terpenoid biosynthetic pathway, linalool- and farnesal-related pathways stood out for being upregulated only in resistant tissues, which suggest their role in mediating the resistance to M. laxa. The in vitro antifungal activity of linalool and farnesol (precursor of farnesal) revealed fungicidal and fungistatic activities against M. laxa, respectively, depending on the concentration tested. Understanding the different responses between resistant and susceptible tissues could be further considered for breeding or developing new strategies to control brown rot in stone fruit.

Isoprenoid alcohols utilization by malaria parasites.

Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical regions. Drug resistance is one of the biggest problems in controlling the disease, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway, which in some cases failed in clinical studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate, which are necessary for Heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and synthases are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. Similarly, to FOH and GGOH, it was observed that phytol (POH), a 20-carbon plant isoprenoid, as well as unsaponifiable lipid extracts from foods rescue parasites from the antimalarial effect of fosmidomycin. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids, derived from GGOH, dolichol, and POH if exogenously added. Furthermore, the results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGPP, phytyl pyrophosphate (PPP), and dolichols, among other substrates not identified here. Thus, further evidence was obtained for dolichols and other isoprenoid products attached to proteins. This study helps to better understand the apicoplast-targeting antimalarial mechanism of action and a novel post-translational modification of proteins in P. falciparum.

Genomic, metabonomic and transcriptomic analyses of sweet osmanthus varieties provide insights into floral aroma formation

Sweet osmanthus is an important ornamental evergreen tree or shrub, which has been widely used in the world's subtropical landscapes and the fragrance industry due to its deliciously fragrant flowers. In this study, whole-genome resequencing, RNA sequencing, and GC-MS analysis were used to compare 30 accessions of Osmanthus fragrans to gain insights into taxonomic classification and the diversity of volatile floral scents. A total of 13,466,389 high confidence SNPs were identified from the accessions with an average sequencing depth of 16.96 × . Phylogenetic analysis of the sequences of the 30 ancient or improved O. fragrans genotypes with diverse origins could be roughly divided into two differentiated sweet osmanthus populations, corresponding to the autumn-flowering and the four-seasons flowering characteristics. The odor activity values and orthogonal projections to latent structures-discriminant analysis of GC-MS metabolomics data indicated that linalool was one of the most important volatile organic compounds (VOCs) which can determine the scent diversity among different sweet osmanthus cultivars. Weighted gene co-expression network analysis (WGCNA) identified 23 distinct modules, four of which were highly associated with important floral VOCs. Based on WGCNA analysis, a critical enzyme gene OfHDR2.1 in the MEP pathway and its 12 co-expression transcription factors from the turquoise module were considered to play important roles in linalool synthesis among differently scented O. fragrans populations. Thus, our study provided new clues for the classification of O. fragrans cultivars and valuable genetic resources for future molecular breeding of O. fragrans accessions improved with respect to floral aroma.

Two apicoplast dwelling glycolytic enzymes provide key substrates for metabolic pathways in the apicoplast and are critical for Toxoplasma growth.

Many apicomplexan parasites harbor a non-photosynthetic plastid called the apicoplast, which hosts important metabolic pathways like the methylerythritol 4-phosphate (MEP) pathway that synthesizes isoprenoid precursors. Yet many details in apicoplast metabolism are not well understood. In this study, we examined the physiological roles of four glycolytic enzymes in the apicoplast of Toxoplasma gondii. Many glycolytic enzymes in T. gondii have two or more isoforms. Endogenous tagging each of these enzymes found that four of them were localized to the apicoplast, including pyruvate kinase2 (PYK2), phosphoglycerate kinase 2 (PGK2), triosephosphate isomerase 2 (TPI2) and phosphoglyceraldehyde dehydrogenase 2 (GAPDH2). The ATP generating enzymes PYK2 and PGK2 were thought to be the main energy source of the apicoplast. Surprisingly, deleting PYK2 and PGK2 individually or simultaneously did not cause major defects on parasite growth or virulence. In contrast, TPI2 and GAPDH2 are critical for tachyzoite proliferation. Conditional depletion of TPI2 caused significant reduction in the levels of MEP pathway intermediates and led to parasite growth arrest. Reconstitution of another isoprenoid precursor synthesis pathway called the mevalonate pathway in the TPI2 depletion mutant partially rescued its growth defects. Similarly, knocking down the GAPDH2 enzyme that produces NADPH also reduced isoprenoid precursor synthesis through the MEP pathway and inhibited parasite proliferation. In addition, it reduced de novo fatty acid synthesis in the apicoplast. Together, these data suggest a model that the apicoplast dwelling TPI2 provides carbon source for the synthesis of isoprenoid precursor, whereas GAPDH2 supplies reducing power for pathways like MEP, fatty acid synthesis and ferredoxin redox system in T. gondii. As such, both enzymes are critical for parasite growth and serve as potential targets for anti-toxoplasmic intervention designs. On the other hand, the dispensability of PYK2 and PGK2 suggest additional sources for energy in the apicoplast, which deserves further investigation.

Microbial (E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate reductase (IspH) and its biotechnological potential: A mini review.

(E)-4-hydroxy-3-methylbut-2-enyl pyrophosphate (HMBPP) reductase (IspH) is a [4Fe-4S] cluster-containing enzyme, involved in isoprenoid biosynthesis as the final enzyme of the methylerythritol phosphate (MEP) pathway found in many bacteria and malaria parasites. In recent years, many studies have revealed that isoprenoid compounds are an alternative to petroleum-derived fuels. Thus, ecofriendly methods harnessing the methylerythritol phosphate pathway in microbes to synthesize isoprenoid compounds and IspH itself have received notable attention from researchers. In addition to its applications in the field of biosynthesis, IspH is considered to be an attractive drug target for infectious diseases such as malaria and tuberculosis due to its survivability in most pathogenic bacterium and its absence in humans. In this mini-review, we summarize previous reports that have systematically illuminated the fundamental and structural properties, substrate binding and catalysis, proposed catalytic mechanism, and novel catalytic activities of IspH. Potential bioengineering and biotechnological applications of IspH are also discussed.

Transcriptome Level Reveals the Triterpenoid Saponin Biosynthesis Pathway of Bupleurum falcatum L.

Bupleurum falcatum L. is frequently used in traditional herbal medicine in Asia. Saikosaponins (SSs) are the main bioactive ingredients of B. falcatum, but the biosynthetic pathway of SSs is unclear, and the biosynthesis of species-specific phytometabolites is little known. Here we resolved the transcriptome profiles of B. falcatum to identify candidate genes that might be involved in the biosynthesis of SSs. By isoform sequencing (Iso-Seq) analyses of the whole plant, a total of 26.98 Gb of nucleotides were obtained and 124,188 unigenes were identified, and 81,594 unigenes were successfully annotated. A total of 1033 unigenes of 20 families related to the mevalonate (MVA) pathway and methylerythritol phosphate (MEP) pathway of the SS biosynthetic pathway were identified. The WGCNA (weighted gene co-expression network analysis) of these unigenes revealed that only the co-expression module of MEmagenta, which contained 343 unigenes, was highly correlated with the biosynthesis of SSs. Comparing differentially expressed gene analysis and the WGCNA indicated that 130 out of 343 genes of the MEmagenta module exhibited differential expression levels, and genes with the most "hubness" within this module were predicted. Manipulation of these genes might improve the biosynthesis of SSs.

The Importance of Mycorrhizal Fungi in the Development and Secondary Metabolite Production of Echinacea purpurea and Relatives (Asteraceae): Current Research Status and Perspectives

The cultivation of Echinacea purpurea for commerce and obtaining high-quality plant material on a large scale remain a challenge for growers. Another challenge for the following decades is to create sustainable agriculture that meets society’s needs, has no environmental impact, and reduces the use of fertilizers and pesticides. The aims of this overview were: (1) to present the importance of the chemical compounds reported in E. purpurea; (1) to synthesize results about cultivation of the E. purpurea with arbuscular mycorrhizal fungi (AMF) and associated microorganisms; (2) to exemplify similar research with plants from the Asteraceae family, due to the limited number of published Echinacea studies; (3) to collect recent findings about how the inoculation with AMF affects gene expressions in the host plants; (4) to propose perspective research directions in the cultivation of E. purpurea, in order to increase biomass and economic importance of secondary metabolite production in plants. The AMF inocula used in the Echinacea experiments was mainly Rhizophagus irregularis. The studies found in the selected period (2012–2022), reported the effects of 21 AMFs used as single inocula or as a mixture on growth and secondary metabolites of 17 plant taxa from the Asteraceae family. Secondary metabolite production and growth of the economic plants were affected by mutualistic, symbiotic or parasitic microorganisms via upregulation of the genes involved in hormonal synthesis, glandular hair formation, and in the mevalonate (MVA), methyl erythritol phosphate (MEP) and phenylpropanoid pathways. However, these studies have mostly been carried out under controlled conditions, in greenhouses or in vitro in sterile environments. Since the effect of AMF depends on the variety of field conditions, more research on the application of different AMF (single and in various combinations with bacteria) to plants growing in the field would be necessary. For the identification of the most effective synergistic combinations of AMF and related bacterial populations, transcriptomic and metabolomic investigations might also be useful.

Selection of an Aptamer against the Enzyme 1-deoxy-D-xylulose-5-phosphate Reductoisomerase from Plasmodium falciparum.

The methyl erythritol phosphate (MEP) pathway of isoprenoid biosynthesis is essential for malaria parasites and also for several human pathogenic bacteria, thus representing an interesting target for future antimalarials and antibiotics and for diagnostic strategies. We have developed a DNA aptamer (D10) against Plasmodium falciparum 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), the second enzyme of this metabolic route. D10 binds in vitro to recombinant DXR from P. falciparum and Escherichia coli, showing at 10 µM a ca. 50% inhibition of the bacterial enzyme. In silico docking analysis indicates that D10 associates with DXR in solvent-exposed regions outside the active center pocket. According to fluorescence confocal microscopy data, this aptamer specifically targets in P. falciparum in vitro cultures the apicoplast organelle where the MEP pathway is localized and is, therefore, a highly specific marker of red blood cells parasitized by Plasmodium vs. naïve erythrocytes. D10 is also selective for the detection of MEP+ bacteria (e.g., E. coli and Pseudomonas aeruginosa) vs. those lacking DXR (e.g., Enterococcus faecalis). Based on these results, we discuss the potential of DNA aptamers in the development of ligands that can outcompete the performance of the well-established antibody technology for future therapeutic and diagnostic approaches.

Multi-omics analysis the differences of VOCs terpenoid synthesis pathway in maintaining obligate mutualism between Ficus hirta Vahl and its pollinators.

Volatile organic compounds (VOCs) emitted by the receptive syconia of Ficus species is a key trait to attract their obligate pollinating fig wasps. Ficus hirta Vahl is a dioecious shrub, which is pollinated by a highly specialized symbiotic pollinator in southern China. Terpenoids are the main components of VOCs in F. hirta and play ecological roles in pollinator attraction, allelopathy, and plant defense. However, it remains unclear that what molecular mechanism difference in terpenoid synthesis pathways between pre-receptive stage (A-phase) and receptive stage (B-phase) of F. hirta syconia. Transcriptome, proteome and Gas Chromatography-Mass Spectrometer (GC-MS) were applied here to analyze these difference. Compared to A-phase syconia, the genes (ACAT2, HMGR3, GGPS2, HDR, GPS2, TPS2, TPS4, TPS10-4, TPS14) related to the terpenoid synthesis pathway had higher expression level in receptive syconia (B-phase) according to transcriptome sequencing. Seven differentially expressed transcription factors were screened, namely bHLH7, MYB1R1, PRE6, AIL1, RF2b, ANT, VRN1. Specifically, bHLH7 was only specifically expressed in B-phase. 235 differentially expressed proteins (DEPs) were mainly located in the cytoplasm and chloroplasts. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs were mainly enriched in the metabolic process. A total of 9 terpenoid synthesis proteins were identified in the proteome. Among them, 4 proteins in methylerythritol phosphate (MEP) pathway were all down-regulated. Results suggested the synthesis of terpenoids precursors in B-phase bracts were mainly accomplished through the mevalonic acid (MVA) pathway in cytoplasm. Correlation analysis between the transcriptome and proteome, we detected a total of 1082 transcripts/proteins, three of which are related to stress. From the VOCs analysis, the average percent of monoterpenoids emitted by A-phase and B-phase syconia were 8.29% and 37.08%, while those of sesquiterpenes were 88.43% and 55.02% respectively. Monoterpenes (camphene, myrcene, camphor, menthol) were only detected in VOCs of B-phase syconia. To attract pollinators, B-phase syconia of F. hirta need more monoterpenoids and less sesquiterpenes. We speculate that transcription factor bHLH7 may regulate the terpenoid synthesis pathway between A- and B-phase syconia. Our research provided the first global analysis of mechanism differences of terpenoid synthesis pathways between A and B phases in F. hirta syconia.

SmDXS5, acting as a molecular valve, plays a key regulatory role in the primary and secondary metabolism of tanshinones in Salvia miltiorrhiza.

Red sage, the dry root and rhizome of the herbaceous plant Salvia miltiorrhiza Bunge, is widely used for treating various diseases. The low content of tanshinones (terpenoids) has always restricted development of the S. miltiorrhiza industry. Here, we found that SmDXS5, a rate-limiting enzyme-coding gene located at the intersection of primary and secondary metabolism, can effectively change the transcription level and secondary metabolome profile of hairy roots of S. miltiorrhiza, and significantly increase the content of tanshinones. Agrobacterium rhizogenes was used to infuse S. miltiorrhiza explants, and hairy roots of S. miltiorrhiza expressing the SmDXS5 gene were obtained successfully. We identified 39 differentially accumulated metabolites (DAMs) by metabolomics based on ultra-high performance liquid chromatography quadrupole exactive mass spectrometry and multivariate statistics. These DAMs might be key metabolites of SmDXS5 gene regulation. RNA sequencing was used to compare gene expression between the hairy roots of the SmDXS5 overexpressing group and the blank control (BC) group. Compared with the BC group, 18,646 differentially expressed genes were obtained: 8994 were upregulated and 9,652 downregulated. The combined transcriptome and metabolome analyses revealed that the mevalonate and methylerythritol phosphate pathways and synthase gene expression levels in the SmDXS5 overexpressing group were upregulated significantly, and the accumulation of tanshinone components was increased significantly, which promoted the process of glycolysis and promoted the transformation of carbohydrates to secondary metabolism. Moreover, the expression of SmPAL, the first rate-limiting enzyme gene of the phenylpropane pathway, decreased, reducing the accumulation of phenolic acid, another secondary metabolite. Therefore, SmDXS5 can be defined as a 'valve' gene, mainly responsible for regulating the distribution of primary and secondary metabolic flow of tanshinones in S. miltiorrhiza, and for other secondary metabolic pathways. The discovery of SmDXS5 and its molecular valve function in regulating primary and secondary metabolism will provide a basis for the industrial production of tanshinone components, and cultivation of high quality S. miltiorrhiza.

Acyloxymethyl and alkoxycarbonyloxymethyl prodrugs of a fosmidomycin surrogate as antimalarial and antibacterial agents

Fosmidomycin is a natural antibiotic with potent IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in most bacteria, including A. baumanii and M. tuberculosis, and apicomplexan parasites, including Plasmodium parasites. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against A. baumannii and M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report on the expansion of the acyloxymethyl and alkoxycarbonyloxymethyl phosphonate ester prodrug series of a fosmidomycin surrogate. Prodrug promoieties were designed based on electronic, lipophilic and siderophoric properties. This investigation led to the discovery of derivatives with two-digit nanomolar and submicromolar IC50-values against P. falciparum and A. baumanii, respectively.

Exon skipping in IspE Gene is associated with abnormal chloroplast development in rice albino leaf 4 mutant.

The formation of leaf color largely depends on the components of pigment accumulation in plastids, which are involved in chloroplast development and division. Here, we isolated and characterized the rice albino leaf 4 (al4) mutant, which exhibited an albino phenotype and eventually died at the three-leaf stage. The chloroplasts in al4 mutant were severely damaged and unable to form intact thylakoid structure. Further analysis revealed that the candidate gene encodes 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), which participates in the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis. We further demonstrated that the mutation at the exon-intron junction site cause alternative splicing factors fail to distinguish the origin of the GT-AG intron, leading to exon skipping and producing a truncated OsIspE in the al4 mutant. Notably, disruption of OsIspE led to the reduced expression of chloroplast-associated genes, including chloroplast biosynthetic and translation related genes and photosynthetic associated nuclear genes (PhANGs). In summary, these findings reveal that OsIspE plays a crucial role in chloroplast biogenesis and provides novel insights into the function of CMK during chloroplast development in rice.

Identification of genes involved in oleoresin biosynthesis in Pinus massoniana through the combination of SMRT and Illumina sequencing

Oleoresin, a terpene with important economic and industrial value, is produced via the secondary metabolism of plants in the genus Pinus. Owing to its adaptability and high economic and ecological value, Pinus massoniana (Masson pine) is widely planted in southern China, and this species is also the main species for oleoresin tapping in the world. However, the differences in the expression of genes related to terpene biosynthesis in needles and xylem tissues at the transcriptional level at different time points during oleoresin tapping are still unclear. By combining SMRT and Illumina sequencing, we revealed the key genes involved in terpene biosynthesis in needles and xylem tissues. A total of 32.07 Gb of polymerase reads and 562.60 Gb of Illumina clean reads were obtained in this study. After annotating them in seven databases, we identified 68 genes involved in terpene biosynthesis, and most of them were highly expressed in needles, implying that needles are the biosynthetic sites of oleoresin. Moreover, we found that the TPS genes of gymnosperms more likely to be ‘DDXYD’, compared to the ‘DDXXD’ motif in angiosperms. The genes involved in the MEP pathway are highly expressed in the needles, implying the different efficiency of use of the MEP and MVA pathways in Masson pine. Furthermore, by performing controlled experiments, we found that PmPMK2, PmHDR1, PmTPS09, PmTPS11, PmTPS16, and PmTPS19 genes may play a critical role in oleoresin biosynthesis. This study provide new insights into terpene and oleoresin biosynthesis.

Bioprocess development for enhancing cell biomass, differential picrosides accumulation, and gene expression profiling at shake flask levels in suspension cultures of Picrorhiza kurroa

Plant cell suspension culture is an alternative and perpetual source for large-scale metabolites production in significantly less time. However, this approach has not yet been executed in Picrorhiza kurroa for their specialized metabolites production. Therefore, the present study aimed to develop the suspension culture process from the rhizome and leaf callus of P. kurroa by optimizing the physical cultivation parameters. Furthermore, the established cultures were subjected to sequential scale-up at 250, 500, 1000, 2000, and 5000 mL shake flask levels to evaluate growth, metabolites accumulation, and gene expression profiling of picrosides biosynthesis. Concurrently, the growth, metabolites content, and gene expression patterns were also assessed at the 4th, 8th, and 12th months of the incubation period. The investigation results revealed that the cell biomass in suspension cultures significantly improved solely by varying the culture parameters. The initial inoculum density (3 g/75 mL), subculture by pouring method, shaking speed (80 rpm), and pH (5.8) were suitable for enhanced production of cell biomass in rhizome and leaf suspension cultures in a 250 mL shake flask. During sequential scale-up, the cell biomass was reached in rhizome suspension (335 g FW; 13.80 g DW) and leaf suspension (263.33 g FW; 10.87 g DW) under optimized parameters at 5000 mL shake flask level. However, a maximum increase in the cell growth index and cell biomass (DW) in g/L of the medium was observed in a 250 mL flask compared to other shake flask volumes. In rhizome and leaf suspension, total picrosides (PI, PII, and PIII) (rhizome: 2.36–2.48 mg/g DW; leaf: 2.50–2.67 mg/g DW) and total precursors (vanillic acid, caffeic acid, cinnamic acid, catalpol, and aucubin) (rhizome: 1.35–1.37 mg/g DW; leaf: 1.20–1.45 mg/g DW) content range was observed during sequential scale-up at 250–5000 mL shake flask levels. Hence, no significant level of variation was observed in picrosides content (PI, PII, and PII in mg/g DW) with respect to different shake flask volumes (250–5000 mL) in rhizome and leaf suspension culture, respectively. Nevertheless, the productivity of metabolites content in mg/L of medium with respect to cell growth in g/L of medium showed a significant level of variation. The 250 mL shake flask volume culture of rhizome and leaf cell suspension accumulated the maximum total picrosides (31.07 and 31.20 mg/L of medium) and total precursors (17.04 and 16.94 mg/L of medium) content with cell biomass (12.53 and 11.69 g/L of medium), respectively. The results also revealed that the shikimate/phenylpropanoid pathway (EPSPS), mevalonate pathway (PMK and GDPS), non-mevalonate pathway (ISPD, DXPS, and ISPE), and iridoid pathway (GS, G10H, UGT, F3D, SQM, and ACT) genes showed upregulated expression in the rhizome and leaf suspension cultures. Analyzing the 1st month cultures with 4th, 8th, and 12th month cultures, the study depicted that growth index, cell viability, metabolites content, productivity in mg/L of the medium, and gene expression pattern were decreased with the age of cultures under one year of incubation. The present investigation delivered a simple yet feasible suspension culture process for metabolites enriched cell biomass production in P. kurroa.

Synthesis and inhibition studies towards the discovery of benzodiazepines as potential antimalarial compounds

The target-based discovery of therapeutics against apicoplast, an all-important organelle is an overriding perspective. MEP pathway, an accredited drug target provides an insight into the importance of apicoplast in the survival of the parasite. In this study, we present the rational design strategy employing sustainable catalysis for the synthesis of benzodiazepine (BDZ) conformers followed by their biological evaluation as prospective inhibitors against the potential target of the IPP pathway, 1-deoxy-D-xylulose-5-phosphatereductoisomerase (DXR). The study reported the inhibitory profile of 8c and 6d against the quintessential step of the only drug target in the erythrocytic stages of parasite development. The potential compounds were identified to represent a novel class of inhibitors that serve as the lead molecules to impede the pathway and further affect the survival of the parasite.

Plastid methylerythritol phosphate pathway participates in the hypersensitive response-related cell death in Nicotiana benthamiana.

Programmed cell death (PCD), a characteristic feature of hypersensitive response (HR) in plants, is an important cellular process often associated with the defense response against pathogens. Here, the involvement of LytB, a gene encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase that participates in the final step of the plastid methylerythritol phosphate (MEP) pathway, in plant HR cell death was studied. In Nicotiana benthmiana plants, silencing of the NbLytB gene using virus-induced gene silencing (VIGS) caused plant growth retardation and albino leaves with severely malformed chloroplasts. In NbLytB-silenced plants, HR-related cell death mediated by the expression of either the human proapoptotic protein gene Bax or an R gene with its cognate Avr effector gene was inhibited, whereas that induced by the nonhost pathogen Pseudomonas syringae pv. syringae 61 was enhanced. To dissect the isoprenoid pathway and avoid the pleiotropic effects of VIGS, chemical inhibitors that specifically inhibit isoprenoid biosynthesis in plants were employed. Treatment of N. benthamiana plants with fosmidomycin, a specific inhibitor of the plastid MEP pathway, effectively inhibited HR-related PCD, whereas treatment with mevinolin (a cytoplasmic mevalonate pathway inhibitor) and fluridone (a carotenoid biosynthesis inhibitor) did not. Together, these results suggest that the MEP pathway as well as reactive oxygen species (ROS) generation in the chloroplast play an important role in HR-related PCD, which is not displaced by the cytosolic isoprenoid biosynthesis pathway.

Cytological, genetic and transcriptomic characterization of a cucumber albino mutant.

Photosynthesis, a fundamental process for plant growth and development, is dependent on chloroplast formation and chlorophyll synthesis. Severe disruption of chloroplast structure results in albinism of higher plants. In the present study, we report a cucumber albino alc mutant that presented white cotyledons under normal light conditions and was unable to produce first true leaf. Meanwhile, alc mutant could grow creamy green cotyledons under dim light conditions but died after exposure to normal light irradiation. No chlorophyll and carotenoid were detected in the alc mutant grown under normal light conditions. Using transmission electron microscopy, impaired chloroplasts were observed in this mutant. The genetic analysis indicated that the albino phenotype was recessively controlled by a single locus. Comparative transcriptomic analysis between the alc mutant and wild type revealed that genes involved in chlorophyll metabolism and the methylerythritol 4-phosphate pathway were affected in the alc mutant. In addition, three genes involved in chloroplast development, including two FtsH genes and one PPR gene, were found to have negligible expression in this mutant. The quality of RNA sequencing results was further confirmed by real-time quantitative PCR analysis. We also examined 12 homologous genes from alc mutant in other plant species, but no genetic variation in the coding sequences of these genes was found between alc mutant and wild type. Taken together, we characterized a cucumber albino mutant with albinism phenotype caused by chloroplast development deficiency and this mutant can pave way for future studies on plastid development.

Role of regulatory pathways and multi-omics approaches for carbon capture and mitigation in cyanobacteria

Cyanobacteria are known for their metabolic potential and carbon capture and sequestration capabilities. These cyanobacteria are not only an effective source for carbon minimization and resource mobilization into value-added products for biotechnological gains. The present review focuses on the detailed description of carbon capture mechanisms exerted by the various cyanobacterial strains, the role of important regulatory pathways, and their subsequent genes responsible for such mechanisms. Moreover, this review will also describe effectual mechanisms of central carbon metabolism like isoprene synthesis, ethylene production, MEP pathway, and the role of Glyoxylate shunt in the carbon sequestration mechanisms. This review also describes some interesting facets of using carbon assimilation mechanisms for valuable bio-products. The role of regulatory pathways and multi-omics approaches in cyanobacteria will not only be crucial towards improving carbon utilization but also will give new insights into utilizing cyanobacterial bioresource for carbon neutrality.

Antibacterial Activity of Allicin-Inspired Disulfide Derivatives against Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri (Xac) belongs to the Gram-negative species, causing citrus canker that seriously affects the fruit yield and quality of many rutaceae plants. Herein, we found that compound 2-(butyldisulfanyl) quinazolin-4(3H)-one exhibited remarkable anti-Xac activity in vitro with a half effective concentration (EC50) of 2.6 μg/mL, while the positive controls thiodiazole-copper with 57 μg/mL and bismerthiazol with 68 μg/mL and this compound showed great anti-citrus canker activity in vivo. This active compound also was confirmed to reduce biofilm formation, increase the level of reactive oxygen species, damage the morphological structure of the bacteria, and cause bacterial death. Proteomics and RT-qPCR analysis results indicated that this compound down-regulated the expression of enzymes in the MEP (2-methyl-D-erythritol 4-phosphate) pathway and might achieve destructive ability of Xac. Overall, this study indicates that such derivatives could be a promising scaffold to develop novel bactericides to control citrus canker.