2-C-Methyl-D-erythritol

Metabolic Engineering of Bacillus subtilis Toward Taxadiene Biosynthesis as the First Committed Step for Taxol Production.[Pubmed:30842758]

Front Microbiol. 2019 Feb 20;10:218.

Terpenoids are natural products known for their medicinal and commercial applications. Metabolic engineering of microbial hosts for the production of valuable compounds, such as artemisinin and Taxol, has gained vast interest in the last few decades. The Generally Regarded As Safe (GRAS) Bacillus subtilis 168 with its broad metabolic potential is considered one of these interesting microbial hosts. In the effort toward engineering B. subtilis as a cell factory for the production of the chemotherapeutic Taxol, we expressed the plant-derived taxadiene synthase (TXS) enzyme. TXS is responsible for the conversion of the precursor geranylgeranyl pyrophosphate (GGPP) to taxa-4,11-diene, which is the first committed intermediate in Taxol biosynthesis. Furthermore, overexpression of eight enzymes in the biosynthesis pathway was performed to increase the flux of the GGPP precursor. This was achieved by creating a synthetic operon harboring the B. subtilis genes encoding the 2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway (dxs, ispD, ispF, ispH, ispC, ispE, ispG) together with ispA (encoding geranyl and farnesyl pyrophosphate synthases) responsible for providing farnesyl pyrophosphate (FPP). In addition, a vector harboring the crtE gene (encoding geranylgeranyl pyrophosphate synthase, GGPPS, of Pantoea ananatis) to increase the supply of GGPP was introduced. The overexpression of the MEP pathway enzymes along with IspA and GGPPS caused an 83-fold increase in the amount of taxadiene produced compared to the strain only expressing TXS and relying on the innate pathway of B. subtilis. The total amount of taxadiene produced by that strain was 17.8 mg/l. This is the first account of the successful expression of taxadiene synthase in B. subtilis. We determined that the expression of GGPPS through the crtE gene is essential for the formation of sufficient precursor, GGPP, in B. subtilis as its innate metabolism is not efficient in producing it. Finally, the extracellular localization of taxadiene production by overexpressing the complete MEP pathway along with IspA and GGPPS presents the prospect for further engineering aiming for semisynthesis of Taxol.

The plastidial metabolite 2-C-methyl-D-erythritol-2,4-cyclodiphosphate modulates defense responses against aphids.[Pubmed:30786032]

Plant Cell Environ. 2019 Feb 20.

Feeding by insect herbivores such as caterpillars and aphids induces plant resistance mechanisms that are mediated by the phytohormones jasmonic acid (JA) and salicylic acid (SA). These phytohormonal pathways often crosstalk. Besides phytohormones, methyl-D-erythriol-2,4-cyclodiphosphate (MEcPP), the penultimate metabolite in the methyl-D-erythritol-4-phosphate (MEP) pathway, has been speculated to regulate transcription of nuclear genes in response to biotic stressors such as aphids. Here, we show that MEcPP uniquely enhances the SA pathway without attenuating the JA pathway. Arabidopsis mutant plants that accumulate high levels of MEcPP (hds3) are highly resistant to the cabbage aphid (Brevicoryne brassicae) while resistance to the large cabbage white caterpillar (Pieris brassicae) remains unaltered. Thus, MEcPP is a distinct signaling molecule that acts beyond phytohormonal crosstalk to induce resistance against the cabbage aphid in Arabidopsis. We dissect the molecular mechanisms of MEcPP mediating plant resistance against the aphid B. brassicae. This shows that MEcPP induces the expression of genes encoding enzymes involved in the biosynthesis of several primary and secondary metabolic pathways contributing to enhanced resistance against this aphid species. A unique ability to regulate multifaceted molecular mechanisms makes MEcPP an attractive target for metabolic engineering in Brassica crop plants to increase resistance to cabbage aphids.

Deciphering structure, function and mechanism of Plasmodium IspD homologs from their evolutionary imprints.[Pubmed:30783866]

J Comput Aided Mol Des. 2019 Apr;33(4):419-436.

Malaria is a life-threatening mosquito-borne blood disease caused by infection with Plasmodium parasites. Anti-malarial drug resistance is a global threat to control and eliminate malaria and therefore, it is very important to discover and evaluate new drug targets. The 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) homolog is a second in vivo target for fosmidomycin within isoprenoid biosynthesis in malarial parasites. In the present study, we have deciphered the sequence-structure-function integrity of IspD homologs based on their evolutionary imprints. The function and catalytic mechanism of them were also intensively studied by using sequence-structure homology, molecular modeling, and docking approach. Results of our study indicated that substrate-binding and dimer interface motifs in their structures were extensively conserved and part of them closely related to eubacterial origins. Amino acid substitutions in their coiled-coil regions found to bring a radical change in secondary structural elements, which in turn may change the local structural environment. Arg or Asp was identified as a catalytic site in plasmodium IspD homologs, contributing a direct role in the cytidylyltransferase activity similar to bacterial IspD. Results of molecular docking studies demonstrated how anti-malarial drugs such as fosmidomycin and FR-900098 have competitively interacted with the substrate-binding site of these homologs. As shown by our analysis, species-specific evolutionary imprints in these homologs determine the sequence-structure-function-virulence integrity and binding site alterations in order to confer anti-malarial drug resistance.

Alternative Carbon Sources for Isoprene Emission.[Pubmed:30472998]

Trends Plant Sci. 2018 Dec;23(12):1081-1101.

Isoprene and other plastidial isoprenoids are produced primarily from recently assimilated photosynthates via the 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway. However, when environmental conditions limit photosynthesis, a fraction of carbon for MEP pathway can come from extrachloroplastic sources. The flow of extrachloroplastic carbon depends on the species and on leaf developmental and environmental conditions. The exchange of common phosphorylated intermediates between the MEP pathway and other metabolic pathways can occur via plastidic phosphate translocators. C1 and C2 carbon intermediates can contribute to chloroplastic metabolism, including photosynthesis and isoprenoid synthesis. Integration of these metabolic processes provide an example of metabolic flexibility, and results in the synthesis of primary metabolites for plant growth and secondary metabolites for plant defense, allowing effective use of environmental resources under multiple stresses.

Molecular regulatory mechanism of isoprene emission under short-term drought stress in the tropical tree Ficus septica.[Pubmed:30445554]

Tree Physiol. 2019 Mar 1;39(3):440-453.

Isoprene is emitted by many plants and is thought to function as an antioxidant under stressful conditions. However, the detailed regulatory mechanism of isoprene emission in relation to the antioxidant system remains unclear. Therefore, in this study, we explored the molecular regulatory mechanism of isoprene emission under short-term drought stress in the tropical tree Ficus septica Burm.f. We found that the soil moisture content gradually decreased from 55% on Day 1 (D1) to 23% (wilting point) on D5 after withholding water for 4 days and then returning to the initial level following re-watering on D6. On D5, drought-stressed plants had more than twofold higher isoprene emission and 90.6% lower photosynthesis rates, 99.5% lower stomatal conductance and 82.3% lower transpiration rates than well-watered control plants. It was also estimated that the isoprene concentration inside the leaf greatly increased on D5 due to the increased isoprene emission rate and reduced stomatal conductance. Among the traits related to the 2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway, which is responsible for isoprene biosynthesis, the isoprene synthase (IspS) protein level was positively correlated with the isoprene emission rate in stressed plants. The transcripts of the antioxidant genes peroxidase 2 (POD2), POD4, copper-zinc superoxide dismutase 2 (Cu-ZnSOD2) and manganese superoxide dismutase 1 (Mn-SOD1) also increased during the drying period, while those of ascorbate peroxidase 1 (APX1) decreased. However, there was only a weak correlation between isoprene emission and antioxidant enzyme gene expression, indicating that the regulation of isoprene biosynthesis is not directly linked to the antioxidant defense network in drought-stressed F. septica. These findings suggest that the post-transcriptional regulation of IspS led to the observed change in isoprene emission rate, which enhanced the quenching of reactive oxygen species (ROS) and, in combination with the increased antioxidant enzyme activity, conferred tolerance to drought stress in this species.

Role of Mitochondria in Regulating Lutein and Chlorophyll Biosynthesis in Chlorella pyrenoidosa under Heterotrophic Conditions.[Pubmed:30274203]

Mar Drugs. 2018 Sep 28;16(10). pii: md16100354.

The green alga Chlorella pyrenoidosa can accumulate lutein and chlorophyll under heterotrophic conditions. We propose that the mitochondrial respiratory electron transport chain (mRET) may be involved in this process. To verify this hypothesis, algal cells were treated with different mRET inhibitors. The biosynthesis of lutein and chlorophyll was found to be significantly stimulated by salicylhydroxamic acid (SHAM), whereas their contents substantially decreased after treatment with antimycin A and sodium azide (NaN(3)). Proteomic studies revealed profound protein alterations related to the redox and energy states, and a network was proposed: The up-regulation of peroxiredoxin reduces oxidized glutathione (GSSG) to reduced glutathione (GSH); phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetic acid to phosphoenolpyruvate, and after entering the methylerythritol phosphate (MEP) pathway, 4-hydroxy-3-methylbut-2-en-1yl diphosphate synthase reduces 2-C-Methyl-D-erythritol-2,4-cyclodiphosphate (ME-Cpp) to 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP), which is closely related to the synthesis of lutein; and coproporphyrinogen III oxidase and ChlI play important roles in the chlorophyll biosynthetic pathway. These results supported that for the heterotrophic C. pyrenoidosa, the signaling, oriented from mRET, may regulate the nuclear genes encoding the enzymes involved in photosynthetic pigment biosynthesis.

Molecular evolution and functional divergence of IspD homologs in malarial parasites.[Pubmed:30118875]

Infect Genet Evol. 2018 Nov;65:340-349.

Malaria is one of the leading parasitic diseases to humans caused by Plasmodium falciparum. It is imperative to discover novel targets for the development of antimalarial drugs. The 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD) in 2-C-Methyl-D-erythritol-4-phosphate pathway has been considered as a second in vivo off-target for antimalarial drugs discovery as its essentiality in malarial parasites and devoid in mammals. Our study was intended to reveal the molecular basis of its functional parts by inferring diversity, origin and evolution across important malarial parasites. Phylogenetic analyses revealed its conservation probability and sequence homology among bacterial IspD homologs. It also indicated that Plasmodium IspD homologs were distantly related to each other and their functional counterparts originated from different progenitor genes. Nucleotide-diphospho-sugar transferase fold and conserved domain of them might have evolved from green sulphur bacteria, whereas coiled-coil region and apicoplast targeting signal derived from protozoal origins. These homologs contained prospectively definable motifs subject to neutral or nearly neutral evolution on a scale that were diverged radically and subsequently converged in making spatial structural arrangements. Our genetic diversity analysis has shown a constructive signal for identifying the evolutionary constraints, which has imposed on their functional divergence in malarial parasites. Thus, this study provides a novel insight into our understanding of the molecular basis of the origin and evolution history of IspD homologs across apicomplexa.

Molecular Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Gene Family in Artemisia annua.[Pubmed:30116250]

Front Plant Sci. 2018 Aug 2;9:952.

Artemisia annua produces artemisinin, an effective antimalarial drug. In recent decades, the later steps of artemisinin biosynthesis have been thoroughly investigated; however, little is known about the early steps of artemisinin biosynthesis. Comparative transcriptomics of glandular and filamentous trichomes and (13)CO2 radioisotope study have shown that the 2-C-Methyl-D-erythritol-4-phosphate (MEP) pathway, rather than the mevalonate pathway, plays an important role in artemisinin biosynthesis. In this study, we have cloned three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) genes from A. annua (AaDXS1, AaDXS2, and AaDXS3); the DXS enzyme catalyzes the first and rate-limiting enzyme of the MEP pathway. We analyzed the expression of these three genes in different tissues in response to multiple treatments. Phylogenetic analysis revealed that each of the three DXS genes belonged to a distinct clade. Subcellular localization analysis indicated that all three AaDXS proteins are targeted to chloroplasts, which is consistent with the presence of plastid transit peptides in their N-terminal regions. Expression analyses revealed that the expression pattern of AaDXS2 in specific tissues and in response to different treatments, including methyl jasmonate, light, and low temperature, was similar to that of artemisinin biosynthesis genes. To further investigate the tissue-specific expression pattern of AaDXS2, the promoter of AaDXS2 was cloned upstream of the beta-glucuronidase gene and was introduced in arabidopsis. Histochemical staining assays demonstrated that AaDXS2 was mainly expressed in the trichomes of Arabidopsis leaves. Together, these results suggest that AaDXS2 might be the only member of the DXS family in A. annua that is involved in artemisinin biosynthesis.

An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid.[Pubmed:30097469]

Plant Physiol. 2018 Oct;178(2):535-551.

Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-Methyl-D-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii.[Pubmed:30017797]

Metab Eng. 2018 Sep;49:116-127.

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm(-1) d(-1) over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Deciphering the role of IspD (2CmethylDerythritol 4phosphate cytidyltransferase) enzyme as a potential therapeutic drug target against Plasmodium vivax.[Pubmed:29958953]

Gene. 2018 Oct 30;675:240-253.

Biosynthesis of isoprenoids (MEP Pathway) in apicoplast has an important role during the erythrocytic stages of Plasmodium, as it is the sole pathway to provide the major isoprene units required as metabolic precursor for various housekeeping activities. With the intensifying need to identify a novel therapeutic drug target against Plasmodium, the MEP pathway and its components are considered as potential therapeutic targets, due to the difference in the isoprenoid synthesis route (MVA) functional in the host cells. While few major components have already been studied from this pathway for their potential as a drug target, IspD (2-C-Methyl-D-erythritol-4-phosphate cytidyltransferase) enzyme, the enzyme catalyzing the third step of the pathway has only been tested against a synthetic compound from Malaria box called MMV008138, which also has not shown adequate inhibitory activity against P. vivax IspD. In the present study, to validate the potential of PvIspD as a drug target, various antimicrobial agents were screened for their inhibition possibilities, using in-vitro High Throughput Screening (HTS) technique. Shortlisted antimicrobial drug molecules like Cefepime, Tunicamycin and Rifampicin were further validated by in-vitro biochemical enzyme inhibition assays where they showed activity at nanomolar concentrations suggesting them or their derivatives as prospective future antimalarials. This study also confirmed the in-vivo expression of PvIspD protein during asexual stages by sub-cellular localization in apicoplast and explores the importance of the IspD enzyme in the development of new therapeutics.

Isolation and characterization of 4-hydroxy-3-methylbut-2-enyl diphosphate reductase gene from Botryococcus braunii, race B.[Pubmed:29725892]

J Plant Res. 2018 Sep;131(5):839-848.

The B race of a green microalga Botryococcus braunii Kutzing produces triterpene hydrocarbons that is a promising source for biofuel. In this algal race, precursors of triterpene hydrocarbons are provided from the 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway. The terminal enzyme of this pathway, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is regarded as one of the key enzymes that affect yields of products in terpene biosynthesis. In order to better understand the MEP pathway of the alga, cDNA and genomic clones of HDR were obtained from B. braunii Showa strain. B. braunii HDR (BbHDR) is encoded on a single copy gene including a 1509-bp open reading frame that was intervened by 6 introns. The exon-intron structure of BbHDR genes did not show clear relation to phylogeny, while its amino acid sequence reflected phyla and classes well. BbHDR sequence was distinctive from that of the HDR protein from Escherichia coli in the residues involved in hydrogen-bond network that surrounds substrate. Introduction of BbHDR cDNA into an E. coli HDR deficient mutant resulted in recovery of its auxotrophy. BbHDR expression level was upregulated from the onset of liquid culture to the 24th day after inoculation with a 2.5-fold increase and retained its level in the subsequent period.

Transcriptome analysis of Hevea brasiliensis in response to exogenous methyl jasmonate provides novel insights into regulation of jasmonate-elicited rubber biosynthesis.[Pubmed:29692543]

Physiol Mol Biol Plants. 2018 May;24(3):349-358.

The phytohomorne methyl jasmonate (MeJA) is known to trigger extensive reprogramming of gene expression leading to transcriptional activation of many secondary metabolic pathways. However, natural rubber is a commercially important secondary metabolite and little is known about the genetic and genomic basis of jasmonate-elicited rubber biosynthesis in rubber tree (Hevea brasiliensis). RNA sequencing (RNA-seq) of H. brasiliensis bark treated with 1 g lanolin paste containing 0.02% w/w MeJA for 24 h (M2) and 0.04% w/w MeJA for 24 h (M4) was performed. A total of 2950 and 2850 differentially expressed genes in M2 and M4 compared with control (C) were respectively detected. Key genes involved in 2-C-Methyl-D-erythritol 4-phosphate, rubber biosynthesis, glycolysis and carbon fixation (Calvin cycle) pathway were found to be up-regulated by MeJA treatment. Particularly, the expression of 3-hydroxy-3-metylglutaryl coenzyme A reductase in MVA pathway was down-regulated by MeJA treatment, but the expression of farnesyl diphosphate synthase (FPS) and cis-prenyltransferase (CPT, or rubber transferase) in rubber biosynthesis pathway were up-regulated by MeJA treatment. Up-regulation of critical genes in JA biosynthesis in response to MeJA treatment exhibited the self-activation of JA biosynthesis. In addition, up-regulated genes of great regulatory importance in cross-talk between JA and other hormone signaling, and of transcriptional regulation were identified. The increased expression levels of FPS and CPT in rubber biosynthesis pathway possibly resulted in an increased latex production in rubber tree treated with MeJA. The present results provide insights into the mechanism by which MeJA activates the rubber biosynthesis and the transcriptome data can also serve as the foundation for future research into the molecular basis for MeJA regulation of other cellular processes.