Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

The methylerythritol phosphate pathway as an oxidative stress sense and response system.

The methylerythritol phosphate (MEP) pathway is responsible for biosynthesis of the precursors of isoprenoid compounds in eubacteria and plastids. It is a metabolic alternative to the well-known mevalonate pathway for isoprenoid production found in archaea and eukaryotes. Recently, a role for the MEP pathway in oxidative stress detection, signalling, and response has been identified. This role is executed in part through the unusual cyclic intermediate, methylerythritol cyclodiphosphate (MEcDP). We postulate that this response is triggered through the oxygen sensitivity of the MEP pathway's terminal iron-sulfur (Fe-S) cluster enzymes. MEcDP is the substrate of IspG, the first Fe-S cluster enzyme in the pathway; it accumulates under oxidative stress conditions and acts as a signalling molecule. It may also act as an antioxidant. Furthermore, evidence is emerging for a broader and highly nuanced role of the MEP pathway in oxidative stress responses, implemented through a complex system of differential regulation and sensitivity at numerous nodes in the pathway. Here, we explore the evidence for such a role (including the contribution of the Fe-S cluster enzymes and different pathway metabolites, especially MEcDP), the evolutionary implications, and the many questions remaining about the behaviour of the MEP pathway in the presence of oxidative stress.

Reviving resilience: MEcPP-mediated ASK1-IMPα-9-TRP2 stress-responsive module.

Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.

Neural responses to oral administration of erythritol vs. sucrose and sucralose explain differences in subjective liking ratings.

INTRODUCTION: High sugar intake is associated with many chronic diseases. However, non-caloric sweeteners (NCSs) might fail to successfully replace sucrose due to the mismatch between their rewarding sweet taste and lack of caloric content. The natural NCS erythritol has been proposed as a sugar substitute due to its satiating properties despite being non-caloric. We aimed to compare brain responses to erythritol vs. sucrose and the artificial NCS sucralose in a priori taste, homeostatic, and reward brain regions of interest (ROIs). METHODS: We performed a within-subject, single-blind, counterbalanced fMRI study in 30 healthy men (mean ± SEM age:24.3 ± 0.8 years, BMI:22.3 ± 0.3 kg/m2). Before scanning, we individually matched the concentrations of both NCSs to the perceived sweetness intensity of a 10% sucrose solution. During scanning, participants received 1 mL sips of the individually titrated equisweet solutions of sucrose, erythritol, and sucralose, as well as water. After each sip, they rated subjective sweetness liking. RESULTS: Liking ratings were significantly higher for sucrose and sucralose vs. erythritol (both pHolm = 0.0037); water ratings were neutral. General Linear Model (GLM) analyses of brain blood oxygen level-depended (BOLD) responses at qFDR<0.05 showed no differences between any of the sweeteners in a priori ROIs, but distinct differences were found between the individual sweeteners and water. These results were confirmed by Bayesian GLM and machine learning-based models. However, several brain response patterns mediating the differences in liking ratings between the sweeteners were found in whole-brain multivariate mediation analyses. Both subjective and neural responses showed large inter-subject variability. CONCLUSION: We found lower liking ratings in response to oral administration of erythritol vs. sucrose and sucralose, but no differences in neural responses between any of the sweeteners in a priori ROIs. However, differences in liking ratings between erythritol vs. sucrose or sucralose are mediated by multiple whole-brain response patterns.

Functional analysis of the methylerythritol phosphate pathway terminal enzymes IspG and IspH from Zymomonas mobilis.

Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions. IMPORTANCE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Metabolic crosstalk between hydroxylated monoterpenes and salicylic acid in tomato defense response against bacteria.

Hydroxylated monoterpenes (HMTPs) are differentially emitted by tomato (Solanum lycopersicum) plants resisting bacterial infection. We have studied the defensive role of these volatiles in the tomato response to bacteria, whose main entrance is through stomatal apertures. Treatments with some HMTPs resulted in stomatal closure and pathogenesis-related protein 1 (PR1) induction. Particularly, α-terpineol induced stomatal closure in a salicylic acid (SA) and abscisic acid-independent manner and conferred resistance to bacteria. Interestingly, transgenic tomato plants overexpressing or silencing the monoterpene synthase MTS1, which displayed alterations in the emission of HMTPs, exhibited changes in the stomatal aperture but not in plant resistance. Measures of both 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) and SA levels revealed competition for MEcPP by the methylerythritol phosphate (MEP) pathway and SA biosynthesis activation, thus explaining the absence of resistance in transgenic plants. These results were confirmed by chemical inhibition of the MEP pathway, which alters MEcPP levels. Treatments with benzothiadiazole (BTH), a SA functional analog, conferred enhanced resistance to transgenic tomato plants overexpressing MTS1. Additionally, these MTS1 overexpressors induced PR1 gene expression and stomatal closure in neighboring plants. Our results confirm the role of HMTPs in both intra- and interplant immune signaling and reveal a metabolic crosstalk between the MEP and SA pathways in tomato plants.

Plant In Vitro Culture Factories for Pentacyclic Triterpenoid Production.

Pentacyclic triterpenoids are a diverse subclass of naturally occurring terpenes with various biological activities and applications. These compounds are broadly distributed in natural plant resources, but their low abundance and the slow growth cycle of plants pose challenges to their extraction and production. The biosynthesis of pentacyclic triterpenoids occurs through two main pathways, the mevalonic acid (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which involve several enzymes and modifications. Plant in vitro cultures, including elicited and hairy root cultures, have emerged as an effective and sustainable system for pentacyclic triterpenoid production, circumventing the limitations associated with natural plant resources. Bioreactor systems and controlling key parameters, such as media composition, temperature, light quality, and elicitor treatments, have been optimized to enhance the production and characterization of specific pentacyclic triterpenoids. These systems offer a promising bioprocessing tool for producing pentacyclic triterpenoids characterized by a low carbon footprint and a sustainable source of these compounds for various industrial applications.

Design and synthesis of fosmidomycin analogs containing aza-linkers and their biological activity evaluation.

BACKGROUND: The enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway are attractive targets of a new mode of action for developing anti-infective drugs and herbicides, and inhibitors against 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), the second key enzyme in the pathway, have been intensively investigated; however, few works are reported regarding IspC inhibitors designed for new herbicide discovery. RESULTS: A series of fosmidomycin (FOS) analogs were designed with nitrogen-containing linkers replacing the trimethylene linker between the two active substructures of FOS, phosphonic acid and hydroxamic acid. Synthesis followed a facile three-step route of sequential aza-Michael addition of α-amino acids to dibenzyl vinylphosphonate, amidation of the amino acid carboxyl with O-benzyl hydroxylamine, and simultaneous removal of the benzyl protective groups. Biological activity evaluation of IspC and model plants revealed that some compounds had moderate enzyme and model plant growth inhibition effects. In particular, compound 10g, which has a N-(4-fluorophenylethyl) nitrogen-containing linker, exhibited the best plant inhibition activities, superior to the control FOS against the model plants Arabidopsis thaliana, Brassica napus L., Amaranthus retroflexus and Echinochloa crus-galli. A dimethylallyl pyrophosphate rescue assay on A. thaliana confirmed that both 10g and FOS exert their herbicidal activity by blocking the MEP pathway. This result consistent with molecular docking, which confirmed 10g and FOS binding to the IspC active site in a similar way. CONCLUSION: Compound 10g has excellent herbicidal activity and represents the first herbicide lead structure of a new mode of action that targets IspC enzyme in the MEP pathway. © 2023 Society of Chemical Industry.

The gibberellic acid derived from the plastidial MEP pathway is involved in the accumulation of Bamboo mosaic virus.

A gene upregulated in Nicotiana benthamiana after Bamboo mosaic virus (BaMV) infection was revealed as 1-deoxy-d-xylulose-5-phosphate reductoisomerase (NbDXR). DXR is the key enzyme in the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway that catalyzes the conversion of 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol-4-phosphate. Knockdown and overexpression of NbDXR followed by BaMV inoculation revealed that NbDXR is involved in BaMV accumulation. Treating leaves with fosmidomycin, an inhibitor of DXR function, reduced BaMV accumulation. Subcellular localization confirmed that DXR is a chloroplast-localized protein by confocal microscopy. Furthermore, knockdown of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase, one of the enzymes in the MEP pathway, also reduced BaMV accumulation. The accumulation of BaMV increased significantly in protoplasts treated with isopentenyl pyrophosphate. Thus, the metabolites of the MEP pathway could be involved in BaMV infection. To identify the critical components involved in BaMV accumulation, we knocked down the crucial enzyme of isoprenoid synthesis, NbGGPPS11 or NbGGPPS2. Only NbGGPPS2 was involved in BaMV infection. The geranylgeranyl pyrophosphate (GGPP) synthesized by NbGGPPS2 is known for gibberellin synthesis. We confirmed this result by supplying gibberellic acid exogenously on leaves, which increased BaMV accumulation. The de novo synthesis of gibberellic acid could assist BaMV accumulation.

YdfD, a Lysis Protein of the Qin Prophage, Is a Specific Inhibitor of the IspG-Catalyzed Step in the MEP Pathway of Escherichia coli.

Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.

SlWRKY35 positively regulates carotenoid biosynthesis by activating the MEP pathway in tomato fruit.

Carotenoids are vital phytonutrients widely recognised for their health benefits. Therefore, it is vital to thoroughly investigate the metabolic regulatory network underlying carotenoid biosynthesis and accumulation to open new leads towards improving their contents in vegetables and crops. The outcome of our study defines SlWRKY35 as a positive regulator of carotenoid biosynthesis in tomato. SlWRKY35 can directly activate the expression of the 1-deoxy-d-xylulose 5-phosphate synthase (SlDXS1) gene to reprogramme metabolism towards the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, leading to enhanced carotenoid accumulation. We also show that the master regulator SlRIN directly regulates the expression of SlWRKY35 during tomato fruit ripening. Compared with the SlLCYE overexpression lines, coexpression of SlWRKY35 and SlLCYE can further enhance lutein production in transgenic tomato fruit, indicating that SlWRKY35 represents a potential target towards designing innovative metabolic engineering strategies for carotenoid derivatives. In addition to providing new insights into the metabolic regulatory network associated with tomato fruit ripening, our data define a new tool for improving fruit content in specific carotenoid compounds.

Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway.

Apicomplexan parasites, such as Toxoplasma gondii, are unusual in that each cell contains a single apicoplast, a plastid-like organelle that compartmentalizes enzymes involved in the essential 2C-methyl-D-erythritol 4-phosphate pathway of isoprenoid biosynthesis. The last two enzymatic steps in this organellar pathway require electrons from a redox carrier. However, the small iron-sulfur cluster-containing protein ferredoxin, a likely candidate for this function, has not been investigated in this context. We show here that inducible knockdown of T. gondii ferredoxin results in progressive inhibition of growth and eventual parasite death. Surprisingly, this phenotype is not accompanied by ultrastructural changes in the apicoplast or overall cell morphology. The knockdown of ferredoxin was instead associated with a dramatic decrease in cellular levels of the last two metabolites in isoprenoid biosynthesis, 1-hydroxy-2-methyl-2-(E)- butenyl-4-pyrophosphate, and isomeric dimethylallyl pyrophosphate/isopentenyl pyrophosphate. Ferredoxin depletion was also observed to impair gliding motility, consistent with isoprenoid metabolites being important for dolichol biosynthesis, protein prenylation, and modification of other proteins involved in motility. Significantly, pharmacological inhibition of isoprenoid synthesis of the host cell exacerbated the impact of ferredoxin depletion on parasite replication, suggesting that the slow onset of parasite death after ferredoxin depletion is because of isoprenoid scavenging from the host cell and leading to partial compensation of the depleted parasite metabolites upon ferredoxin knockdown. Overall, these findings show that ferredoxin has an essential physiological function as an electron donor for the 2C-methyl-D-erythritol 4-phosphate pathway and is a potential drug target for apicomplexan parasites.

Targeting the IspD Enzyme in the MEP Pathway: Identification of a Novel Fragment Class.

The enzymes of the 2-C-methylerythritol-d-erythritol 4-phosphate (MEP) pathway (MEP pathway or non-mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high-throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity-stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure-guided optimization.

Protein-Ribofuranosyl Interactions Activate Orotidine 5'-Monophosphate Decarboxylase for Catalysis.

The role of a global, substrate-driven, enzyme conformational change in enabling the extraordinarily large rate acceleration for orotidine 5'-monophosphate decarboxylase (OMPDC)-catalyzed decarboxylation of orotidine 5'-monophosphate (OMP) is examined in experiments that focus on the interactions between OMPDC and the ribosyl hydroxyl groups of OMP. The D37 and T100' side chains of OMPDC interact, respectively, with the C-3' and C-2' hydroxyl groups of enzyme-bound OMP. D37G and T100'A substitutions result in 1.4 kcal/mol increases in the activation barrier ΔG⧧ for catalysis of decarboxylation of the phosphodianion-truncated substrate 1-(ß-d-erythrofuranosyl)orotic acid (EO) but result in larger 2.1-2.9 kcal/mol increases in ΔG⧧ for decarboxylation of OMP and for phosphite dianion-activated decarboxylation of EO. This shows that these substitutions reduce transition-state stabilization by the Q215, Y217, and R235 side chains at the dianion binding site. The D37G and T100'A substitutions result in <1.0 kcal/mol increases in ΔG⧧ for activation of OMPDC-catalyzed decarboxylation of the phosphoribofuranosyl-truncated substrate FO by phosphite dianions. Experiments to probe the effect of D37 and T100' substitutions on the kinetic parameters for d-glycerol 3-phosphate and d-erythritol 4-phosphate activators of OMPDC-catalyzed decarboxylation of FO show that ΔG⧧ for sugar phosphate-activated reactions is increased by ca. 2.5 kcal/mol for each -OH interaction eliminated by D37G or T100'A substitutions. We conclude that the interactions between the D37 and T100' side chains and ribosyl or ribosyl-like hydroxyl groups are utilized to activate OMPDC for catalysis of decarboxylation of OMP, EO, and FO.

CO2 -responsiveness of leaf isoprene emission: Why do species differ?

Leaf isoprene emission rate, I, decreases with increasing atmospheric CO2 concentration with major implications for global change. There is a significant interspecific variability in [CO2 ]-responsiveness of I, but the extent of this variation is unknown and its reasons are not understood. We hypothesized that the magnitude of emission reduction reflects the size and changeability of precursor pools responsible for isoprene emission (dimethylallyl diphosphate, DMADP and 2-methyl-erythritol 2,4-cyclodiphosphate, MEcDP). Changes in I and intermediate pool sizes upon increase of [CO2 ] from 400 to 1500 µmol/mol were studied in nine woody species spanning boreal to tropical ecosystems. I varied 10-fold, total substrate pool size 37-fold and the ratio of DMADP/MEcDP pool sizes 57-fold. At higher [CO2 ], I was reduced on average by 65%, but [CO2 ]-responsiveness varied an order of magnitude across species. The increase in [CO2 ] resulted in concomitant reductions in both substrate pools. The variation in [CO2 ]-responsiveness across species scaled with the reduction in pool sizes, the substrate pool size supported and the share of DMADP in total substrate pool. This study highlights a major interspecific variation in [CO2 ]-responsiveness of isoprene emission and conclusively links this variation to interspecific variability in [CO2 ] effects on substrate availability and intermediate pool size.

Transcriptomics Integrated with Free and Bound Terpenoid Aroma Profiling during "Shine Muscat" (Vitis labrusca × V. vinifera) Grape Berry Development Reveals Coordinate Regulation of MEP Pathway and Terpene Synthase Gene Expression.

Terpenes and their derivatives are important biomarkers of grape quality as they contribute to the flavor and aroma of grapes. However, the molecular basis of terpene biosynthesis throughout the grapevine phenological developmental cycle remains elusive. Our current study investigates the free and bound terpene biosynthesis of berries at different phenological stages from preveraison to harvest. Detailed gene expression (transcriptomics) analysis, terpenoid volatile production by gas chromatography-mass spectrometry (GC-MS), and in planta transient expression were employed. Our results show that concentrations of most individual terpenes at different stages are distinctive and increase from preveraison to the veraison stage followed by a decrease from veraison to maturity. The combined transcriptomic analysis and terpene profiling revealed that 22 genes belonging to the MEP pathway and multiple classes of transcription factor family members including bHLH and several hormone biosynthesis- or signaling-related genes likely participate in the regulation of terpenoid biosynthesis according to their specific expression patterns in berries. Quantitative real-time polymerase chain expression analysis of 8 key differentially expressed genes in MEP pathways and further 12 randomly selected genes was performed during 8 sampling stages and validated the RNA-seq-derived expression profiles. To further confirm the function of a subset of the differentially expressed genes, we investigated the effects of combined overexpression of 1-deoxy-d-xylulose-5-phosphate synthase (VvDXS1-LOC100249323), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (VvDXR-LOC100248516), and terpene synthase (VvTPS56-LOC100266449) on the production of terpenes by transient overexpression in Nicotiana benthamiana leaves. The overall developmental patterns of total terpenes and gene expression profiles will help guide the functional analyses of further candidate genes important for terpene biosynthesis of grape as well as identifying the master transcriptional and hormonal regulators of this pathway in the future.

Balancing glucose and oxygen uptake rates to enable high amorpha-4,11-diene production in Escherichia coli via the methylerythritol phosphate pathway.

Amorpha-4,11-diene (AMD4,11) is a precursor to artemisinin, a potent antimalarial drug that is traditionally extracted from the shrubs of Artemisia annua. Despite significant prior efforts to produce artemisinin and its precursors through biotechnology, there remains a dire need for more efficient biosynthetic routes for its production. Here, we describe the optimization of key process conditions for an Escherichia coli strain producing AMD4,11 via the native methylerythritol phosphate (MEP) pathway. By studying the interplay between glucose uptake rates and oxygen demand, we were able to identify optimal conditions for increasing carbon flux through the MEP pathway by manipulating the availability of NADPH required for terpenoid production. Installation of an optimal qO2 /qglucose led to a 6.7-fold increase in product titers and a 6.5-fold increase in carbon yield.

Heat priming improved heat tolerance of photosynthesis, enhanced terpenoid and benzenoid emission and phenolics accumulation in Achillea millefolium.

The mechanism of heat priming, triggering alteration of secondary metabolite pathway fluxes and pools to enhance heat tolerance is not well understood. Achillea millefolium is an important medicinal herbal plant, rich in terpenoids and phenolics. In this study, the potential of heat priming treatment (35°C for 1 hr) to enhance tolerance of Achillea plants upon subsequent heat shock (45°C for 5 min) stress was investigated through recovery (0.5-72 hr). The priming treatment itself had minor impacts on photosynthesis, led to moderate increases in the emission of lipoxygenase (LOX) pathway volatiles and isoprene, and to major elicitation of monoterpene and benzaldehyde emissions in late stages of recovery. Upon subsequent heat shock, in primed plants, the rise in LOX and reduction in photosynthetic rate (A) was much less, stomatal conductance (gs ) was initially enhanced, terpene emissions were greater and recovery of A occurred faster, indicating enhanced heat tolerance. Additionally, primed plants accumulated higher contents of total phenolics and condensed tannins at the end of the recovery. These results collectively indicate that heat priming improved photosynthesis upon subsequent heat shock by enhancing gs and synthesis of volatile and non-volatile secondary compounds with antioxidative characteristics, thereby maintaining the integrity of leaf membranes under stress.

Positioning Bacillus subtilis as terpenoid cell factory.

Increasing demands for bioactive compounds have motivated researchers to employ micro-organisms to produce complex natural products. Currently, Bacillus subtilis has been attracting lots of attention to be developed into terpenoids cell factories due to its generally recognized safe status and high isoprene precursor biosynthesis capacity by endogenous methylerythritol phosphate (MEP) pathway. In this review, we describe the up-to-date knowledge of each enzyme in MEP pathway and the subsequent steps of isomerization and condensation of C5 isoprene precursors. In addition, several representative terpene synthases expressed in B. subtilis and the engineering steps to improve corresponding terpenoids production are systematically discussed. Furthermore, the current available genetic tools are mentioned as along with promising strategies to improve terpenoids in B. subtilis, hoping to inspire future directions in metabolic engineering of B. subtilis for further terpenoid cell factory development.