A pyruvate transporter in the apicoplast of apicomplexan parasites.

Pyruvate lies at a pivotal node of carbon metabolism in eukaryotes. It is involved in diverse metabolic pathways in multiple organelles, and its interorganelle shuttling is crucial for cell fitness. Many apicomplexan parasites harbor a unique organelle called the apicoplast that houses metabolic pathways like fatty acid and isoprenoid precursor biosyntheses, requiring pyruvate as a substrate. However, how pyruvate is supplied in the apicoplast remains enigmatic. Here, deploying the zoonotic parasite Toxoplasma gondii as a model apicomplexan, we identified two proteins residing in the apicoplast membranes that together constitute a functional apicoplast pyruvate carrier (APC) to mediate the import of cytosolic pyruvate. Depletion of APC results in reduced activities of metabolic pathways in the apicoplast and impaired integrity of this organelle, leading to parasite growth arrest. APC is a pyruvate transporter in diverse apicomplexan parasites, suggesting a common strategy for pyruvate acquisition by the apicoplast in these clinically relevant intracellular pathogens.

Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission.

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.

A picomolar inhibitor of the Plasmodium falciparum IPP pathway.

We identified MMV026468 as a picomolar inhibitor of blood-stage Plasmodium falciparum. Phenotyping assays, including isopentenyl diphosphate rescue of parasite growth inhibition, demonstrated that it targets MEP isoprenoid precursor biosynthesis. MMV026468-treated parasites showed an overall decrease in MEP pathway intermediates, which could result from inhibition of the first MEP enzyme DXS or steps prior to DXS such as regulation of the MEP pathway. Selection of MMV026468-resistant parasites lacking DXS mutations suggested that other targets are possible. The identification of MMV026468 could lead to a new class of antimalarial isoprenoid inhibitors.

Divergent contribution of the MVA and MEP pathways to the formation of polyprenols and dolichols in Arabidopsis.

Isoprenoids, including dolichols (Dols) and polyprenols (Prens), are ubiquitous components of eukaryotic cells. In plant cells, there are two pathways that produce precursors utilized for isoprenoid biosynthesis: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. In this work, the contribution of these two pathways to the biosynthesis of Prens and Dols was addressed using an in planta experimental model. Treatment of plants with pathway-specific inhibitors and analysis of the effects of various light conditions indicated distinct biosynthetic origin of Prens and Dols. Feeding with deuteriated, pathway-specific precursors revealed that Dols, present in leaves and roots, were derived from both MEP and MVA pathways and their relative contributions were modulated in response to precursor availability. In contrast, Prens, present in leaves, were almost exclusively synthesized via the MEP pathway. Furthermore, results obtained using a newly introduced here 'competitive' labeling method, designed so as to neutralize the imbalance of metabolic flow resulting from feeding with a single pathway-specific precursor, suggest that under these experimental conditions one fraction of Prens and Dols is synthesized solely from endogenous precursors (deoxyxylulose or mevalonate), while the other fraction is synthesized concomitantly from endogenous and exogenous precursors. Additionally, this report describes a novel methodology for quantitative separation of 2H and 13C distributions observed for isotopologues of metabolically labeled isoprenoids. Collectively, these in planta results show that Dol biosynthesis, which uses both pathways, is significantly modulated depending on pathway productivity, while Prens are consistently derived from the MEP pathway.

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.

Recent advances in understanding the regulation of plant secondary metabolite biosynthesis by ethylene-mediated pathways.

Plants produce a large repertoire of secondary metabolites. The pathways that lead to the biosynthesis of these metabolites are majorly conserved in the plant kingdom. However, a significant portion of these metabolites are specific to certain groups or species due to variations in the downstream pathways and evolution of the enzymes. These metabolites show spatiotemporal variation in their accumulation and are of great importance to plants due to their role in development, stress response and survival. A large number of these metabolites are in huge industrial demand due to their potential use as therapeutics, aromatics and more. Ethylene, as a plant hormone is long known, and its biosynthetic process, signaling mechanism and effects on development and response pathways have been characterized in many plants. Through exogenous treatments, ethylene and its inhibitors have been used to manipulate the production of various secondary metabolites. However, the research done on a limited number of plants in the last few years has only started to uncover the mechanisms through which ethylene regulates the accumulation of these metabolites. Often in association with other hormones, ethylene participates in fine-tuning the biosynthesis of the secondary metabolites, and brings specificity in the regulation depending on the plant, organ, tissue type and the prevailing conditions. This review summarizes the related studies, interprets the outcomes, and identifies the gaps that will help to breed better varieties of the related crops and produce high-value secondary metabolites for human benefits.

Plastidial engineering with coupled farnesyl diphosphate pool reconstitution and enhancement for sesquiterpene biosynthesis in tomato fruit.

Sesquiterpenes represent a large class of terpene compounds found in plants with broad applications such as pharmaceuticals and biofuels. The plastidial MEP pathway in ripening tomato fruit is naturally optimized to provide the 5-carbon isoprene building blocks of all terpenes for production of the tetraterpene pigment lycopene and other carotenoids, making it an excellent plant system to be engineered for production of high-value terpenoids. We reconstituted and enhanced the pool of sesquiterpene precursor farnesyl diphosphate (FPP) in plastids of tomato fruit by overexpressing the fusion gene DXS-FPPS encoding a fusion protein of 1-deoxy-D-xylulose 5-phosphate synthase (DXS) linked with farnesyl diphosphate synthase (originally called farnesyl pyrophosphate synthase, and abbreviated as FPPS) under the control of fruit-ripening specific polygalacturonase (PG) promoter concomitant with substantial reduction in lycopene content and large production of FPP-derived squalene. The supply of precursors achieved by the fusion gene expression can be harnessed by an engineered sesquiterpene synthase that is retargeted to plastid to engineer high-yield sesquiterpene production in tomato fruit, offering an effective production system for high-value sesquiterpene ingredients.

A Programmable CRISPR/Cas9 Toolkit Improves Lycopene Production in Bacillus subtilis.

Bacillus subtilis has been widely used and generally recognized as a safe host for the production of recombinant proteins, high-value chemicals, and pharmaceuticals. Thus, its metabolic engineering attracts significant attention. Nevertheless, the limited availability of selective markers makes this process difficult and time-consuming, especially in the case of multistep biosynthetic pathways. Here, we employ CRISPR/Cas9 technology to build an easy cloning toolkit that addresses commonly encountered obstacles in the metabolic engineering of B. subtilis, including the chromosomal integration locus, promoter, terminator, and guide RNA (gRNA) target. Six promoters were characterized, and the promoter strengths ranged from 0.9- to 23-fold that of the commonly used strong promoter P43. We characterized seven terminators in B. subtilis, and the termination efficiencies (TEs) of the seven terminators are all more than 90%. Six gRNA targets were designed upstream of the promoter and downstream of the terminator. Using a green fluorescent protein (GFP) reporter, we confirmed integration efficiency with the single-locus integration site is up to 100%. We demonstrated the applicability of this toolkit by optimizing the expression of a challenging but industrially important product, lycopene. By heterologous expression of the essential genes for lycopene synthesis on the B. subtilis genome, a total of 13 key genes involved in the lycopene biosynthetic pathway were manipulated. Moreover, our findings showed that the gene cluster ispG-idi-dxs-ispD could positively affect the production of lycopene, while the cluster dxr-ispE-ispF-ispH had a negative effect on lycopene production. Hence, our multilocus integration strategy can facilitate the pathway assembly for production of complex chemicals and pharmaceuticals in B. subtilis. IMPORTANCE We present a toolkit that allows for rapid cloning procedures and one-step subcloning to move from plasmid-based expression to stable chromosome integration and expression in a production strain in less than a week. The utility of the customized tool was demonstrated by integrating the MEP (2C-methyl-d-erythritol-4-phosphate) pathway, part of the pentose phosphate pathway (PPP), and the hetero-lycopene biosynthesis genes by stable expression in the genome. The tool could be useful to engineer B. subtilis strains through diverse recombination events and ultimately improve its potential and scope of industrial application as biological chassis.

Combining in silico and in vitro approaches to understand the involvement of methylerythritol 4-phosphate and shikimate pathways in Agrobacterium tumefaciens for enhanced coenzyme Q10 production.

AIMS: To perform an integrated comparative analysis of metabolic pathway to understand coenzyme Q10 (CoQ10) production in Agrobacterium tumefaciens. METHODS AND RESULTS: Comparative analysis of the CoQ10 metabolic pathway in 10 organisms using a genome to KEGG orthology program (G2KO) and the KEGG database elucidated the completeness of the production pathway in A. tumefaciens. The specific roles of the key precursors and the enzymes in the metabolic network were subsequently confirmed using pathway inhibitors and enhancers. While the use of fosmidomycin and glyphosate was found to inhibit CoQ10 production by 54.54% to 99%, the supplementation of polyprenyl pyrophosphate of the methylerythritol 4-phosphate pathway and 4-hydroxybenzoate precursor of the shikimate pathway did increse the production of CoQ10 by 2.3-fold. CONCLUSIONS: The present study provides a comprehensive understanding of the CoQ10 biosynthetic pathway in A. tumefaciens, which would assist rational metabolic engineering strategies for augmenting CoQ10 biosynthesis.

Uncovering the functional residues of Arabidopsis isoprenoid biosynthesis enzyme HDS.

The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives.

Isoprenoids, a diverse class of natural products, are present in all living organisms. Their two universal building blocks are synthesized via two independent pathways: the mevalonate pathway and the 2-C-methyl-ᴅ-erythritol 4-phosphate (MEP) pathway. The presence of the latter in pathogenic bacteria and its absence in humans make all its enzymes suitable targets for the development of novel antibacterial drugs. (E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), the last intermediate of this pathway, is a natural ligand for the human Vγ9Vδ2 T cells and the most potent natural phosphoantigen known to date. Moreover, 5-hydroxypentane-2,3-dione, a metabolite produced by Escherichia coli 1-deoxy-ᴅ-xylulose 5-phosphate synthase (DXS), the first enzyme of the MEP pathway, structurally resembles (S)-4,5-dihydroxy-2,3-pentanedione, a signal molecule implied in bacterial cell communication. In this review, we shed light on the diversity of potential uses of the MEP pathway in antibacterial therapies, starting with an overview of the antibacterials developed for each of its enzymes. Then, we provide insight into HMBPP, its synthetic analogs, and their prodrugs. Finally, we discuss the potential contribution of the MEP pathway to quorum sensing mechanisms. The MEP pathway, providing simultaneously antibacterial drug targets and potent immunostimulants, coupled with its potential role in bacterial cell-cell communication, opens new therapeutic perspectives.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production.

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L-1 with a maximal productivity of 78 mg L-1 day-1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

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.

Quantitative insight into the metabolism of isoprene-producing Synechocystis sp. PCC 6803 using steady state 13C-MFA.

Cyanobacteria are photosynthetic bacteria, widely studied for the conversion of atmospheric carbon dioxide to useful platform chemicals. Isoprene is one such industrially important chemical, primarily used for production of synthetic rubber and biofuels. Synechocystis sp. PCC 6803, a genetically amenable cyanobacterium, produces isoprene on heterologous expression of isoprene synthase gene, albeit in very low quantities. Rationalized metabolic engineering to re-route the carbon flux for enhanced isoprene production requires in-dept knowledge of the metabolic flux distribution in the cell. Hence, in the present study, we undertook steady state 13C-metabolic flux analysis of glucose-tolerant wild-type (GTN) and isoprene-producing recombinant (ISP) Synechocystis sp. to understand and compare the carbon flux distribution in the two strains. The R-values for amino acids, flux analysis predictions and gene expression profiles emphasized predominance of Calvin cycle and glycogen metabolism in GTN. Alternatively, flux analysis predicted higher activity of the anaplerotic pathway through phosphoenolpyruvate carboxylase and malic enzyme in ISP. The striking difference in the Calvin cycle, glycogen metabolism and anaplerotic pathway activity in GTN and ISP suggested a possible role of energy molecules (ATP and NADPH) in regulating the carbon flux distribution in GTN and ISP. This claim was further supported by the transcript level of selected genes of the electron transport chain. This study provides the first quantitative insight into the carbon flux distribution of isoprene-producing cyanobacterium, information critical for developing Synechocystis sp. as a single cell factory for isoprenoid/terpenoid production.

Metabolic engineering strategies for sesquiterpene production in microorganism.

Sesquiterpenes are a large variety of terpene natural products, widely existing in plants, fungi, marine organisms, insects, and microbes. Value-added sesquiterpenes are extensively used in industries such as: food, drugs, fragrances, and fuels. With an increase in market demands and the price of sesquiterpenes, the biosynthesis of sesquiterpenes by microbial fermentation methods from renewable feedstocks is acquiring increasing attention. Synthetic biology provides robust tools of sesquiterpene production in microorganisms. This review presents a summary of metabolic engineering strategies on the hosts and pathway engineering for sesquiterpene production. Advances in synthetic biology provide new strategies on the creation of desired hosts for sesquiterpene production. Especially, metabolic engineering strategies for the production of sesquiterpenes such as: amorphadiene, farnesene, bisabolene, and caryophyllene are emphasized in: Escherichia coli, Saccharomyces cerevisiae, and other microorganisms. Challenges and future perspectives of the bioprocess for translating sesquiterpene production into practical industrial work are also discussed.

Irreversible Inhibition of IspG, a Target for the Development of New Antimicrobials, by a 2-Vinyl Analogue of its MEcPP Substrate.

IspG (also called GcpE) is an oxygen-sensitive [4Fe-4S] enzyme catalyzing the penultimate step of the methylerythritol phosphate (MEP) pathway, a validated target for drug development. It converts 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl-1-diphosphate (HMBPP). The reaction, assimilated to a reductive dehydration, involves redox partners responsible for the formal transfer of two electrons to substrate MEcPP. The 2-vinyl analogue of MEcPP was designed to generate conjugated species during enzyme catalysis, with the aim of providing new reactive centers to be covalently trapped by neighboring amino acid residues. The synthesized substrate analogue displayed irreversible inhibition towards IspG. Furthermore, we have shown that electron transfer occurs prior to inhibition; this might designate conjugated intermediates as probable affinity tags through covalent interaction at the catalytic site. This is the first report of an irreversible inhibitor of the IspG metalloenzyme.

Engineering plant family TPS into cyanobacterial host for terpenoids production.

Terpenoids are synthesized naturally by plants as secondary metabolites, and are diverse and complex in structure with multiple applications in bioenergy, food, cosmetics, and medicine. This makes the production of terpenoids such as isoprene, ß-phellandrene, farnesene, amorphadiene, and squalene valuable, owing to which their industrial demand cannot be fulfilled exclusively by plant sources. They are synthesized via the Methylerythritol phosphate pathway (MEP) and the Mevalonate pathway (MVA), both existing in plants. The advent of genetic engineering and the latest accomplishments in synthetic biology and metabolic engineering allow microbial synthesis of terpenoids. Cyanobacteria manifest to be the promising hosts for this, utilizing sunlight and CO2. Cyanobacteria possess MEP pathway to generate precursors for terpenoid synthesis. The terpenoid synthesis can be amplified by overexpressing the MEP pathway and engineering MVA pathway genes. According to the desired terpenoid, terpene synthases unique to the plant kingdom must be incorporated in cyanobacteria. Engineering an organism to be used as a cell factory comes with drawbacks such as hampered cell growth and disturbance in metabolic flux. This review set forth a comparison between MEP and MVA pathways, strategies to overexpress these pathways with their challenges.

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.

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.

Comparative Transcriptome Analysis Reveals Coordinated Transcriptional Regulation of Central and Secondary Metabolism in the Trichomes of Cannabis Cultivars.

Cannabis is one of the few plant genera capable of producing cannabinoids, the effects of which are synergized by terpene interactions. As the biosynthesis of both metabolite classes requires the same intracellular feedstocks, this work describes the coordinated regulation of global metabolic pathways that allows for their joint copious production in vivo. To this end, a transcriptomics-based approach to characterize the glandular trichomes of five Cannabis cultivars was pursued. Besides revealing metabolic traits that enhanced and proportionated the supply of critical carbon precursors, in-depth analysis showed significantly increased gene expression of two particular enzymes to meet the huge nicotinamide adenine dinucleotide phosphate (NADPH) demand of secondary metabolite production. Furthermore, it led to a hypothesis that the methyl-d-erythritol 4-phosphate pathway might be utilized more than the mevalonic acid pathway in Cannabis trichomes. While both pathways were found to be activated in a modular and calibrated way that reflected their broad participation in physiological processes, the genes for hexanoate, cannabinoid, and terpene biosynthesis were, in contrast, up-regulated in an en bloc and multi-loci manner due to their specific roles in secondary metabolite production. In addition, three new terpene synthases were characterized based on both in silico and experimental assays. Altogether, the study enhances the current understanding of secondary metabolite production in Cannabis cultivars, which may assist in their characterization and development.