A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

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.

Synthesis of the Carba-Analogs of the α-Pyranose and ß-Pyranose Forms of Sedoheptulose 7-Phosphate and Probing the Stereospecificity of Sedoheptulose 7-Phosphate Cyclases.

Sedoheptulose 7-phosphate (SH7P) cyclases are a subset of sugar phosphate cyclases that are known to catalyze the first committed step in many biosynthetic pathways in primary and secondary metabolism. Among them are 2-epi-5-epi-valiolone synthase (EEVS) and 2-epi-valiolone synthase (EVS), two closely related SH7P cyclases that catalyze the conversion of SH7P to 2-epi-5-epi-valiolone and 2-epi-valiolone, respectively. However, how these two homologous enzymes use a common substrate to produce stereochemically different products is unknown. Two competing hypotheses have been proposed for the stereospecificity of EEVS and EVS: (1) variation in aldol acceptor geometry during enzyme catalysis, and (2) preselection of the α-pyranose or ß-pyranose forms of the substrate by the enzymes. Yet, there is no direct evidence to support or rule out either of these hypotheses. Here we report the synthesis of the carba-analogs of the α-pyranose and ß-pyranose forms of SH7P and their use in probing the stereospecificity of ValA (EEVS from Streptomyces hygroscopicus subsp. jinggangensis) and Amir_2000 (EVS from Actinosynnema mirum DSM 43827). Kinetic studies of the enzymes in the presence of the synthetic compounds as well as docking studies of the enzymes with the α- and ß-pyranose forms of SH7P suggest that the inverted configuration of the products of EEVS and EVS is not due to the preselection of the different forms of the substrate by the enzymes.

The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

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.

Reversing the directionality of reactions between non-oxidative pentose phosphate pathway and glycolytic pathway boosts mycosporine-like amino acid production in Saccharomyces cerevisiae.

BACKGROUND: Mycosporine-like amino acids (MAAs) are a class of strongly UV-absorbing compounds produced by cyanobacteria, algae and corals and are promising candidates for natural sunscreen components. Low MAA yields from natural sources, coupled with difficulties in culturing its native producers, have catalyzed synthetic biology-guided approaches to produce MAAs in tractable microbial hosts like Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, the MAA titres obtained in these hosts are still low, necessitating a thorough understanding of cellular factors regulating MAA production. RESULTS: To delineate factors that regulate MAA production, we constructed a shinorine (mycosporine-glycine-serine) producing yeast strain by expressing the four MAA biosynthetic enzymes from Nostoc punctiforme in Saccharomyces cerevisiae. We show that shinorine is produced from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P), and not from the shikimate pathway intermediate 3-dehydroquinate (3DHQ) as previously suggested. Deletions of transaldolase (TAL1) and phosphofructokinase (PFK1/PFK2) genes boosted S7P/shinorine production via independent mechanisms. Unexpectedly, the enhanced S7P/shinorine production in the PFK mutants was not entirely due to increased flux towards the pentose phosphate pathway. We provide multiple lines of evidence in support of a reversed pathway between glycolysis and the non-oxidative pentose phosphate pathway (NOPPP) that boosts S7P/shinorine production in the phosphofructokinase mutant cells. CONCLUSION: Reversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

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.

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.

A Visual Compendium of Principal Modifications within the Nucleic Acid Sugar Phosphate Backbone.

Nucleic acid chemistry is a huge research area that has received new impetus due to the recent explosive success of oligonucleotide therapy. In order for an oligonucleotide to become clinically effective, its monomeric parts are subjected to modifications. Although a large number of redesigned natural nucleic acids have been proposed in recent years, the vast majority of them are combinations of simple modifications proposed over the past 50 years. This review is devoted to the main modifications of the sugar phosphate backbone of natural nucleic acids known to date. Here, we propose a systematization of existing knowledge about modifications of nucleic acid monomers and an acceptable classification from the point of view of chemical logic. The visual representation is intended to inspire researchers to create a new type of modification or an original combination of known modifications that will produce unique oligonucleotides with valuable characteristics.

The trehalose 6-phosphate phosphatase family in plants.

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signalling metabolite linking plant growth and development to carbon metabolism. While recent work has focused predominantly on the enzymes that produce Tre6P, little is known about the proteins that catalyse its degradation, the trehalose 6-phosphate phosphatases (TPPs). Often occurring in large protein families, TPPs exhibit cell-, tissue- and developmental stage-specific expression patterns, suggesting important regulatory functions in controlling local levels of Tre6P and trehalose as well as Tre6P signalling. Furthermore, growing evidence through gene expression studies and transgenic approaches shows that TPPs play an important role in integrating environmental signals with plant metabolism. This review highlights the large diversity of TPP isoforms in model and crop plants and identifies how modulating Tre6P metabolism in certain cell types, tissues, and at different developmental stages may promote stress tolerance, resilience and increased crop yield.

Sucrose homeostasis: Mechanisms and opportunity in crop yield improvement.

Sugar homeostasis is a critical feature of biological systems. In humans, raised and dysregulated blood sugar is a serious health issue. In plants, directed changes in sucrose homeostasis and allocation represent opportunities in crop improvement. Plant tissue sucrose varies more than blood glucose and is found at higher concentrations (cytosol and phloem ca. 100 mM v 3.9-6.9 mM for blood glucose). Tissue sucrose varies with developmental stage and environment, but cytosol and phloem exhibit tight sucrose control. Sucrose homeostasis is a consequence of the integration of photosynthesis, synthesis of storage end-products such as starch, transport of sucrose to sinks and sink metabolism. Trehalose 6-phosphate (T6P)-SnRK1 and TOR play central, still emerging roles in regulating and coordinating these processes. Overall, tissue sucrose levels are more strongly related to growth than to photosynthesis. As a key sucrose signal, T6P regulates sucrose levels, transport and metabolic pathways to coordinate source and sink at a whole plant level. Emerging evidence shows that T6P interacts with meristems. With careful targeting, T6P manipulation through exploiting natural variation, chemical intervention and genetic modification is delivering benefits for crop yields. Regulation of cereal grain set, filling and retention may be the most strategically important aspect of sucrose allocation and homeostasis for food security.

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.

Molecular mechanism of trehalose 6-phosphate inhibition of the plant metabolic sensor kinase SnRK1.

SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), a central plant metabolic sensor kinase, phosphorylates its target proteins, triggering a global shift from anabolism to catabolism. Molecular modeling revealed that upon binding of KIN10 to GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1), KIN10's activation T-loop reorients into GRIK1's active site, enabling its phosphorylation and activation. Trehalose 6-phosphate (T6P) is a proxy for cellular sugar status and a potent inhibitor of SnRK1. T6P binds to KIN10, a SnRK1 catalytic subunit, weakening its affinity for GRIK1. Here, we investigate the molecular details of T6P inhibition of KIN10. Molecular dynamics simulations and in vitro phosphorylation assays identified and validated the T6P binding site on KIN10. Under high-sugar conditions, T6P binds to KIN10, blocking the reorientation of its activation loop and preventing its phosphorylation and activation by GRIK1. Under these conditions, SnRK1 maintains only basal activity levels, minimizing phosphorylation of its target proteins, thereby facilitating a general shift from catabolism to anabolism.

Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum

In Plasmodium falciparum, the formation of isopentenyl diphosphate and dimethylallyl diphosphate, central intermediates in the biosynthesis of isoprenoids, occurs via the methylerythritol phosphate (MEP) pathway. Fosmidomycin is a specific inhibitor of the second enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate reductoisomerase. We analyzed the effect of fosmidomycin on the levels of each intermediate and its metabolic requirement for the isoprenoid biosynthesis, such as dolichols and ubiquinones, throughout the intraerythrocytic cycle of P. falciparum. The steady-state RNA levels of the MEP pathway-associated genes were quantified by real-time polymerase chain reaction and correlated with the related metabolite levels. Our results indicate that MEP pathway metabolite peak precede maximum transcript abundance during the intraerythrocytic cycle. Fosmidomycin-treatment resulted in a decrease of the intermediate levels in the MEP pathway as well as in ubiquinone and dolichol biosynthesis. The MEP pathway associated transcripts were modestly altered by the drug, indicating that the parasite is not strongly responsive at the transcriptional level. This is the first study that compares the effect of fosmidomycin on the metabolic and transcript profiles in P. falciparum, which has only the MEP pathway for isoprenoid biosynthesis.

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule