Aldehyde-based activation of C2α-lactylthiamin diphosphate decarboxylation on bacterial 1-deoxy-d-xylulose 5-phosphate synthase.

1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate (donor substrate) and d-glyceraldehyde 3-phosphate (d-GAP, acceptor substrate) in bacterial central metabolism. DXPS uses a ligand-gated mechanism in which binding of a small molecule "trigger" activates the first enzyme-bound intermediate, C2α-lactylThDP (LThDP), to form the reactive carbanion via LThDP decarboxylation. d-GAP is the natural acceptor substrate for DXPS and also serves a role as a trigger to induce LThDP decarboxylation in the gated step. Additionally, we have shown that O2 and d-glyceraldehyde (d-GA) can induce LThDP decarboxylation. We hypothesize this ligand-gated mechanism poises DXPS to sense and respond to cellular cues in metabolic remodeling during bacterial adaptation. Here we sought to characterize features of small molecule inducers of LThDP decarboxylation. Using a combination of CD, NMR and biochemical methods, we demonstrate that the α-hydroxy aldehyde moiety of d-GAP is sufficient to induce LThDP decarboxylation en route to DXP formation. A variety of aliphatic aldehydes also induce LThDP decarboxylation. The study highlights the capacity of DXPS to respond to different molecular cues, lending support to potential multifunctionality of DXPS and its metabolic regulation by this mechanism.

Metabolism-Based Nontarget-Site Mechanism Is the Main Cause of a Four-Way Resistance in Shortawn Foxtail (Alopecurus aequalis Sobol.).

Alopecurus aequalis Sobol. is a predominant grass weed in Chinese winter wheat fields, posing a substantial threat to crop production owing to its escalating herbicide resistance. This study documented the initial instance of an A. aequalis population (AHFT-3) manifesting resistance to multiple herbicides targeting four distinct sites: acetyl-CoA carboxylase (ACCase), acetolactate synthase, photosystem II, and 1-deoxy-d-xylulose-5-phosphate synthase. AHFT-3 carried an Asp-to-Gly mutation at codon 2078 of ACCase, with no mutations in the remaining three herbicide target genes, and exhibited no overexpression of any target gene. Compared with the susceptible population AHFY-3, AHFT-3 metabolized mesosulfuron-methyl, isoproturon, and bixlozone faster. The inhibition and comparison of herbicide-detoxifying enzyme activities indicated the participation of cytochrome P450s in the resistance to all four herbicides, with glutathione S-transferases specifically linked to mesosulfuron-methyl. Three CYP72As and a Tau class glutathione S-transferase, markedly upregulated in resistant plants, potentially played pivotal roles in the multiple-herbicide-resistance phenotype.

Functional Characterization of PmDXR, a Critical Rate-Limiting Enzyme, for Turpentine Biosynthesis in Masson Pine (Pinus massoniana Lamb.).

As one of the largest and most diverse classes of specialized metabolites in plants, terpenoids (oprenoid compounds, a type of bio-based material) are widely used in the fields of medicine and light chemical products. They are the most important secondary metabolites in coniferous species and play an important role in the defense system of conifers. Terpene synthesis can be promoted by regulating the expressions of terpene synthase genes, and the terpene biosynthesis pathway has basically been clarified in Pinus massoniana, in which there are multiple rate-limiting enzymes and the rate-limiting steps are difficult to determine, so the terpene synthase gene regulation mechanism has become a hot spot in research. Herein, we amplified a PmDXR gene (GenBank accession no. MK969119.1) of the MEP pathway (methyl-erythritol 4-phosphate) from Pinus massoniana. The DXR enzyme activity and chlorophyll a, chlorophyll b and carotenoid contents of overexpressed Arabidopsis showed positive regulation. The PmDXR gene promoter was a tissue-specific promoter and can respond to ABA, MeJA and GA stresses to drive the expression of the GUS reporter gene in N. benthamiana. The DXR enzyme was identified as a key rate-limiting enzyme in the MEP pathway and an effective target for terpene synthesis regulation in coniferous species, which can further lay the theoretical foundation for the molecularly assisted selection of high-yielding lipid germplasm of P. massoniana, as well as provide help in the pathogenesis of pine wood nematode disease.

Reverse N-Substituted Hydroxamic Acid Derivatives of Fosmidomycin Target a Previously Unknown Subpocket of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase (DXR).

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog.

New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS) converts pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)-based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure-activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

Investigating inhibitors of 1-deoxy-d-xylulose 5-phosphate synthase in a mouse model of UTI.

The rising rate of antimicrobial resistance continues to threaten global public health. Further hastening antimicrobial resistance is the lack of new antibiotics against new targets. The bacterial enzyme, 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), is thought to play important roles in central metabolism, including processes required for pathogen adaptation to fluctuating host environments. Thus, impairing DXPS function represents a possible new antibacterial strategy. We previously investigated a DXPS-dependent metabolic adaptation as a potential target in uropathogenic Escherichia coli (UPEC) associated with urinary tract infection (UTI), using the DXPS-selective inhibitor butyl acetylphosphonate (BAP). However, investigations of DXPS inhibitors in vivo have not been conducted. The goal of the present study is to advance DXPS inhibitors as in vivo probes and assess the potential of inhibiting DXPS as a strategy to prevent UTI in vivo. We show that BAP was well-tolerated at high doses in mice and displayed a favorable pharmacokinetic profile for studies in a mouse model of UTI. Further, an alkyl acetylphosphonate prodrug (homopropargyl acetylphosphonate, pro-hpAP) was significantly more potent against UPEC in urine culture and exhibited good exposure in the urinary tract after systemic dosing. Prophylactic treatment with either BAP or pro-hpAP led to a partial protective effect against UTI, with the prodrug displaying improved efficacy compared to BAP. Overall, our results highlight the potential for DXPS inhibitors as in vivo probes and establish preliminary evidence that inhibiting DXPS impairs UPEC colonization in a mouse model of UTI.IMPORTANCENew antibiotics against new targets are needed to prevent an antimicrobial resistance crisis. Unfortunately, antibiotic discovery has slowed, and many newly FDA-approved antibiotics do not inhibit new targets. Alkyl acetylphosphonates (alkyl APs), which inhibit the enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS), represent a new possible class of compounds as there are no FDA-approved DXPS inhibitors. To our knowledge, this is the first study demonstrating the in vivo safety, pharmacokinetics, and efficacy of alkyl APs in a urinary tract infection mouse model.

The non-mevalonate pathway requires a delicate balance of intermediates to maximize terpene production.

Terpenes are valuable industrial chemicals whose demands are increasingly being met by bioengineering microbes such as E. coli. Although the bioengineering efforts commonly involve installing the mevalonate (MVA) pathway in E. coli for terpene production, the less studied methylerythritol phosphate (MEP) pathway is a more attractive target due to its higher energy efficiency and theoretical yield, despite its tight regulation. In this study, we integrated an additional copy of the entire MEP pathway into the E. coli genome for stable, marker-free terpene production. The genomically integrated strain produced more monoterpene geraniol than a plasmid-based system. The pathway genes' transcription was modulated using different promoters to produce geraniol as the reporter of the pathway flux. Pathway genes, including dxs, idi, and ispDF, expressed from a medium-strength promoter, led to the highest geraniol production. Quantifying the MEP pathway intermediates revealed that the highest geraniol producers had high levels of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), but moderate levels of the pathway intermediates upstream of these two building blocks. A principal component analysis demonstrated that 1-deoxy-D-xylulose 5-phosphate (DXP), the product of the first enzyme of the pathway, was critical for determining the geraniol titer, whereas MEP, the product of DXP reductoisomerase (Dxr or IspC), was the least essential. This work shows that an intricate balance of the MEP pathway intermediates determines the terpene yield in engineered E. coli. The genetically stable and intermediate-balanced strains created in this study will serve as a chassis for producing various terpenes. KEY POINTS: • Genome-integrated MEP pathway afforded higher strain stability • Genome-integrated MEP pathway produced more terpene than the plasmid-based system • High monoterpene production requires a fine balance of MEP pathway intermediates.

Identification of novel compounds and repurposing of FDA drugs for 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme of Plasmodium falciparum to combat malaria resistance.

The rise of Plasmodium falciparum resistance to Artemisinin-based combination therapies (ACTs) is a significant concern in the fight against malaria. This situation calls for the search for novel anti-malarial candidates. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) is a potential target involved in various cellular processes in P. falciparum (Pf). We screened ∼0.69 billion novel compounds from the ZINC20 library and repurposed ∼1400 FDA drugs using computational drug discovery methods against PfIspC. Following our computational pipeline, we found five novel ZINC20 compounds (Z-2, Z-3, Z-10, Z-13, and Z-14) and three FDA drugs (Aliskiren, Ceftolozane, and Ombitasvir) that showed striking docking energy (ranging from -8.405 to -10.834 kcal/mol), and strong interactions with key binding site residues (Ser269, Ser270, Ser306, Asn311, Lys312, and Met360) of PfIspC. The novel anti-malarial compounds also exhibited favorable pharmacokinetics and physicochemical properties. Furthermore, through molecular dynamics simulation, we observed the stable dynamics of PfIspC-inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. Notably, the binding free energy estimation confirmed high binding affinity (varied from -11.68 to -33.16 kcal/mol) of these compounds for PfIspC. Our findings could contribute to the ongoing efforts in combating malaria and invite experimental-lab researchers for validation.

Differential roles of Cassia tora 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase in trade-off between plant growth and drought tolerance.

Due to global climate change, drought is emerging as a major threat to plant growth and agricultural productivity. Abscisic acid (ABA) has been implicated in plant drought tolerance, however, its retarding effects on plant growth cannot be ignored. The reactions catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) proteins are critical steps within the isoprenoid biosynthesis in plants. Here, five DXS (CtDXS1-5) and two DXR (CtDXR1-2) genes were identified from Cassia tora genome. Based on multiple assays including the phylogeny, cis-acting element, expression pattern, and subcellular localization, CtDXS1 and CtDXR1 genes might be potential candidates controlling the isoprenoid biosynthesis. Intriguingly, CtDXS1 transgenic plants resulted in drought tolerance but retardant growth, while CtDXR1 transgenic plants exhibited both enhanced drought tolerance and increased growth. By comparison of ß-carotene, chlorophyll, abscisic acid (ABA) and gibberellin 3 (GA3) contents in wild-type and transgenic plants, the absolute contents and (or) altered GA3/ABA levels were suggested to be responsible for the balance between drought tolerance and plant growth. The transcriptome of CtDXR1 transgenic plants suggested that the transcript levels of key genes, such as DXS, 9-cis-epoxycarotenoid dioxygenases (NCED), ent-kaurene synthase (KS) and etc, involved with chlorophyll, ß-carotene, ABA and GA3 biosynthesis were induced and their contents increased accordingly. Collectively, the trade-off effect induced by CtDXR1 was associated with redesigning architecture in phytohormone homeostasis and thus was highlighted for future breeding purposes.

1-deoxy-D-xylulose-5-phosphate synthase from Pseudomonas aeruginosa and Klebsiella pneumoniae reveals conformational changes upon cofactor binding.

The ESKAPE bacteria are the six highly virulent and antibiotic-resistant pathogens that require the most urgent attention for the development of novel antibiotics. Detailed knowledge of target proteins specific to bacteria is essential to develop novel treatment options. The methylerythritol-phosphate (MEP) pathway, which is absent in humans, represents a potentially valuable target for the development of novel antibiotics. Within the MEP pathway, the enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) catalyzes a crucial, rate-limiting first step and a branch point in the biosynthesis of the vitamins B1 and B6. We report the high-resolution crystal structures of DXPS from the important ESKAPE pathogens Pseudomonas aeruginosa and Klebsiella pneumoniae in both the co-factor-bound and the apo forms. We demonstrate that the absence of the cofactor thiamine diphosphate results in conformational changes that lead to disordered loops close to the active site that might be important for the design of potent DXPS inhibitors. Collectively, our results provide important structural details that aid in the assessment of DXPS as a potential target in the ongoing efforts to combat antibiotic resistance.

Intraspecific divergence in essential oil content, composition and genes expression patterns of monoterpene synthesis in Origanum vulgare subsp. vulgare and subsp. gracile under salinity stress.

BACKGROUND: Oregano (Origanum vulgare L.), one of the important medicinal plants in the world, has valuable pharmacological compounds with antimicrobial, antiviral, antioxidant, anti-inflammatory, antispasmodic, antiurolithic, antiproliferative and neuroprotective activities. Phenolic monoterpenes such as thymol and carvacrol with many medical importance are found in Oregano essential oil. The biosynthesis of these compounds is carried out through the methyl erythritol-4 phosphate (MEP) pathway. Environmental stresses such as salinity might improve the secondary metabolites in medicinal plants. The influence of salinity stress (0 (control), 25, 50 and 100 mM NaCl) on the essential oil content, composition and expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), γ-terpinene synthase (Ovtps2) and cytochrome P450 monooxygenases (CYP71D180) genes involved in thymol and carvacrol biosynthesis, was investigated in two oregano subspecies (vulgare and gracile). RESULTS: Essential oil content was increased at low NaCl concentration (25 mM) compared with non-stress conditions, whereas it was decreased as salinity stress intensified (50 and 100 mM). Essential oil content was significantly higher in subsp. gracile than subsp. vulgare. The highest (0.20 mL pot-1) and lowest (0.06 mL pot-1) amount of essential oil yield was obtained in subsp. gracile at 25 and 100 mM NaCl, respectively. The content of carvacrol, as the main component of essential oil, decreased with increasing salinity level in subsp. gracile, but increased in subsp. vulgare. The highest expression of DXR, Ovtps2 and CYP71D180 genes was observed at 50 mM NaCl in subsp. vulgare. While, in subsp. gracile, the expression of the mentioned genes decreased with increasing salinity levels. A positive correlation was obtained between the expression of DXR, Ovtps2 and CYP71D180 genes with carvacrol content in both subspecies. On the other hand, a negative correlation was found between the expression of CYP71D180 and carvacrol content in subsp. gracile. CONCLUSIONS: The findings of this study demonstrated that both oregano subspecies can tolerate NaCl salinity up to 50 mM without significant reduction in essential oil yield. Also, moderate salinity stress (50 mM NaCl) in subsp. vulgare might increase the carvacrol content partly via increment the expression levels of DXR, Ovtps2 and CYP71D180 genes.

Design of thiamine analogues for inhibition of thiamine diphosphate (ThDP)-dependent enzymes: Systematic investigation through Scaffold-Hopping and C2-Functionalisation.

Thiamine diphosphate (ThDP), the bioactive form of vitamin B1, is an essential coenzyme needed for processes of cellular metabolism in all organisms. ThDP-dependent enzymes all require ThDP as a coenzyme for catalytic activity, although individual enzymes vary significantly in substrate preferences and biochemical reactions. A popular way to study the role of these enzymes through chemical inhibition is to use thiamine/ThDP analogues, which typically feature a neutral aromatic ring in place of the positively charged thiazolium ring of ThDP. While ThDP analogues have aided work in understanding the structural and mechanistic aspects of the enzyme family, at least two key questions regarding the ligand design strategy remain unresolved: 1) which is the best aromatic ring? and 2) how can we achieve selectivity towards a given ThDP-dependent enzyme? In this work, we synthesise derivatives of these analogues covering all central aromatic rings used in the past decade and make a head-to-head comparison of all the compounds as inhibitors of several ThDP-dependent enzymes. Thus, we establish the relationship between the nature of the central ring and the inhibitory profile of these ThDP-competitive enzyme inhibitors. We also demonstrate that introducing a C2-substituent onto the central ring to explore the unique substrate-binding pocket can further improve both potency and selectivity.

DXP Synthase Function in a Bacterial Metabolic Adaptation and Implications for Antibacterial Strategies.

Pathogenic bacteria possess a remarkable ability to adapt to fluctuating host environments and cause infection. Disturbing bacterial central metabolism through inhibition of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) has the potential to hinder bacterial adaptation, representing a new antibacterial strategy. DXPS functions at a critical metabolic branchpoint to produce the metabolite DXP, a precursor to pyridoxal-5-phosphate (PLP), thiamin diphosphate (ThDP) and isoprenoids presumed essential for metabolic adaptation in nutrient-limited host environments. However, specific roles of DXPS in bacterial adaptations that rely on vitamins or isoprenoids have not been studied. Here we investigate DXPS function in an adaptation of uropathogenic E. coli (UPEC) to d-serine (d-Ser), a bacteriostatic host metabolite that is present at high concentrations in the urinary tract. UPEC adapt to d-Ser by producing a PLP-dependent deaminase, DsdA, that converts d-Ser to pyruvate, pointing to a role for DXPS-dependent PLP synthesis in this adaptation. Using a DXPS-selective probe, butyl acetylphosphonate (BAP), and leveraging the toxic effects of d-Ser, we reveal a link between DXPS activity and d-Ser catabolism. We find that UPEC are sensitized to d-Ser and produce sustained higher levels of DsdA to catabolize d-Ser in the presence of BAP. In addition, BAP activity in the presence of d-Ser is suppressed by ß-alanine, the product of aspartate decarboxylase PanD targeted by d-Ser. This BAP-dependent sensitivity to d-Ser marks a metabolic vulnerability that can be exploited to design combination therapies. As a starting point, we show that combining inhibitors of DXPS and CoA biosynthesis displays synergy against UPEC grown in urine where there is increased dependence on the TCA cycle and gluconeogenesis from amino acids. Thus, this study provides the first evidence for a DXPS-dependent metabolic adaptation in a bacterial pathogen and demonstrates how this might be leveraged for development of antibacterial strategies against clinically relevant pathogens.

Isolation, characterisation, and expression profiling of DXS and DXR genes in Atractylodes lancea.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

Not Every Hit-Identification Technique Works on 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign.

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.

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

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

The Identification of ß-Ocimene Biosynthetic Pathway Through Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) Pathways Using Crude Enzyme Extracts in Indonesian Bay Leaf/Salam Leaf (Syzygium polyanthum).

Salam leaf has a ß-ocimene as a key volatile compound that gives a fresh aroma to the food when the salam leaves are involved in the cooking process. As a secondary metabolic product, enzymatic biosynthesis as the early stage of ß-ocimene is a factor that needs to be known. Thus, this study was done to identify the mechanism of the two well-known terpenoid biosynthetic pathways, namely Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathways, in the biosynthesis of ß-ocimene in salam leaves. The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-MVA pathway-determining enzyme and 1-deoxy-D-xylulose-5-phosphate synthase (DXS)-DXP pathway-determining enzyme in the crude enzyme and their derivative products of salam leaves were analysed for their changes by differences of substrate ratios and enzyme inhibitors. The results showed that the activity of the HMGR enzyme was lower significantly than the DXS enzyme based on the addition of variations to the substrate ratio. These results were also supported by the enzyme and substrate reaction products, MVA and Isopentenyl diphosphate (IPP) intermediates from the MVA pathway, which were significantly lower when compared to DXP and IPP intermediates from the DXP pathway. As the end product of the reaction, ß-ocimene gave a significantly higher value of the DXP pathway than the MVA pathway. Therefore, it can conclude that the mechanism of the biosynthetic pathway of ß-ocimene in salam leaves was synthesised via the DXP pathway. The production of ß-ocimene could have crosstalk-pathway through the MVA pathway, especially when the DXP pathway was blocked.

The modulation of light quality on carotenoids in maize (Zea mays L.) sprouts.

The present study aimed to identify the regulatory mechanisms of red, blue, and white light on carotenoid biosynthesis in maize sprouts. Determinations of carotenoid, chlorophyll and phytohormone profiles, as well as relative gene expression, were explored. The results identified enhancement of carotenoid and chlorophyll production as well as gene expression. Most notably, the expression levels of CRY, HY5, and beta-carotene 3-hydroxylase genes peaked under blue light. Photomorphogene-related hormone, auxins and strigolactone production was also altered under different lights and might have a role in carotenoid metabolism. Gibberellins competed with carotenoids for the precursor geranylgeranyl diphosphate and were hindered by certain light characteristics, probably via DELLA-PIF4 signalling. ERF021 and MYB68 were negative regulators of carotenoid biosynthesis in maize sprouts. These findings provide new insights into the light-regulated mechanism and biofortification of carotenoids in maize sprouts.

Dissecting carbon metabolism of Yarrowia lipolytica type strain W29 using genome-scale metabolic modelling.

Yarrowia lipolytica is a widely-used chassis cell in biotechnological applications. It has recently gained extensive research interest owing to its extraordinary ability of producing industrially valuable biochemicals from a variety of carbon sources. Genome-scale metabolic models (GSMMs) enable analyses of cellular metabolism for engineering various industrial hosts. In the present study, we developed a high-quality GSMM iYli21 for Y. lipolytica type strain W29 by extensive manual curation with Biolog experimental data. The model showed a high accuracy of 85.7% in predicting nutrient utilization. Transcriptomics data were integrated to delineate cellular metabolism of utilizing six individual metabolites as sole carbon sources. Comparisons showed that 302 reactions were commonly used, including those from TCA cycle, oxidative phosphorylation, and purine metabolism for energy and material supply. Whereas glycolytic reactions were employed only when glucose and glycerol used as sole carbon sources, gluconeogenesis and fatty acid oxidation reactions were specifically employed when fatty acid, alkane and glycerolipid were the sole carbon sources. Further test of 46 substrates for generating 5 products showed that hexanoate outcompeted other compounds in terms of maximum theoretical yield owing to the lowest carbon loss for energy supply. This newly generated model iYli21 will be a valuable tool in dissecting metabolic mechanism and guiding metabolic engineering of this important industrial cell factory.

Transcriptomic profiling of the floral fragrance biosynthesis pathway of Liriodendron and functional characterization of the LtuDXR gene.

Floral fragrance, which has the function of attracting pollinators, is a class of volatile secondary metabolites mainly released by the secretory tissue of petals. Terpenoids are key components of floral volatile substances. Previous studies have shown that there are significant differences in the concentration and composition of volatile floral fragrances, especially terpenoids, between Liriodendron chinense and L. tulipifera. At present, the mechanism by which the synthesis of floral fragrance is regulated in Liriodendron remains unexplored. In this study, we analyzed the differentially expressed genes (DEGs) of L. chinense and L. tulipifera, and identified 130 DEGs related to terpenoid synthesis. A KEGG enrichment analysis of DEGs related to terpenoid biosynthesis revealed that the monoterpenoid biosynthesis pathway was the most significant. We cloned the LtuDXR gene from L. tulipifera using RACE technology. RT-qPCR results showed that the expression of the LtuDXR gene was the highest in the early florescence petals, indicating that the LtuDXR gene may play a role in the synthesis of volatile terpenoids. Subcellular localization showed that the LtuDXR protein is mainly localized in the chloroplast. Overexpression of LtuDXR in Arabidopsis thaliana significantly increased the plant height, DXR enzyme activity, and carotenoid content. In this study, we identified and functionally characterized LtuDXR, which is involved in terpenoid synthesis in Liriodendron. Our work lays the foundation for further exploration of the molecular mechanism by which terpenoid biosynthesis is regulated in Liriodendron.