Synergism with Shikimic Acid Restores ß-Lactam Antibiotic Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved into a dangerous pathogen resistant to beta-lactam antibiotics (BLAs) and has become a worrisome superbug. In this study, a strategy in which shikimic acid (SA), which has anti-inflammatory and antibacterial activity, is combined with BLAs to restart BLA activity was proposed for MRSA treatment. The synergistic effects of oxacillin combined with SA against oxacillin resistance in vitro and in vivo were investigated. The excellent synergistic effect of the oxacillin and SA combination was confirmed by performing the checkerboard assay, time-killing assay, live/dead bacterial cell viability assay, and assessing protein leakage. SEM showed that the cells in the control group had a regular, smooth, and intact surface. In contrast, oxacillin and SA or the combination treatment group exhibited different degrees of surface collapse. q-PCR indicated that the combination treatment group significantly inhibited the expression of the mecA gene. In vivo, we showed that the combination treatment increased the survival rate and decreased the bacterial load in mice. These results suggest that the combination of oxacillin with SA is considered an effective treatment option for MRSA, and the combination of SA with oxacillin in the treatment of MRSA is a novel strategy.

Plant-Derived Caffeic Acid and Its Derivatives: An Overview of Their NMR Data and Biosynthetic Pathways.

In recent years, caffeic acid and its derivatives have received increasing attention due to their obvious physiological activities and wide distribution in nature. In this paper, to clarify the status of research on plant-derived caffeic acid and its derivatives, nuclear magnetic resonance spectroscopy data and possible biosynthetic pathways of these compounds were collected from scientific databases (SciFinder, PubMed and China Knowledge). According to different types of substituents, 17 caffeic acid and its derivatives can be divided into the following classes: caffeoyl ester derivatives, caffeyltartaric acid, caffeic acid amide derivatives, caffeoyl shikimic acid, caffeoyl quinic acid, caffeoyl danshens and caffeoyl glycoside. Generalization of their 13C-NMR and 1H-NMR data revealed that acylation with caffeic acid to form esters involves acylation shifts, which increase the chemical shift values of the corresponding carbons and decrease the chemical shift values of the corresponding carbons of caffeoyl. Once the hydroxyl group is ester, the hydrogen signal connected to the same carbon shifts to the low field (1.1~1.6). The biosynthetic pathways were summarized, and it was found that caffeic acid and its derivatives are first synthesized in plants through the shikimic acid pathway, in which phenylalanine is deaminated to cinnamic acid and then transformed into caffeic acid and its derivatives. The purpose of this review is to provide a reference for further research on the rapid structural identification and biofabrication of caffeic acid and its derivatives.

High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit.

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

Two New Sesquiterpenoids and a New Shikimic Acid Metabolite from Mangrove Sediment-Derived Fungus Roussoella sp. SCSIO 41427.

Two new sesquiterpenoid derivatives, elgonenes M (1) and N (2), and a new shikimic acid metabolite, methyl 5-O-acetyl-5-epi-shikimate (3), were isolated from the mangrove sediment-derived fungus Roussoella sp. SCSIO 41427 together with fourteen known compounds (4-17). The planar structures were elucidated through nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses. The relative configurations of 1-3 were ascertained by NOESY experiments, while their absolute configurations were determined by electronic circular dichroism (ECD) calculation. Elgonene M (1) exhibited inhibition of interleukin-1ß (IL-1ß) mRNA, a pro-inflammatory cytokine, at a concentration of 5 µM, with an inhibitory ratio of 31.14%. On the other hand, elgonene N (2) demonstrated inhibition at a concentration of 20 µM, with inhibitory ratios of 27.57%.

Reconstructing curcumin biosynthesis in yeast reveals the implication of caffeoyl-shikimate esterase in phenylpropanoid metabolic flux.

Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2-19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.

Metabolic engineering of Synechocystis sp. PCC 6803 for the improved production of phenylpropanoids.

BACKGROUND: Phenylpropanoids are a large group of plant secondary metabolites with various biological functions, derived from aromatic amino acids. Cyanobacteria are promising host organisms for sustainable production of plant phenylpropanoids. We have previously engineered Synechocystis sp. PCC 6803 to produce trans-cinnamic acid (tCA) and p-coumaric acid (pCou), the first intermediates of phenylpropanoid pathway, by overexpression of phenylalanine- and tyrosine ammonia lyases. In this study, we aimed to enhance the production of the target compounds tCA and pCou in Synechocystis. RESULTS: We eliminated the 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, which is a competing pathway consuming tyrosine and, possibly, phenylalanine for tocopherol synthesis. Moreover, several genes of the terminal steps of the shikimate pathway were overexpressed alone or in operons, such as aromatic transaminases, feedback insensitive cyclohexadienyl dehydrogenase (TyrC) from Zymomonas mobilis and the chorismate mutase (CM) domain of the fused chorismate mutase/prephenate dehydratase enzyme from Escherichia coli. The obtained engineered strains demonstrated nearly 1.5 times enhanced tCA and pCou production when HPPD was knocked out compared to the parental production strains, accumulating 138 ± 3.5 mg L-1 of tCA and 72.3 ± 10.3 mg L-1 of pCou after seven days of photoautotrophic growth. However, there was no further improvement when any of the pathway genes were overexpressed. Finally, we used previously obtained AtPRM8 and TsPRM8 Synechocystis strains with deregulated shikimate pathway as a background for the overexpression of synthetic constructs with ppd knockout. CONCLUSIONS: HPPD elimination enhances the tCA and pCou productivity to a similar extent. The use of PRM8 based strains as a background for overexpression of synthetic constructs, however, did not promote tCA and pCou titers, which indicates a tight regulation of the terminal steps of phenylalanine and tyrosine synthesis. This work contributes to establishing cyanobacteria as hosts for phenylpropanoid production.

A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora.

BACKGROUND: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora. RESULTS: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp. CONCLUSIONS: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Metabolic engineering with adaptive laboratory evolution for phenylalanine production by Corynebacterium glutamicum.

The mutants resistant to a phenylalanine analog, 4-fluorophenylalanine (4FP), were obtained for metabolic engineering of Corynebacterium glutamicum for producing aromatic amino acids synthesized through the shikimate pathway by adaptive laboratory evolution. Culture experiments of the C. glutamicum strains which carry the mutations found in the open reading frame from the 4FP-resistant mutants revealed that the mutations in the open reading frames of aroG (NCgl2098), pheA (NCgl2799) and aroP (NCgl1062) encoding 3-deoxy-d-arabino-heptulosonate-7-phosphate, prephenate dehydratase, and aromatic amino acid transporter are responsible for 4FP resistance and higher concentration of aromatic amino acids in their culture supernatants in the 4FP-resistant strains. It was expected that aroG and pheA mutations would release feedback inhibition of the enzymes involved in the shikimate pathway by phenylalanine and that aroP mutations would prevent intracellular uptake of aromatic amino acids. Therefore, we conducted metabolic engineering of the C. glutamicum wild-type strain for aromatic amino acid production and found that phenylalanine production at 6.11 ± 0.08 g L-1 was achieved by overexpressing the mutant pheA and aroG genes from the 4FP-resistant mutants and deleting aroP gene. This study demonstrates that adaptive laboratory evolution is an effective way to obtain useful mutant genes related to production of target material and to establish metabolic engineering strategies.

Exploiting the synergistic antibacterial activity of shikimic acid and ceftiofur against methicillin-resistant Staphylococcus aureus.

Efforts to curtail the escalating health threat posed by methicillin-resistant Staphylococcus aureus (MRSA), a formidable superbug, necessitate the development of innovative treatment strategies. Leveraging potential compounds from natural sources in tandem with antibiotics has emerged as a promising approach against MRSA. These strategies should enhance the antibiotic efficacy, reduce dosage and toxicity, and bypass MRSA resistance. In this study, we used a checkerboard assay to illustrate the significant synergistic anti-MRSA effect of shikimic acid (SA), a naturally occurring compound, and ceftiofur (CF). Time-kill curves further revealed that a combination of 1/4 of the minimum inhibitory concentration (MIC) of SA and 1/8 MIC of the sodium CF eradicated MRSA within 2 h, with no noticeable toxicity observed with these concentrations. In vivo experiments confirmed that this combination therapy demonstrated robust antimicrobial activity against MRSA-induced bacteremia in mice, significantly reducing bacterial loads in the kidneys, liver, and spleen, attenuating inflammatory cell infiltration, and alleviating pathological damage. This study not only offers a compelling strategy, capitalizing on the synergistic potential of SA and CF, to rapidly address antibiotic resistance but also contributes significantly to the refinement of antimicrobial therapeutic strategies.

Metabolic engineering of Corynebacterium glutamicum for the production of anthranilate from glucose and xylose.

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof.

Reproducible growth of Brachypodium in EcoFAB 2.0 reveals that nitrogen form and starvation modulate root exudation.

Understanding plant-microbe interactions requires examination of root exudation under nutrient stress using standardized and reproducible experimental systems. We grew Brachypodium distachyon hydroponically in fabricated ecosystem devices (EcoFAB 2.0) under three inorganic nitrogen forms (nitrate, ammonium, and ammonium nitrate), followed by nitrogen starvation. Analyses of exudates with liquid chromatography-tandem mass spectrometry, biomass, medium pH, and nitrogen uptake showed EcoFAB 2.0's low intratreatment data variability. Furthermore, the three inorganic nitrogen forms caused differential exudation, generalized by abundant amino acids-peptides and alkaloids. Comparatively, nitrogen deficiency decreased nitrogen-containing compounds but increased shikimates-phenylpropanoids. Subsequent bioassays with two shikimates-phenylpropanoids (shikimic and p-coumaric acids) on soil bacteria or Brachypodium seedlings revealed their distinct capacity to regulate both bacterial and plant growth. Our results suggest that (i) Brachypodium alters exudation in response to nitrogen status, which can affect rhizobacterial growth, and (ii) EcoFAB 2.0 is a valuable standardized plant research tool.

Evaluation of neurological behavior alterations and metabolic changes in mice under chronic glyphosate exposure.

Glyphosate is a widely used active ingredient in agricultural herbicides, inhibiting the biosynthesis of aromatic amino acids in plants by targeting their shikimate pathway. Our gut microbiota also facilitates the shikimate pathway, making it a vulnerable target when encountering glyphosate. Dysbiosis in the gut microbiota may impair the gut-brain axis, bringing neurological outcomes. To evaluate the neurotoxicity and biochemical changes attributed to glyphosate, we exposed mice with the reference dose (RfD) set by the U.S. EPA (1.75 mg/Kg-BW/day) and its hundred-time-equivalence (175 mg/Kg-BW/day) chronically via drinking water, then compared a series of neurobehaviors and their fecal/serum metabolomic profile against the non-exposed vehicles (n = 10/dosing group). There was little alteration in the neurobehavior, including motor activities, social approach, and conditioned fear, under glyphosate exposure. Metabolomic differences attributed to glyphosate were observed in the feces, corresponding to 68 and 29 identified metabolites with dysregulation in the higher and lower dose groups, respectively, compared to the vehicle-control. There were less alterations observed in the serum metabolome. Under 175 mg/Kg-BW/day of glyphosate exposure, the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) were reduced in the feces but not in the serum of mice. We further focused on how tryptophan metabolism was dysregulated based on the pathway analysis, and identified the indole-derivatives were more altered compared to the serotonin and kynurenine derivatives. Together, we obtained a three-dimensional data set that records neurobehavioral, fecal metabolic, and serum biomolecular dynamics caused by glyphosate exposure at two different doses. Our data showed that even under the high dose of glyphosate irrelevant to human exposure, there were little evidence that supported the impairment of the gut-brain axis.

Improved production of the antidiabetic metabolite montbretin A in Nicotiana benthamiana: discovery, characterization, and use of Crocosmia shikimate shunt genes.

The plant-specialized metabolite montbretin A (MbA) is being developed as a new treatment option for type-2 diabetes, which is among the ten leading causes of premature death and disability worldwide. MbA is a complex acylated flavonoid glycoside produced in small amounts in below-ground organs of the perennial plant Montbretia (Crocosmia × crocosmiiflora). The lack of a scalable production system limits the development and potential application of MbA as a pharmaceutical or nutraceutical. Previous efforts to reconstruct montbretin biosynthesis in Nicotiana benthamiana (Nb) resulted in low yields of MbA and higher levels of montbretin B (MbB) and montbretin C (MbC). MbA, MbB, and MbC are nearly identical metabolites differing only in their acyl moieties, derived from caffeoyl-CoA, coumaroyl-CoA, and feruloyl-CoA, respectively. In contrast to MbA, MbB and MbC are not pharmaceutically active. To utilize the montbretia caffeoyl-CoA biosynthesis for improved MbA engineering in Nb, we cloned and characterized enzymes of the shikimate shunt of the general phenylpropanoid pathway, specifically hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (CcHCT), p-coumaroylshikimate 3'-hydroxylase (CcC3'H), and caffeoylshikimate esterase (CcCSE). Gene expression patterns suggest that CcCSE enables the predominant formation of MbA, relative to MbB and MbC, in montbretia. This observation is supported by results from in vitro characterization of CcCSE and reconstruction of the shikimate shunt in yeast. Using CcHCT together with montbretin biosynthetic genes in multigene constructs resulted in a 30-fold increase of MbA in Nb. This work advances our understanding of the phenylpropanoid pathway and features a critical step towards improved MbA production in bioengineered Nb.

Meroterpenoids with divers' rings systems from Phyllosticta capitalensis and their anti-inflammatory activity.

Four undescribed sesquiterpene-shikimates (1-4), eight undescribed monoterpene-shikimates (5-12), together with two known ones were isolated and identified from the 95% ethanol extract of the plant endophytic fungus Phyllosticta capitalensis cultured in rice medium. Capitalensis A (1) was identified as the first sesquiterpene-shikimate-conjugated spirocyclic meroterpenoid degradation product, while capitalensis B (2) is a sesquiterpene-shikimate-conjugated spirocyclic meroterpenoid with a unique D-ring formed by a C-2-O-C-9' connection. The structures of these previously undescribed compounds were elucidated by multiple techniques, including IR, HR-ESI-MS, and NMR analysis. Furthermore, their absolute configurations were established through the comprehensive approach that involved the calculations of ECD spectra, optical rotation values, and single-crystal X-ray analysis. Moreover, the anti-inflammatory activity of all isolated compounds was evaluated using a lipopolysaccharide (LPS)-induced inflammation model in BV2 microglial cells. Meanwhile, these compounds exhibited activity in inhibiting NO production. Four compounds, capitalensis C (3), capitalensis D (4), 15-hydroxyl tricycloalternarene 5b (13) and guignarenone A (14) showed strong inhibitory effects with IC50 values of 21.6 ± 1.33, 12.2 ± 1.08, 18.6 ± 1.27, and 15.8 ± 1.20 µM, respectively. In addition, the structure-activity relationship of the anti-inflammatory activity of the compounds was discussed.

From dysbiosis to neuropathologies: Toxic effects of glyphosate in zebrafish.

Glyphosate, a globally prevalent herbicide known for its selective inhibition of the shikimate pathway in plants, is now implicated in physiological effects on humans and animals, probably due to its impacts in their gut microbiomes which possess the shikimate pathway. In this study, we investigate the effects of environmentally relevant concentrations of glyphosate on the gut microbiota, neurotransmitter levels, and anxiety in zebrafish. Our findings demonstrate that glyphosate exposure leads to dysbiosis in the zebrafish gut, alterations in central and peripheral serotonin levels, increased dopamine levels in the brain, and notable changes in anxiety and social behavior. While the dysbiosis can be attributed to glyphosate's antimicrobial properties, the observed effects on neurotransmitter levels leading to the reported induction of oxidative stress in the brain indicate a novel and significant mode of action for glyphosate, namely the impairment of the microbiome-gut-axis. While further investigations are necessary to determine the relevance of this mechanism in humans, our findings shed light on the potential explanation for the contradictory reports on the safety of glyphosate for consumers.

Enhancing menaquinone-7 biosynthesis through strengthening precursor supply and product secretion.

Menaquinone-7 (MK-7) is an important class of vitamin K2 that is essential in human health and can prevent osteoporosis and cardiovascular disease. However, due to the complex synthesis pathway, the synthesis efficiency is low. The main objective of this study was to explore the effect of enhanced supply of precursors in Bacillus natto. Three precursors of pyruvate, shikimic acid, and sodium glutamate were chosen to investigate the effect of enhanced supply of precursors on MK-7 synthesis. Then, the optimal concentrations, different combinations, and different adding times were systematically studied, respectively. Results showed that the combination of shikimic acid and sodium glutamate could boost MK-7 production by 2 times, reaching 50 mg/L of MK-7 titer and 0.52 mg/(L·h) of MK-7 productivity. Furthermore, adding shikimic acid and sodium glutamate initially and feeding pyruvate at 48 h and 72 h increased MK-7 production to 58 mg/L. At the same time, the expression of the three related genes was also significantly upregulated. Subsequently, a new fermentation strategy combining the precursors enhancement and product secretion was proposed to enhance MK-7 yield and MK-7 productivity to 63 mg/L and 0.45 mg/(L·h). This study proposed a new fermentation regulation strategy for the enhancement of vitamin K2 biosynthesis.

A Comprehensive Review of the Pharmacology, Chemistry, Traditional Uses and Quality Control of Star Anise (Illicium verum Hook. F.): An Aromatic Medicinal Plant.

Illicium verum Hook. F., also known as star anise, is one of the most important plants of the genus Anise in the family Magnoliaceae. I. verum not only has the functions of warming Yang, dispersing cold, regulating Qi and relieving pain but can also be used as a condiment to increase flavor as well as reconcile and remove fish smells. Currently, 201 chemical constituents have been identified from star anise; among these, star anise oil and shikimic acid are the two most widely used and studied chemical components in star anise, with the oil accounting for a large proportion of the total. This review integrates, classifies and updates studies related to the botany, pharmacology, phytochemistry, traditional and modern uses and quality control of star anise, with a special reference to its phytochemical composition and pharmacological activity. It will provide a reference for further research on this important medicinal plant. In addition, the broad applications and research profiles of star anise essential oil and shikimic acid are highlighted. Our review indicates that the research prospects regarding star anise are very broad and worthy of further investigation.

Shikimate-Derived Meroterpenoids from the Ascidian-Derived Fungus Amphichorda felina SYSU-MS7908 and Their Anti-Glioma Activity.

Glioma is a clinically heterogeneous type of brain tumor with a poor prognosis. Current treatment approaches have limited effectiveness in treating glioma, highlighting the need for novel drugs. One approach is to explore marine natural products for their therapeutic potential. In this study, we isolated nine shikimate-derived diisoprenyl-cyclohexene/ane-type meroterpenoids (1-9), including four new ones, amphicordins A-D (1-4) from the ascidian-derived fungus Amphichorda felina SYSU-MS7908, and further semisynthesized four derivatives (10-13). Their structures were extensively characterized using 1D and 2D NMR, modified Mosher's method, HR-ESIMS, NMR and ECD calculations, and X-ray crystallography. Notably, amphicordin C (3) possesses a unique benzo[g]chromene (6/6/6) skeleton in this meroterpenoid family. In an anti-glioma assay, oxirapentyn A (7) effectively inhibited the proliferation, migration, and invasion of glioma cells and induced their apoptosis. Furthermore, an in silico analysis suggested that oxirapentyn A has the potential to penetrate the blood-brain barrier. These findings highlight the potential of oxirapentyn A as a candidate for the development of novel anti-glioma drugs.

Challenges and opportunities for engineered Escherichia coli as a pivotal chassis toward versatile tyrosine-derived chemicals production.

Growing concerns over limited fossil resources and associated environmental problems are motivating the development of sustainable processes for the production of high-volume fuels and high-value-added compounds. The shikimate pathway, an imperative pathway in most microorganisms, is branched with tyrosine as the rate-limiting step precursor of valuable aromatic substances. Such occurrence suggests the shikimate pathway as a promising route in developing microbial cell factories with multiple applications in the nutraceutical, pharmaceutical, and chemical industries. Therefore, an increasing number of studies have focused on this pathway to enable the biotechnological manufacture of pivotal and versatile aromatic products. With advances in genome databases and synthetic biology tools, genetically programmed Escherichia coli strains are gaining immense interest in the sustainable synthesis of chemicals. Engineered E. coli is expected to be the next bio-successor of fossil fuels and plants in commercial aromatics synthesis. This review summarizes successful and applicable genetic and metabolic engineering strategies to generate new chassis and engineer the iterative pathway of the tyrosine route in E. coli, thus addressing the opportunities and current challenges toward the realization of sustainable tyrosine-derived aromatics.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation in Arabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.