Sedoheptulose 7-phosphate Research Articles

Sustainable Production of Shinorine from Agricultural Wastes Using Engineered Saccharomyces cerevisiae Expressing Novel d-Alanine-d-alanine Ligase from Pseudonocardia pini.

Shinorine, a compound known for its protective properties against UV radiation, is widely used in cosmetics and pharmaceuticals. Despite the construction of various recombinant Saccharomyces cerevisiae strains for shinorine production, achieving industrial-scale yields remains a challenge. In this study, genes encoding enzymes (DDGS, O-MT, and ATP-grasp enzyme) from Actinosynnema mirum were introduced into S. cerevisiae DXdT to enable the heterologous conversion of sedoheptulose 7-phosphate to mycosporine-glycine─the direct biosynthetic precursor of shinorine. Subsequently, a novel d-alanine-d-alanine ligase from Pseudonocardia pini was introduced to produce shinorine. The engineered strain (DXdT-MG-mi89-PP.ddl) produced 267.9 mg/L shinorine with a 48.6 mg/g dry cell weight (DCW) content in a medium supplemented with lignocellulosic hydrolysate derived from rice straw. Notably, the recombinant strain produced 1.7 g/L shinorine with a 79.1 mg/g DCW content from a corn steep liquor medium with a mixture of glucose and xylose. These results support the idea that sustainable shinorine production from agricultural wastes holds significant promise for industrial applications.

Synthesis and properties of the natural ultraviolet absorber gadusol in Komagataella phaffii

Gadusol, an efficient natural ultraviolet (UV) absorbing substance with antioxidant capacity, is ubiquitous in aquatic organisms such as microorganisms, algae, and fish eggs. In order to address issues such as its low natural extraction yield and environmental unfriendliness, we introduced the gadusol synthesis pathway from zebrafish into Komagataella phaffii and successfully constructed the recombinant strain capable of synthesizing gadusol. The xylose assimilation genes derived from Scheffersomyces stipitis were further introduced into the recombinant strain to increase the content of the key substrate sedoheptulose 7-phosphate (S7P). The results showed that the utilization of xylose was an effective strategy to increase the yield of gadusol. In the medium with only xylose as the substrate, the yield of gadusol reached 141.8 mg/L (32.3 mg/g dry cell weight, DCW), which was about 46 times of that in the medium with only glucose as the substrate. The product showed obvious absorption in the range of 275-305 nm, with the maximum absorption at 290 nm. Moreover, the product demonstrated antioxidant capacity. After reaction for 5 h, the ferric ion reducing antioxidant power (FRAP), 2, 2'-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rates changed by 90.83%, 25.50%, and 131.80%, respectively, which suggested that the product may be used as a long-acting antioxidant. This study demonstrated the great potential of K. phaffii as a chassis for the biosynthesis of natural UV absorbers. The biosynthesized gadusol has good UV absorption and antioxidant properties, which provides a theoretical basis for the industrial production and application of gadusol.

The mitigating effects and mechanisms of Bacillus cereus on chronic cadmium poisoning in Litopenaeus vannamei based on histopathological, transcriptomic, and metabolomic analyses

Shrimp are non-negligible victims of cadmium (Cd) contamination, and there is still a lack of strategies for mitigating Cd toxicity in shrimp. Bacillus cereus, with its significant heavy metal (HM) tolerance and chelating effects, is a representative beneficial bacterium to be investigated for mitigating the toxicity of Cd exposure. This study revealed the effects and potential mechanisms of B. cereus in mitigating chronic Cd toxicity in shrimp by analyzing growth performance, hepatopancreatic Cd accumulation, pathology, as well as comprehensive hepatopancreatic transcriptomics and metabolomics in Litopenaeus vannamei. The results showed that shrimp's growth inhibition, hepatopancreatic Cd accumulation and physiological structure damage in B. cereus+chronic Cd group were effectively alleviated compared with the chronic Cd treatment group. The pathways related to amino acid metabolism, glycolipid metabolism, immune response, and antioxidant stress were significantly activated in the B. cereus+chronic Cd group, including glycolysis, pentose phosphate pathway, oxidative phosphorylation, biosynthesis of amino acids, and biosynthesis of unsaturated fatty acids pathways. The key differentially expressed genes (e.g., macrophage migration inhibitory factor, glycine cleavage system H protein, glycine dehydrogenase, phosphoglucomutase-2, asparaginase, ATP synthase subunit, cytochrome c, and 4-hydroxyphenylpyruvate dioxygenase) and metabolites (e.g., L-leucine, D-ribose, gluconic acid, 6-Phosphogluconic acid, sedoheptulose 7-phosphate, 1-Kestose, glyceric acid, arachidic acid, prostaglandins, 12-Keto-tetrahydro-leukotriene B4, and gamma-glutamylcysteine) associated with the above pathways were significantly altered. This study demonstrated that B. cereus is an effective mitigator for the treatment of chronic Cd poisoning in shrimp. B. cereus may play a role in alleviating the toxicity of Cd by enhancing the antioxidant performance, immune defense ability, metabolic stability, and energy demand regulation of shrimp. The study provides reference materials for the study of B. cereus in alleviating Cd toxicity of shrimp and broadens the application of probiotics in treating HM toxicity.

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

BackgroundMycosporine-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.ResultsTo 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.ConclusionReversing the direction of flux between glycolysis and the NOPPP offers a novel metabolic engineering strategy in Saccharomyces cerevisiae.

Synthesis of the Carba-Analogs of the α-Pyranose and β-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 β-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 β-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 β-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.

Mass spectrometry-based pseudotargeted metabolomics reveals metabolic variations in a2-induced gastric cancer cell.

Gastric cancer (GC) is one of the most malignant tumors with high morbidity and mortality in the world. Compound a2, a Jiyuan oridonin derivative, exhibited excellent anti-proliferative activity against GC cells. To investigate the gastric cellular response to a2 therapy as a novel drug candidate, we adopted a pseudotargeted metabolomics method to explore metabolic variation in a2-induced MGC-803 gastric cells using liquid chromatography tandem mass spectrometry combined with multivariate statistical analysis. The results showed that a2 treatment induced significant metabolic changes in the levels of aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, pyrimidine metabolism, and tricarboxylic acid cycle, approximately 80% of the metabolites were down-regulated in the low-dose and high-dose groups including aspartate, tryptophan, sedoheptulose 7-phosphate, succinate, 2'-deoxyadenosine, uridine, cytidine, etc. which can provide evidence for a new therapy of GC.

Potentiating Activity of GmhA Inhibitors on Gram-Negative Bacteria.

Inhibition of the biosynthesis of bacterial heptoses opens novel perspectives for antimicrobial therapies. The enzyme GmhA responsible for the first committed biosynthetic step catalyzes the conversion of sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate and harbors a Zn2+ ion in the active site. A series of phosphoryl- and phosphonyl-substituted derivatives featuring a hydroxamate moiety were designed and prepared from suitably protected ribose or hexose derivatives. High-resolution crystal structures of GmhA complexed to two N-formyl hydroxamate inhibitors confirmed the binding interactions to a central Zn2+ ion coordination site. Some of these compounds were found to be nanomolar inhibitors of GmhA. While devoid of HepG2 cytotoxicity and antibacterial activity of their own, they demonstrated in vitro lipopolysaccharide heptosylation inhibition in Enterobacteriaceae as well as the potentiation of erythromycin and rifampicin in a wild-type Escherichia coli strain. These inhibitors pave the way for a novel treatment of Gram-negative infections.

A novel gluconeogenic route enables efficient use of erythritol in zoonotic Brucella.

Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we re-examined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp-Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals.

Insulin and glycolysis dependency of cardioprotection by nicotinamide riboside

Decreased nicotinamide adenine dinucleotide (NAD+) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia–reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD+ precursor due to efficient NAD+ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and 13C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) (“mild glycolysis group”), (b) lactate + pyruvate + FA (“no glycolysis group”), or (c) glucose + FA + insulin (“high glycolysis group”). NR increased cardiac NAD+ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the “no glycolysis” hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high 13C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining “no glycolysis” hearts or “high glycolysis” hearts, NR protection was abolished. The data suggest that NR’s acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

Abstract C050: The unique metabolic signatures of keratin-17 expressing pancreatic ductal adenocarcinomas

Abstract Background: Overexpression of Keratin 17 (K17) is associated with upregulation of de novo pyrimidine synthesis, which is linked to resistance to gemcitabine (dC analog) and 5-fluorouracil (dT analog) in pancreatic ductal adenocarcinomas (PDAC). The metabolic rewiring associated with K17 in upstream pathways has not been defined, and the spatial relationships between K17 overexpression and the metabolome are unknown. Understanding these relationships may elucidate the roles of K17 in drug resistance and metabolic phenotypes of PDACs. We test the hypothesis that K17 is spatially linked to alterations of various metabolites, and that there exists an association with the upregulation of upstream pathways to de novo pyrimidine synthesis. Methods: KPC cells transduced to express wtK17 versus an empty vector were orthotopically implanted into the head of the pancreas of mice. Mice were administered 5-FU at 50 mg/kg twice a week. To evaluate the metabolic profile in situ, MALDI-MSI (Matrix-assisted laser desorption/ionization mass spectrometry imaging) was performed on snap-frozen tumor specimens. Following removal of the MALDI matrix, immunohistochemistry (IHC) was performed on the same sections for K17 to define elevated or low expressing regions. ROIs were selected within the MALDI-MSI for High K17, Low K17, and empty vector viable tissue. Principal component analysis was then performed on the mass spectra of these regions to provide unbiased pattern recognition. Results: The principal component analysis segregated K17-positive ROIs from empty vector (EV) ROIs, revealing that K17-positive tissue has a unique metabolic signature compared to EV tissue. Metabolites with loadings outside the 95th percentiles in the 5-component PCA included UMP, UDP, orotate, sedoheptulose 7-phosphate, glucose 6-phosphate, glutamate, malate, phosphoglycerate, and lactate. Conclusion: These metabolites, therefore, distinguish between the EV and K17-positive clusters. Further hierarchical clustering will be performed to provide a more comprehensive evaluation of the rewired pathways in pancreatic cancer. Citation Format: Mahmoud M. Salem, Michael Horowitz, Lyanne Oblein, Chun-Hao Pan, Natalia Marchenko, Luisa Escobar-Hoyos, Bo Chen, Kenneth Shroyer. The unique metabolic signatures of keratin-17 expressing pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C050.

Nicotinamide riboside cardioprotection is mediated through glycolysis activation

Abstract Purpose Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection. Methods Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate. Results NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2). Conclusions NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts

Activation of Purine Biosynthesis Suppresses the Sensitivity of E. coli gmhA Mutant to Antibiotics.

Inactivation of enzymes responsible for biosynthesis of the cell wall component of ADP-glycero-manno-heptose causes the development of oxidative stress and sensitivity of bacteria to antibiotics of a hydrophobic nature. The metabolic precursor of ADP-heptose is sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative branch of the pentose phosphate pathway (PPP), in which ribose-5-phosphate and NADPH are generated. Inactivation of the first stage of ADP-heptose synthesis (ΔgmhA) prevents the outflow of S7P from the PPP, and this mutant is characterized by a reduced biosynthesis of NADPH and of the Glu-Cys-Gly tripeptide, glutathione, molecules known to be involved in the resistance to oxidative stress. We found that the derepression of purine biosynthesis (∆purR) normalizes the metabolic equilibrium in PPP in ΔgmhA mutants, suppressing the negative effects of gmhA mutation likely via the over-expression of the glycine-serine pathway that is under the negative control of PurR and might be responsible for the enhanced synthesis of NADPH and glutathione. Consistently, the activity of the soxRS system, as well as the level of glutathionylation and oxidation of proteins, indicative of oxidative stress, were reduced in the double ΔgmhAΔpurR mutant compared to the ΔgmhA mutant.

Efficient production of mycosporine-like amino acids, natural sunscreens, in Yarrowia lipolytica

BackgroundMycosporine-like amino acids (MAAs), including shinorine and porphyra-334, are gaining attention as safe natural sunscreens. The production of MAAs has been achieved in diverse microbial hosts, including Saccharomyces cerevisiae. While S. cerevisiae is the most extensively studied model yeast, the oleaginous yeast Yarrowia lipolytica has emerged as a promising candidate for the synthesis of valuable products. In this study, we explored the potential of Y. lipolytica as a host for producing MAAs, utilizing its advantages such as a robust pentose phosphate pathway flux and versatile carbon source utilization.ResultsWe produced MAAs in Y. lipolytica by introducing the MAA biosynthetic genes from cyanobacteria Nostoc punctiforme and Anabaena variabilis. These genes include mysA, mysB, and mysC responsible for producing mycosporine-glycine (MG) from sedoheptulose 7-phosphate (S7P). The two strains utilize different enzymes, D-Ala-D-Ala ligase homologue (MysD) in N. punctiforme and NRPS-like enzyme (MysE) in A. variabilis, for amino acid conjugation to MG. MysE specifically generated shinorine, a serine conjugate of MG, while MysD exhibited substrate promiscuity, yielding both shinorine and a small amount of porphyra-334, a threonine conjugate of MG. We enhanced MAAs production by selecting mysA, mysB, and mysC from A. variabilis and mysD from N. punctiforme based on their activities. We further improved production by strengthening promoters, increasing gene copies, and introducing the xylose utilization pathway. Co-utilization of xylose with glucose or glycerol increased MAAs production by boosting the S7P pool through the pentose phosphate pathway. Overexpressing GND1 and ZWF1, key genes in the pentose phosphate pathway, further enhanced MAAs production. The highest achieved MAAs level was 249.0 mg/L (207.4 mg/L shinorine and 41.6 mg/L of porphyra-334) in YP medium containing 10 g/L glucose and 10 g/L xylose.ConclusionsY. lipolytica was successfully engineered to produce MAAs, primarily shinorine. This achievement involved the introduction of MAA biosynthetic genes from cyanobacteria, establishing xylose utilizing pathway, and overexpressing the pentose phosphate pathway genes. These results highlight the potential of Y. lipolytica as a promising yeast chassis strain for MAAs production, notably attributed to its proficient expression of MysE enzyme, which remains non-functional in S. cerevisiae, and versatile utilization of carbon sources like glycerol.

Effects of Aeromonas hydrophila infection on the intestinal microbiota, transcriptome, and metabolomic of common carp (Cyprinus carpio).

Aeromonas hydrophila frequently has harmful effects on aquatic organisms. The intestine is an important defense against stress. In this study, we investigated the intestinal microbiota and transcriptomic and metabolomic responses of Cyprinus carpio subjected to A. hydrophila infection. The results showed that obvious variation in the intestinal microbiota was observed after infection, with increased levels of Firmicutes and Bacteroidetes and decreased levels of Proteobacteria. Several genera of putatively beneficial microbiota (Cetobacterium, Bacteroides, and Lactobacillus) were abundant, while Demequina, Roseomonas, Rhodobacter, Pseudoxanthomonas, and Cellvibrio were decreased; pathogenic bacteria of the genus Vibrio were increased after microbiota infection. The intestinal transcriptome revealed several immune-related differentially expressed genes associated with the cytokines and oxidative stress. The metabolomic analysis showed that microbiota infection disturbed the metabolic processes of the carp, particularly amino acid metabolism. This study provides insight into the underlying mechanisms associated with the intestinal microbiota, immunity, and metabolism of carp response to A. hydrophila infection; eleven stress-related metabolite markers were identified, including N-acetylglutamic acid, capsidiol, sedoheptulose 7-phosphate, prostaglandin B1, 8,9-DiHETrE, 12,13-DHOME, ADP, cellobiose, 1H-Indole-3-carboxaldehyde, sinapic acid and 5,7-dihydroxyflavone.

Metabolic Fingerprints of Effective Fluoxetine Treatment in the Prefrontal Cortex of Chronically Socially Isolated Rats: Marker Candidates and Predictive Metabolites.

The increasing prevalence of depression requires more effective therapy and the understanding of antidepressants' mode of action. We carried out untargeted metabolomics of the prefrontal cortex of rats exposed to chronic social isolation (CSIS), a rat model of depression, and/or fluoxetine treatment using liquid chromatography-high resolution mass spectrometry. The behavioral phenotype was assessed by the forced swim test. To analyze the metabolomics data, we employed univariate and multivariate analysis and biomarker capacity assessment using the receiver operating characteristic (ROC) curve. We also identified the most predictive biomarkers using a support vector machine with linear kernel (SVM-LK). Upregulated myo-inositol following CSIS may represent a potential marker of depressive phenotype. Effective fluoxetine treatment reversed depressive-like behavior and increased sedoheptulose 7-phosphate, hypotaurine, and acetyl-L-carnitine contents, which were identified as marker candidates for fluoxetine efficacy. ROC analysis revealed 4 significant marker candidates for CSIS group discrimination, and 10 for fluoxetine efficacy. SVM-LK with accuracies of 61.50% or 93.30% identified a panel of 7 or 25 predictive metabolites for depressive-like behavior or fluoxetine effectiveness, respectively. Overall, metabolic fingerprints combined with the ROC curve and SVM-LK may represent a new approach to identifying marker candidates or predictive metabolites for ongoing disease or disease risk and treatment outcome.

Metabolic alterations of the gut–liver axis induced by cholic acid contribute to hepatic steatosis in rats

12α-Hydroxylated (12αOH) bile acids (BAs) selectively increase with high-fat diet intake. Dietary supplementation with cholic acid (CA) in rats is a possible strategy to reveal the causal link between 12αOH BAs and hepatic steatosis. The present study aimed to investigate the metabolic mechanism underlying the effect of 12αOH BAs on hepatic steatosis. Male WKAH rats were fed either a control (Ct) or CA-supplemented diet (0.5 g/kg). After the 12-week intervention, the CA diet elevated the 12αOH BA levels in the gut–liver axis. CA-fed rats showed greater hepatic lipid accumulation than in the Ct group, regardless of the dietary energy balance. Untargeted metabolomics suggested marked differences in the fecal metabolome of rats subjected to the CA diet compared with that of Ct, characterized by the depletion of fatty acids and enrichment of amino acids and amines. Moreover, the liver metabolome differed in the CA group, characterized by an alteration in redox-related pathways. The CA diet elevated nicotinamide adenine dinucleotide consumption owing to the activation of poly(ADP-ribose) polymerase 1, resulting in impaired peroxisome proliferator-activated receptor α signaling in the liver. The CA diet increased sedoheptulose 7-phosphate, and enhanced glucose-6-phosphate dehydrogenase activity, suggesting promotion of the pentose phosphate pathway that generates reducing equivalents. Integrated analysis of the gut–liver metabolomic data revealed the role of deoxycholic acid and its liver counterpart in mediating these metabolic alterations. These observations suggest that alterations in metabolites induced by 12αOH BAs in the gut–liver axis contribute to the enhancement of liver lipid accumulation.

Multi-Omics Analysis Reveals the Regulatory and Metabolic Mechanisms Underlying Low-Nitrogen Tolerance at the Flowering Stage in Rice

Crop productivity depends on nitrogen fertilization, but plants take up only an average of 30–50% of the applied nitrogen. Developing rice cultivars with improved nitrogen use efficiency or low-nitrogen (LN) tolerance is critical for sustainable agriculture. In this study, a backcross introgression line (G9) with 15 introgressed regions from donor parent and its recurrent parent Shuhui 527 (G1), which are differentially responsive to LN stress, were used to investigate the mechanism mediating rice LN tolerance at the flowering stage based on metabolome and transcriptome profiles. Three genes (LOC_Os02g40010, LOC_Os11g25260 and LOC_Os11g47300) involved in purine metabolism, which are located in the introgressed regions, were detected with significantly higher expression levels in G9 than in G1 under LN stress, and the contents of two relative metabolites (uric acid and guanine) were significantly different between the two genotypes. Additionally, two genes (LOC_Os02g36880 and LOC_Os08g05830) located in the introgressed regions and relative metabolites (3-phosphoglyceric acid and sedoheptulose 7-phosphate) involved in glycosis and pentose phosphate pathway are differentially expressed between G9 and G1. In addition to the two nitrogen metabolism-related genes (OsLHT1 and OsACR9) located in the introgressed regions, 23 differentially expressed genes mainly involved in nitrogen metabolism were identified between genotypes or treatments. With the comprehensive analysis of transcriptomes and metabolomes, our results reveal that the active purine metabolism may be the main factor contributing to LN tolerance in rice at the flowering stage, and also provide five new candidate genes for improving LN tolerance during the molecular breeding of rice.

Sustainable Production of Shinorine from Lignocellulosic Biomass by Metabolically Engineered Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs) have been used in cosmetics and pharmaceuticals. The purpose of this work was to develop yeast strains for sustainable and economical production of MAAs, especially shinorine. First, genes involved in MAA biosynthetic pathway from Actinosynnema mirum were introduced into Saccharomyces cerevisiae for heterologous shinorine production. Second, combinatorial expression of wild and mutant xylose reductase was adopted in the engineered S. cerevisiae to facilitate xylose utilization in the pentose phosphate pathway. Finally, the accumulation of sedoheptulose 7-phosphate (S7P) was attempted by deleting transaldolase-encoding TAL1 in the pentose phosphate pathway to increase carbon flux toward shinorine production. In fed-batch fermentation, the engineered strain (DXdT-M) produced 751 mg/L shinorine in 71 h. Ultimately, 54 mg/L MAAs was produced by DXdT-M from rice straw hydrolysate. The results suggest that shinorine production by S. cerevisiae might be a promising process for sustainable production and industrial applications.

In silico analysis of enzymes involved in mycosporine-like amino acids biosynthesis in Euhalothece sp.: Structural and functional characterization

Mycosporine-like amino acids (MAAs) are ultraviolet-absorbing compounds synthesized by photoautotrophic microorganisms such as cyanobacteria. They have applications as UV protectants and antioxidants in the cosmetic and pharmaceutical sectors. To date, >30 different types of MAAs have been identified, having an absorption range between 310 and 365 nm that covers most of the UVR spectrum (~295–400 nm). In the study, MAAs were extracted and partially purified from a euryhaline Euhalothece sp. Thereafter, in silico analysis of the MAAs biosynthetic genes was conducted to determine the physicochemical characteristics, structural properties, functional analysis and homology model. High salinity stress (120 g L−1) significantly enhanced the production of MAAs (~56 %) in Euhalothece sp. Bioinformatics analysis of the genome of Euhalothece sp. revealed a distinctive mys gene cluster that contained six genes (mysA to mysE), compared to four genes commonly found in MAA-producing cyanobacteria. Interestingly, both EEVS and DHQ synthase were present, indicating the potential to synthesize MAAs via both the shikimate and sedoheptulose 7-phosphate pathways. Secondary structure analysis confirmed that in all the biosynthetic enzymes, the major components were the alpha-helices with random coils. This study emphasized the unique genes for MAAs in Euhalothece sp. as well as highlighted the potential metabolite pathways for commercial production. Elucidation of the protein structure, physicochemical properties, and interactions provides scientific insight into the biosynthesis of MAAs from hypersaline cyanobacteria. Additionally, it is essential for determining the applications of these novel photoprotective compounds.

A novel variant of the Calvin\u2013Benson cycle bypassing fructose bisphosphate

The Calvin–Benson cycle (CB cycle) is quantitatively the most important metabolic pathway for CO2 fixation. In the canonical CB cycle, fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FBP), sedoheptulose 7-phosphate (S7P), and sedoheptulose 1,7-bisphosphate (SBP) appear as essential intermediates, where F6P is formed from FBP by the fructose 1,6-bisphosphatase (FBPase) reaction, and S7P is formed from SBP by the sedoheptulose 1,7-bisphosphatase (SBPase) reaction. Although the involvement of SBP and SBPase in the canonical CB cycle is consistent with the reported dependency of photosynthetic carbon metabolism on SBPase, the involvement of FBP and FBPase is not completely consistent with the reported FBP- or FBPase-related findings such as, although with a diminished growth rate, an Arabidopsis mutant lacking FBPase grew photoautotrophically in soil. Here, we show a novel variant of the CB cycle involving SBP, SBPase, and transaldolase, but neither FBP nor FBPase. This novel variant, named the S7P-removing transaldolase variant, bypasses FBP. This variant explains the FBP- or FBPase-related findings more easily than the canonical CB cycle as well as the dependency of photosynthetic carbon metabolism on SBPase and further suggests that co-overexpression of SBPase and transaldolase can be a strategy for enhancing photosynthetic carbon metabolism, which is important for the global environment.