Amino Acid Synthesis Pathways Research Articles

The albino mechanism of a new theanine-rich tea cultivar 'Fuhuang 2'

To explore the mechanism of tea albino variation and high theanine formation, 'Fuyun 6' and a new theanine-rich tea cultivar 'Fuhuang 2' were as materials in this study, pigment content, metabolome and transcriptome of the two cultivars were analyzed by ultramicroelectron microscopy, widely targeted metabolomics, targeted metabolomics and transcriptomics. The results showed that five catechins, theobromine, caffeine, and 20 free amino acids, including theanine, glutamine, arginine, etc., were identified by targeted metabolomics. The amino acid content of 'Fuhuang 2' was significantly higher than that of 'Fuyun 6', and the theanine content was as high as 57.37 mg/g in 'Fuhuang 2'. The ultrastructure of leaves showed that the chloroplast cell structure of 'Fuhuang 2' was fuzzy, most of the grana lamellae were arranged in disorder, with large gaps, and the thylakoids were filiform. The determination of pigments showed that compared with 'Fuyun 6', the contents of chlorophyll A and B, carotenoids, flavonoids and other pigments of 'Fuhuang 2' decreased significantly, some important pigment-related-genes, such as chlorophyllase (CLH), 9-cis-epoxycarotenoid dioxygenase (NCED), flavonoid 3β-hydroxylase (F3H) and flavonoid 3', 5'-hydroxylase (F3'5'H) were significantly changed. Compared with 'Fuyun 6', 'Fuhuang 2' identified 138 significantly changed metabolites (SCMs) and 658 differentially expressed genes (DEGs). KEGG enrichment analysis showed that SCMs and DEGs were significantly enriched in amino acid biosynthesis, glutathione metabolism and TCA cycle. In general, the albino phenotype of 'Fuhuang 2' may be caused by a deficiency in photosynthetic proteins, chlorophyll metabolism genes and chlorophyll content. The accumulation of high theanine in 'Fuhuang 2' may be due to the low nitrogen consumption in yellowed leaves and the lack of carbon skeleton, amino and nitrogen resources are stored more effectively, resulting in the up regulation of metabolites and related gene expression in the amino acid synthesis pathway, theanine has become a significant accumulation of nitrogen-containing compounds in yellowed leaves.

Different Types of Non-Starch Polysaccharides Alter the Growth, Intestinal Flora and Serum Metabolite Profile of Grass Carp, Ctenopharyngodon idella.

Dietary non-starch polysaccharides (NSPs) broadly influence fish intestinal flora and physiological metabolism, but limited information is available on grass carp (Ctenopharyngodon idella). This study investigated the effects of different types of NSPs on the growth, nutrient metabolism status, gut microbiota, and serum metabolome of grass carp. Fish were fed with diets containing 4.4% insoluble NSPs (INSP), 9.24% soluble NSPs (SNSP), 13.64% NSPs (4.4% INSP + 9.24% SNSP, NSP) and non NSPs (FM), respectively, for 9 weeks. Results showed that dietary SNSP decreased protein efficiency ratio and serum protein content, but increased feed coefficient ratio, feed intake, plasma blood urea nitrogen content, and plasma aspartate aminotransferase activity (AST); conversely, dietary INSP decreased plasma AST activity. Dietary INSP and SNSP increased serum free cholesterol content. Dietary NSPs altered the abundance of dominant bacteria and serum metabolite profiles. The differential metabolites between groups were significantly enriched in amino acid synthesis and metabolic pathways. In conclusion, dietary INSP exhibited a growth-promoting effect compared to SNSP. Dietary INSP is beneficial for improving nutrient metabolism and intestinal health. Moreover, dietary NSPs may regulate the physiological metabolism and feeding behavior of grass carp by altering amino acid synthesis and metabolism.

Combined analysis of transcriptome and metabolome reveals the molecular mechanism and candidate genes of Haloxylon drought tolerance.

Haloxylon ammodendron and Haloxylon persicum, as typical desert plants, show strong drought tolerance and environmental adaptability. They are ideal model plants for studying the molecular mechanisms of drought tolerance. Transcriptomic and metabolomic analyses were performed to reveal the response mechanisms of H. ammodendron and H. persicum to a drought environment at the levels of transcription and physiological metabolism. The results showed that the morphological structures of H. ammodendron and H. persicum showed adaptability to drought stress. Under drought conditions, the peroxidase activity, abscisic acid content, auxin content, and gibberellin content of H. ammodendron increased, while the contents of proline and malondialdehyde decreased. The amino acid content of H. persicum was increased, while the contents of proline, malondialdehyde, auxin, and gibberellin were decreased. Under drought conditions, 12,233 and 17,953 differentially expressed genes (DEGs) were identified in H. ammodendron and H. persicum , respectively, including members of multiple transcription factor families such as FAR1, AP2/ERF, C2H2, bHLH, MYB, C2C2, and WRKY that were significantly up-regulated under drought stress. In the positive ion mode, 296 and 452 differential metabolites (DEMs) were identified in H. ammodendron and H. persicum, respectively; in the negative ion mode, 252 and 354 DEMs were identified, primarily in carbohydrate and lipid metabolism. A combined transcriptome and metabolome analysis showed that drought stress promoted the glycolysis/gluconeogenesis pathways of H. ammodendron and H. persicum and increased the expression of amino acid synthesis pathways, consistent with the physiological results. In addition, transcriptome and metabolome were jointly used to analyze the expression changes of the genes/metabolites of H. ammodendron and H. persicum that were associated with drought tolerance but were regulated differently in the two plants. This study identified drought-tolerance genes and metabolites in H. ammodendron and H. persicum and has provided new ideas for studying the drought stress response of Haloxylon.

In silico analysis of metabolic effects of bipolar disorder on prefrontal cortex identified altered GABA, glutamate-glutamine cycle, energy metabolism and amino acid synthesis pathways.

Bipolar disorder (BP) is a lifelong psychiatric condition, which often disrupts the daily life of the patients. It is characterized by unstable and periodic mood changes, which cause patients to display unusual shifts in mood, energy, activity levels, concentration and the ability to carry out day-to-day tasks. BP is a major psychiatric condition, and it is still undertreated. The causes and neural mechanisms of bipolar disorder are unclear, and diagnosis is still mostly based on psychiatric examination, furthermore the unstable character of the disorder makes diagnosis challenging. Identification of the molecular mechanisms underlying the disease may improve the diagnosis and treatment rates. Single nucleotide polymorphisms (SNP) and transcriptome profiles of patients were studied along with signalling pathways that are thought to be associated with bipolar disorder. Here, we present a computational approach that uses publicly available transcriptome data from bipolar disorder patients and healthy controls. Along with statistical analyses, data are integrated with a genome-scale metabolic model and protein-protein interaction network. Healthy individuals and bipolar disorder patients are compared based on their metabolic profiles. We hypothesize that energy metabolism alterations in bipolar disorder relate to perturbations in amino-acid metabolism and neuron-astrocyte exchange reactions. Due to changes in amino acid metabolism, neurotransmitters and their secretion from neurons and metabolic exchange pathways between neurons and astrocytes such as the glutamine-glutamate cycle are also altered. Changes in negatively charged (-1) KIV and KMV molecules are also observed, and it indicates that charge balance in the brain is highly altered in bipolar disorder. Due to this fact, we also hypothesize that positively charged lithium ions may stabilize the disturbed charge balance in neurons in addition to its effects on neurotransmission. To the best of our knowledge, our approach is unique as it is the first study using genome-scale metabolic models in neuropsychiatric research.

Identify metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 as potential prognostic markers and correlate with immune infiltrates in head and neck squamous cell carcinoma

Hypopharyngeal squamous cell carcinoma (HSCC) is a kind of head and neck squamous cell carcinoma (HNSCC) with poor prognosis. Metabolic reprogramming may regulate the tumor microenvironment (TME) by adapting quickly to cellular stress and regulating immune response, but its role in HSCC has not been reported. We used the nCounter® Metabolic Pathways Panel to investigate metabolic reprogramming, cellular stress, and their relationship in HSCC tissues and adjacent normal tissues. Metabolism-related pathways nucleotide synthesis and glycolysis pathways were significantly upregulated, while amino acid synthesis and fatty acid oxidation pathways were significantly downregulated in HSCC tissues compared to adjacent normal tissues. There is a significant correlation between metabolism-related pathways and cellular stress pathways. Enrichment of immune cell and tumor infiltrating lymphocyte (TIL) analysis showed changes in immune responses between HSCC tissues and adjacent normal tissues. Overall survival analysis showed that upregulated genes CD276, LDHB, SLC3A2, EGFR, SLC7A5, and HPRT1 are potential unfavorable prognostic markers in HNSCC, while downregulated genes EEA1, IDO1, NCOA2, REST, CCL19, and ALDH2 are potential favorable prognostic markers in HNSCC. Moreover, metabolism-related genes IDO1, ALDH2, NCOA2, SLC7A5, SLC3A2, LDHB, and HPRT1 are correlated with immune infiltrates in HNSCC. These results suggest that metabolic reprogramming occurs and correlates with cellular stress and immune response in HSCC, which may help researchers understand mechanisms of metabolic reprogramming and develop effective immunotherapeutic strategies in HNSCC.

An expanded whole-cell model of E. coli links cellular physiology with mechanisms of growth rate control

Growth and environmental responses are essential for living organisms to survive and adapt to constantly changing environments. In order to simulate new conditions and capture dynamic responses to environmental shifts in a developing whole-cell model of E. coli, we incorporated additional regulation, including dynamics of the global regulator guanosine tetraphosphate (ppGpp), along with dynamics of amino acid biosynthesis and translation. With the model, we show that under perturbed ppGpp conditions, small molecule feedback inhibition pathways, in addition to regulation of expression, play a role in ppGpp regulation of growth. We also found that simulations with dysregulated amino acid synthesis pathways provide average amino acid concentration predictions that are comparable to experimental results but on the single-cell level, concentrations unexpectedly show regular fluctuations. Additionally, during both an upshift and downshift in nutrient availability, the simulated cell responds similarly with a transient increase in the mRNA:rRNA ratio. This additional simulation functionality should support a variety of new applications and expansions of the E. coli Whole-Cell Modeling Project.

Emergent expression of fitness-conferring genes by phenotypic selection.

Genotypic and phenotypic adaptation is the consequence of ongoing natural selection in populations and is key to predicting and preventing drug resistance. Whereas classic antibiotic persistence is all-or-nothing, here we demonstrate that an antibiotic resistance gene displays linear dose-responsive selection for increased expression in proportion to rising antibiotic concentration in growing Escherichia coli populations. Furthermore, we report the potentially wide-spread nature of this form of emergent gene expression (EGE) by instantaneous phenotypic selection process under bactericidal and bacteriostatic antibiotic treatment, as well as an amino acid synthesis pathway enzyme under a range of auxotrophic conditions. We propose an analogy to Ohm's law in electricity (V = IR), where selection pressure acts similarly to voltage (V), gene expression to current (I), and resistance (R) to cellular machinery constraints and costs. Lastly, mathematical modeling using agent-based models of stochastic gene expression in growing populations and Bayesian model selection reveal that the EGE mechanism requires variability in gene expression within an isogenic population, and a cellular "memory" from positive feedbacks between growth and expression of any fitness-conferring gene. Finally, we discuss the connection of the observed phenomenon to a previously described general fluctuation-response relationship in biology.

On the rate of phytoplankton respiration in the light

The rate of algal and cyanobacterial respiration in the light is an important ecophysiological term that remains to be completely characterized and quantified. To address this issue, we exploited process-specific decarboxylation rates from flux balance analysis and isotopically nonstationary metabolic flux analysis. Our study, based on published data, suggested that decarboxylation is about 22% of net CO2 assimilation when the tricarboxylic acid cycle is completely open (characterized by the commitment of alpha ketoglutarate to amino acid synthesis and very low rates of succinate formation). This estimate was supported by calculating the decarboxylation rates required to synthesize the major components of biomass (proteins, lipids, and carbohydrates) at their typical abundance. Of the 22 CO2 molecules produced by decarboxylation (normalized to net assimilation = 100), approximately 13 were from pyruvate and 3 were from isocitrate. The remaining six units of decarboxylation were in the amino acid synthesis pathways outside the tricarboxylic acid cycle. A small additional flux came from photorespiration, decarboxylations of six phosphogluconate in the oxidative pentose phosphate pathway, and decarboxylations in the syntheses of lower-abundance compounds, including pigments and ribonucleic acids. This general approach accounted for the high decarboxylation rates in algae and cyanobacteria compared to terrestrial plants. It prompts a simple speculation for the origin of the Kok effect and helps constrain the photoautotrophic respiration rate, in the light, in the euphotic zone of the ocean and lakes.

In silico target-based strain engineering of Saccharomyces cerevisiae for terpene precursor improvement.

Systems-based metabolic engineering enables cells to enhance product formation by predicting gene knockout and overexpression targets using modeling tools. FOCuS, a novel metaheuristic tool, was used to predict flux improvement targets in terpenoid pathway using the genome-scale model of Saccharomyces cerevisiae, iMM904. Some of the key knockout target predicted includes LYS1, GAP1, AAT1, AAT2, TH17, KGD-m, MET14, PDC1 and ACO1. It was also observed that the knockout reactions belonged either to fatty acid biosynthesis, amino acid synthesis pathways or nucleotide biosynthesis pathways. Similarly, overexpression targets such as PFK1, FBA1, ZWF1, TDH1, PYC1, ALD6, TPI1, PDX1 and ENO1 were established using three different existing gene amplification algorithms. Most of the overexpression targets belonged to glycolytic and pentose phosphate pathways. Each of these targets had plausible role for improving flux toward sterol pathway and were seemingly not artifacts. Moreover, an in vitro study as validation was carried with overexpression of ALD6 and TPI1. It was found that there was an increase in squalene synthesis by 2.23- and 4.24- folds, respectively, when compared with control. In general, the rationale for predicting these in silico targets was attributed to either increasing the acetyl-CoA precursor pool or regeneration of NADPH, which increase the sterol pathway flux.

Correlation between intestinal flora disruption and protein\u2013energy wasting in patients with end-stage renal disease

BackgroundDifferent dialysis treatments may affect the composition and structure of the intestinal flora of dialysis-treated chronic kidney disease (CKD) patients. This study aimed to analyze the correlations between the different flora and the nutritional indexes and further explore the potential metabolic pathways in patients with CKD in end-stage renal disease (ESRD).MethodsAltogether, 102 patients with ESRD were recruited and categorized into the hemodialysis (HD) group (N = 49) and the peritoneal dialysis (PD) group (N = 53). Their biochemical indexes, anthropometric indicators, and inflammatory markers were determined. The total genomic DNA was extracted for 16S ribosomal DNA sequencing. Furthermore, bioinformatics analysis was employed for functional analysis.ResultsAnthropometric indicators, including handgrip strength, mid-upper arm circumference, mid-upper arm muscle circumference, and body mass index, in the HD and PD groups showed a positive correlation with butyric acid-producing bacteria (Rosella and Phascolarctobacterium) and a negative correlation with conditional pathogens (Escherichia spp.). Meanwhile, the inflammatory markers, including high-sensitivity C-reactive protein and interleukin-6, were significantly higher in the PD-protein–energy wasting (PEW) group than in the PD-non-protein–energy wasting (NPEW) group; although they showed an increasing trend in the HD-PEW group, no significant difference was noted. Rosella was considerably scarce in the HD-PEW group than in the HD-NPEW group, whereas Escherichia was substantially more abundant in the PD-PEW group than in the PD-NPEW group. Compared with the HD group, the essential amino acid synthesis pathway, amino acid metabolism-related enzyme pathways, and aminoacyl-transfer RNA biosynthesis pathways were weakened in the PD group. Most carbohydrate metabolic pathways were weakened, although the tricarboxylic acid cycle was slightly enhanced. Concurrently, the fatty acid metabolism was enhanced, whereas fatty acid synthesis was weakened; the metabolic pathways of B vitamins were also weakened. These potential metabolic pathways of the various compounds released by intestinal flora showed a significant correlation with blood biochemical indexes, anthropometric indicators, and inflammatory markers.ConclusionIn patients with ESRD, different dialysis treatments affected the abundance of butyric acid-producing bacteria (Rosella and Phascolarctobacterium) and conditional pathogens (Escherichia spp.). Butyric acid-producing bacteria showed a positive correlation with PEW and showed a negative correlation with Escherichia. Improving the intestinal diversity and increasing the amount of butyric acid-producing bacteria, such as Blautella, Faecococcus, and Phascolarctobacterium, are potential therapeutic approaches to enhance protein–energy consumption in patients with ESRD.

Blocking the 2,3-butanediol synthesis pathway of Klebsiella pneumoniae resulted in L-valine production.

Klebsiella pneumoniae is a 2,3-butanediol producing bacterium. Nevertheless, a design and construction of L-valine production strain was studied in this paper. The first step of 2,3-butanediol synthesis and branched-chain amino acid synthesis pathways share the same step of α-acetolactate synthesis from pyruvate. However, the two pathways are existing in parallel and do not interfere with each other in the wild-type strain. A knockout of budA blocked the 2,3-butanediol synthesis pathway and resulted in the L-valine production. The budA coded an α-acetolactate decarboxylase and catalyzed the acetoin formation from α-acetolactate. Furthermore, blocking the lactic acid synthesis by knocking out of ldhA, which is encoding a lactate dehydrogenase, improved the L-valine synthesis. 2-Ketoisovalerate is the precursor of L-valine, it is also an intermediate of the isobutanol synthesis pathway, while indole-3-pyruvate decarboxylase (ipdC) is responsible for isobutyraldehyde formation from 2-ketoisovalerate. Production of L-valine has been improved by knocking out of ipdC. On the other side, the ilvE, encoding a transaminase B, reversibly transfers one amino group from glutamate to α-ketoisovalerate. Overexpression of ilvE exhibited a distinct improvement of L-valine production. The brnQ encodes a branched-chain amino acid transporter, and L-valine production was further improved by disrupting brnQ. It is also revealed that weak acidic and aerobic conditions favor L-valine production. Based on these findings, L-valine production by metabolically engineered K. pneumonia was examined. In fed-batch fermentation, 22.4g/L of L-valine was produced by the engineered K. pneumoniae ΔbudA-ΔldhA-ΔipdC-ΔbrnQ-ilvE after 55h of cultivation, with a substrate conversion ratio of 0.27mol/mol glucose.

Insights from shotgun metagenomics into bacterial species and metabolic pathways associated with NAFLD in obese youth.

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease and is often the precursor for more serious liver conditions such as nonalcoholic steatohepatitis and cirrhosis. Although the gut microbiome has been implicated in the development of NAFLD, the strong association of obesity with NAFLD and its effect on microbiome structure has made interpreting study outcomes difficult. In the present study, we examined the taxonomic and functional differences between the microbiomes of youth with obesity and with and without NAFLD. Shotgun metagenome sequencing was performed to profile the microbiomes of 36 subjects, half of whom were diagnosed with NAFLD using abdominal magnetic resonance imaging. Beta diversity analysis showed community‐wide differences between the groups (p = 0.002). Specific taxonomic differences included increased relative abundances of the species Fusicatenibacter saccharivorans (p = 0.042), Romboutsia ilealis (p = 0.046), and Actinomyces sp. ICM47 (p = 0.0009), and a decrease of Bacteroides thetaiotamicron (p = 0.0002), in the NAFLD group as compared with the non‐NAFLD group. At the phylum level, Bacteroidetes (p < 0.0001) was decreased in the NAFLD group. Functionally, branched‐chain amino acid (p = 0.01343) and aromatic amino acid (p = 0.01343) synthesis pathways had increased relative abundances in the NAFLD group along with numerous energy use pathways, including pyruvate fermentation to acetate (p = 0.01318). Conclusion: Community‐wide differences were noted based on NAFLD status, and individual bacterial species along with specific metabolic pathways were identified as potential drivers of these differences. The results of the present study support the idea that the NAFLD phenotype displays a differentiated microbial and functional signature from the obesity phenotype.

Combined Analysis of the Metabolome and Transcriptome to Explore Heat Stress Responses and Adaptation Mechanisms in Celery (Apium graveolens L.).

Celery is an important leafy vegetable that can grow during the cool season and does not tolerate high temperatures. Heat stress is widely acknowledged as one of the main abiotic stresses affecting the growth and yield of celery. The morphological and physiological indices of celery were investigated in the present study to explore the physiological mechanisms in response to high temperatures. Results showed that the antioxidant enzyme activity, proline, relative conductivity, and malondialdehyde were increased, while chlorophyll and the water content of leaves decreased under high-temperature conditions. Short-term heat treatment increased the stomatal conductance to cool off the leaves by transpiration; however, long-term heat treatment led to stomatal closure to prevent leaf dehydration. In addition, high temperature caused a disordered arrangement of palisade tissue and a loose arrangement of spongy tissue in celery leaves. Combined metabolomic and transcriptomic analyses were further used to reveal the regulatory mechanisms in response to heat stress at the molecular level in celery. A total of 1003 differential metabolites were identified and significantly enriched in amino acid metabolism and the tricarboxilic acid (TCA) cycle. Transcriptome sequencing detected 24,264 different genes, including multiple transcription factor families such as HSF, WRKY, MYB, AP2, bZIP, and bHLH family members that were significantly upregulated in response to heat stress, suggesting that these genes were involved in the response to heat stress. In addition, transcriptional and metabolic pathway analyses showed that heat stress inhibited the glycolysis pathway and delayed the TCA cycle but increased the expression of most amino acid synthesis pathways such as proline, arginine, and serine, consistent with the results of physiological indicators. qRT-PCR further showed that the expression pattern was similar to the expression abundance in the transcriptome. The important metabolites and genes in celery that significantly contributed to the response to high temperatures were identified in the present study, which provided the theoretical basis for breeding heat-resistant celery.

Integrated Analysis of Metabolome and Transcriptome of Bambusa emeiensis Shoots in Response to Bamboo Snout Beetle Cyrtotrachelus buqueti (Coleoptera: Curculionidae)

Plants have evolved constitutive and inductive defense mechanisms to protect themselves from herbivorous insects. Metabolites in plants are thought to be involved in response to herbivores. Bambusa emeiensis is an important bamboo species widely distributed in Southwest China. It is easy to attract herbivores such as Cyrtotrachelus buqueti. Through the detection and analysis of metabolites in B. emeiensis metabolomic data, 35 differential metabolites (biomarkers) were finally identified from 206 detection peaks, mainly primary metabolites. Subsequently, we conducted an association analysis between 35 biomarkers that annotated to be involved in 71 metabolic pathways in the metabolome and 324 differentially expressed genes (DEGs) in 207 gene expression pathways distributed in the transcriptome of B. emeiensis after feeding by C. buqueti. We also discussed the relationship between the changes in gene expression levels and metabolite content variation. A total of 47 integrated pathways containing the corresponding DEGs and biomarkers were screened out, with the amino acid synthesis pathway (ko01230) containing the most DEGs and biomarkers. In these integrated pathways, the changes in biomarkers content and the expression levels of the corresponding genes were generally consistent. For example, the increase in tryptophan content was accompanied by an increase in the expression of the corresponding catalase in the tryptophan synthesis pathway. Similar to this was glucose and trehalose in carbohydrate metabolism. Therefore, this study further deepened our understanding of the defense mechanism of B. emeiensis against bamboo pests and provided new insights for the prevention and control of bamboo diseases and pests.

Carbon flux through photosynthesis and central carbon metabolism show distinct patterns between algae, C3 and C4 plants

Photosynthesis-related pathways are regarded as a promising avenue for crop improvement. Whilst empirical studies have shown that photosynthetic efficiency is higher in microalgae than in C3 or C4 crops, the underlying reasons remain unclear. Using a tailor-made microfluidics labelling system to supply 13CO2 at steady state, we investigated in vivo labelling kinetics in intermediates of the Calvin Benson cycle and sugar, starch, organic acid and amino acid synthesis pathways, and in protein and lipids, in Chlamydomonas reinhardtii, Chlorella sorokiniana and Chlorella ohadii, which is the fastest growing green alga on record. We estimated flux patterns in these algae and compared them with published and new data from C3 and C4 plants. Our analyses identify distinct flux patterns supporting faster growth in photosynthetic cells, with some of the algae exhibiting faster ribulose 1,5-bisphosphate regeneration and increased fluxes through the lower glycolysis and anaplerotic pathways towards the tricarboxylic acid cycle, amino acid synthesis and lipid synthesis than in higher plants.

Adjunctive Probiotic Lactobacillus rhamnosus Probio-M9 Administration Enhances the Effect of Anti-PD-1 Antitumor Therapy via Restoring Antibiotic-Disrupted Gut Microbiota.

Emerging evidence supports that the efficacy of immune checkpoint blockade (ICB) therapy is associated with the host’s gut microbiota, as prior antibiotic intake often leads to poor outcome and low responsiveness toward ICB treatment. Therefore, we hypothesized that the efficacy of ICB therapy like anti-programmed cell death protein-1 (PD-1) treatment required an intact host gut microbiota, and it was established that probiotics could enhance the recovery of gut microbiota disruption by external stimuli. Thus, the present study aimed to evaluate the effect of the probiotics, Lactobacillus rhamnosus Probio-M9, on recovering antibiotic-disrupted gut microbiota and its impact on the outcome of ICB therapy in tumor-bearing mice. We first disrupted the mouse microbiota by antibiotics and then remediated the gut microbiota by probiotics or naturally. Tumor transplantation was then performed, followed by anti-PD-1-based antitumor therapy. Changes in the fecal metagenomes and the tumor suppression effect were monitored during different stages of the experiment. Our results showed that Probio-M9 synergized with ICB therapy, significantly improving tumor inhibition compared with groups not receiving the probiotic treatment (P < 0.05 at most time points). The synergistic effect was accompanied by effective restoration of antibiotic-disrupted fecal microbiome that was characterized by a drastically reduced Shannon diversity value and shifted composition of dominating taxa. Moreover, probiotic administration significantly increased the relative abundance of beneficial bacteria (e.g., Bifidobacterium pseudolongum, Parabacteroides distasonis, and some Bacteroides species; 0.0001 < P < 0.05). The gut microbiome changes were accompanied by mild reshaping of the functional metagenomes characterized by enrichment in sugar degradation and vitamin and amino acid synthesis pathways. Collectively, this study supported that probiotic administration could enhance the efficacy and responsiveness of anti-PD-1-based immunotherapy, and Probio-M9 could be a potential candidate of microbe-based synergistic tumor therapeutics. The preclinical data obtained here would support the design of future human clinical trials for further consolidating the current findings and for safety assessment of probiotic adjunctive treatment in ICB therapy.

Yeast interaction on Chardonnay wine composition: Impact of strain and inoculation time

It is of great importance to understand the molecular characteristics and substantial chemical transformations due to yeast-yeast interaction. Non-targeted metabolomics was used to unravel must in fermentation composition, inoculated with non-Saccharomyces (NS) yeasts and Saccharomyces cerevisiae (S) for sequential fermentation. ultrahigh-resolution mass spectrometry was able to distinguish thousands of metabolites and provides deep insights into grape must composition allowing better understanding of the yeast-yeast interactome. The dominance of S, characterized by a metabolic richness not found with NS, is dependent on inoculation time and on the yeast species present. Co-inoculation leads to the formation of new compounds, reflecting a reshuffling of yeast metabolism linked to interaction mechanisms. Among the modifications observed, metabolomic unravels deep changes in nitrogen metabolism due to yeast-yeast interactions and suggests that the redistribution pattern affects two different routes, the pentose phosphate and the amino acid synthesis pathways.

Comparison of the Nutritional Properties and Transcriptome Profiling Between the Two Different Harvesting Periods of Auricularia polytricha.

Auricularia polytricha (A. polytricha), regarded as an edible and medical mushroom, has attracted toward the research interests because of the high nutrition and bioactivity. The nutritional and medical properties of A. polytricha have been well-studied; however, research about the difference of the nutritional properties and transcriptome profiling between the two different harvesting periods of A. polytricha was limited. In this study, the nutritional properties and transcriptome profiling were compared between the two different harvesting periods of A. polytricha: AP_S1 (the stage for the first harvesting period) and AP_S2 (the stage for the third harvesting period). This study showed that AP_S1 had the more growth advantages than AP_S2 including biomass, auricle area and thickness, protein and calcium contents, and most species of the amino acid contents, which contributed to the higher sensory evaluation and acceptability of AP_S1. Transcriptome profiling showed that a total of 30,298 unigenes were successfully annotated in the two different harvesting periods of A. polytricha. At a threshold of two-fold change, 1,415 and 3,213 unigenes were up- and downregulated, respectively. All the differentially expressed genes (DEGs) analysis showed that the some synthesis and metabolic processes were strengthened in AP_S1, especially the synthesis and metabolism of the amino acids and protein. The enhanced energy metabolism pathways could provide more energy for AP_S1 to synthesize the nutritional substance. Moreover, the expressions of 10 selected DEGs involved in the amino acid and protein synthesis pathways and energy metabolism pathways were higher in AP_S1 compared to AP_S2, consistent with Illumina analysis. To the best of our knowledge, this is the first study that compares the nutritional properties and transcriptome profiling between the two different harvesting periods of A. polytricha and the results can present insights into the growth and genetic characteristics of A. polytricha.

Gut microbiome in people living with HIV is associated with impaired thiamine and folate syntheses

People living with HIV have a high incidence of cardiovascular and neurological diseases as comorbid disorders that are commonly linked to inflammation. While microbial translocation can augment inflammation during HIV infection, functional microbiome shifts that may increase pro-inflammatory responses have not been fully characterized. In addition, defining HIV-induced microbiome changes has been complicated by high variability among individuals. Here we conducted functional annotation of previously-published 16S ribosomal RNA gene sequences of 305 HIV positive and 249 negative individuals, with adjustment for geographic region, sex, sexual behavior, and age. Metagenome profiles were inferred from these individuals' 16S data. HIV infection was associated with impaired microbial vitamin B synthesis; around half of the gene families in thiamine and folate biosynthesis pathways were significantly less abundant in the HIV positive group than the negative control. These results are consistent with the high prevalence of thiamine and folate deficiencies in HIV infections. These HIV-induced microbiota shifts have the potential to influence cardiovascular and neurocognitive diseases, given the documented associations between B-vitamin deficiencies, inflammation, and these diseases. We also observed that most essential amino acid biosynthesis pathways were downregulated in the microbiome of HIV-infected individuals. Microbial vitamin B and amino acid synthesis pathways were not significantly recovered by antiretroviral treatment when we compared 262 ART positive and 184 ART negative individuals. Our meta-analysis provides a new outlook for understanding vitamin B and amino acid deficiencies in HIV patients, suggesting that interventions for reversing HIV-induced microbiome shifts may aid in lessening the burdens of HIV comorbidities.

Construction of the Five-point Mutant in Novel Aspartokinase and Its Enzymatic Characterization from Corynebacterium pekinense

Purpose: To improve the activity of the first key allosteric enzyme-aspartate kinase (AK) in the aspartate amino acid synthesis pathway, and to weaken or remove the terminal products lysine (Lys) and threonine (Thr) synergistic feed back inhibition of AK. Methods: On this basis of the four-point mutant T379N/A380C/G171I/Y198N AK(NCIN AK) of Corynebacterium pekinense, it was found that the highly conserved mutation sites of Gly295 was bound with inhibitor Thr by hydrogen bonds. The site of Gly295 were selected for site-directed mutation. The plasmid was extracted and transformed into E. coli competent BL21 to induce expression. High-throughput screening was used to obtain mutant strains with significantly improved enzyme activity. Enzyme kinetic study and characterization of enzyme properties were performed on the wild type(WT) and the five-point mutant T379N/A380C/G171I/Y198N/G295L AK(NCINL AK). Results: The five-point mutant NCINL AK was successfully constructed, and its maximum reaction rate Vmax was 259 U/(mg·min). The Km value of the Michaelis constant of the mutant reduced from 3.44 mmol/L to 0.93 mmol/L, and the substrate affinity was enhanced compared with the wild type enzyme. The hill coefficient n value was reduced from 2.73 to 1.21, and the positive synergy was decreased. The optimum temperature of NCINL AK increased from 25 ℃ to 28 ℃, the optimum pH value increased from 8.0 to 8.5, and the half-life was extended from 4.7 h to 5.2 h. Compared with the wild type WT AK, the five-point mutant NCINL AK had different inhibitory effects at different substrate inhibitors with concentration of 0.2, 1.0, 5.0, 10.0 mmol/L and even the inhibitory effect of Lys was completely lifted, and it had an activation effect under the condition of 10 mmol/L Thr. Conclusion: In this experiment, the five-point mutant NCINL AK with enzyme activity increased by 87.20 times was obtained, and the enzymatic properties were also significantly improved. Within a certain inhibitor concentration range, the feedback-inhibition effect of Lys on AK was basically lifted, and to a certain extent releasing Thr's feedback inhibition provided the possibility for the accumulation of large amounts of aspartate amino acids and provided reference for constructing high-yield aspartate amino acid strains. It was possible for us to get the pollution-free and high-efficiency amino acids such as methionine (Met).