Aromatic Amino Acid Catabolism Research Articles

Key bacterial taxa determine longitudinal dynamics of aromatic amino acid catabolism in infants’ gut

ABSTRACT The development of the gut microbiota in early life is linked to metabolic, neuronal, and immunological development. Recent studies have shown that bacterial production of short-chain fatty acids (SCFAs) and aromatic amino acid (AAA) catabolites in the gut can mediate host–microbe interactions. However, the dynamics of these microbiota-derived metabolites and the key bacterial taxa producing AAA catabolites during infancy are largely unknown. Here, we investigated the longitudinal dynamics of the microbiota and microbiota-derived SCFAs and AAA catabolites in more than 200 fecal samples from 25 healthy breast- or mixed-fed Danish infants during the first 6 months of life. We found that the gut microbiota composition and metabolism were highly individual but showed significant development over time. SCFAs and specific groups of AAA catabolites showed distinct temporal abundance patterns. Furthermore, we identified bacterial taxa responsible for the generation of AAA catabolites by associating the dynamics of gut microbial taxa and AAA catabolites and subsequently validating these associations in vitro by cultivation of strains representing the associated taxa. In addition to specific Bifidobacterium species being the main producers of aromatic lactic acids, we identified Peptostreptococcus anaerobius as the main producer of aromatic propionic acids, Ruminococcus gnavus as a main producer of tryptamine, and Enterococcus species as main tyramine producers in infants’ gut. Thus, our results showcase the temporal dynamics of key gut microbial metabolites in early life and demonstrate that the appearance and abundance of specific AAA catabolites result from the appearance and abundance of specific key bacterial taxa in infants’ gut.

Identification of candidate metabolite biomarkers for metabolic syndrome and its five components in population-based human cohorts

BackgroundMetabolic Syndrome (MetS) is characterized by risk factors such as abdominal obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol (HDL-C), hypertension, and hyperglycemia, which contribute to the development of cardiovascular disease and type 2 diabetes. Here, we aim to identify candidate metabolite biomarkers of MetS and its associated risk factors to better understand the complex interplay of underlying signaling pathways.MethodsWe quantified serum samples of the KORA F4 study participants (N = 2815) and analyzed 121 metabolites. Multiple regression models adjusted for clinical and lifestyle covariates were used to identify metabolites that were Bonferroni significantly associated with MetS. These findings were replicated in the SHIP-TREND-0 study (N = 988) and further analyzed for the association of replicated metabolites with the five components of MetS. Database-driven networks of the identified metabolites and their interacting enzymes were also constructed.ResultsWe identified and replicated 56 MetS-specific metabolites: 13 were positively associated (e.g., Val, Leu/Ile, Phe, and Tyr), and 43 were negatively associated (e.g., Gly, Ser, and 40 lipids). Moreover, the majority (89%) and minority (23%) of MetS-specific metabolites were associated with low HDL-C and hypertension, respectively. One lipid, lysoPC a C18:2, was negatively associated with MetS and all of its five components, indicating that individuals with MetS and each of the risk factors had lower concentrations of lysoPC a C18:2 compared to corresponding controls. Our metabolic networks elucidated these observations by revealing impaired catabolism of branched-chain and aromatic amino acids, as well as accelerated Gly catabolism.ConclusionOur identified candidate metabolite biomarkers are associated with the pathophysiology of MetS and its risk factors. They could facilitate the development of therapeutic strategies to prevent type 2 diabetes and cardiovascular disease. For instance, elevated levels of lysoPC a C18:2 may protect MetS and its five risk components. More in-depth studies are necessary to determine the mechanism of key metabolites in the MetS pathophysiology.

Tyrosol induces multiple drug resistance in yeast Saccharomyces cerevisiae.

In yeast, multiple (pleiotropic) drug resistance (MDR) transporters efflux xenobiotics from the cytoplasm to the environment. Additionally, upon the accumulation of xenobiotics in the cells, MDR genes are induced. At the same time, fungal cells can produce secondary metabolites with physico-chemical properties similar to MDR transporter substrates. Nitrogen limitation in yeast Saccharomyces cerevisiae leads to the accumulation of phenylethanol, tryptophol, and tyrosol, which are products of aromatic amino acid catabolism. In this study, we investigated whether these compounds could induce or inhibit MDR in yeast. Double deletion of PDR1 and PDR3 genes, which are transcription factors that upregulate the expression of PDR genes, reduced yeast resistance to high concentrations of tyrosol (4-6 g/L) but not to the other two tested aromatic alcohols. PDR5 gene, but not other tested MDR transporter genes (SNQ2, YOR1, PDR10, PDR15) contributed to yeast resistance to tyrosol. Tyrosol inhibited the efflux of rhodamine 6G (R6G), a substrate for MDR transporters. However, preincubating yeast cells with tyrosol induced MDR, as evidenced by increased Pdr5-GFP levels and reduced yeast ability to accumulate Nile red, another fluorescent MDR-transporter substrate. Moreover, tyrosol inhibited the cytostatic effect of clotrimazole, the azole antifungal. Our results demonstrate that a natural secondary metabolite can modulate yeast MDR. We speculate that intermediates of aromatic amino acid metabolites coordinate cell metabolism and defense mechanisms against xenobiotics.

Strain-Level Dynamics Reveal Regulatory Roles in Atopic Eczema by Gut Bacterial Phages.

The vast population of bacterial phages or viruses (virome) plays pivotal roles in the ecology of human microbial flora and health conditions. Obstacles, including poor viral sequence inference, strain-sensitive virus-host relationship, and the high diversity among individuals, hinder the in-depth understanding of the human virome. We conducted longitudinal studies of the virome based on constructing a high-quality personal reference metagenome (PRM). By applying long-read sequencing for representative samples, we could build a PRM of high continuity that allows accurate annotation and abundance estimation of viruses and bacterial species in all samples of the same individual by aligning short sequencing reads to the PRM. We applied this approach to a series of fecal samples collected for 6 months from a 2-year-old boy who had experienced a 2-month flare-up of atopic eczema (dermatitis) in this period. We identified 31 viral strains in the patient's gut microbiota and deciphered their strain-level relationship to their bacterial hosts. Among them, a lytic crAssphage developed into a dozen substrains and coordinated downregulation in the catabolism of aromatic amino acids (AAAs) in their host bacteria which govern the production of immune-active AAA derivates. The metabolic alterations confirmed based on metabolomic assays cooccurred with symptom remission. Our PRM-based analysis provides an easy approach for deciphering the dynamics of the strain-level human gut virome in the context of entire microbiota. Close temporal correlations among virome alteration, microbial metabolism, and disease remission suggest a potential mechanism for how bacterial phages in microbiota are intimately related to human health. IMPORTANCE The vast populations of viruses or bacteriophages in human gut flora remain mysterious. However, poor annotation and abundance estimation remain obstacles to strain-level analysis and clarification of their roles in microbiome ecology and metabolism associated with human health and diseases. We demonstrate that a personal reference metagenome (PRM)-based approach provides strain-level resolution for analyzing the gut microbiota-associated virome. When applying such an approach to longitudinal samples collected from a 2-year-old boy who has experienced a 2-month flare-up of atopic eczema, we observed thriving substrains of a lytic crAssphage, showing temporal correlation with downregulated catabolism of aromatic amino acids, lower production of immune-active metabolites, and remission of the disease. The PRM-based approach is practical and powerful for strain-centric analysis of the human gut virome, and the underlying mechanism of how strain-level virome dynamics affect disease deserves further investigation.

Microbial paracetamol degradation involves a high diversity of novel amidase enzyme candidates

Pharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 h while excreting 4-aminophenol, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and the Pseudomonas spp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs.

Development and validation of a sensitive and reliable targeted metabolomics method for the quantification of cardiovascular disease-related biomarkers in plasma using ultrahigh-performance liquid chromatography-tandem mass spectrometry.

Cardiovascular disease, as a multifactorial disease caused by genetics and environment, has emerged as a leading cause of mortality. The discovery of metabolic biomarkers for the clinical diagnosis, early warning and elucidation of the molecular pathogenesis of cardiovascular disease, using metabolomics, has attracted broad interest. Therefore, this work aimed to develop a sensitive and reliable targeted metabolomics method for the quantification of cardiovascular disease-related biomarkers in plasma. The method was developed and validated using ultrahigh-performance liquid chromatography augmented with tandem mass spectrometry (UHPLC/MS/MS). The LC conditions and MS parameters were optimized using selected reaction monitoring scanning mode to high-throughput and sensitive separation, and could detect 20 metabolic biomarkers in a single experiment. And the linearity, selectivity, accuracy, precision, stability and recovery of the developed method were assessed according to the Bioanalytical Method Validation guidelines of the United States Food and Drug Administration. These quantified metabolic biomarkers are involved in pathways such as aromatic amino acid catabolism (e.g. phenylalanine, tryptophan, tyrosine), trimethylamine N-oxide (TMAO) biosynthesis (e.g. TMAO, choline, carnitine, betaine) and histidine metabolism (e.g. histidine), among others. All analytes exhibited excellent linearities with coefficients of determination greater than 0.99. Accuracies deviated by less than 15% for medium- and high-concentration samples and less than 20% for low-concentration samples, with intra- and inter-day precisions of 1.12-14.12% and 0.30-13.74%, respectively. Recoveries and stabilities also met the analysis requirements of biological samples. The targeted metabolomics method was shown to have a powerful ability to accurately analyze metabolic biomarkers, thereby providing valuable information for large-scale biomarker validation and clarifying the potential material basis of cardiovascular disease for clinical diagnosis or early warning.

Computational modeling of the gut microbiota reveals putative metabolic mechanisms of recurrent Clostridioides difficile infection.

Approximately 30% of patients who have Clostridioides difficile infection (CDI) will suffer at least one incident of reinfection. While the underlying causes of CDI recurrence are poorly understood, interactions between C. difficile and commensal gut bacteria are thought to play an important role. In this study, an in silico pipeline was used to process 16S rRNA gene amplicon sequence data of 225 stool samples from 93 CDI patients into sample-specific models of bacterial community metabolism. Clustered metabolite production rates generated from post-diagnosis samples generated a high Enterobacteriaceae abundance cluster containing disproportionately large numbers of recurrent samples and patients. This cluster was predicted to have significantly reduced capabilities for secondary bile acid synthesis but elevated capabilities for aromatic amino acid catabolism. When applied to 16S sequence data of 40 samples from fecal microbiota transplantation (FMT) patients suffering from recurrent CDI and their stool donors, the community modeling method generated a high Enterobacteriaceae abundance cluster with a disproportionate large number of pre-FMT samples. This cluster also was predicted to exhibit reduced secondary bile acid synthesis and elevated aromatic amino acid catabolism. Collectively, these in silico predictions suggest that Enterobacteriaceae may create a gut environment favorable for C. difficile spore germination and/or toxin synthesis.

Elucidating aromatic acid tolerance at low pH in Saccharomyces cerevisiae using adaptive laboratory evolution

Toxicity from the external presence or internal production of compounds can reduce the growth and viability of microbial cell factories and compromise productivity. Aromatic compounds are generally toxic for microorganisms, which makes their production in microbial hosts challenging. Here we use adaptive laboratory evolution to generate Saccharomyces cerevisiae mutants tolerant to two aromatic acids, coumaric acid and ferulic acid. The evolution experiments were performed at low pH (3.5) to reproduce conditions typical of industrial processes. Mutant strains tolerant to levels of aromatic acids near the solubility limit were then analyzed by whole genome sequencing, which revealed prevalent point mutations in a transcriptional activator (Aro80) that is responsible for regulating the use of aromatic amino acids as the nitrogen source. Among the genes regulated by Aro80, ESBP6 was found to be responsible for increasing tolerance to aromatic acids by exporting them out of the cell. Further examination of the native function of Esbp6 revealed that this transporter can excrete fusel acids (byproducts of aromatic amino acid catabolism) and this role is shared with at least one additional transporter native to S. cerevisiae (Pdr12). Besides conferring tolerance to aromatic acids, ESBP6 overexpression was also shown to significantly improve the secretion in coumaric acid production strains. Overall, we showed that regulating the activity of transporters is a major mechanism to improve tolerance to aromatic acids. These findings can be used to modulate the intracellular concentration of aromatic compounds to optimize the excretion of such products while keeping precursor molecules inside the cell.

Effects of Azole Fungicides on Secreted Metabolomes of Botrytis cinerea.

Botrytis cinerea, gray mold, is one of the most notorious phytopathgens, causing serious economic loss in the agricultural industry. The phytotoxic effects are mainly derived from secreted virulence proteins and terpenoid-type secondary metabolites. Azole fungicides are commonly used to manage the disease. However, their biochemical effects other than sterol biosynthesis were not documented, especially toxic secreted metabolites. In this study, six azole fungicides were treated with in vitro and in vivo conditions. Comprehensive profiles of primary and secondary metabolites in culture media were evaluated to assess the fungal metabolomes under pesticide-stressed conditions. The results indicated that extensive metabolic differentiation was induced by azole fungicides. Epoxiconazole clearly reduced the extracellular phytotoxin concentrations, while the level of indole-3-acetic acid was increased. In addition, significant differentiation of primary metabolism could be deduced from secreted metabolite profiles, including the tricarboxylic acid cycle and aromatic amino acid catabolism. Cellular lipid profiles, including fatty acids and sterol, have been altered drastically by azoles, which indicate extensive changes of cellular lipid metabolism. These system-wide metabolic alterations resulted in reduced plant damages, proven by the in vivo assay with tomato. Overall, azole fungicides induced significant changes of endo- and exometabolomes and could reduce the fungal infection. The experimental results will provide a more detailed understanding of physiological changes of phytopathogens under pesticide treatment and information for new pesticide development.

Metabolomics Associated with Genome-Wide Association Study Related to the Basal Metabolic Rate in Overweight/Obese Korean Women.

A previous genome-wide association study (GWAS) on obese/overweight Korean women reported five new genetic loci associated with the basal metabolic rate (BMR) and body mass index (BMI), NRG3, OR8U8, BCL2L2-PABPN1, PABPN1, and SLC22A17. This metabolite GWAS (mGWAS) aimed to identify the key metabolites and metabolic pathways regulated by these genes. Potential metabolic pathways associated with leanness and obesity were identified by detecting metabolites in association with GWAS. Waist circumference, lean body mass, and body fat mass were strongly associated with BMI rather than BMR. However, plasma triglyceride and total cholesterol were significantly higher in obese individuals with low BMR than in lean individuals with high BMR. Upon analyzing NRG3, BCL2L2-PABPN1, and SLC22A17, uric acid, succinic acid, arginine, uridine, and aspartic acid were the metabolites positively associated with obesity. Uric acid and arginine were both identified through general metabolomics targeting of obesity genes classified on the basis of BMI or BMR. Metabolites associated with disruption in beta-oxidation, lipid metabolism, branched-chain amino acid and aromatic amino acid catabolism, the urea cycle, and purine/pyrimidine metabolism play important roles in obesity classified on the basis of either BMI or BMR in middle-aged Korean women. These results further the current understanding of obesity and the predictability of obesity-related risks using mGWAS.

Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans.

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2 Å resolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

Versatility of putative aromatic aminotransferases from Candida albicans

Amino acids constitute the key sources of nitrogen for growth of Candida albicans. In order to survive inside the host in different and rapidly changing environments, this fungus must be able to adapt via its expression of genes for amino acid metabolism. We analysed the ARO8, ARO9, YER152C, and BNA3 genes with regards to their role in the nutritional flexibility of C. albicans. CaAro8p is undoubtedly the most versatile enzyme among the aminotransferases investigated. It is involved in the catabolism of histidine, lysine, and aromatic amino acids as well as in l-Lys, Phe and Tyr biosynthesis. CaAro9p participates in the catabolism of aromatic amino acids and lysine at high concentrations of these compounds, with no biosynthetic role. Conversely, the CaYer152Cp catalytic potential for aromatic amino acid catabolism observed in vitro appears to be of little importance in vivo. Neither biosynthetic nor catabolic roles of CaBan3p were observed for any proteinogenic amino acid. Finally, none of the analysed aminotransferases was solely responsible for the catabolism of a single particular amino acid or its biosynthesis.

Oxidative Stickland reactions in an obligate aerobic organism - amino acid catabolism in the Crenarchaeon Sulfolobus solfataricus.

The thermoacidophilic Crenarchaeon Sulfolobus solfataricus is a model organism for archaeal adaptation to extreme environments and renowned for its ability to degrade a broad variety of substrates. It has been well characterised concerning the utilisation of numerous carbohydrates as carbon source. However, its amino acid metabolism, especially the degradation of single amino acids, is not as well understood. In this work, we performed metabolic modelling as well as metabolome, transcriptome and proteome analysis on cells grown on caseinhydrolysate as carbon source in order to draw a comprehensive picture of amino acid metabolism in S. solfataricus P2. We found that 10 out of 16 detectable amino acids are imported from the growth medium. Overall, uptake of glutamate, methionine, leucine, phenylalanine and isoleucine was the highest of all observed amino acids. Our simulations predict an incomplete degradation of leucine and tyrosine to organic acids, and in accordance with this, we detected the export of branched-chain and aromatic organic acids as well as amino acids, ammonium and trehalose into the culture supernatants. The branched-chain amino acids as well as phenylalanine and tyrosine are degraded to organic acids via oxidative Stickland reactions. Such reactions are known for prokaryotes capable of anaerobic growth, but so far have never been observed in an obligate aerobe. Also, 3-methyl-2-butenoate and 2-methyl-2-butenoate are for the first time found as products of modified Stickland reactions for the degradation of branched-chain amino acids. This work presents the first detailed description of branched-chain and aromatic amino acid catabolism in S. solfataricus.

Reduced Glucose Sensation Can Increase the Fitness of Saccharomyces cerevisiae Lacking Mitochondrial DNA.

Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction.

Aromatic Amino Acid Network: Biosynthesis, Regulation and Transport

Aromatic amino acids are essential constituents of proteins in all living organisms, while in plants phenylalanine (Phe) and tyrosine (Tyr) additionally serve as precursors for thousands of specialized compounds that have a profound impact on plant growth, development, reproduction and defense. Plants direct 20‐30% of photosynthetically fixed carbon to the production of Phe and Phe‐derived compounds, which constitute approximately 30‐45% of plant organic matter. Humans are incapable of synthesizing Phe and must primarily obtain it directly or indirectly from plants. Phe biosynthesis in plants occurs via two alternative routes with either arogenate or phenylpyruvate as a key intermediate. Using petunia flowers as a model system, we have shown that plants predominantly synthesize Phe in plastids via the arogenate pathway while they can also utilize the microbial‐like phenylpyruvate pathway. Moreover, flux through the phenylpyruvate route is increased when the entry point to the arogenate pathway is limiting. Interestingly, the phenylpyruvate pathway utilizes a cytosolic aminotransferase that strongly favors Tyr as the amino donor. These results demonstrate that Phe biosynthesis is not limited to plastids and that there is an interconnection between aromatic amino acid catabolism and biosynthesis in planta. Another aspect of plant metabolism to be discussed is its compartmentalization and metabolite transport within the cell and how this intracellular transport contributes to the interconnections between and within metabolic networks.

Promoters inducible by aromatic amino acids and γ-aminobutyrate (GABA) for metabolic engineering applications in Saccharomyces cerevisiae.

A wide range of promoters with different strengths and regulatory mechanisms are valuable tools in metabolic engineering and synthetic biology. While there are many constitutive promoters available, the number of inducible promoters is still limited for pathway engineering in Saccharomyces cerevisiae. Here, we constructed aromatic amino-acid-inducible promoters based on the binding sites of Aro80 transcription factor, which is involved in the catabolism of aromatic amino acids through transcriptional activation of ARO9 and ARO10 genes in response to aromatic amino acids. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. Using low and high copy number plasmid vectors and different tryptophan concentrations, a 29-fold range of fluorescence intensities of enhanced green fluorescent protein (EGFP) reporter could be achieved from a synthetic U4C ARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element. The U4C ARO9 promoter was applied to express alsS and alsD genes from Bacillus subtilis for acetoin production in S. cerevisiae, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, we demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter, regulated by Uga3, can also be used in metabolic engineering as a dose-dependent inducible promoter. The wide range of controllable expression levels provided by these tryptophan- and GABA-inducible promoters might contribute to fine-tuning gene expression levels and timing for the optimization of pathways in metabolic engineering.

Alterations of urinary metabolite profile in model diabetic nephropathy

Countering the diabetes pandemic and consequent complications, such as nephropathy, will require better understanding of disease mechanisms and development of new diagnostic methods. Animal models can be versatile tools in studies of diabetic renal disease when model pathology is relevant to human diabetic nephropathy (DN). Diabetic models using endothelial nitric oxide synthase (eNOS) knock-out mice develop major renal lesions characteristic of human disease. However, it is unknown whether they can also reproduce changes in urinary metabolites found in human DN. We employed Type 1 and Type 2 diabetic mouse models of DN, i.e. STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db, with the goal of determining changes in urinary metabolite profile using proton nuclear magnetic resonance (NMR). Six urinary metabolites with significantly lower levels in diabetic compared to control mice have been identified. Specifically, major changes were found in metabolites from tricarboxylic acid (TCA) cycle and aromatic amino acid catabolism including 3-indoxyl sulfate, cis-aconitate, 2-oxoisocaproate, N-phenyl-acetylglycine, 4-hydroxyphenyl acetate, and hippurate. Levels of 4-hydroxyphenyl acetic acid and hippuric acid showed the strongest reverse correlation to albumin-to-creatinine ratio (ACR), which is an indicator of renal damage. Importantly, similar changes in urinary hydroxyphenyl acetate and hippurate were previously reported in human renal disease. We demonstrated that STZ-eNOS−/− C57BLKS and eNOS−/− C57BLKS db/db mouse models can recapitulate changes in urinary metabolome found in human DN and therefore can be useful new tools in metabolomic studies relevant to human pathology.

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source.

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium-chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched-chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO2 /O2 balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α-keto-acids and/or NADH pools. High-availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2-phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon-exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2-phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase.

Major contribution of the Ehrlich pathway for 2-phenylethanol/rose flavor production in Ashbya gossypii.

Aroma alcohols of fermented food and beverages are derived from fungal amino acids catabolism via the Ehrlich pathway. This linear pathway consists of three enzymatic reactions to form fusel alcohols. Regulation of some of the enzymes occurs on the transcriptional level via Aro80. The riboflavin overproducer Ashbya gossypii produces strong fruity flavours in contrast to its much less aromatic relative Eremothecium cymbalariae. Genome comparisons indicated that A.gossypii harbors genes for aromatic amino acid catabolism (ARO8a, ARO8b, ARO10, and ARO80) while E.cymbalariae only encodes ARO8a and thus lacks major components of aromatic amino acid catabolism. Volatile compound (VOC) analysis showed that both Eremothecium species produce large amounts of isoamyl alcohol while A.gossypii also produces high levels of 2-phenylethanol. Deletion of the A.gossypii ARO-genes did not confer any growth deficiencies. However, A.gossypii ARO-mutants (except Agaro8a) were strongly impaired in aroma production, particularly in the production of the rose flavour 2-phenylethanol. Conversely, overexpression of ARO80 via the AgTEF1 promoter resulted in 50% increase in VOC production. Together these data indicate that A.gossypii is a very potent flavour producer and that amongst the non-Saccharomyces biodiversity strains can be identified that could provide positive sensory properties to fermented beverages.

Activation of Aro80 transcription factor by heat-induced aromatic amino acid influx in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, transcription of ARO9 and ARO10 genes, involved in the catabolism of aromatic amino acids, is activated by Aro80 transcription factor in response to aromatic amino acids. Here we show that the transcription of ARO9 and ARO10 is also induced by heat shock in an Aro80-dependent manner. However, heat shock-related signaling pathways including PKA, PKC, and HOG pathways are not involved in the heat shock activation of Aro80. We elucidate that heat-induced increase in aromatic amino acid influx can lead to the inducer-dependent activation of Aro80 upon heat shock. Known aromatic amino acid permeases play an insignificant role in the heat-induced expression of ARO9 and ARO10, suggesting that an increase in plasma membrane fluidity might be responsible for the influx of aromatic amino acids during heat shock stress.