Tremella fuciformis Polysaccharides Inhibited Colonic Inflammation in Dextran Sulfate Sodium-Treated Mice via Foxp3+ T Cells, Gut Microbiota, and Bacterial Metabolites.

Tremella fuciformis is an edible medicinal mushroom, and its polysaccharide components are found to confer various health benefits. This study identified the protective effects of polysaccharides of Tremella fuciformis (TPs) against dextran sulfate sodium (DSS)-induced colitis in mice. High dose of TPs (HTPs) could prevent the colon from shortening, reduce activity of colonic myeloperoxidase and serum diamine oxidase (DAO), decrease the concentration of D-lactate, and alleviate the colonic tissue damage in colitic mice. HTPs treatment stimulated Foxp3+T cells, and promoted the production of anti-inflammatory cytokines whereas it reduced the production of pro-inflammatory and the portion of immunoglobulin A (IgA)-coated bacteria, which was related to modulation of immune responses. 16S rRNA sequencing analysis showed that TPs could significantly increase gut community diversity, and restore the relative abundances of Lactobacillus, Odoribacter, Helicobacter, Ruminococcaceae, and Marinifilaceae. According to metabolomic analysis, HTPs induced specific microbial metabolites akin to that in normal mice. Tyrosine biosynthesis, tryptophan metabolism, and bile acid metabolism were influenced in the HTPs group compared with those in the DSS group. HTPs could alleviate DSS-induced colitis by immunoregulation and restored the gut microbiota and microbial metabolites. The results indicated that HTPs have potential to be developed as a food supplement to ameliorate intestinal diseases.

Improving the Level of the Tyrosine Biosynthesis Pathway in Saccharomyces cerevisiae through HTZ1 Knockout and Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis.

In recent years, many studies have been conducted on the expression of multiple aromatic compounds by Saccharomyces cerevisiae. The concentration of l-tyrosine, as a precursor of such valuable compounds, is crucial for the biosynthesis of aromatic metabolites. In this study, a novel function of HTZ1 was found to be related to tyrosine biosynthesis, which has not yet been reported. Knockout of this gene could significantly improve the ability of yeast cells to synthesize tyrosine, and its p-coumaric acid (p-CA) titer was approximately 3.9-fold higher than that of the wild-type strain BY4742. Subsequently, this strain was selected for random mutagenesis through an emerging mutagenesis technique, namely, atmospheric and room temperature plasma (ARTP). After two rounds of mutagenesis, five tyrosine high-producing mutants were obtained. The highest production of p-CA was 7.6-fold higher than that of the wild-type strain. Finally, transcriptome data of the htz1Δ strain and the five mutants were analyzed. The genome of mutagenic strains was also resequenced to reveal the mechanism underlying the high titer of tyrosine. This system of target engineering combined with random mutagenesis to screen excellent mutants provides a new basis for synthetic biology.

Accessing p-Hydroxycinnamic Acids: Chemical Synthesis, Biomass Recovery, or Engineered Microbial Production?

p-Hydroxycinnamic acids (i. e., p-coumaric, ferulic, sinapic, and caffeic acids) are phenolic compounds involved in the biosynthesis pathway of lignin. These naturally occurring molecules not only exhibit numerous attractive properties, such as antioxidant, anti-UV, and anticancer activities, but they also have been used as building blocks for the synthesis of tailored monomers and functional additives for the food/feed, cosmetic, and plastics sectors. Despite their numerous high value-added applications, the sourcing of p-hydroxycinnamic acids is not ensured at the industrial scale except for ferulic acid, and their production cost remains too high for commodity applications. These compounds can be either chemically synthesized or extracted from lignocellulosic biomass, and recently their production through bioconversion emerged. Herein the different strategies described in the literature to produce these valuable molecules are discussed.

Microbial Upgrading of Acetate into Value-Added Products-Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches.

Ecological concerns have recently led to the increasing trend to upgrade carbon contained in waste streams into valuable chemicals. One of these components is acetate. Its microbial upgrading is possible in various species, with Escherichia coli being the best-studied. Several chemicals derived from acetate have already been successfully produced in E. coli on a laboratory scale, including acetone, itaconic acid, mevalonate, and tyrosine. As acetate is a carbon source with a low energy content compared to glucose or glycerol, energy- and redox-balancing plays an important role in acetate-based growth and production. In addition to the energetic challenges, acetate has an inhibitory effect on microorganisms, reducing growth rates, and limiting product concentrations. Moreover, extensive metabolic engineering is necessary to obtain a broad range of acetate-based products. In this review, we illustrate some of the necessary energetic considerations to establish robust production processes by presenting calculations of maximum theoretical product and carbon yields. Moreover, different strategies to deal with energetic and metabolic challenges are presented. Finally, we summarize ways to alleviate acetate toxicity and give an overview of process engineering measures that enable sustainable acetate-based production of value-added chemicals.

A laccase-catalysed tyrosine click reaction.

A laccase-catalysed tyrosine click reaction proceeded between the tyrosine modification reagent N-methyl luminol and tyrosine residues in peptides/proteins. Laccase-catalysed tyrosine-specific modification under mild reaction conditions (shaking at 37 °C) was more efficient than previously reported tyrosine click reactions using hemin, horseradish peroxidase (HRP) or electrochemistry.

Changes of para-, meta- and ortho-tyrosine over time in burned patients.

PURPOSE: Serious burn injury leads to oxidative stress resulting in production of meta- and ortho-tyrosine, while para-tyrosine is the physiological isoform. Our aim was to investigate the metabolism of these tyrosine isoforms following major burn injury. METHODS: Fifteen patients requiring intensive care were followed for 5 consecutive days after major burn injury. Serum and urine concentrations of para-, meta-, and ortho-tyrosine were measured with high performance liquid chromatography. Fifteen healthy matching individuals were invited as control group. RESULTS: Median serum concentration of normal isoform para-tyrosine decreased in burned patients between days 2 and 5 (p < 0.01). Mean meta-, and ortho-tyrosine levels were significantly higher in patients compared to controls in the same time period (p < 0.05). Renal excretion of para-tyrosine increased significantly in our observation period (p < 0.01). CONCLUSIONS: Pathologic isoforms of tyrosine accumulate in serum meanwhile the level of normal isoform decreases possibly due to belated enhanced renal excretion or, to decreased synthesis after major burn injury.

Construction of a switchable synthetic Escherichia coli for aromatic amino acids by a tunable switch.

Escherichia coli, a model microorganism for which convenient metabolic engineering tools are available and that grows quickly in cheap media, has been widely used in the production of valuable chemicals, including aromatic amino acids. As the three aromatic amino acids, L-tryptophan, L-tyrosine, and L-phenylalanine, share the same precursors, to increase the titer of a specific aromatic amino acid, the branch pathways to the others are usually permanently inactivated, which leads to the generation of auxotrophic strains. In this study, a tunable switch that can toggle between different states was constructed. Then, a switchable and non-auxotrophic E. coli strain for synthesis of aromatic amino acids was constructed using this tunable switch. By adding different inducers to cultures, three different production patterns of aromatic amino acids by the engineered strain could be observed. This tunable switch can also be applied in regulating other branch pathways and in other bacteria.

Comparative transcriptome analysis of genomic region deletion strain with enhanced L-tyrosine production in Saccharomyces cerevisiae.

OBJECTIVE: To determine the effect of large genomic region deletion in a Saccharomyces cerevisiae strain on tyrosine yield and to identify new genetic modification targets through transcriptome analysis. RESULTS: TAL was used to produce p-coumaric acid (p-CA) from tyrosine to quantity tyrosine yield. S. cerevisiae mutant strain NK14 with deletion of a 23.8 kb genomic region was identified to have p-CA production of 10.3 mg L- 1, while the wild-type strain BY4741 had p-CA production of 1.06 mg L- 1. Analysis of growth patterns and stress tolerance showed that the deletion did not affect the growth phenotype of NK14. Transcriptome analysis suggested that, compared to BY4741, genes related to glycolysis (ENO2, TKL1) and the tyrosine pathway (ARO1, ARO2, ARO4, ARO7, TYR1) were upregulated in NK14 at different levels. Besides genes related to the tyrosine biosynthetic pathway, amino acid transporters (AVT6, VBA5, THI72) and transcription factor (ARO80) also showed changes in transcription levels. CONCLUSIONS: We developed a strain with improved tyrosine yield and identified new genetic modification candidates for tyrosine production.

Metabolic engineering of Synechocystis sp. PCC 6803 for the production of aromatic amino acids and derived phenylpropanoids.

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ±â€¯53 mg/gDW (580 ±â€¯34 mg/L) of phenylalanine and 64 ±â€¯3.7 mg/gDW (41 ±â€¯2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ±â€¯70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ±â€¯31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ±â€¯13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.

Role of aspirin-triggered lipoxin A4, aspirin, and salicylic acid in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia.

PROBLEM: Oxidative stress and inflammation are key events leading to pre-eclampsia, involved in several maternal deaths. Low doses of acetylsalicylic acid (ASA) are used in the prevention and treatment of pre-eclampsia. The synthesis of aspirin-triggered lipoxin (ATL) by cyclooxygenase-2 acetylation is an alternative mechanism of ASA, which could explain the effectiveness of ASA treatments. The aim of this study was to evaluate the role of ASA, salicylates, and ATL in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia. METHOD OF STUDY: Plasma from 14 women with pre-eclampsia and 17 normotensive pregnant women was probed for inducing oxidative and inflammatory responses on endothelial cells and U937 promonocytes. The role of ATL, ASA, and salicylic acid (SA) on these events was evaluated. RESULTS: Plasma from women with pre-eclampsia induced TBARS and nitrotyrosine production on endothelial and U937 cells. Pre-treatment with both ATL and ASA decreased the TBARS production, while ATL decreased the nitrotyrosine. Pre-eclamptic plasma augmented the translocation of NF-kB on U937 cells, which decreased by a high dose of ASA and SA. Finally, the pre-eclamptic plasma increased the adhesion of leukocytes-PMN and monocytes-to endothelium, and we were able to determine a state of resolution of inflammation, since ATL decreased the PMN adhesion, and conversely, it increased the monocytes adhesion to endothelium. CONCLUSION: Together, these results suggest that ATL could explain the beneficial actions of ASA and support further research on mechanisms, real efficacy, and rational use of ASA in pre-eclampsia.

In vivo biosynthesis of tyrosine analogs and their concurrent incorporation into a residue-specific manner for enzyme engineering.

Herein we report the development of an efficient cellular system for the in vivo biosynthesis of Tyr-analogs and their concurrent incorporation into target proteins by the residue-specific approach. This system makes use of common phenol derivatives and the tyrosine phenol lyase machinery to create various tyrosine analogues that impart desired properties on the target proteins. Biosynthesized 2-fluorotyrosine was incorporated into three industrially important enzymes which resulted in enhanced thermostability.

Unraveling the specific regulation of the shikimate pathway for tyrosine accumulation in Bacillus licheniformis.

L-Tyrosine serves as a common precursor for multiple valuable secondary metabolites. Synthesis of this aromatic amino acid in Bacillus licheniformis occurs via the shikimate pathway, but the underlying mechanisms involving metabolic regulation remain unclear. In this work, improved L-tyrosine accumulation was achieved in B. licheniformis via co-overexpression of aroGfbr and tyrAfbr from Escherichia coli to yield strain 45A12, and the L-tyrosine titer increased to 1005 mg/L with controlled glucose feeding. Quantitative RT-PCR results indicated that aroA, encoding DAHP synthase, and aroK, encoding shikimate kinase, were feedback-repressed by the end product L-tyrosine in the modified strain. Therefore, the native aroK was first expressed with multiple copies to yield strain 45A13, which could accumulate 1201 mg/L L-tyrosine. Compared with strain 45A12, the expression of aroB and aroF in strain 45A13 was upregulated by 21% and 27%, respectively, which may also have resulted in the improvement of L-tyrosine production. Furthermore, supplementation with 5 g/L shikimate enhanced the L-tyrosine titers of 45A12 and 45A13 by 29.1% and 24.0%, respectively. However, the yield of L-tyrosine per unit of shikimate decreased from 0.365 to 0.198 mol/mol after aroK overexpression in strain 45A12, which suggested that the gene product was also involved in uncharacterized pathways. This study provides a good starting point for further modification to achieve industrial-scale production of L-tyrosine using B. licheniformis, a generally recognized as safe workhorse.

How Legionella defend their turf.

Communities of bacteria that cause Legionnaires' disease repel other bacteria by secreting an acid called HGA.

Production of clovamide and its analogues in Saccharomyces cerevisiae and Lactococcus lactis.

Clovamide and its analogues are N-hydroxycinnamoyl-L-amino acids (HAA) that exhibit antioxidant activities. For environmental and economic reasons, biological synthesis of these plant-derived metabolites has garnered interest. In this study, we exploited HDT1, a BAHD acyltransferase recently isolated from red clover, for the production of clovamide and derivatives in S. cerevisiae and L. lactis. HDT1 catalyses the transfer of hydroxycinnamoyl-coenzyme A (CoA) onto aromatic amino acids. Therefore, by heterologously co-expressing HDT1 with 4-coumarate:CoA ligase (4CL), we succeeded in the biological production of clovamide and more than 20 other HAA, including halogenated ones, upon feeding the engineered micro-organisms with various combinations of cinnamates and amino acids. To the best of our knowledge, this is the first report on the biological synthesis of HAA and, more generally, on the synthesis of plant-derived antioxidant phenolic compounds in L. lactis. The production of these health beneficial metabolites in Generally Recognized As Safe (GRAS) micro-organisms such as S. cerevisiae and L. lactis provides new options for their delivery as therapeutics. SIGNIFICANCE AND IMPACT OF THE STUDY: N-hydroxycinnamoyl-L-amino acids such as clovamide are bioactive plant-derived phenolic compounds with health beneficial effects. Relying on chemical synthesis or direct extraction from plant sources for the supply of these valuable molecules poses challenges to environmental sustainability. As an alternative route, this work demonstrates the potential for biological synthesis of N-hydroxycinnamoyl-L-amino acids using engineered microbial hosts such as Saccharomyces cerevisiae and Lactococcus lactis. Besides being more eco-friendly, this approach should also provide more structurally diverse compounds and offer new methods for their delivery to the human body.

Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate.

BACKGROUND: Acetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production. RESULTS: Here, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain. CONCLUSIONS: Tyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

Tyrosine metabolism: identification of a key residue in the acquisition of prephenate aminotransferase activity by 1ß aspartate aminotransferase.

Alternative routes for the post-chorismate branch of the biosynthetic pathway leading to tyrosine exist, the 4-hydroxyphenylpyruvate or the arogenate route. The arogenate route involves the transamination of prephenate into arogenate. In a previous study, we found that, depending on the microorganisms possessing the arogenate route, three different aminotransferases evolved to perform prephenate transamination, that is, 1ß aspartate aminotransferase (1ß AAT), N-succinyl-l,l-diaminopimelate aminotransferase, and branched-chain aminotransferase. The present work aimed at identifying molecular determinant(s) of 1ß AAT prephenate aminotransferase (PAT) activity. To that purpose, we conducted X-ray crystal structure analysis of two PAT competent 1ß AAT from Arabidopsis thaliana and Rhizobium meliloti and one PAT incompetent 1ß AAT from R. meliloti. This structural analysis supported by site-directed mutagenesis, modeling, and molecular dynamics simulations allowed us to identify a molecular determinant of PAT activity in the flexible N-terminal loop of 1ß AAT. Our data reveal that a Lys/Arg/Gln residue in position 12 in the sequence (numbering according to Thermus thermophilus 1ß AAT), present only in PAT competent enzymes, could interact with the 4-hydroxyl group of the prephenate substrate, and thus may have a central role in the acquisition of PAT activity by 1ß AAT.

An efficient colorimetric high-throughput screening method for synthetic activity of tyrosine phenol-lyase.

Tyrosine phenol-lyase (TPL) naturally catalyzes the reversible ß-elimination of l-tyrosine to phenol, pyruvate and ammonium. With its reverse reaction (synthetic activity), l-tyrosine and its derivatives could be synthesized with high atom economy, which are widely used in pharmaceutical industries. In this study, a high-throughput screening method for synthetic activity of TPL was developed. One of the substrate, sodium pyruvate was found to react with salicylaldehyde under alkali condition, forming a yellow color compound. The concentration of sodium pyruvate can be quantified according to the absorbance of the colorimetric compound at wavelength of 465 nm and the activity of TPL could be screened according to the decrease of the absorbance. After optimization of the colorimetric reaction conditions, the established high-throughput screening method was successfully used for screening of TPL with enhanced activity for l-DOPA synthesis. The confirmed sensitivity and accuracy demonstrated the feasibility and application potential of this screening method.

Branched-chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation.

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro-inflammatory responses through the transcription factor NF-κB that resulted in the release of intracellular adhesion molecule-1 and E-selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA-induced pro-oxidant and pro-inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre-contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.

Metabolic engineering of Escherichia coli for the enhanced production of l-tyrosine.

Despite wide applications of l-tyrosine in the market, microbial overproduction of l-tyrosine has been a great challenge due to the complex gene regulations involved in its biosynthetic pathway. To this end, effects of knocking out tyrR on the l-tyrosine production were further explored during the strain development. Also, blocking cellular uptake of l-tyrosine by knocking out tyrosine transporters was examined with respect to l-tyrosine production. Using feedback-resistant aroG and tyrA genes (aroGfbr and tyrAfbr hereafter) as initial overexpression targets, which encode 3-deoxy-7-phosphoheptulonate synthase and chorismate mutase or prephenate dehydrogenase, respectively, various combinations of genes were subsequently overexpressed in the Escherichia coli wild-type and tyrR knockout strain, and their effects on the l-tyrosine production were examined. Co-overexpression of aroGfbr , aroL and tyrC, a gene from Zymomonas mobilis functionally similar to tyrAfbr , but insensitive to l-tyrosine, led to the greatest l-tyrosine production regardless of the strains and plasmid constructs examined in this study. The strain BTY2.13 overexpressing the abovementioned three genes together with the removal of the l-tyrosine-specific transporter (tyrP) produced 43.14 g/L of l-tyrosine by fed-batch fermentation using the exponential feeding followed by DO-stat feeding method. This outcome suggested that the tyrR gene knockout was not mandatory for the l-tyrosine overproduction, but the production performance of strains having tyrR appeared to be highly affected by vector systems and feeding methods. With an optimal vector system and a feeding method, tyrP knockout appeared to be more effective in enhancing the l-tyrosine than tyrR knockout.

A high-throughput method for screening of L-tyrosine high-yield strains by Saccharomyces cerevisiae.

A biosensor screening assay based on the synthesis of betaxanthin was applied to relatively high throughput screening of the L-tyrosine mutant library. In the assays, fluorescence output showed a linear relationship between extracellular L-tyrosine content and yellow pigment formation. In addition, the yellow pigment accumulation of the L-tyrosine high-yield strain can be easily distinguished with the naked eye compared with the wild-type strain. As a result, numerous mutants that exhibited significantly increased coloration, were screened out after random mutagenesis, and p-coumaric acid production in mutants NK-A3 and NK-B4, were remarkably improved by 4-fold more than that of the wild-type strain. In general, this study provides a novel strategy for screening mutant libraries in the search for highly L-tyrosine-producing strains.