Tryptophan Biosynthetic Pathway Research Articles

TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection.

Response mechanism of Cynodon dactylon to flooding stress based on integrating metabonomics and transcriptomics analysis

Cynodon dactylon, a perennial herbaceous of Poaceae, possessing unique abilities in flooding tolerance and clonal propagation. However, evidences on molecular response mechanism from field are missing. Here, the differential responses of above-ground (stolon) and below-ground (root) organs of C. dactylon under flooding and non-flooding (CK) conditions were compared at the transcriptional and metabolomic levels. Under flooding stress, significant enrichment of differentially expressed genes (DEGs) related to dehydration responses, including cell water homeostasis and ion transmembrane transport were observed in both roots and stolons. The stolons were more sensitive to flooding, while enrichment in energy metabolism in roots were higher, such as the glycolytic process and glycogen biosynthetic process. The DEGs of photosynthesis in stolons were significantly down-regulated compared to roots. Under flooding stress, oxidative phosphorylation (OXPHOS) is inactivated, and the terminal oxidase of the mitochondrial electron transport chain (mtETC), cytochrome c oxidase (COX), is inhibited. Consequently, the expression of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) involved in fermentative metabolism is upregulated. The combined analysis indicated that phenylalanine, tyrosine, and tryptophan biosynthetic pathways in roots were significantly enriched in the flooding group. In contrast, there was a significant increase in phenylalanine biosynthesis in stolons, and the metabolite phenol exhibited up-regulated expression. In summary, phenylalanine metabolism and phenylalanine biosynthesis are the major responses to flooding stress, indicating the crucial role of these pathways in the flood tolerance of C. dactylon. These findings provide valuable evidence for understanding plant regulatory mechanisms underlying flooding tolerance.

Highly Reduced Complementary Genomes of Dual Bacterial Symbionts in the Mulberry Psyllid Anomoneura mori.

The genomes of obligately host-restricted bacteria suffer from accumulating mildly deleterious mutations, resulting in marked size reductions. Psyllids (Hemiptera) are phloem sap-sucking insects with a specialized organ called the bacteriome, which typically harbors two vertically transmitted bacterial symbionts: the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria) and a secondary symbiont that is phylogenetically diverse among psyllid lineages. The genomes of several Carsonella lineages were revealed to be markedly reduced (158-174‍ ‍kb), AT-rich (14.0-17.9% GC), and structurally conserved with similar gene inventories devoted to synthesizing essential amino acids that are scarce in the phloem sap. However, limited genomic information is currently available on secondary symbionts. Therefore, the present study investigated the genomes of the bacteriome-associated dual symbionts, Secondary_AM (Gammaproteobacteria) and Carsonella_AM, in the mulberry psyllid Anomoneura mori (Psyllidae). The results obtained revealed that the Secondary_AM genome is as small and AT-rich (229,822 bp, 17.3% GC) as those of Carsonella lineages, including Carsonella_AM (169,120 bp, 16.2% GC), implying that Secondary_AM is an evolutionarily ancient obligate mutualist, as is Carsonella. Phylogenomic ana-lyses showed that Secondary_AM is sister to "Candidatus Psyllophila symbiotica" of Cacopsylla spp. (Psyllidae), the genomes of which were recently reported (221-237‍ ‍kb, 17.3-18.6% GC). The Secondary_AM and Psyllophila genomes showed highly conserved synteny, sharing all genes for complementing the incomplete tryptophan biosynthetic pathway of Carsonella and those for synthesizing B vitamins. However, sulfur assimilation and carotenoid-synthesizing genes were only retained in Secondary_AM and Psyllophila, respectively, indicating ongoing gene silencing. Average nucleotide identity, gene ortholog similarity, genome-wide synteny, and substitution rates suggest that the Secondary_AM/Psyllophila genomes are more labile than Carsonella genomes.

Efficient production of phenyllactic acid in Escherichia coli via metabolic engineering and fermentation optimization strategies.

Phenyllactic acid (PhLA), an important natural organic acid, can be used as a biopreservative, monomer of the novel polymeric material poly (phenyllactic acid), and raw material for various medicines. Herein, we achieved a high-level production of PhLA in Escherichia coli through the application of metabolic engineering and fermentation optimization strategies. First, the PhLA biosynthetic pathway was established in E. coli CGSC4510, and the phenylalanine biosynthetic pathway was disrupted to improve the carbon flux toward PhLA biosynthesis. Then, we increased the copy number of the key genes involved in the synthesis of the PhLA precursor phenylpyruvic acid. Concurrently, we disrupted the tryptophan biosynthetic pathway and enhanced the availability of phosphoenolpyruvate and erythrose 4-phosphate, thereby constructing the genetically engineered strain MG-P10. This strain was capable of producing 1.42 ± 0.02 g/L PhLA through shake flask fermentation. Furthermore, after optimizing the dissolved oxygen feedback feeding process and other conditions, the PhLA yield reached 52.89 ± 0.25 g/L in a 6 L fermenter. This study successfully utilized metabolic engineering and fermentation optimization strategies to lay a foundation for efficient PhLA production in E. coli as an industrial application.

In silico prediction of mutation sites for anthranilate synthase from Serratia marcesens to deregulate tryptophan feedback inhibition

Allosteric feedback inhibition of the committed step in amino acid biosynthetic pathways is a major concern for production of amino acids at industrial scale. Anthranilate synthase (AS) catalyzes the first reaction of tryptophan biosynthetic pathway found in microorganisms and is feedback inhibited by its own product i.e. tryptophan. Here, we identified new mutant sites in AS using computational mutagenesis approach. MD simulations (20 ns) followed by MMPBSA and per residue decomposition energy analysis identified seven amino acid residues with best binding affinity for tryptophan. All 19 mutant structures were generated for each identified amino acid residue followed by simulation to evaluate effect of mutation on protein stability. Later, molecular docking studies were employed to generate mutant-tryptophan complex and structures with binding energies (kcal/mol) much higher than wild-type AS were selected. Finally, two mutants i.e., S37W and S37H were identified on the basis of positive binding scores and loss of tryptophan binding inside pocket. Further, MD simulations run for 200 ns were performed over these mutant-tryptophan complexes followed by RMSD, RMSF, radius of gyration , solvent accessible surface area , intra-protein hydrogen bond numbers, principal component analysis, free energy landscape (FEL) and secondary structure analysis to rationale effect of mutations on stability of protein. Cross correlation analysis of mutant site amino acids (S37W) with key residues of catalytic site (G325, T326, H395 and G482) was done to evaluate the effect of mutations on catalytic site conformation. Current computational mutagenesis approach predicted two mutants S37W and S37H with proposed deregulated feedback inhibition by tryptophan and retained catalytic activity. Communicated by Ramaswamy H. Sarma

Genome‐based analysis of Pseudomonas paracarnisRQ057, a strain responsible for blue discoloration spoilage in processed cheese

A strain of Pseudomonas causing blue discoloration was isolated from processed cheese ‘Requeijão em Barra’ and studied by whole‐genome sequencing. The strain RQ057 was identified as Pseudomonas paracarnis using the TYGS web server and also by in silico DNA–DNA hybridisation and ANIb calculation. Pyoverdine biosynthetic clusters, integrative and conjugative elements (ICE), prophage regions and genomic islands (GIs) were predicted. Furthermore, we observed that copies of genes related to the tryptophan biosynthetic pathway (trpCBAFD), which are exclusive to strains causing blue discoloration, are contained in the region identified as ICE and which was also identified in GIs. This genomic region also contains genes for transposase, type IV transport system (T4SS) and pili construction. Thereby, we can assume that this region contains potential conjugative‐transposon‐like mobile genetic elements and it has plasmid‐like conjugative properties.

Mechanism of Metabolic Response to Hepatectomy by Integrated Analysis of Gut Microbiota, Metabolomics, and Proteomics.

Hepatectomy is a common clinical procedure for the treatment of many liver diseases, and the successful recovery of a patient's liver metabolism and function after surgery is crucial for a good prognosis. The objective of this study was to elucidate the metabolic response to hepatectomy using high-throughput sequencing analysis of 16S rRNA gene, metabolomics, and proteomics data. Fecal and serum samples from beagle dogs were collected on day 0 (LH0), day 7 (LH7), and day 28 (LH28) after laparoscopic partial hepatectomy. Liver tissue samples were taken on LH0 and LH7. Dysbiosis in the fecal microbiota was explored, and host-microbiome interactions based on global metabolic and protein profiles and inflammatory processes were determined. Results showed that the relative abundance of Allobaculum and Turicibacter was decreased and that of Escherichia-Shigella was increased after hepatectomy (P < 0.05); the phenylalanine, tyrosine, and tryptophan biosynthetic pathway, along with the phenylalanine and aminoacyl-tRNA biosynthetic pathway, was significantly associated with liver injury. The serum metabolites l-phenylalanine and l-arginine were useful as biomarkers, and the fecal metabolite l-threonine was a signature target monitor for liver recovery. The proteomics profile revealed 412 significantly different proteins and further highlighted two key signaling pathways (mitogen-activated protein kinase [MAPK] and peroxisome proliferator-activated receptor [PPAR]) involved in the response to liver injury. We systematically explored the metabolic mechanism of liver injury and recovery, providing new insights into effective ways to promote recovery after hepatectomy and improve liver function and long-term survival. These fundamental studies on hepatectomy will provide the basis for future advances in treatment and recovery from common liver diseases. IMPORTANCE As the largest parenchymal organ, the liver is a target for bacterial and viral infections, nonalcoholic fatty liver disease (NAFLD), cirrhosis, cancer, and many other diseases, constituting a serious worldwide problem. The treatment for many of these diseases involves hepatectomy. Here, we show that aberrant inflammatory processes after hepatectomy of the liver as reflected in the association between liver metabolism and gut microbiota create a grave risk. This study investigated the mechanisms of gut microbiota and host metabolism involved in liver injury and recovery after hepatectomy, using proteomics to reveal the mechanisms of postoperative liver injury and a comprehensive multi-omics approach to identify changes in metabolism after hepatectomy.

Tryptophan synthase ß subunit 1 affects stomatal phenotypes in Arabidopsis thaliana.

Stomata open in response to several environmental stimuli, such as light and low CO2. Plasma membrane (PM) H+-ATPase in guard cells plays a pivotal role for light-induced stomatal opening. In contrast, stomata close in response to the dark or plant hormone abscisic acid (ABA). However, molecular mechanisms of stomatal movements remain unclear. To elucidate the molecular mechanism of stomatal movements, we performed a genetic screen based on stomatal aperture-dependent weight decrease of detached leaves from EMS-treated Arabidopsis thaliana and isolated a rapid transpiration in detached leaves 2 (rtl2). The rtl2 mutant showed constitutive open-stomata phenotype with lower leaf temperature. ABA had no effect on stomatal aperture in rtl2. The rtl2 mutant also showed increased stomatal density, severe dwarf phenotype with pale green leaves and dark veins. Map-based analysis of the RTL2 locus revealed that the rtl2 mutant possesses a single nucleotide substitution, which induces amino acid substitution Gly162 to Glu in the tryptophan synthase ß subunit 1 (TSB1). The TSB1 encodes an enzyme in tryptophan (Trp) biosynthetic pathway. Amount of TSB1 protein was drastically reduced in rtl2 mutant. A different allele of tsb1 mutant (tsb1-1) also showed constitutive open-stomata phenotype with reduced TSB1 protein as in rtl2. Analyses of test-crossed plants of rtl2 and tsb1-1 showed open-stomata and dwarf phenotypes. These results indicate that a responsible gene for rtl2 is TSB1. We further investigated stomatal phenotype in mutants from Trp biosynthetic pathway, such as wei2-1 wei7-1, trp3-1, and tsb2-1. The trp3-1 mutant showed significant wider stomatal aperture as well as tsb1-1. Trp biosynthetic pathway closely relates to auxin biosynthesis. Then, we investigated auxin responsible genes and found that an expression of AUR3 was up in rtl2. In contrast, auxin had no effect on stomatal aperture in Arabidopsis and the phosphorylation status of PM H+-ATPase in guard cell protoplasts from Vicia faba. In addition, auxin antagonist had no effect on stomatal aperture. Interestingly, tsb1-1 grown under hydroponic culture system showed normal stomatal aperture by exogenously application of Trp. These results suggest that open stomata phenotype in tsb1-1 is due to Trp deficiency but not auxin.

High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae.

Hydroxytyrosol (HT) is a valuable aromatic compound with numerous applications. Herein, we enabled the efficient and scalable de novo HT production in engineered Saccharomyces cerevisiae (S. cerevisiae) from glucose. Starting from a tyrosol-overproducing strain, six HpaB/HpaC combinations were investigated, and the best catalytic performance was acquired with HpaB from Pseudomonas aeruginosa (PaHpaB) and HpaC from Escherichia coli (EcHpaC), resulting in 425.7 mg/L HT in shake flasks. Next, weakening the tryptophan biosynthetic pathway through downregulating the expression of TRP2 (encoding anthranilate synthase) further improved the HT titer by 27.2% compared to the base strain. Moreover, the cytosolic NADH supply was improved through introducing the feedback-resistant mutant of the TyrA (the NAD+-dependent chorismate mutase/prephenate dehydrogenase, TyrA*) from E. coli, which further increased the HT titer by 36.9% compared to the base strain. The best performing strain was obtained by optimizing the biosynthesis of HT in S. cerevisiae through a screening for an effective HpaB/HpaC combination, biosynthetic flux rewiring, and cofactor engineering, which enabled the titer of HT reaching 1120.0 mg/L in the shake flask. Finally, the engineered strain produced 6.97 g/L of HT by fed-batch fermentation, which represents the highest titer for de novo HT biosynthesis in microorganisms reported to date.

Insights into the catalytic mechanism of M. tuberculosis indole‐3‐glycerol phosphate synthase

The pathogen M. tuberculosis causes the lung disease tuberculosis (TB). Emerging TB drug resistance worldwide indicates the need for new inhibitors and targets. Recent research suggests that the tryptophan biosynthetic pathway in M. tuberculosis can be targeted to treat TB. Indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) is an α/β barrel enzyme that catalyzes the fourth step in the tryptophan biosynthetic pathway: the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose 5’‐phosphate (CdRP) into indole‐glycerol‐phosphate (IGP) via cyclization, decarboxylation and dehydration steps. The pH profile of MtIGPS was observed to have a bell shaped curve with pKa1 and pKa2 values or 6.7 ± 0.14 and 8.7 ± 0.12, supporting the involvement of both a general acid and a general base in MtIGPS catalysis. Mutations were introduced to three residues that had been previously proposed to serve as the catalytic base: E57, E168 and E219. The E57Q variant had a &gt;200‐fold reduced catalytic activity compared to the wildtype and the E168Q variant had a &gt;187‐fold increased KM. The E219 sidechain interacts with K119, which is proposed to be the catalytic acid. The E219D variant had a &gt;233‐fold decreased catalytic activity. Combined with docking predictions, these data were consistent with E57 as the catalytic base, E168 playing a role in substrate binding, and E219 in orienting the catalytic acid K119. These data together provide an increased understanding of the structure‐function relationship in the MtIGPS active site and insights in the IGPS enzyme family.

Indole‐3‐glycerol phosphate synthase (IGPS) serving as a drug target in M. tuberculosis

Tuberculosis (TB) is a disease that is caused by a bacteria called Mycobacterium tuberculosis and primarily attacks the lungs but can spread to other areas of the body as well. TB can develop resistance to drugs that may be used to cure the disease. Multi‐drug resistant TB, for example, does not respond to the two most powerful anti‐TB drugs, which are isoniazid and rifampicin. For this reason, finding new ways to target TB are highly needed. One possible way to do this is to target the enzymes in the tryptophan biosynthetic pathway of M. tuberculosis. Studies have suggested that the enzyme indole‐3‐glycerol phosphate synthase (IGPS) in M. tuberculosis(MtIGPS) coded for by the trpC gene could be a target (Shen et al. 2009). The interactions between ligands and MtIGPS were thus investigated by introducing mutations to residues that had been proposed to play a role in catalysis or binding. Specifically, E168 was mutated to Q and K119 was mutated to R. Predictions for these mutations were that K119 is a catalytic acid and that E168 plays a role in substrate binding. We investigated the impact of these mutations on steady state kinetics and rate‐pH profiles to obtain insights into the roles of these two residues in substrate binding and catalysis. These findings will facilitate the design of MtIGPS inhibitor candidates.

Proposed pathway of degradation of indigo carmine and its co-metabolism by white-rot fungus Cyathus bulleri

The stimulation of growth by the addition of indigo carmine (5, 5 indigo disulfonic acid sodium salt, IC) was studied in the white-rot fungus Cyathus bulleri. C. bulleri was grown on a variety of solid medium and addition of IC (5000 mg/L) resulted in enhancement of growth, particularly on malt-extract agar. Stimulation of growth by IC was also observed in liquid cultures grown in potato dextrose broth. IC was degraded into anthranilic acid by oxidoreductases, particularly laccases, present in the culture broth of the fungus grown on wheat bran and orange peelings, which was confirmed by treatment of IC with purified laccase. Anthranilic acid was proposed to further enter into the tryptophan (TRP) biosynthetic pathway, as demonstrated by up-regulation of newly identified trpD, trpC and trp1 genes from this fungus. These genes encode anthranilate phosphoribosyl transferase, a multi-functional enzyme with phosphoribosyl anthranilate isomerase/other activities and tryptophan synthase respectively. No such increased transcription of these genes was observed in the absence of IC.

Virtual high-throughput screening: potential inhibitors for the mycobacterial α-subunit of tryptophan synthase

ABSTRACT Mycobacterium tuberculosis (Mtb), which causes tuberculosis (TB), is the world’s most successful pathogen. If it is to survive in phagosomes, the tryptophan biosynthetic pathway is essential. The α-subunit of tryptophan synthase (TRPS) executes the irreversible catalysis of indole-3-glyceral-3-phosphate to generate indole. The β-subunit catalyses the conversion of indole and serine to tryptophan. Targeting the TRPS α-subunit may, therefore, render the enzyme inactive. The crystal structure of α-subunit downloaded from the protein data bank (PDB: 5OCW) has gaps at the amino acid sequence positions in three places. We, therefore, retrieved the entire amino acid sequence of the protein from the UniProt and built a complete protein structure by adding the missing amino acids using a Modeller. The 3D model of the α-subunit was then used as the target to develop inhibitors for Mtb. We downloaded 58,699 natural products from the Maybridge database. After space filtering, molecular docking, molecular dynamic simulation and binding free energy calculation were done to develop inhibitors for the TRPS α-subunit. Out of the 18 compounds selected, five virtual hit compounds, namely MFCD00103482, MFCD00118123, MFCD04110719, MFCD01005315 and MFCD04112426, bind well to the TRPS α-subunit. These five potential inhibitors for Mtb will be taken for preclinical testing.

Effects of Lactobacillus plantarum FZU3013-Fermented Laminaria japonica on Lipid Metabolism and Gut Microbiota in Hyperlipidaemic Rats.

In this study, we explored the effect of Lactobacillus plantarum FZU3013-fermented Laminaria japonica (LPLJ) supplementation to prevent hyperlipidaemia in rats fed with a high-fat diet (HFD). The results indicate that LPLJ supplementation improved serum and hepatic biochemical indicators (p < 0.05), elevated short-chain fatty acid levels, reduced HFD-induced accumulation of lipid droplets in the liver, modulated the relative abundance of some microbial phylotypes, and reduced hyperlipidaemia in HFD-fed rats by adjusting the aminoacyl-tRNA, phenylalanine, tyrosine, and tryptophan biosynthetic pathways, as well as the phenylalanine, D-glutamine and D-glutamate, and glutathione metabolic pathways. Additionally, hepatic mRNA levels of the genes involved in lipid metabolism and bile acid homeostasis were significantly reduced by LPLJ intervention (p < 0.05). These results suggest that LPLJ has a positive effect on modulating lipid metabolism and has the potential to be a functional food that can help prevent hyperlipidaemia.

Cover Feature: Indole‐3‐Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target (ChemBioChem 2/2022)

Indole-3-glycerol phosphate synthase from Mycobacterium tuberculosis (MtIGPS) is a fascinating indole-forming enzyme in the bacterial tryptophan biosynthetic pathway. MtIGPS appears to have important untapped potential as both a novel target for new antibacterial agents and a scaffold for protein engineering. This cover feature art highlights the structure, unique chemistry, and role of MtIGPS in tryptophan biosynthesis. More information can be found in the Minireview by N. M. Goodey et al.

Indole-3-Glycerol Phosphate Synthase From Mycobacterium tuberculosis: A Potential New Drug Target.

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, affects millions of people worldwide. Several TB drugs have lost efficacy due to emerging drug resistance and new anti-TB targets are needed. Recent research suggests that indole-3-glycerol phosphate synthase (IGPS) in M. tuberculosis (MtIGPS) could be such a target. IGPS is a (β/α)8 -barrel enzyme that catalyzes the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP) into indole-glycerol-phosphate (IGP) in the bacterial tryptophan biosynthetic pathway. M. tuberculosis over expresses the tryptophan pathway genes during an immune response and inhibition of MtIGPS allows CD4 T-cells to more effectively fight against M. tuberculosis. Here we review the published data on MtIGPS expression, kinetics, mechanism, and inhibition. We also discuss MtIGPS crystal structures and compare them to other IGPS structures to reveal potential structure-function relationships of interest for the purposes of drug design and biocatalyst engineering.

Metabolic mechanism of Prunella vulgaris in treatment of ethanol-induced oxidative stress in rats based on metabonomics

Prunella vulgaris(PV) is an edible and traditional medicinal herb which has a wide range application in fighting inflammation and oxidative stress, and protecting liver. Now it has been used to treat various types of liver diseases and has significant clinical efficacy. This study aims to investigate the effects of PV on ethanol-induced oxidative stress injury in rats and its metabolic mechanism. The rats were divided into control group, model group, PV group, and VC group. The liver protection of PV was identified by measuring pharmacological indexes such as antioxidant and anti-inflammatory activity. The metabolic mechanism of long-term ethanol exposure and the metabolic regulation mechanism of PV treatment were studied by LS-MS metabonomics. The pharmacological investigation indicated that ethanol could significantly decrease the contents of SOD, GSH-Px, CAT and other antioxidant enzymes in liver and increase the content of MDA. At the same time, PV could significantly reduce the contents of inflammatory factors(TNF-α, IL-6 and IL-1β) and liver function markers(ALT, AST, ALP) in serum. What's more, long-term ethanol exposure could significantly cause liver injury, while PV could protect liver. Metabolomics based on multiple statistical analyses showed that long-term ethanol exposure could cause significant metabolic disorder, and fatty acids, phospholipids, carnitines and sterols were the main biomarkers. Meanwhile, pathway analysis and enrichment analysis showed that the β oxidation of branched fatty acids was the main influencing pathway. Also, PV could improve metabolic disorder of liver injury induced by ethanol, and amino acids, fatty acids, and phospholi-pids were the main biomarkers in PV treatment. Metabolic pathway analysis showed that PV mainly regulated metabolic disorder of ethanol-induced liver injury through phenylalanine, tyrosine and tryptophan biosynthetic pathways. This study could provide a new perspective on the hepatoprotective effect of natural medicines, such as PV.

Engineering of Saccharomyces cerevisiae for anthranilate and methyl anthranilate production

BackgroundAnthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce.ResultsWe subjected Saccharomyces cerevisiae, a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l−1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula. When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l−1.ConclusionsIn this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.

Flux redistribution of central carbon metabolism for efficient production of l-tryptophan in Escherichia coli.

Microbial production of l-tryptophan (l-trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate-limiting enzymes and blockage of competing pathways can effectively promote microbial production of l-trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of l-trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) pools in an l-trp producing strain of Escherichia coli for efficient l-trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the l-trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP-independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved l-trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP-pyruvate-oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L l-trp with an overall yield of 0.227 g/g glucose after 40 h fed-batch fermentation in 5-L bioreactor. This is the highest overall yield of l-trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient l-trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.

The tryptophan biosynthetic pathway is essential for Mycobacterium tuberculosis to cause disease.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the most significant cause of death from a single infectious agent worldwide. Antibiotic-resistant strains of M. tuberculosis represent a threat to effective treatment, and the long duration, toxicity and complexity of current chemotherapy for antibiotic-resistant disease presents a need for new therapeutic approaches with novel modes of action. M. tuberculosis is an intracellular pathogen that must survive phagocytosis by macrophages, dendritic cells or neutrophils to establish an infection. The tryptophan biosynthetic pathway is required for bacterial survival in the phagosome, presenting a target for new classes of antitubercular compound. The enzymes responsible for the six catalytic steps that produce tryptophan from chorismate have all been characterised in M. tuberculosis, and inhibitors have been described for some of the steps. The innate immune system depletes cellular tryptophan in response to infection in order to inhibit microbial growth, and this effect is likely to be important for the efficacy of tryptophan biosynthesis inhibitors as new antibiotics. Allosteric inhibitors of both the first and final enzymes in the pathway have proven effective, including by a metabolite produced by the gut biota, raising the intriguing possibility that the modulation of tryptophan biosynthesis may be a natural inter-bacterial competition strategy.