Aromatic Amino Acid Biosynthesis Pathway Research Articles

Comparative analysis of metabolome and transcriptome in the fruits and leaves of Elaeocarpus braceanus

The utilization of Elaeocarpus braceanus fruits and leaves as medicinal and dietary resources by ethnic minorities in southwestern China has been well-documented throughout history. However, due to the lack of understanding regarding the specific chemical constituents and synthetic processes involved, comprehensive metabolome and transcriptomic assays were conducted on the fruits and leaves. The findings revealed the presence of 749 compounds in these plant parts, with 424 metabolites displaying differential accumulation between fruits and leaves. Among these, 316 metabolites were up-regulated while 108 were down-regulated. A total of 100 phenolic acids and 104 flavonoids exhibited distinct accumulation patterns in fruits and leaves, respectively. Subsequent KEGG enrichment analysis revealed alterations in 77 pathways, with significant differential expression observed in genes associated with phenylpropanoid biosynthesis, aromatic amino acid biosynthesis, flavonoid biosynthesis, and flavonol biosynthesis pathways between fruits and leaves. These findings contribute to our comprehension of metabolite accumulation and offer valuable insights for investigating the food and medicinal properties of E. braceanus.

Metabolomic Responses of Lettuce (Lactuca sativa) to Allelopathic Benzoquinones from Iris sanguinea Seeds.

Weed management is important in modern crop protection. Chemical weed control using synthetic herbicides, however, suffers from resistance and ecotoxicity. Metabolomic investigation of allelopathy (or allelochemicals) may provide novel alternatives to synthetic herbicides. This study aimed to investigate the detailed metabolomic responses of plants to allelochemicals in Iris seed extracts. The seed extracts of Iris sanguinea showed the strongest growth inhibitory activity against alfalfa, barnyard grass, lettuce, and mustard. 3-Hydroxyirisquinone (3-[10(Z)-heptadecenyl]-2-hydroxy-5-methoxy-1,4-benzoquinone) was isolated as a major allelochemical from I. sanguinea seeds through bioassay-guided fractionation. The compound inhibited the growth of shoots and roots by browning root tips. Discriminant analysis identified 33 differentially regulated lettuce metabolites after treatment with 3-hydroxyirisquinone (3HIQ). Metabolic pathway analysis revealed that several metabolic pathways, including aromatic amino acid biosynthesis and respiratory pathways, were affected by the compounds. Differential responses of membrane lipids (accumulation of unsaturated fatty acids) and extensive formation of reactive oxygen species were observed in root tissues following treatment with 3HIQ. Overall, alkylbenzoquinone from I. sanguinea induced extensive metabolic modulation, oxidative stress, and growth inhibition. The metabolomic responses to allelochemicals may provide fundamental information for the development of allelochemical-based herbicides.

Effect of calcium peroxide assisted microwave irradiation pretreatment on humus formation and microbial community in straw and dairy manure composting

In this study, the effects of four pretreatment methods on the crystallinity of maize straw were compared, and the CaO2 assisted microwave pretreatment was selected for straw and dairy manure composting. The humification and microbial community were investigated. Results showed that the pretreatment increased the initial water-soluble carbon, which favored the microbial activity, and the CO2 release increased by 15.71%. Pretreatment promoted the lignocellulose degradation, with total degradation ratio of 37.06%. The final humic acid content was 11.39 g/kg higher than the control. Spearman correlation analysis indicated that polyphenols and amino acids were significantly related to humus formation. In addition, pretreatment rendered the Firmicutes the most dominant phylum, and increased the metabolic intensity of reducing sugar metabolism, aromatic amino acid biosynthesis and carbon fixation pathways. Redundancy analysis revealed that the dominant genus of Firmicutes was significantly positively correlated with humus, while that of Actinobacteriota was correlated with CO2 release.

A systems level approach to study metabolic networks in prokaryotes with the aromatic amino acid biosynthesis pathway.

Metabolism of an organism underlies its phenotype, which depends on many factors, such as the genetic makeup, habitat, and stresses to which it is exposed. This is particularly important for the prokaryotes, which undergo significant vertical and horizontal gene transfers. In this study we have used the energy-intensive Aromatic Amino Acid (Tryptophan, Tyrosine and Phenylalanine, TTP) biosynthesis pathway, in a large number of prokaryotes, as a model system to query the different levels of organization of metabolism in the whole intracellular biochemical network, and to understand how perturbations, such as mutations, affects the metabolic flux through the pathway - in isolation and in the context of other pathways connected to it. Using an agglomerative approach involving complex network analysis and Flux Balance Analyses (FBA), of the Tryptophan, Tyrosine and Phenylalanine and other pathways connected to it, we identify several novel results. Using the reaction network analysis and Flux Balance Analyses of the Tryptophan, Tyrosine and Phenylalanine and the genome-scale reconstructed metabolic pathways, many common hubs between the connected networks and the whole genome network are identified. The results show that the connected pathway network can act as a proxy for the whole genome network in Prokaryotes. This systems level analysis also points towards designing functional smaller synthetic pathways based on the reaction network and Flux Balance Analyses analysis.

Microbiota transplantation from younger to older mice could restore lost immunity to effectively clear salmonella infection in Th2-biased BALB/c mice

AimsThe composition, overtly abundance, and diversity of gut microbiota, play a significant role in maintaining physiological homeostasis with age. Reports revealed that the gut microbial profile might be correlated with immunity and metabolism. It is, therefore, tantamount to know if an older individual can achieve the immunity and metabolic profile of a younger individual by receiving the gut microbiome of a younger individual. In the current report, we have studied the effects of cecal microbiota transplantation (CMT) from younger to older mice. Materials and methodsIn this study, older BALB/c mice (23 weeks) received CMT from younger BALB/c mice (3 weeks). Key findingsCMT recipient mice showed altered expressions of immune and tight junction protein genes in the colon of mice, while the non-CMT recipient mice did not. Older mice were treated with AVNM to make them compatible with CMT. Further data from metabolite studies revealed that AVNM treatment mainly affected the aromatic amino acid biosynthesis pathway while CMT mostly affected the metabolism of different carbohydrates. We repeated the analysis in C57BL/6 mice without any significant effects of CMT. SignificanceResults revealed that mice who received CMT showed more efficient restoration of gut microbiota than non-CMT recipient mice. CMT caused the alleviation of Salmonella infection and efficient recovery of the cecal index in the mice following antibiotics treatment.

Biotechnological Production of the Sunscreen Pigment Scytonemin in Cyanobacteria: Progress and Strategy.

Scytonemin is a promising UV-screen and antioxidant small molecule with commercial value in cosmetics and medicine. It is solely biosynthesized in some cyanobacteria. Recently, its biosynthesis mechanism has been elucidated in the model cyanobacterium Nostoc punctiforme PCC 73102. The direct precursors for scytonemin biosynthesis are tryptophan and p-hydroxyphenylpyruvate, which are generated through the shikimate and aromatic amino acid biosynthesis pathway. More upstream substrates are the central carbon metabolism intermediates phosphoenolpyruvate and erythrose-4-phosphate. Thus, it is a long route to synthesize scytonemin from the fixed atmospheric CO2 in cyanobacteria. Metabolic engineering has risen as an important biotechnological means for achieving sustainable high-efficiency and high-yield target metabolites. In this review, we summarized the biochemical properties of this molecule, its biosynthetic gene clusters and transcriptional regulations, the associated carbon flux-driving progresses, and the host selection and biosynthetic strategies, with the aim to expand our understanding on engineering suitable cyanobacteria for cost-effective production of scytonemin in future practices.

Computational investigations of allostery in aromatic amino acid biosynthetic enzymes.

Allostery, in which binding of ligands to remote sites causes a functional change in the active sites, is a fascinating phenomenon observed in enzymes. Allostery can occur either with or without significant conformational changes in the enzymes, and the molecular basis of its mechanism can be difficult to decipher using only experimental techniques. Computational tools for analyzing enzyme sequences, structures, and dynamics can provide insights into the allosteric mechanism at the atomic level. Combining computational and experimental methods offers a powerful strategy for the study of enzyme allostery. The aromatic amino acid biosynthesis pathway is essential in microorganisms and plants. Multiple enzymes involved in this pathway are sensitive to feedback regulation by pathway end products and are known to use allostery to control their activities. To date, four enzymes in the aromatic amino acid biosynthesis pathway have been computationally investigated for their allosteric mechanisms, including 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, anthranilate synthase, chorismate mutase, and tryptophan synthase. Here we review the computational studies and findings on the allosteric mechanisms of these four enzymes. Results from these studies demonstrate the capability of computational tools and encourage future computational investigations of allostery in other enzymes of this pathway.

H2O2-Inactivated Salmonella typhimurium RE88 Strain as a New Cancer Vaccine Carrier: Evaluation in a Mouse Model of Cancer.

PurposeThis study aimed to describe a novel cancer vaccine developed using H2O2-inactivated Salmonella typhimurium RE88 [with deletions of AroA (the first enzyme in the aromatic amino acid biosynthesis pathway) and DNA adenine methylase] as the carrier.MethodsThe pVLT33 plasmid was used to engineer an RE88 strain induced to express ovalbumin (OVA) by isopropylthiogalactoside (RE88-pVLT33-OVA). The immune responses and anticancer effects of H2O2-inactivated RE88-pVLT33-OVA were compared with those of non-inactivated RE88-pVLT33-OVA and OVA (positive control) in mice carrying OVA-expressing tumors (EG7-OVA) cells.ResultsAnti-ovalbumin IgG (immunoglobulin G) titer following vaccination with H2O2-inactivated RE88-pVLT33-OVA was higher for subcutaneous than for intragastric vaccination. When subcutaneous administration was used, H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU (colony forming units)/mouse) achieved an anti-ovalbumin IgG titer higher than that for the same dose of RE88-pVLT33-OVA and comparable to that for 10 µg ovalbumin (positive control). The binding of mouse serum antibodies to EG7-OVA cells was stronger for H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) than for 10 µg ovalbumin. Furthermore, subcutaneous vaccination with H2O2-inactivated RE88-pVLT33-OVA (2 × 109 CFU/mouse) induced greater activation of splenic T cells and more extensive tumor infiltration with CD4+/CD8+ T cells compared with 10 µg ovalbumin (positive control). The mice vaccinated subcutaneously with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 108 or 6 × 108 CFU/mouse had smaller tumors compared with mice in the negative control groups. Tumor weight in mice vaccinated with H2O2-inactivated RE88-pVLT33-OVA at a dose of 2 × 109 CFU/mouse was significantly lower than that in both negative control groups (P < 0.05) and decreased with the increasing dose of H2O2-inactivated RE88-pVLT33-OVA. H2O2-inactivated RE88-pVLT33-OVA was potentially safer than the non-inactivated strain, could carry exogenous antigens, and had specific epitopes that could be exploited as natural adjuvants to facilitate the induction of cellular and humoral immune responses.ConclusionIt was anticipated that H2O2-inactivated RE88-pVLT33-OVA could be used as a novel delivery system for new cancer vaccines.

Multi-modular engineering of Saccharomyces cerevisiae for high-titre production of tyrosol and salidroside.

Tyrosol and its glycosylated product salidroside are important ingredients in pharmaceuticals, nutraceuticals and cosmetics. Despite the ability of Saccharomyces cerevisiae to naturally synthesize tyrosol, high yield from de novo synthesis remains a challenge. Here, we used metabolic engineering strategies to construct S. cerevisiae strains for high-level production of tyrosol and salidroside from glucose. First, tyrosol production was unlocked from feedback inhibition. Then, transketolase and ribose-5-phosphate ketol-isomerase were overexpressed to balance the supply of precursors. Next, chorismate synthase and chorismate mutase were overexpressed to maximize the aromatic amino acid flux towards tyrosol synthesis. Finally, the competing pathway was knocked out to further direct the carbon flux into tyrosol synthesis. Through a combination of these interventions, tyrosol titres reached 702.30 ± 0.41 mg l-1 in shake flasks, which were approximately 26-fold greater than that of the WT strain. RrU8GT33 from Rhodiola rosea was also applied to cells and maximized salidroside production from tyrosol in S. cerevisiae. Salidroside titres of 1575.45 ± 19.35 mg l-1 were accomplished in shake flasks. Furthermore, titres of 9.90 ± 0.06 g l-1 of tyrosol and 26.55 ± 0.43 g l-1 of salidroside were achieved in 5 l bioreactors, both are the highest titres reported to date. The synergistic engineering strategies presented in this study could be further applied to increase the production of high value-added aromatic compounds derived from the aromatic amino acid biosynthesis pathway in S. cerevisiae.

Independent regulation of age associated fat accumulation and longevity

Age-dependent changes in metabolism can manifest as cellular lipid accumulation, but how this accumulation is regulated or impacts longevity is poorly understood. We find that Saccharomyces cerevisiae accumulate lipid droplets (LDs) during aging. We also find that over-expressing BNA2, the first Biosynthesis of NAD+ (kynurenine) pathway gene, reduces LD accumulation during aging and extends lifespan. Mechanistically, this LD accumulation during aging is not linked to NAD+ levels, but is anti-correlated with metabolites of the shikimate and aromatic amino acid biosynthesis (SA) pathways (upstream of BNA2), which produce tryptophan (the Bna2p substrate). We provide evidence that over-expressed BNA2 skews glycolytic flux from LDs towards the SA-BNA pathways, effectively reducing LDs. Importantly, we find that accumulation of LDs does not shorten lifespan, but does protect aged cells against stress. Our findings reveal how lipid accumulation impacts longevity, and how aging cell metabolism can be rewired to modulate lipid accumulation independently from longevity.

Differential Impacts of Gingerols- and Shogaols-Enriched Ginger Root Extracts on Fecal Metabolites in Rats with Neuropathic Pain

Abstract Objectives Emerging evidence suggests that gut microbiota-derived mediators play key roles in the development of chronic pain, particularly, neuropathic pain (NP), by serving as critical modulators. We evaluated the effects of two ginger root extracts, gingerols and shogaols respectively, on gut microbiota-derived metabolites in the feces of animals with NP. Methods Sixteen male rats were randomly divided into 4 groups: sham group, SNL group, SNL+gingerols-enriched ginger (GEG) extract group, and SNL+shogoals-enriched ginger (SEG) extract group. At 30 days, fecal samples were processed or LC-MS/MS analysis. Principle Component Analysis was performed to assess the different profiling of the metabolites detected and quantified among 4 groups. Data were analyzed using compound discover software (3.1) to identify and quantify metabolites. t-test was performed to determine the number of statistically different metabolites in SNL group compared to the other groups. Results Among all identified pathways, three amino acid- associated pathways (namely, branched chain amino acid biosynthesis (BCAAB), anaerobic aromatic compound degradation (AACD), and aromatic amino acid biosynthesis (AAAB)) were significantly affected in the paired comparisons. The SNL group tended to have higher concentration of phenylacetadehyde (AACD pathway) than that in the sham group (P = 0.09). In BCAAB pathway, relative to the SNL group, (i) GEG group had an increase in L-glutamate, 4-methyl-2-oxopentanoate, and (S)-3-methyl-2-oxopentanoate, and a decrease in (S)-2-acetolactate, and (ii) SEG group showed increased L-valine and decreased 2-oxoglutarate levels. In AACD pathway, compared to the SNL group, R-2-benzylsuccinate, phenylacetate, phenlacetadehyde, and L-glutamate levels were increased in the GEG group, while only phenlacetate level was increased in the SEG group. In AAAB pathway, compared to the SNL group, increased L-glutamate and decreased D-erythrose 4-phospahte level was seen in the GEG group; whereas the SEG group tended to have lower fecal L-glutamine level. Conclusions Based on the results of fecal metabolite analyses, gingerols and shogaols supplementation had significant but differential impacts on the amino acid-associated pathways. Funding Sources Texas Tech University Health Sciences Center.

Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae

The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.

Metabolic engineering of Escherichia coli W3110 strain by incorporating genome-level modifications and synthetic plasmid modules to enhance L-Dopa production from glycerol

L-Tyrosine which is one of the terminal metabolites of highly regulated aromatic amino-acid biosynthesis pathway in Escherichia coli is a precursor for synthesis of L-Dopa. In this study, we report over production of L-Dopa by enhancing expression of rate limiting isoenzyme of shikimate kinase (aroL), chorismate synthase (aroC), aromatic-amino-acid aminotransferase (tyrB) and 3-phosphoshikimate 1-carboxyvinyltransferase (aroA) form a plasmid module harboring five enzymes under two inducible promoters converting shikimate to tyrosine. 4-hydroxyphenylacetate-3-hydrolase (hpaBC) which converts L-Tyrosine to L-Dopa was expressed constitutively from a separate plasmid module. Feedback deregulated expression of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (aroG*) replacing wild type aroG under its natural promoter led to enhancement of L-Dopa production. Deletion of transcriptional repressor tyrR and links to other competing pathways improved titers of L-Dopa. We focused on having a balanced flux by constitutive expression of pathway enzymes from plasmid constructs rather than achieving higher amounts of catalytic protein by induction. We observed glycerol when used as a carbon source for the final strain led to low acid production. The best performing strain led to decoupling of acid production and product formation in bioreactor. Fed batch analysis of the final strain led to 12.5 g/L of L-Dopa produced in bioreactor.

Induction of the PDH bypass and upregulation of the ALDH7B4 in plants treated with herbicides inhibiting amino acid biosynthesis

Imazamox and glyphosate represent two classes of herbicides that inhibit the activity of acetohydroxyacid synthase in the branched-chain amino acid biosynthesis pathway and the activity of 5-enolpyruvylshikimate-3-phosphate synthase in the aromatic amino acid biosynthesis pathway, respectively. However, it is still unclear how imazamox and glyphosate lead to plant death. Both herbicides inhibit amino-acid biosynthesis and were found to induce ethanol fermentation in plants, but an Arabidopsis mutant deficient in alcohol dehydrogenase 1 was neither more susceptible nor more resistant than the wild-type to the herbicides. In this study, we investigated the effects of the amino acid biosynthesis inhibitors, imazamox and glyphosate, on the pyruvate dehydrogenase bypass reaction and fatty acid metabolism in A. thaliana. We found that the pyruvate dehydrogenase bypass was upregulated following the treatment by the two herbicides. Our results suggest that the Arabidopsis aldehyde dehydrogenase 7B4 gene might be participating in the pyruvate dehydrogenase bypass reaction. We evaluated the potential role of the aldehyde dehydrogenase 7B4 upon herbicide treatment in the plant defence mechanism. Plants that overexpressed the ALDH7B4 gene accumulated less soluble sugars, starch, and fatty acids and grew better than the wild-type after herbicide treatment. We discuss how the upregulation of the ALDH7B4 alleviates the effects of the herbicides, potentially through the detoxification of the metabolites produced in the pyruvate dehydrogenase bypass.

Multilevel engineering of the upstream module of aromatic amino acid biosynthesis in Saccharomyces cerevisiae for high production of polymer and drug precursors

A multilevel approach was implemented in Saccharomyces cerevisiae to optimize the precursor module of the aromatic amino acid biosynthesis pathway, which is a rich resource for synthesizing a great variety of chemicals ranging from polymer precursor, to nutraceuticals and pain-relief drugs. To facilitate the discovery of novel targets to enhance the pathway flux, we incorporated the computational tool YEASTRACT for predicting novel transcriptional repressors and OptForce strain-design for identifying non-intuitive pathway interventions. The multilevel approach consisted of (i) relieving the pathway from strong transcriptional repression, (ii) removing competing pathways to ensure high carbon capture, and (iii) rewiring precursor pathways to increase the carbon funneling to the desired target. The combination of these interventions led to the establishment of a S. cerevisiae strain with shikimic acid (SA) titer reaching as high as 2.5gL−1, 7-fold higher than the base strain. Further expansion of the platform led to the titer of 2.7gL−1 of muconic acid (MA) and its intermediate protocatechuic acid (PCA) together. Both the SA and MA production platforms demonstrated increases in titer and yield nearly 300% from the previously reported, highest-producing S. cerevisiae strains. Further examination elucidated the diverged impacts of disrupting the oxidative branch (ZWF1) of the pentose phosphate pathway on the titers of desired products belonging to different portions of the pathway. The investigation of other non-intuitive interventions like the deletion of the Pho13 enzyme also revealed the important role of the transaldolase in determining the fate of the carbon flux in the pathways of study. This integrative approach identified novel determinants at both transcriptional and metabolic levels that constrain the flux entering the aromatic amino acid pathway. In the future, this platform can be readily used for engineering the downstream modules toward the production of important plant-sourced aromatic secondary metabolites.

Improvement of shikimic acid production in Escherichia coli with growth phase-dependent regulation in the biosynthetic pathway from glycerol.

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.

Yeast factories for the production of aromatic compounds: from building blocks to plant secondary metabolites.

The aromatic amino acid biosynthesis pathway is a source to a plethora of commercially relevant chemicals with very diverse industrial applications. Tremendous efforts in microbial engineering have led to the production of compounds ranging from small aromatic molecular building blocks all the way to intricate plant secondary metabolites. Particularly, the yeast Saccharomyces cerevisiae has been a great model organism given its superior capability to heterologously express long metabolic pathways, especially the ones containing cytochrome P450 enzymes. This review contains a collection of state-of-the-art metabolic engineering work devoted towards unraveling the mechanisms for enhancing the flux of carbon into the aromatic pathway. Some of the molecules discussed include the polymer precursor muconic acid, as well as important nutraceuticals (flavonoids and stilbenoids), and opium-derived drugs (benzylisoquinoline alkaloids).

Mechanism of glyphosate control of Phelipanche aegyptiaca.

Despite its total reliance on its host plant, the holoparasite Phelipanche aegyptiaca suffers from a deficiency of aromatic amino acids upon exposure to glyphosate. The herbicide glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the biosynthesis of aromatic amino acids. However, the functionality of the EPSPS pathway in the obligate root holoparasite Phelipanche aegyptiaca is not straightforward because of the parasite's total dependence on the host plant. Despite the importance of glyphosate as a means of controlling P. aegyptiaca, the mechanism of action of the herbicide in this parasite is not clearly understood. We characterized glyphosate control of P. aegyptiaca by using a glyphosate-resistant tomato (GRT) genotype as the host plant and evaluating the activity of EPSPS and the levels of free aromatic amino acids in the parasite. The viability of the parasite's tissues deteriorated within the first 40h after treatment (HAT) with glyphosate. In parallel, shikimate accumulation in the parasite was first detected at 24 HAT and increased over time. However, shikimate levels in the GRT host did not increase, indicating that the host was indeed glyphosate tolerant. Free phenylalanine and tyrosine levels decreased by 48 HAT in the parasite, indicating a deficiency of aromatic amino acids. The use of GRT as the host enabled us to observe, in an in situ experimental system, both endogenous EPSPS inhibition and a deficiency of aromatic amino acids in the parasite. We thus provided evidence for the presence of an active EPSPS and aromatic amino acid biosynthesis pathway in P. aegyptiaca and pinpointed this pathway as the target of glyphosate action in this parasite.

Paradoxical performance of tryptophan synthase gene trp1 (+) in transformations of the basidiomycete Coprinopsis cinerea.

Several transformation strains of Coprinopsis cinerea carry the defective tryptophan synthase allele trp1-1,1-6 which can be complemented by introduction of the trp1 (+) wild-type gene. Regularly in C. cinerea, single-trp1 (+)-vector transformations yield about half the numbers of clones than cotransformations with a non-trp1 (+)-plasmid done in parallel. The effect is also observed with the orthologous Schizophyllum commune trpB (+) gene shown here to function as a selection marker in C. cinerea. Parts ofsingle-trp1 (+) - or single-trpB (+) -vector transformants are apparently lost. This paradoxical phenomenon relates to de-regulation of aromatic amino acid biosynthesis pathways. Adding tryptophan precursors to protoplast regeneration agar or feeding with other aromatic amino acids increases loss of single-trp1 (+)-vector transformants and also sets off loss of clones in cotransformation with a non-trp1 (+)-plasmid. Feedback control by tryptophan and cross-pathway control by tyrosine and phenylalanine are both active in the process. We deduce from the observations that more cotransformants than single-vector transformants are obtained by in average less disturbance of the tryptophan biosynthesis pathway. DNA in C. cinerea transformation usually integrates into the genome at multiple ectopic places. Integration events for a single vector per nucleus should statistically be 2-fold higher in single-vector transformations than in cotransformations in which the two different molecules compete for the same potential integration sites. Integration of more trp1 (+) copies into the genome might more likely lead to sudden tryptophan overproduction with subsequent rigid shut-down of the pathway. Blocking ectopic DNA integration in a Δku70 mutant abolished the effect of doubling clone numbers in cotransformations due to preferred single trp1 (+) integration by homologous recombination at its native genomic site.

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii.

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.