Amino Acid Biosynthetic Pathways Research Articles

A simplified liquid chromatography-mass spectrometry methodology to probe the shikimate and aromatic amino acid biosynthetic pathways in plants.

Plants direct substantial amounts of carbon toward the biosynthesis of aromatic amino acids (AAAs), particularly phenylalanine to produce lignin and other phenylpropanoids. Yet, we have a limited understanding of how plants regulate AAA metabolism, partially because of a scarcity of robust analytical methods. Here, we established a simplified workflow for simultaneous quantification of AAAs and their pathway intermediates from plant tissues, based on extraction at two alternative pH and analysis by Zwitterionic hydrophilic interaction liquid chromatography coupled to mass spectrometry. This workflow was then used to analyze metabolic responses to elevated or reduced carbon flow through the shikimate pathway in plants. Increased flow upon expression of a feedback-insensitive isoform of the first shikimate pathway enzyme elevated all AAAs and pathway intermediates, especially arogenate, the last common precursor within the post-chorismate pathway of tyrosine and phenylalanine biosynthesis. Additional overexpression of an arogenate dehydrogenase enzyme increased tyrosine levels and depleted phenylalanine and arogenate pools; however, the upstream shikimate pathway intermediates remained accumulated at high levels. Glyphosate treatment, which restricts carbon flow through the shikimate pathway by inhibiting its penultimate step, led to a predictable accumulation of shikimate and other precursors upstream of its target enzyme but also caused an unexpected accumulation of downstream metabolites, including arogenate. These findings highlight that the shikimate pathway and the downstream post-chorismate AAA pathways function as independently regulated modules in plants. The method developed here paves the way for a deeper understanding of the shikimate and AAA biosynthetic pathways in plants.

Mammalian colonic contribution of amino acids to whole-body homeostasis.

The colon is an effective bioreactor with auxotrophic microbiota that can serve by hydrolyzing dietary and endogenous protein, as well as by synthesizing essential amino acids through nitrogen salvage. Due to assumed negligible amino acid absorption, this colonic contribution was thought to be minimal, but this may not be true. Several studies that examined the colonic environment in health and disease, show the presence of proteases in the colonic lumen, which are of both host and microbial origin, along with indirect evidence of amino acid transporters in the colonic epithelium. There are also amino acid biosynthetic pathways in the microflora, and the contribution of colonic amino acid to host amino acid nutrition has been shown in wild animals. Yet, current direct and quantitative evidence on amino acid absorption in human colon is minimal. Although amino acid absorption in colon is not very well established, current studies show that substantial amounts of amino acid could possibly be contributed to the host by the colon. There is a need for assessing this contribution quantitatively using direct isotopic methods under different nutritional conditions, dietary intakes, and clinical conditions.

The impact of alternate-day fasting on the salivary gland stem cell compartments in non-obese diabetic mice with newly established Sjögren's syndrome.

Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAMhi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16INK4a, a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS.

Effect of Lactiplantibacillus plantarum cell-free supernatant on the physiology, quorum sensing, transcription, and enhanced GABA production of Enterococcus faecium

A co-culture strategy has been proposed for the production of desired products. However, the interaction effect on enhancing γ-aminobutyric acid (GABA) production requires further investigation. In this study, the mechanism by which Lactiplantibacillus plantarum BC112 cell-free supernatant (CFS) enhances GABA production in Enterococcus faecium AB157 was investigated, including physiological characteristics, quorum sensing (QS), and mRNA expression. The results showed CFS can significantly enhance the production of γ-aminobutyric acid (GABA) in E. faecium AB157, with a maximum increase of 4.6 times compared to the strain without CFS. CFS promoted the growth of E. faecium AB157, enhanced the activity of glutamate decarboxylase (GAD) and ATPase, and reduced the pH. The effect of CFS on QS was demonstrated through changes in Autoinducer-2 (AI-2) activity and luxS expression. Moreover, 16 amino acids were detected in CFS, with glutamic acid having the highest content. Additionally, 974 genes of E. faecium AB157 differed significantly in cultures with added CFS, which involved glycolytic, amino acid, and GABA biosynthetic pathways. Therefore, Lb. plantarum BC112 CFS enhances GABA production in E. faecium AB157 by affecting energy metabolism, the QS system, and the GABA biosynthesis system.

Control of three-carbon amino acid homeostasis by promiscuous importers and exporters in Bacillus subtilis: role of the "sleeping beauty" amino acid exporters.

The Gram-positive model bacterium Bacillus subtilis can acquire amino acids by import, de novo biosynthesis, or degradation of proteins and peptides. The accumulation of several amino acids inhibits the growth of B. subtilis, probably due to misincorporation into cellular macromolecules such as proteins or peptidoglycan or due to interference with other amino acid biosynthetic pathways. Here, we studied the adaptation of B. subtilis to toxic concentrations of the three-carbon amino acids L-alanine, β-alanine, and 2,3-diaminopropionic acid, as well as the two-carbon amino acid glycine. Resistance to the non-proteinogenic amino acid β-alanine, which is a precursor for coenzyme A biosynthesis, is achieved by mutations that either activate a cryptic amino acid exporter, AexA (previously YdeD), or inactivate the amino acid importers AimA, AimB (previously YbxG), and BcaP. The aexA gene is very poorly expressed under most conditions studied. However, mutations affecting the transcription factor AerA (previously YdeC) can result in strong constitutive aexA expression. AexA is the first characterized member of a group of amino acid exporters in B. subtilis, which are all very poorly expressed. Therefore, we suggest to call this group "sleeping beauty amino acid exporters." 2,3-Diaminopropionic acid can also be exported by AexA, and this amino acid also seems to be a natural substrate of AerA/AexA, as it can cause a slight but significant induction of aexA expression, and AexA also provides some natural resistance toward 2,3-diaminopropionic acid. Moreover, our work shows how low-specificity amino acid transporters contribute to amino acid homeostasis in B. subtilis.IMPORTANCEEven though Bacillus subtilis is one of the most-studied bacteria, amino acid homeostasis in this organism is not fully understood. We have identified import and export systems for the C2 and C3 amino acids. Our work demonstrates that the responsible amino acid permeases contribute in a rather promiscuitive way to amino acid uptake. In addition, we have discovered AexA, the first member of a group of very poorly expressed amino acid exporters in B. subtilis that we call "sleeping beauty amino acid exporters." The expression of these transporters is typically triggered by mutations in corresponding regulator genes that are acquired upon exposure to toxic amino acids. These exporters are ubiquitous in all domains of life. It is tempting to speculate that many of them are not expressed until the cells experience selective pressure by toxic compounds, and they protect the cells from rare but potentially dangerous encounters with such compounds.

Structural Bases of Dihydroxy Acid Dehydratase Inhibition and Biodesign for Self-Resistance.

Dihydroxy acid dehydratase (DHAD) is the third enzyme in the plant branched-chain amino acid biosynthetic pathway and the target for commercial herbicide development. We have previously reported the discovery of fungal natural product aspterric acid (AA) as a submicromolar inhibitor of DHAD through self-resistance gene directed genome mining. Here, we reveal the mechanism of AA inhibition on DHAD and the self-resistance mechanism of AstD, which is encoded by the self-resistance gene astD. As a competitive inhibitor, the hydroxycarboxylic acid group of AA mimics the binding of the natural substrate of DHAD, while the hydrophobic moiety of AA occupies the substrate entrance cavity. Compared to DHAD, AstD has a relatively narrow substrate channel to prevent AA from binding. Several mutants of DHAD were generated and assayed to validate the self-resistance mechanism and to confer Arabidopsis thaliana DHAD with AA resistance. These results will lead to the engineering of new type of herbicides targeting DHAD and provide direction for the ecological construction of herbicide-resistant crops.

243 Translating Maternal Vitamin and Mineral Supplementation Effect on Early Developing Fetus Through Hepatic Long Non-Coding Rnas Profile in Beef Heifers

Abstract Maternal nutrition is critical during pregnancy as the fetus relies on the nutrients from the dam. Fetal organogenesis starts during the first trimester. Thus, an appropriate supply of nutrients, including vitamins and minerals, is indispensable. In the field of fetal developmental programming, there is still a lack of knowledge based on appropriate livestock models to explain the role of regulatory factors in translating the effect of maternal minerals and vitamins on fetal development and metabolic function. Therefore, this study was designed to investigate the effect of maternal vitamin and mineral supplementation (VTM -d 71 periconceptual to d 83 of pregnancy) on fetal liver expression of long non-coding RNAs (lncRNAs) and their potential regulatory metabolic pathways. Angus crossbreed beef heifers (n = 16) were supplemented (VTM, n = 8) or not (NoVTM, n = 8) with 113 g· heifer-1· day-1 of mineral premix (Purina Wind & Rain Storm All-Season 7.5 Complete) and all heifers were bred with female sexed semen from a single sire. Transcriptomic profiles were determined with RNA-Seq in fetal livers collected on d 83 ± 0.27 of gestation. After data quality control and read mapping, lncRNAs were predicted using the FEELnc pipeline. Using the DESeq2 software, we identified 50 differentially expressed novel lncRNAs (expression of 32 lncRNAs was upregulated and 18 were downregulated; FDR < 0.05) located either downstream, upstream, or in exonic, or intronic regions of 51 genes (including PFKP, SLC30A10, KCNH8, ZNF451, MINPP1, BCL9, PAH, ITPR2, AHCY, and ABCG8), potentially regulating their expression. These genes could affect pathways (FDR < 0.1) such as PI3K signaling, mineral storage and transport, and amino acids biosynthetic pathways (i.e., cysteine and methionine biosynthesis). Collectively, these results suggest that periconceptual maternal vitamin and mineral supplementation affects the expression of lncRNAs and potentially regulates the expression of genes involved in fetal liver development and function.

Reconstitution and Optimization of the Marmesin Biosynthetic Pathway in Yeast.

Marmesin is essential in plant defense systems and exhibits various biological activities. In this study, we reconstituted the marmesin biosynthetic pathway in the Saccharomyces cerevisiae BY4741 chassis. We engineered the aromatic amino acid (AAA) biosynthetic pathways by introducing Escherichia coli-derived ppsA to improve the availability of the AAA precursor phosphoenolpyruvate, overexpressing the feedback inhibition resistance genes ARO4K229L and ARO7G141S to direct the metabolic flux toward the tyrosine branch, and deleting ARO10, PDC5, and PDC6 to reduce the byproducts from the Ehrlich pathway. The umbelliferone 6-dimethylallyltransferase (U6DT) and marmesin synthase (MS) involved in marmesin synthesis were optimized to increase marmesin production. Marmesin production was improved by truncating the transmembrane domains of PcU6DT, FcMS, and AtCPR1 and increasing the copy numbers of the genes encoding the truncated enzymes. Finally, a marmesin titer of 27.7 mg/L was obtained in shake flasks using the engineered yeast strain MU5. The constructed marmesin-producing strain provides the foundation for the green and large-scale production of pharmaceutically important furanocoumarins.

Customizing amino acid metabolism of Pichia pastoris for recombinant protein production.

Amino acids are the building blocks of proteins. In this respect, a reciprocal effect of recombinant protein production on amino acid biosynthesis as well as the impact of the availability of free amino acids on protein production can be anticipated. In this study, the impact of engineering the amino acid metabolism on the production of recombinant proteins was investigated in the yeast Pichia pastoris (syn Komagataella phaffii). Based on comprehensive systems-level analyses of the metabolomes and transcriptomes of different P. pastoris strains secreting antibody fragments, cell engineering targets were selected. Our working hypothesis that increasing intracellular amino acid levels could help unburden cellular metabolism and improve recombinant protein production was examined by constitutive overexpression of genes related to amino acid metabolism. In addition to 12 genes involved in specific amino acid biosynthetic pathways, the transcription factor GCN4 responsible for regulation of amino acid biosynthetic genes was overexpressed. The production of the used model protein, a secreted carboxylesterase (CES) from Sphingopyxis macrogoltabida, was increased by overexpression of pathway genes for alanine and for aromatic amino acids, and most pronounced, when overexpressing the regulator GCN4. The analysis of intracellular amino acid levels of selected clones indicated a direct linkage of improved recombinant protein production to the increased availability of intracellular amino acids. Finally, fed batch cultures showed that overexpression of GCN4 increased CES titers 2.6-fold, while the positive effect of other amino acid synthesis genes could not be transferred from screening to bioreactor cultures.

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

Cysteine proteases are activated in sensitive Amaranthus palmeri populations upon treatment with herbicides inhibiting amino acid biosynthesis.

The herbicides glyphosate and pyrithiobac inhibit the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the aromatic amino acid biosynthetic pathway and acetolactate synthase (ALS) in the branched-chain amino acid biosynthetic pathway, respectively. Here we characterise the protease activity profiles of a sensitive (S), a glyphosate-resistant (GR) and a multiple-resistant (MR) population of Amaranthus palmeri in response to glyphosate and pyrithiobac. Amino acid accumulation and cysteine protease activities were induced with both herbicides in the S population and with pyrithiobac in the GR population, suggesting that the increase in cysteine proteases is responsible for the increased degradation of the available proteins and the observed increase in free amino acids. Herbicides did not induce any changes in the proteolytic activities in the populations with target-site resistance, indicating that this effect was only induced in sensitive plants.

Role of TRDMT1/DNMT2 in stress adaptation and its influence on transcriptome and proteome dynamics under osmotic stress in Physcomitrium patens.

Early land plants such as the moss Physcomitrium patens lack several morphological traits that offer protection to tracheophytes from environmental stresses. These plants instead have evolved several physiological and biochemical mechanisms that facilitate them to adapt to terrestrial stresses such as drought. We have previously shown that loss-of-function mutants of tRNA (cytosine(38)-C(5))-methyltransferase TRDMT1/DNMT2 in P. patens are highly sensitive to oxidative and osmotic stress. To gain insight into the role of PpTRDMT1/PpDNMT2 in modulating genetic networks under osmotic stress, genome-wide transcriptome and proteome studies were undertaken in wild-type and ppdnmt2 plants. Transcriptome analysis revealed 375 genes to be differentially expressed in the ppdnmt2 under stress compared to the WT. Most of these genes are affiliated with carbohydrate metabolic pathways, photosynthesis, cell wall biogenesis, pathways related to isotropic and polarised cell growth and transcription factors among others. Histochemical staining showed elevated levels of reactive oxygen species in ppdnmt2 while transmission electron microscopy revealed no distinct defects in the ultrastructure of chloroplasts. Immunoprecipitation using PpDNMT2-specific antibody coupled with mass spectrometry revealed core proteins of the glycolytic pathway, antioxidant enzymes, proteins of amino acid biosynthetic pathways and photosynthesis-related proteins among others to co-purify with PpTRDMT1/PpDNMT2 under osmotic stress. Yeast two-hybrid assays, protein deletion and α-galactosidase assays showed the cytosol glycolytic protein glyceraldehyde 3-phosphate dehydrogenase to bind to the catalytic motifs in PpTRDMT1/PpDNMT2. Results presented in this study allow us to better understand genetic networks linking enzymes of energy metabolism, epigenetic processes and RNA pol II-mediated transcription during osmotic stress tolerance in P. patens.

Facile immobilization of pyridoxal 5′-phosphate using p-diazobenzoyl-derivatized Sepharose 4B

Pyridoxal 5′-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ∼2.6 µmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors.

Interaction between rhizobacterial community and host root determines poplar salt tolerance

AbstractRhizosphere microbes play important roles in plant tolerance to abiotic stresses. Plants of different genetic backgrounds acquire stress resistance by assembling specific rhizosphere microbial communities or typical beneficial microbiota. However, the molecular mechanisms by which plants recruit microbiota during plant acclimation to environmental stresses are unclear. Here, we investigated transcription pattern in three poplar genotypes, namely Populus davidiana × P. bolleana Loucne (SXY), P. deltoides × P. euramericana ‘Nanlin 895’ (NL895) and P. alba × P. glandulosa ‘84K’ (84K), and their effect on the rhizosphere microbial community under salinity. The results showed that SXY exhibited salt tolerance characterized by the best performance of photosynthesis and antioxidant system upon salt stress, while salt stress severely damaged the growth and membrane system of 84K. 16S rRNA sequencing revealed the lowest rhizobacterial community diversity associated with SXY compared to 84K and NL895, implying strong enrichment of certain bacterial taxa by the salt tolerant cultivar. Specifically, SXY recruited higher abundances of Hydrogenophaga and Pseudomonas. Furthermore, RNA‐seq analysis of roots combined with weighted gene co‐expression network analysis (WGCNA) identified module eigengene (ME)yellow, a module positively correlated to the SXY‐enriched OTUs. KEGG analysis revealed significant enrichment of lipid metabolic and amino acid biosynthetic pathways in MEyellow. Twenty‐four genes selected from the pathways enriched in MEyellow showed the highest expression levels in SXY. Our results suggest a “gene expression‐rhizosphere‐microbiota‐salt tolerance” regulatory process in the poplar under salinity and provide new insights into the mechanisms by which plants shape the soil microbiome to improve salt tolerance.

Compositional and functional variability of the gut microbiome in children with infantile colic

The inconsolable crying of a child for no apparent reason at an early age is a source of excitement and anxiety for parents. Previous studies have reported that crying may be caused by discomfort associated with the occupation of the intestines of the newborn by microbiota and its vital activity. We conducted a prospective observational study in which 62 newborns and their mothers were recruited. The study comprised two groups, each consisting of 15 infants with colic and 21 controls. Colic and control groups were vaginally born and exclusively breastfed. Fecal samples from children were collected over time from day 1 to 12 months. Full metagenomic sequencing of fecal samples from children and their mothers was carried out. It was determined that the trajectory of the development of the intestinal microbiome of children with colic was different from the group without colic. In the colic group, a depleted relative abundance of Bifidobacterium and enrichment of Bacteroides Clostridiales was found, while the microbial biodiversity in this group was enriched. Metabolic pathway profiling showed that the non-colic group was enriched by amino acid biosynthetic pathways, while the feces microbiome of the colic group was enriched by glycolysis metabolic pathways that correlated with the Bacteroides taxon. This study shows that infantile colic has a definite relationship with the microbiome structure of infants.

Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

Adenylyl-Sulfate Kinase (Met14)-Dependent Cysteine and Methionine Biosynthesis Pathways Contribute Distinctively to Pathobiological Processes in Cryptococcus neoformans.

Blocking of nutrient uptake and amino acid biosynthesis are considered potential targets for next-generation antifungal drugs against pathogenic fungi, including Cryptococcus neoformans. In this regard, the sulfate assimilation pathway is particularly attractive, as it is only present in eukaryotes such as plants and fungi, yet not in mammals. Here, we demonstrated that the adenylyl sulfate kinase (Met14) in the sulfate assimilation pathway is not essential yet is required for the viability of C. neoformans due to its involvement in biosynthesis of two sulfur-containing amino acids, cysteine and methionine. Met14-dependent cysteine and methionine biosynthesis was found to significantly contribute to a diverse range of pathobiological processes in C. neoformans. Met14-dependent cysteine rather than methionine biosynthesis was also found to play pivotal roles in cell growth and tolerance to environmental stresses and antifungal drugs. In contrast, the Met14-dependent methionine biosynthesis was found to be more important than cysteine biosynthesis for the production of major cryptococcal virulence factors of melanin pigments and polysaccharide capsules. Finally, we also found that despite its attenuated virulence in an insect model, Galleria mellonella, the met14Δ mutant yielded no difference in virulence in a murine model of systemic cryptococcosis. Hence, clinical inhibition of Met14-dependent amino acid biosynthetic pathways may not be advantageous for the treatment of systemic cryptococcosis. IMPORTANCE Current antifungal drugs have several limitations, such as drug resistance, severe side effects, and a narrow spectrum. Therefore, novel antifungal targets are urgently needed. To this end, fungal sulfur amino acid biosynthetic pathways are considered potential targets for development of new antifungal agents. Here, we demonstrated that Met14 in the sulfate assimilation pathway promotes growth, stress response, and virulence factor production in C. neoformans via synthesis of sulfur-containing amino acids methionine and cysteine. Met14-dependent cysteine rather than methionine synthesis was found to be critical for growth and stress responses, whereas Met14-dependent methionine synthesis was more important for the production of antiphagocytic capsules and antioxidant melanin in C. neoformans. Surprisingly, deletion of the MET14 gene was found to attenuate cryptococcal virulence in an insect model, yet not in a murine model. Collectively, our results showed that Met14-dependent cysteine and methionine biosynthesis play roles that are distinct from each other in C. neoformans. Moreover, Met14 is unlikely to be a suitable anticryptococcal drug target.

Amino acid metabolic reprogramming in tumor metastatic colonization.

Metastasis is considered as the major cause of cancer death. Cancer cells can be released from primary tumors into the circulation and then colonize in distant organs. How cancer cells acquire the ability to colonize in distant organs has always been the focus of tumor biology. To enable survival and growth in the new environment, metastases commonly reprogram their metabolic states and therefore display different metabolic properties and preferences compared with the primary lesions. For different microenvironments in various colonization sites, cancer cells must transfer to specific metabolic states to colonize in different distant organs, which provides the possibility of evaluating metastasis tendency by tumor metabolic states. Amino acids provide crucial precursors for many biosynthesis and play an essential role in cancer metastasis. Evidence has proved the hyperactivation of several amino acid biosynthetic pathways in metastatic cancer cells, including glutamine, serine, glycine, branched chain amino acids (BCAAs), proline, and asparagine metabolism. The reprogramming of amino acid metabolism can orchestrate energy supply, redox homeostasis, and other metabolism-associated pathways during cancer metastasis. Here, we review the role and function of amino acid metabolic reprogramming in cancer cells colonizing in common metastatic organs, including lung, liver, brain, peritoneum, and bone. In addition, we summarize the current biomarker identification and drug development of cancer metastasis under the amino acid metabolism reprogramming, and discuss the possibility and prospect of targeting organ-specific metastasis for cancer treatment.

Effects of low-energy diet supplemented with betaine on growth performance, nutrient digestibility, and serum metabolomic profiles in growing pigs.

Two experiments were carried out to evaluate the effects of betaine (BET) supplementation in diets with reduced net energy (NE) levels on growth performance, nutrient digestibility, and serum metabolomic profiles in growing pigs. In experiment 1, 24 growing pigs (initial body weight, BW, 30.83 ± 2.50 kg) were allotted to one of the four treatments (six replications with 1 pig per pen) in a 2 × 2 factorial arrangement, including two dietary NE levels (2475 [N-NE] or 2395 [R80-NE] kcal/kg) and two BET doses (0 or 1500 mg/kg). In experiment 2, 72 growing pigs were used in a 2 × 3 factorial arrangement, including three dietary NE levels (2475 [N-NE], 2415 [R60-NE], or 2355 [R120-NE] kcal/kg) and two BET doses (0 or 1500 mg/kg). Pigs with initial BW of 31.44 ± 1.65 kg were divided to one of the six treatments (six replications with 2 pigs per pen). In experiment 1, lowing NE concentrations increased average daily feed intake (ADFI) by 10.69% in pigs fed the diet without BET (P > 0.05). BET significantly increased ADFI in N-NE diet (P < 0.05) but had no influence on ADFI in R80-NE diet (P > 0.05). BET enhanced the apparent digestibility of crude protein (CP), dry matter (DM), organic matter (OM), gross energy (GE), and ether extract (EE) in R80-NE diet (P < 0.05). In experiment 2, lowing NE concentrations enhanced ADFI (P > 0.05) and decreased average daily gain (ADG; P < 0.05). The reduction in feed intake by BET was further enhanced as NE concentrations decreased from 2415 to 2355 kcal/kg (P < 0.10). BET reversed the elevation of serum triglyceride, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase levels caused by R120-NE diet (P < 0.05). The concentrations of cholecystokinin and glucagon-like peptide 1 were increased by BET in pigs fed the R120-NE diet (P < 0.05). Serum metabolomics reveals that lowing dietary NE concentrations affected mainly amino acid biosynthetic pathways (P < 0.05). BET supplementation in R120-NE diet up-regulated serum BET levels and down-regulated homocysteine, DL-carnitine, and four amino acid secondary metabolites (P < 0.05). In conclusion, lowing dietary NE contents reduced the growth performance and caused metabolic abnormalities in growing pigs. However, BET decreased feed intake to a certain extent and improved the metabolic health of pigs fed the low-NE diets, which may be related to the dual regulation of amino acid metabolism and the secretion of appetite related hormones by BET.

Transcriptomic Analysis of Aromatic Amino Acid Biosynthesis Under Nitrogen Starvation in Saccharomyces Cerevisiae

Yeast Saccharomyces cerevisiae has been a preferred organism for scientific research on many sensing and signaling processes. Its ability to adapt to changes in reproductive conditions, especially its fermentative properties have made this yeast species an industrially important species. Because of that S. cerevisiae has been genetically modified to create many kinds of beneficial chemicals from biomass. S. cerevisiae cells have the ability to detect the quality of nitrogen sources in their environment. And It has many regulatory mechanisms to utilize alternative nitrogen sources after the favored sources have been depleted. And proline is the least desired nitrogen source for many laboratory yeast strains. Usage of proline results in the slowest growth rates. The yeast S. cerevisiae can only get cellular nitrogen from tryptophan, tyrosine, or phenylalanine. The major products of this catabolism are tryptophol, phenylethanol, and tyrosol, which are elements of the mixture of alcohols known as fusel oil and this makes yeast S. cerevisiae important also in the production of seconder metabolites. Next Generation Sequencing allows deep analysis of gene expression. The effect of growth in media containing poor nitrogen source, proline, on the aromatic amino acid biosynthetic pathway was observed using NGS technologies in this work. According to the transcriptomic analysis results, we found that 14 genes are responsible for aromatic amino acid biosynthesis and 12 genes (ARO1, ARO2, ARO4, ARO7, ARO8, ARO9, PHA2, TRP1, TRP2, TRP3, TRP4, and TYR1) were down-regulated, while 2 genes (ARO3 and TRP1) were up-regulated. In addition, ARO1, ARO2, ARO4, ARO7, ARO9, PHA2, TRP1, TRP2, TRP3, TRP4, and TYR1 genes have showed 1.5-fold or more expression.