L-arginine Biosynthesis Research Articles

Association between the Maternal Gut Microbiome and Macrosomia.

Fetal macrosomia is defined as a birthweight ≥4000 g and causes harm to pregnant women and fetuses. Studies reported that the maternal intestinal microbiome plays a key role in the establishment, growth, and development of the fetal intestinal microbiome. However, whether there is a relationship between maternal gut microbiota and macrosomia remains unclear. Our study aimed to identify gut microbiota that may be related to the occurrence of macrosomia, explore the possible mechanisms by which it causes macrosomia, and establish a prediction model to determine the feasibility of predicting macrosomia by early maternal gut microbiota. We conducted a nested case-control study based on an early pregnancy cohort (ChiCTR1900020652) in the Maternity and Child Health Hospital of Hunan Province on fecal samples of 93 women (31 delivered macrosomia as the case group and 62 delivered normal birth weight newborns as the control group) collected and included in this study. We performed metagenomic analysis to compare the composition and function of the gut microbiome between cases and controls. Correlation analysis was used to explore the association of differential species and differential functional pathways. A random forest model was used to construct an early pregnancy prediction model for macrosomia. At the species level, there were more Bacteroides salyersiae, Bacteroides plebeius, Ruminococcus lactaris, and Bacteroides ovatus in the intestinal microbiome of macrosomias' mothers compared with mothers bearing fetuses that had normal birth weight. Functional pathways of the gut microbiome including gondoate biosynthesis, L-histidine degradation III, cis-vaccenate biosynthesis, L-arginine biosynthesis III, tRNA processing, and mannitol cycle, which were more abundant in the macrosomia group. Significant correlations were found between species and functional pathways. Bacteroides plebeius was significantly associated with the pathway of cis-vaccenate biosynthesis (r = 0.28, p = 0.005) and gondoate biosynthesis (r = 0.28, p < 0.001) and Bacteroides ovatus was positively associated with the pathway of cis-vaccenate biosynthesis (r = 0.29, p = 0.005) and gondoate biosynthesis (r = 0.32, p = 0.002). Bacteroides salyersiae was significantly associated with the pathway of cis-vaccenate biosynthesis (r = 0.24, p = 0.018), gondoate biosynthesis (r = 0.31, p = 0.003), and L-histidine degradation III (r = 0.22, p = 0.291). Finally, four differential species and four clinical indicators were included in the random forest model for predicting macrosomia. The areas under the working characteristic curves of the training and validation sets were 0.935 (95% CI: 0.851~0.979) and 0.909 (95% CI: 0.679~0.992), respectively. Maternal gut microbiota in early pregnancy may play an important role in the development of macrosomia and can be used as potential predictors to prevent macrosomia.

Regulation of Gut Microbiota and Microbial Metabolome of Kefir Supernatant against Fusobacterium nucleatum and DSS-Coinduced Colitis.

The aim of this study was to investigate the intervention effect of kefir supernatant (KS) on the initiation and progression of an ulcerative colitis (UC) murine model. We established an UC murine model by orally administrating with 109 CFUs of Fusobacterium nucleatum for 3 weeks and 3% dextran sulfate sodium (DSS) treatment in the third week. KS was used to intervene in this colitis model. Our results showed that KS supplementation ameliorated the symptoms, restrained the secretion of pro-inflammatory cytokines (TNF-α, IL-6, and IL-17F), promoted the release of anti-inflammatory cytokines (IL-4 and IL-10), and ameliorated oxidative stress. Furthermore, the increased number of goblet cells and upregulated expression of MUC2, occludin and claudin-1 indicated that the colon barrier was protected by KS. Additionally, KS supplementation mitigated gut microbiota dysbiosis in the UC murine model, leading to an increase in the abundance of Blautia and Akkermansia and a decrease in the level of Bacteroides. The altered gut microbiota also affected colon metabolism, with differential metabolites mainly associated with the biosynthesis of the l-arginine pathway. This study revealed that KS supplementation restored the community structure of gut microbiota, altered the biosynthesis of l-arginine, and thereby modulated the process of colonic inflammation.

Role of Argininosuccinate Synthase 1 -Dependent L-Arginine Biosynthesis in the Protective Effect of Endothelial Sirtuin 3 Against Atherosclerosis.

Atherosclerosis is initiated with endothelial cell (EC) dysfunction and vascular inflammation under hyperlipidemia. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase. However, the specific role of endothelial SIRT3 during atherosclerosis remains poorly understood. The present study aims to study the role and mechanism of SIRT3 in EC function during atherosclerosis. Wild-type Sirt3f/f mice and endothelium-selective SIRT3 knockout Sirt3f/f; Cdh5Cre/+ (Sirt3EC-KO) mice are injected with adeno-associated virus (AAV)to overexpress PCSK9 and fed with high-cholesterol diet (HCD)for 12 weeks to induce atherosclerosis. Sirt3EC-KO mice exhibit increased atherosclerotic plaque formation, along with elevated macrophage infiltration, vascular inflammation, and reduced circulating L-argininelevels. In human ECs, SIRT3 inhibition resulted in heightened vascular inflammation, reduced nitric oxide (NO) production, increased reactive oxygen species (ROS), and diminished L-arginine levels. Silencing of SIRT3 results in hyperacetylation and deactivation of Argininosuccinate Synthase 1 (ASS1), a rate-limiting enzyme involved in L-arginine biosynthesis, and this effect is abolished in mutant ASS1. Furthermore, L-arginine supplementation attenuates enhanced plaque formation and vascular inflammation in Sirt3EC-KO mice. This study provides compelling evidence supporting the protective role of endothelial SIRT3 in atherosclerosis and also suggests a critical role of SIRT3-induced deacetylation of ASS1 by ECs for arginine synthesis.

A novel strategy for l-arginine production in engineered Escherichia coli

Backgroundl-arginine is an important amino acid with applications in diverse industrial and pharmaceutical fields. n-acetylglutamate, synthesized from l-glutamate and acetyl-CoA, is a precursor of the l-arginine biosynthetic branch in microorganisms. The enzyme that produces n-acetylglutamate, n-acetylglutamate synthase, is allosterically inhibited by l-arginine. l-glutamate, as a central metabolite, provides carbon backbone for diverse biological compounds besides l-arginine. When glucose is the sole carbon source, the theoretical maximum carbon yield towards l-arginine is 96.7%, but the experimental highest yield was 51%. The gap of l-arginine yield indicates the regulation complexity of carbon flux and energy during the l-arginine biosynthesis. Besides endogenous biosynthesis, n-acetylglutamate, the key precursor of l-arginine, can be obtained by chemical acylation of l-glutamate with a high yield of 98%. To achieve high-yield production of l-arginine, we demonstrated a novel approach by directly feeding precursor n-acetylglutamate to engineered Escherichia coli.ResultsWe reported a new approach for the high yield of l-arginine production in E. coli. Gene argA encoding n-acetylglutamate synthase was deleted to disable endogenous biosynthesis of n-acetylglutamate. The feasibility of external n-acetylglutamate towards l-arginine was verified via growth assay in argA− strain. To improve l-arginine production, astA encoding arginine n-succinyltransferase, speF encoding ornithine decarboxylase, speB encoding agmatinase, and argR encoding an arginine responsive repressor protein were disrupted. Based on overexpression of argDGI, argCBH operons, encoding enzymes of the l-arginine biosynthetic pathway, ~ 4 g/L l-arginine was produced in shake flask fermentation, resulting in a yield of 0.99 mol l-arginine/mol n-acetylglutamate. This strain was further engineered for the co-production of l-arginine and pyruvate by removing genes adhE, ldhA, poxB, pflB, and aceE, encoding enzymes involved in the conversion and degradation of pyruvate. The resulting strain was shown to produce 4 g/L l-arginine and 11.3 g/L pyruvate in shake flask fermentation.ConclusionsHere, we developed a novel approach to avoid the strict regulation of l-arginine on ArgA and overcome the metabolism complexity in the l-arginine biosynthesis pathway. We achieve a high yield of l-arginine production from n-acetylglutamate in E. coli. Co-production pyruvate and l-arginine was used as an example to increase the utilization of input carbon sources.

Unique microbial landscape in the human oropharynx during different types of acute respiratory tract infections

BackgroundSecondary bacterial infections and pneumonia are major mortality causes of respiratory viruses, and the disruption of the upper respiratory tract (URT) microbiota is a crucial component of this process. However, whether this URT dysbiosis associates with the viral species (in other words, is viral type-specific) is unclear.ResultsHere, we recruited 735 outpatients with upper respiratory symptoms, identified the infectious virus types in 349 participants using multiplex RT-PCR, and profiled their upper respiratory microbiome using the 16S ribosomal RNA gene and metagenomic gene sequencing. Microbial and viral data were subsequently used as inputs for multivariate analysis aimed at revealing viral type-specific disruption of the upper respiratory microbiota. We found that the oropharyngeal microbiota shaped by influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (RSV), and human rhinovirus (HRV) infections exhibited three distinct patterns of dysbiosis, and Veillonella was identified as a prominent biomarker for any type of respiratory viral infections. Influenza virus infections are significantly correlated with increased oropharynx microbiota diversity and enrichment of functional metabolic pathways such as L-arginine biosynthesis and tetracycline resistance gene tetW. We used the GRiD algorithm and found the predicted growth rate of common respiratory pathogens was increased upon influenza virus infection, while commensal bacteria, such as Streptococcus infantis and Streptococcus mitis, may act as a colonization resistance to the overgrowth of these pathogens.ConclusionsWe found that respiratory viral infections are linked with viral type-specific disruption of the upper respiratory microbiota, particularly, influenza infections uniquely associated with increased microbial diversity and growth rates of specific pathogens in URT. These findings are essential for clarifying the differences and dynamics of respiratory microbiota in healthy participants and acute respiratory viral infections, which contribute to elucidating the pathogenesis of viral-host-bacterial interactions to provide insights into future studies on effective prevention and treatment of respiratory tract infections.AxMbpzHUJuHXyJKsbr5ywRVideo

Phenotype-genotype mapping reveals the betaine-triggered L-arginine overproduction mechanism in Escherichia coli

The production phenotype improvement of industrial microbes is extremely needed and challenging. Environmental factors optimization provides insightful ideas to trigger the superior production phenotype by activating potential genetic determiners. Here, phenotype-genotype mapping was used to dissect the betaine-triggered L-arginine overproduction mechanism and mine beneficial genes for further improving production phenotype. The comparative transcriptomic analysis revealed a novel role for betaine in modulating global gene transcription. Guided by this finding, 4 novel genes (cynX, cynT, pyrB, and rhaB) for L-arginine biosynthesis were identified via reverse engineering. Moreover, the rhaB deletion was demonstrated as a common metabolic engineering strategy to improve ATP pool in E. coli. By combinatorial genes manipulation, the L-arginine titer and yield increased by 17.9% and 28.9% in a 5-L bioreactor without betaine addition. This study revealed the molecular mechanism of gene transcription regulation by betaine and developed a superior L-arginine overproducer that does not require betaine.

Glyoxylate Shunt and Pyruvate-to-Acetoin Shift Are Specific Stress Responses Induced by Colistin and Ceragenin CSA-13 in Enterobacter hormaechei ST89.

Ceragenins, including CSA-13, are cationic antimicrobials that target the bacterial cell envelope differently than colistin. However, the molecular basis of their action is not fully understood. Here, we examined the genomic and transcriptome responses by Enterobacter hormaechei after prolonged exposure to either CSA-13 or colistin. Resistance of the E. hormaechei 4236 strain (sequence type 89 [ST89]) to colistin and CSA-13 was induced in vitro during serial passages with sublethal doses of tested agents. The genomic and metabolic profiles of the tested isolates were characterized using a combination of whole-genome sequencing (WGS) and transcriptome sequencing (RNA-seq), followed by metabolic mapping of differentially expressed genes using Pathway Tools software. The exposure of E. hormaechei to colistin resulted in the deletion of the mgrB gene, whereas CSA-13 disrupted the genes encoding an outer membrane protein C and transcriptional regulator SmvR. Both compounds upregulated several colistin-resistant genes, such as the arnABCDEF operon and pagE, including genes coding for DedA proteins. The latter proteins, along with beta-barrel protein YfaZ and VirK/YbjX family proteins, were the top overexpressed cell envelope proteins. Furthermore, the l-arginine biosynthesis pathway and putrescine-ornithine antiporter PotE were downregulated in both transcriptomes. In contrast, the expression of two pyruvate transporters (YhjX and YjiY) and genes involved in pyruvate metabolism, as well as genes involved in generating proton motive force (PMF), was antimicrobial specific. Despite the similarity of the cell envelope transcriptomes, distinctly remodeled carbon metabolism (i.e., toward fermentation of pyruvate to acetoin [colistin] and to the glyoxylate pathway [CSA-13]) distinguished both antimicrobials, which possibly reflects the intensity of the stress exerted by both agents. IMPORTANCE Colistin and ceragenins, like CSA-13, are cationic antimicrobials that disrupt the bacterial cell envelope through different mechanisms. Here, we examined the genomic and transcriptome changes in Enterobacter hormaechei ST89, an emerging hospital pathogen, after prolonged exposure to these agents to identify potential resistance mechanisms. Interestingly, we observed downregulation of genes associated with acid stress response as well as distinct dysregulation of genes involved in carbon metabolism, resulting in a switch from pyruvate fermentation to acetoin (colistin) and the glyoxylate pathway (CSA-13). Therefore, we hypothesize that repression of the acid stress response, which alkalinizes cytoplasmic pH and, in turn, suppresses resistance to cationic antimicrobials, could be interpreted as an adaptation that prevents alkalinization of cytoplasmic pH in emergencies induced by colistin and CSA-13. Consequently, this alteration critical for cell physiology must be compensated via remodeling carbon and/or amino acid metabolism to limit acidic by-product production.

Effect of Probiotic Yogurt Supplementation(Bifidobacterium animalis ssp. lactis BB-12) on Gut Microbiota of Female Taekwondo Athletes and Its Relationship with Exercise-Related Psychological Fatigue.

The gut microbiota plays a critical role in regulating human health and athletic performance. Probiotic supplementation has been shown to modulate gut microbiota composition and improve exercise performance. This study aimed to investigate the effect of probiotic yogurt supplementation on gut microbiota and its relationship with exercise-related psychological fatigue in female taekwondo athletes. Twenty female taekwondo athletes were randomly assigned to either a dietary intervention group (DK) or a control group (CK). The athletes' exercise-related psychological fatigue was measured using the Athlete Burnout Questionnaire (ABQ) before and after an 8-week intervention. High-throughput sequencing was used to profile the gut microbiota, and functional prediction of the microbial community was performed. The effect of the dietary intervention on the athletes' exercise-related psychological fatigue clearance rate and its relationship with the gut microbiota were explored. (1) The probiotic supplementation of Bifidobacterium animalis ssp. lactis BB-12 for 8 weeks significantly increased the ABQ scores of the DK group compared to the CK group (p < 0.05). (2) The abundances of Bifidobacterium, Bacteroides, Lachnospiraceae, family _Lactobacillaceae, and genus _Lactobacillus were significantly higher in the DK group than in the CK group after probiotic supplementation, while Escherichia coli was significantly lower in the DK group than in the CK group. (3) The ABQa scores were positively correlated with Proteus; ABQb scores were positively correlated with Streptococcus and Enterococcus; and ABQc scores were positively correlated with Klebsiella, Bacteroides, and Streptomyces. (4) The DK group had significantly higher levels of L-arginine biosynthesis I (via L-ornithine), fatty acid biosynthesis and oxidation, and L-isoleucine biosynthesis III pathways compared to the CK group. Tyrosine degradation I (via 2,3-dihydroxyphenylpropionate) was significantly lower in the DK group than in the CK group. Probiotic yogurt supplementation of Bifidobacterium animalis ssp. lactis can promote the clearance of exercise-related psychological fatigue in female taekwondo athletes by upregulating beneficial gut microbiota, inhibiting harmful gut microbiota, and regulating relevant metabolic pathways.

Whole intestinal microbiota transplantation is more effective than fecal microbiota transplantation in reducing the susceptibility of DSS-induced germ-free mice colitis.

Fecal microbiota transplantation (FMT) is an emerging and effective therapy for the treatment of inflammatory bowel disease (IBD). Previous studies have reported that compared with FMT, whole intestinal microbiota transplantation (WIMT) can more precisely replicate the community structure and reduce the inflammatory response of the host. However, it remains unclear whether WIMT is more effective in alleviating IBD. To examine the efficacy of WIMT and FMT in the intervention of IBD, GF (Germ-free) BALB/c mice were pre-colonized with whole intestinal microbiota or fecal microbiota before being treated with dextran sodium sulfate (DSS). As expected, the symptoms of colitis were alleviated by both WIMT and FMT, as demonstrated by the prevention of body weight loss and decreased the Disease activity index and histological scores in mice. However, WIMT's anti-inflammatory effect was superior to that of FMT. In addition, the inflammatory markers myeloperoxidase (MPO) and eosinophil peroxidase were dramatically downregulated by WIMT and FMT. Furthermore, the use of two different types of donors facilitated the regulation of cytokine homeostasis in colitis mice; the level of the pro-inflammatory cytokine IL-1β in the WIMT group was significantly lower than that in the FMT group, while the level of the anti-inflammatory factor IL-10 was significantly higher than that in the FMT group. Both groups showed enhanced expression of occludin to protect the intestinal barrier in comparison with the DSS group, and the WIMT group demonstrated considerably increased levels of ZO-1. The sequencing results showed that the WIMT group was highly enriched in Bifidobacterium, whereas the FMT group was significantly enriched in Lactobacillus and Ochrobactrum. Correlation analysis revealed that Bifidobacterium was negatively correlated with TNF-α, whereas Ochrobactrum was positively correlated with MPO and negatively correlated with IL-10, which might be related to different efficacies. Functional prediction using PICRUSt2 revealed that the FMT group was considerably enriched in the L-arginine biosynthesis I and L-arginine biosynthesis IV pathway, whereas the WIMT group was enriched in the L-lysine fermentation to acetate and butanoate pathway. In conclusion, the symptoms of colitis were subsided to varying degrees by the two different types of donors, with the WIMT group being more effective than the FMT group. This study provides new information on clinical interventions for IBD.

The involvement of Th1 cell differentiation in the anti-tumor effect of purified polysaccharide from Sanghuangporus vaninii in colorectal cancer via multi-omics analysis

Sanghuangporus vaninii is a medicinal mushroom, which has been used as a treatment for various diseases; however, the therapeutic potential and mechanism of action of S. vaninii in colorectal cancer (CRC) remain unknown. Herein, human colon adenocarcinoma cells were used to analyze the anti-CRC effects of the purified polysaccharide of S. vaninii (SVP-A-1) in vitro. In SVP-A-1-treated B6/JGpt-Apcem1Cin (Min)/Gpt male (ApcMin/+) mice, 16S rRNA sequencing was performed on cecal feces, metabolites were examined in serum, and LC-MS/MS protein detection was performed in colorectal tumors. Protein changes were further confirmed by various biochemical detection methods. Water-soluble SVP-A-1 with a molecular weight of 22.5 kDa was first obtained. SVP-A-1 prevented gut microbiota dysbiosis related to metabolic pathways of L-arginine biosynthesis, increased L-citrulline levels in the serum of ApcMin/+ mice, mediated L-arginine synthesis, and improved antigen presentation in dendritic cells and activated CD4+ T cells; the resulting Th1 cells released IFN-γ and TNF-α to act on tumor cells and promoted the sensitivity of tumor cells to cytotoxic T lymphocytes. In summary, SVP-A-1 exerted anti-CRC effects and has excellent potential for CRC treatment.

Metabolic reprogramming and biosensor-assisted mutagenesis screening for high-level production of L-arginine in Escherichia coli

L-arginine is a value-added amino acid with promising applications in the pharmaceutical and nutraceutical industries. Further unleashing the potential of microbial cell factories to make L-arginine production more competitive remains challenging due to the sophisticated intracellular interaction networks and the insufficient knowledge of global metabolic regulation. Here, we combined multilevel rational metabolic engineering with biosensor-assisted mutagenesis screening to exploit the L-arginine production potential of Escherichia coli. First, multiple metabolic pathways were systematically reprogrammed to redirect the metabolic flux into L-arginine synthesis, including the L-arginine biosynthesis, TCA cycle, and L-arginine export. Specifically, a toggle switch responding to special cellular physiological conditions was designed to dynamically control the expression of sucA and pull more carbon flux from the TCA cycle toward L-arginine biosynthesis. Subsequently, a biosensor-assisted high-throughput screening platform was designed and applied to further exploit the L-arginine production potential. The best-engineered ARG28 strain produced 132 g/L L-arginine in a 5-L bioreactor with a yield of 0.51 g/g glucose and productivity of 2.75 g/(L ⋅ h), which were the highest values reported so far. Through whole genome sequencing and reverse engineering, Frc frameshift mutant, PqiB A78P mutant, and RpoB P564T mutant were revealed for enhancing the L-arginine biosynthesis. Our study exhibited the power of coupling rational metabolic reprogramming and biosensor-assisted mutagenesis screening to unleash the cellular potential for value-added metabolite production.

The role of trehalose biosynthesis on mycolate composition and L-glutamate production in Corynebacterium glutamicum

Corynebacterium glutamicum has been widely utilized for the industrial production of various amino acids. Trehalose is a prerequisite for the biosynthesis of mycolates which are structurally important constituents of the cell envelope in C. glutamicum. In this study, C. glutamicum mutant ΔSYA, which is unable to synthesize trehalose was constructed by deleting genes treS, treY and otsA in the three pathways of trehalose biosynthesis. In the fermentation medium, ΔSYA grew as well as the control C. glutamicum ATCC13869, synthesized similar levels of glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids to ATCC13869, but produced 12.5 times more L-glutamate than ATCC13869. Transcriptional levels of the genes relevant to L-arginine biosynthesis, encoding ODHC and relevant to the biosynthesis of sulfur-containing amino acids were down-regulated in ΔSYA. In minimal medium with different concentrations of glucose, ΔSYA grew worse than ATCC13869 but excreted more L-glutamate. When grown in minimal medium, phospholipids are the major lipid in ΔSYA, while glucose monocorynomycolate, trehalose dicorynomycolate, and phospholipids are the major lipid in ATCC13869. The transcriptional levels of mscCG in ΔSYA was significantly up-regulated when grown in minimal medium.

The Alteration of Tomato Chloroplast Vesiculation Positively Affects Whole-Plant Source-Sink Relations and Fruit Metabolism under Stress Conditions.

Changes in climate conditions can negatively affect the productivity of crop plants. They can induce chloroplast degradation (senescence), which leads to decreased source capacity, as well as decreased whole-plant carbon/nitrogen assimilation and allocation. The importance, contribution and mechanisms of action regulating source-tissue capacity under stress conditions in tomato (Solanum lycopersicum) are not well understood. We hypothesized that delaying chloroplast degradation by altering the activity of the tomato chloroplast vesiculation (CV) under stress would lead to more efficient use of carbon and nitrogen and to higher yields. Tomato CV is upregulated under stress conditions. Specific induction of CV in leaves at the fruit development stage resulted in stress-induced senescence and negatively affected fruit yield, without any positive effects on fruit quality. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/CAS9) knockout CV plants, generated using a near-isogenic tomato line with enhanced sink capacity, exhibited stress tolerance at both the vegetative and the reproductive stages, leading to enhanced fruit quantity, quality and harvest index. Detailed metabolic and transcriptomic network analysis of sink tissue revealed that the l-glutamine and l-arginine biosynthesis pathways are associated with stress-response conditions and also identified putative novel genes involved in tomato fruit quality under stress. Our results are the first to demonstrate the feasibility of delayed stress-induced senescence as a stress-tolerance trait in a fleshy fruit crop, to highlight the involvement of the CV pathway in the regulation of source strength under stress and to identify genes and metabolic pathways involved in increased tomato sink capacity under stress conditions.

The stressosome is required to transduce low pH signals leading to increased transcription of the amino acid-based acid tolerance mechanisms in Listeria monocytogenes.

Increasing proton concentration in the environment represents a potentially lethal stress for single-celled microorganisms. To survive in an acidifying environment, the foodborne pathogen Listeria monocytogenes quickly activates the alternative sigma factor B (σB), resulting in upregulation of the general stress response (GSR) regulon. Activation of σB is regulated by the stressosome, a multi-protein sensory complex involved in stress detection and signal transduction. In this study, we used L. monocytogenes strains harbouring two stressosome mutants to investigate the role of this complex in triggering expression of known amino acid-based resistance mechanisms in response to low pH. We found that expression of glutamate decarboxylase (gadD3) and arginine and agmatine deiminases (arcA and aguA1, respectively) were upregulated upon acid shock (pH 5 for 15 min) in a stressosome-dependent manner. In contrast, transcription of the arg operons (argGH and argCJBDF), which encode enzymes for the l-arginine biosynthesis pathway, were upregulated upon acid shock in a stressosome-independent manner. Finally, we found that transcription of argR, which encodes a transcriptional regulator of the arc and arg operons, was largely unaffected by acidic shock. Thus, our findings suggest that the stressosome plays a role in activating amino acid-based pH homeostatic mechanisms in L. monocytogenes . Additionally, we show that genes encoding the l-arginine biosynthesis pathway are highly upregulated under acidic conditions, suggesting that intracellular arginine can help withstand environmental acidification in this pathogen.

Extracellular Vesicle Subproteome Differences among Filifactor alocis Clinical Isolates

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the Firmicutes phylum, which has recently been implicated in oral infections. Extracellular vesicles (EVs) are crucial conveyors of microbial virulence in bacteria and archaea. Previously, in highly purified EVs from the F. alocis reference strain ATCC 35896 (CCUG 47790), 28 proteins were identified. The present study aimed to use label-free quantification proteomics in order to chart these EV proteins, in the reference strain, and in nine less-well-characterized clinical F. alocis isolates. In total, 25 of the EV proteins were identified and 24 were quantified. Sixteen of those were differentially expressed between the ten strains and the novel FtxA RTX toxin and one lipoprotein were among them. Consistent expression was observed among ribosomal proteins and proteins involved in L-arginine biosynthesis and type IV pilin, demonstrating a degree of EV protein expression preservation among strains. In terms of protein–protein interaction analysis, 21 functional associations were revealed between 19 EV proteins. Interestingly, FtxA did not display predicted interactions with any other EV protein. In conclusion, the present study charted 25 EV proteins in ten F. alocis strains. While most EV proteins were consistently identified among the strains, several of them were also differentially expressed, which justifies that there may be potential variations in the virulence potential among EVs of different F. alocis strains.

Systems metabolic engineering of Corynebacterium glutamicum eliminates all by-products for selective and high-yield production of the platform chemical 5-aminovalerate

5-aminovalerate (AVA) is a platform chemical of substantial commercial value to derive nylon-5 and five-carbon derivatives like δ-valerolactam, 1,5-pentanediol, glutarate, and 5-hydroxyvalerate. Denovo bio-production synthesis of AVA using metabolically engineered cell factories is regarded as exemplary route to provide this chemical in a sustainable way. So far, this route is limited by low titers, rates and yields and suffers from high levels of by-products. To overcome these limitations, we developed a novel family of AVA producing C. glutamicum cell factories. Stepwise optimization included (i) improved AVA biosynthesis by expression balancing of the heterologous davBA genes from P. putida, (ii) reduced formation of the by-product glutarate by disruption of the catabolic y-aminobutyrate pathway (iii), increased AVA export, and (iv) reduced AVA re-import via native and heterologous transporters to account for the accumulation of intracellular AVA up to 300 mM. Strain C. glutamicum AVA-5A, obtained after several optimization rounds, produced 48.3 g L−1 AVA in a fed-batch process and achieved a high yield of 0.21 g g−1. Surprisingly in later stages, the mutant suddenly accumulated glutarate to an extent equivalent to 30% of the amount of AVA formed, tenfold more than in the early process, displaying a severe drawback toward industrial production. Further exploration led to the discovery that ArgD, naturally aminating N-acetyl-l-ornithine during l-arginine biosynthesis, exhibits deaminating side activity on AVA towards glutarate formation. This promiscuity became relevant because of the high intracellular AVA level and the fact that ArgD became unoccupied with the gradually stronger switch-off of anabolism during production. Glutarate formation was favorably abolished in the advanced strains AVA-6A, AVA-6B, and AVA-7, all lacking argD. In a fed-batch process, C. glutamicum AVA-7 produced 46.5 g L−1 AVA at a yield of 0.34 g g−1 and a maximum productivity of 1.52 g L−1 h−1, outperforming all previously reported efforts and stetting a milestone toward industrial manufacturing of AVA. Notably, the novel cell factories are fully genome-based, offering high genetic stability and requiring no selection markers

Metagenomics analyses of microbial dynamics associated with putative flavor development in mash fermentation of sake

Sake is a traditional alcoholic beverage in Japan. To examine the impact of co-fermentation on microbial composition, fermentation with and without pineapple was conducted. The dynamics of microbial communities during fermentation were investigated using the metagenomics of 16S rRNA and internal transcribed spacers. In regular (RS) and co-fermented (CS) sakes, the fungal microbiota was dominated by Saccharomyces. Hierarchical clustering and non-metric multidimensional scaling identified two phases of bacterial succession. Compared with the dividing point between phases in RS, the transition in CS was delayed by five days. In both experiments, Leuconostoc dominated the premature stage and was replaced by Erwinia in the maturation stage; the alcohol tolerators of Staphylococcus and Bacillus were persistent. In RS, bacterial succession was characterized by a dramatic decrease in Pseudomonas in the maturation stage, whereas it occurred earlier in the premature stage in CS. Nineteen genera were exclusively found in CS, suggesting that co-fermentation may nurture diverse bacteria that enrich the sake flavor. Analysis of functional pathways by PICRUSt2 revealed that sugar alcohol degradation was fundamental to fermentation, whereas flavor-rich CS was attributable to the activation of l-arginine biosynthesis and amine and polyamine degradation. Aerococcus was positively correlated with enzymes involved in phenylethyl metabolism.

Acetylornithine aminotransferase TM1785 performs multiple functions in the hyperthermophile Thermotoga maritima.

The hyperthermophilic bacterium Thermotoga maritima peptidoglycan contains unusual d-lysine alongside typical d-alanine and d-glutamate. We previously identified lysine racemase and threonine dehydratase, but knowledge of d-amino acid metabolism remains limited. Herein, we identified and characterized T. maritima acetylornithine aminotransferase TM1785. The enzyme was most active towards acetyl-l-ornithine, but also utilized l-glutamate, l-ornithine and acetyl-l-lysine as amino donors, and 2-oxoglutarate was the preferred amino acceptor. TM1785 also displayed racemase activity towards four amino acids and lyase activity towards l-cysteine, but no dehydratase activity towards l-serine, l-threonine or corresponding d-amino acids. Catalytic efficiency (kcat /Km ) was highest for aminotransferase activity and lowest for racemase activity. TM1785 is a novel acetylornithine aminotransferase associated with l-arginine biosynthesis that possesses two additional distinct activities.

Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 μM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

Preventing mycolic acid reduction in Corynebacterium glutamicum can efficiently increase L-glutamate production

Corynebacterium glutamicum has been widely utilized for industrial production of various amino acids, especially glutamate, a commonly used food condiment. It has a specific mycolate outer membrane. In this study, the gene cmrA encoding the ketoacyl reductase in the biosynthetic pathway of mycolate was deleted in C. glutamicum ATCC13869, resulting the mutant strain ΔcmrA. As expected, ΔcmrA grown in glucose rich media mainly synthesized keto-glucose monocorynomycolate. Interestingly, L-glutamate production in ΔcmrA increased 10.77-fold, comparing to the control ATCC13869. Based on transcriptomic analysis, the L-glutamate overproduction in C. glutamicum ΔcmrA might be mainly caused by the up-regulation of mscCG expression and the down-regulation of expression of the genes relevant to L-arginine biosynthesis and ODHC. This study provides a novel strategy to improve L-glutamate production in C. glutamicum.