Synthetic Minimal Medium Research Articles

Engineering the carbon and redox metabolism of Paenibacillus polymyxa for efficient isobutanol production.

Paenibacillus polymyxa is a non-pathogenic, Gram-positive bacterium endowed with a rich and versatile metabolism. However interesting, this bacterium has been seldom used for bioproduction thus far. In this study, we engineered P. polymyxa for isobutanol production, a relevant bulk chemical and next-generation biofuel. A CRISPR-Cas9-based genome editing tool facilitated the chromosomal integration of a synthetic operon to establish isobutanol production. The 2,3-butanediol biosynthesis pathway, leading to the main fermentation product of P. polymyxa, was eliminated. A mutant strain harbouring the synthetic isobutanol operon (kdcA from Lactococcus lactis, and the native ilvC, ilvD and adh genes) produced 1 g L-1 isobutanol under microaerobic conditions. Improving NADPH regeneration by overexpression of the malic enzyme subsequently increased the product titre by 50%. Network-wide proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. Glucose-6-phosphate 1-dehydrogenase, the key enzyme in the pentose phosphate pathway, was identified as a bottleneck that hindered efficient NADPH regeneration through this pathway. Furthermore, we conducted culture optimization towards cultivating P. polymyxa in a synthetic minimal medium. We identified biotin (B7), pantothenate (B5) and folate (B9) to be mutual essential vitamins for P. polymyxa. Our rational metabolic engineering of P. polymyxa for the production of a heterologous chemical sheds light on the metabolism of this bacterium towards further biotechnological exploitation.

Reconstructing curcumin biosynthesis in yeast reveals the implication of caffeoyl-shikimate esterase in phenylpropanoid metabolic flux

Curcumin is a polyphenolic natural product from the roots of turmeric (Curcuma longa). It has been a popular coloring and flavoring agent in food industries with known health benefits. The conventional phenylpropanoid pathway is known to proceed from phenylalanine via p-coumaroyl-CoA intermediate. Although hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (HCT) plays a key catalysis in the biosynthesis of phenylpropanoid products at the downstream of p-coumaric acid, a recent discovery of caffeoyl-shikimate esterase (CSE) showed that an alternative pathway exists. Here, the biosynthetic efficiency of the conventional and the alternative pathway in producing feruloyl-CoA was examined using curcumin production in yeast. A novel modular multiplex genome-edit (MMG)-CRISPR platform was developed to facilitate rapid integrations of up to eight genes into the yeast genome in two steps. Using this MMG-CRISPR platform and metabolic engineering strategies, the alternative CSE phenylpropanoid pathway consistently showed higher titers (2–19 folds) of curcumin production than the conventional pathway in engineered yeast strains. In shake flask cultures using a synthetic minimal medium without phenylalanine, the curcumin production titer reached up to 1.5 mg/L, which is three orders of magnitude (∼4800-fold) improvement over non-engineered base strain. This is the first demonstration of de novo curcumin biosynthesis in yeast. Our work shows the critical role of CSE in improving the metabolic flux in yeast towards the phenylpropanoid biosynthetic pathway. In addition, we showcased the convenience and reliability of modular multiplex CRISPR/Cas9 genome editing in constructing complex synthetic pathways in yeast.

Effect of ATG8 or SAC1 deficiency on the cell proliferation and lifespan of the long-lived PMT1 deficiency yeast cells.

Autophagy is pivotal in maintaining intracellular homeostasis, which involves various biological processes, including cellular senescence and lifespan modulation. Being an important member of the protein O-mannosyltransferase (PMT) family of enzymes, Pmt1p deficiency can significantly extend the replicative lifespan (RLS) of yeast cells through an endoplasmic reticulum (ER) unfolded protein response (UPR) pathway, which is participated in protein homeostasis. Nevertheless, the mechanisms that Pmt1p regulates the lifespan of yeast cells still need to be explored. In this study, we found that the long-lived PMT1 deficiency strain (pmt1Δ) elevated the expression levels of most autophagy-related genes, the expression levels of total GFP-Atg8 fusion protein and free GFP protein compared with wild-type yeast strain (BY4742). Moreover, the long-lived pmt1Δ strain showed the greater dot-signal accumulation from GFP-Atg8 fusion protein in the vacuole lumen through a confocal microscope. However, deficiency of SAC1 or ATG8, two essential components of the autophagy process, decreased the cell proliferation ability of the long-lived pmt1Δ yeast cells, and prevented the lifespan extension. In addition, our findings demonstrated that overexpression of ATG8 had no potential effect on the RLS of the pmt1Δ yeast cells, and the maintained incubation of minimal synthetic medium lacking nitrogen (SD-N medium as starvation-induced autophagy) inhibited the cell proliferation ability of the pmt1Δ yeast cells with the culture time, and blocked the lifespan extension, especially in the SD-N medium cultured for 15 days. Our results suggest that the long-lived pmt1Δ strain enhances the basal autophagy activity, while deficiency of SAC1 or ATG8 decreases the cell proliferation ability and shortens the RLS of the long-lived pmt1Δ yeast cells. Moreover, the maintained starvation-induced autophagy impairs extension of the long-lived pmt1Δ yeast cells, and even leads to the cell death.

Aromatic secondary metabolite production from glycerol was enhanced by amino acid addition in Pichia pastoris

Aromatic secondary metabolites are widely used in various industries, including the nutraceutical, dietary supplement, and pharmaceutical industries. Their production currently relies on plant extraction. Microbe-based processes have recently attracted attention as sustainable alternatives to plant-based processes. We previously showed that the yeast Pichia pastoris (Komagataella phaffii) is an optimal host for producing aromatic secondary metabolites. Additionally, titers of resveratrol, an aromatic secondary metabolite, increased by 156 % when glycerol was used as a carbon source instead of glucose. However, the mechanisms by which glycerol resulted in higher production has remained unclear. In this study, we aimed to elucidate how P. pastoris produces higher levels of aromatic secondary metabolites from glycerol than from glucose. Titers of p-coumarate, naringenin, and resveratrol increased by 103 %, 118 %, and 157 %, respectively, in natural complex media containing glycerol compared with that in media containing glucose. However, the titers decreased in minimal synthetic medium without amino acids, indicating that P. pastoris cells used the amino acids only when glycerol was the carbon source. Fermentation with the addition of single amino acids showed that resveratrol titers from glycerol varied depending on the amino acid supplemented. In particular, addition of aspartate or tryptophan into the medium improved resveratrol titers by 146 % and 156 %, respectively. These results suggest that P. pastoris could produce high levels of aromatic secondary metabolites from glycerol with enhanced utilization of specific amino acids. This study provides a basis for achieving high-level production of aromatic secondary metabolites by P. pastoris.Key points•P. pastoris can produce high levels of aromatic metabolites from glycerol•P. pastoris cells use amino acids only when glycerol is the carbon source•Aromatic metabolite titers from glycerol increase with amino acids utilization

Serine metabolism contributes to cell survival by regulating extracellular pH and providing an energy source in Saccharomyces cerevisiae.

Changes in extracellular pH affect the homeostasis and survival of unicellular organisms. Supplementation of culture media with amino acids can extend the lifespan of budding yeast, Saccharomyces cerevisiae, by alleviating the decrease in pH. However, the optimal amino acids to use to achieve this end, and the underlying mechanisms involved, remain unclear. Here, we describe the specific role of serine metabolism in the regulation of pH in a medium. The addition of serine to synthetic minimal medium suppressed acidification, and at higher doses increased the pH. CHA1, which encodes a catabolic serine hydratase that degrades serine into ammonium and pyruvate, is essential for serine-mediated alleviation of acidification. Moreover, serine metabolism supports extra growth after glucose depletion. Therefore, medium supplementation with serine can play a prominent role in the batch culture of budding yeast, controlling extracellular pH through catabolism into ammonium and acting as an energy source after glucose exhaustion.

The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus.

Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in α-proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis.

S6.3c Adaptive dynamics in experimental populations of Aspergillus nidulans

S6.3 Fungal adaptation and evolution, September 22, 2022, 4:45 PM - 6:15 PM A total of 12 replicate populations initiated with a laboratory strain of the ascomycete fungus Aspergillus nidulans evolved on synthetic minimal glucose agar medium for 1 year, using weekly transfers of 1% of the produced asexual spores to fresh medium. This Aspergillus short-term evolution experiment (ASEX) was designed to understand how filamentous fungi adapt to growth on limited carbon in a spatially structured environment. We observed no systematic improvement in the fitness components tested and neither in the competitive fitness relative to the ancestor. Instead, we observed the repeated evolution of at least two morphotypes, with a fluffy-like (FL) or an ancestor-like (AL) colony morphology, leading to non-transitive fitness interactions among isolates in two selected populations. The genomic analyses of clones from all 12 populations at an early (week 10) and the final time point (week 52), show a clear role of natural selection during ASEX. We also observed a shared genetic basis and different timing of adaptation of AL and FL types. In addition, in most populations, both morphotypes do not form monophyletic groups, but they frequently disappear and re-evolve from ancestral forms of both types. Reduction in asexual spore yield, the most evident parallel phenotypic change found in all our evolved populations, is not due to the direct selection of genes involved in asexual reproduction. Instead, we argue that reduced spore yield is a pleiotropic effect of adaptive changes in metabolism.

Temperature- and Nutrients-Induced Phenotypic Changes of Antarctic Green Snow Bacteria Probed by High-Throughput FTIR Spectroscopy.

Temperature fluctuations and nutrient composition are the main parameters influencing green snow microbiome. In this study we investigated the influence of temperature and nutrient conditions on the growth and cellular chemical profile of bacteria isolated from green snow. Chemical profiling of the green snow bacteria was done by high-throughput FTIR spectroscopy combined with multivariate data analysis. We showed that temperature and nutrients fluctuations strongly affect growth ability and chemical profile of the green snow bacteria. The size of colonies for green snow bacteria grown at higher (25 °C) and lower (4 °C and 10 °C) than optimal temperature (18 °C) was smaller. All isolates grew on rich medium, and only 19 isolates were able to grow on synthetic minimal media. Lipid and mixed spectral regions showed to be phylogeny related. FTIR fingerprinting indicates that lipids are often affected by the temperature fluctuations. Growth on different media resulted in the change of the whole chemical profile, where lipids showed to be more affected than proteins and polysaccharides. Correlation analysis showed that nutrient composition is clearly strongly influencing chemical changes in the cells, followed by temperature.

Optimization of Propagation Medium for Enhanced Polyhydroxyalkanoate Production by Pseudomonas oleovorans

Polyhydroxyalkanoates (PHAs) represent a promising alternative to commercially used petroleum-based plastics. Pseudomonas oleovorans is a natural producer of medium-chain-length PHA (mcl-PHA) under cultivation conditions with nitrogen limitation and carbon excess. Two-step cultivation appears to be an efficient but more expensive method of PHA production. Therefore, the aim of this work was to prepare a minimal synthetic medium for maximum biomass yield and to optimize selected independent variables by response surface methodology (RSM). The highest biomass yield (1.71 ± 0.04 g/L) was achieved in the optimized medium containing 8.4 g/L glucose, 5.7 g/L sodium ammonium phosphate and 35.4 mM phosphate buffer. Under these conditions, both carbon and nitrogen sources were completely consumed after 48 h of the cultivation and the biomass yield was 1.7-fold higher than in the conventional medium recommended by the literature. This approach demonstrates the possibility of using two-stage PHA cultivation to obtain the maximum amount of biomass and PHA.

Towards valorization of pectin-rich agro-industrial residues: Engineering of Saccharomyces cerevisiae for co-fermentation of d-galacturonic acid and glycerol

Pectin-rich plant biomass residues represent underutilized feedstocks for industrial biotechnology. The conversion of the oxidized monomer d-galacturonic acid (d-GalUA) to highly reduced fermentation products such as alcohols is impossible due to the lack of electrons. The reduced compound glycerol has therefore been considered an optimal co-substrate, and a cell factory able to efficiently co-ferment these two carbon sources is in demand. Here, we inserted the fungal d-GalUA pathway in a strain of the yeast S. cerevisiae previously equipped with an NAD-dependent glycerol catabolic pathway. The constructed strain was able to consume d-GalUA with the highest reported maximum specific rate of 0.23 g gCDW−1 h−1 in synthetic minimal medium when glycerol was added. By means of a 13C isotope-labelling analysis, carbon from both substrates was shown to end up in pyruvate. The study delivers the proof of concept for a co-fermentation of the two ‘respiratory’ carbon sources to ethanol and demonstrates a fast and complete consumption of d-GalUA in crude sugar beet pulp hydrolysate under aerobic conditions. The future challenge will be to achieve co-fermentation under industrial, quasi-anaerobic conditions.

Regulation of the Pseudomonas syringae Type III Secretion System by Host Environment Signals.

Pseudomonas syringae are Gram-negative, plant pathogenic bacteria that use a type III secretion system (T3SS) to disarm host immune responses and promote bacterial growth within plant tissues. Despite the critical role for type III secretion in promoting virulence, T3SS-encoding genes are not constitutively expressed by P. syringae and must instead be induced during infection. While it has been known for many years that culturing P. syringae in synthetic minimal media can induce the T3SS, relatively little is known about host signals that regulate the deployment of the T3SS during infection. The recent identification of specific plant-derived amino acids and organic acids that induce T3SS-inducing genes in P. syringae has provided new insights into host sensing mechanisms. This review summarizes current knowledge of the regulatory machinery governing T3SS deployment in P. syringae, including master regulators HrpRS and HrpL encoded within the T3SS pathogenicity island, and the environmental factors that modulate the abundance and/or activity of these key regulators. We highlight putative receptors and regulatory networks involved in linking the perception of host signals to the regulation of the core HrpRS–HrpL pathway. Positive and negative regulation of T3SS deployment is also discussed within the context of P. syringae infection, where contributions from distinct host signals and regulatory networks likely enable the fine-tuning of T3SS deployment within host tissues. Last, we propose future research directions necessary to construct a comprehensive model that (a) links the perception of host metabolite signals to T3SS deployment and (b) places these host–pathogen signaling events in the overall context of P. syringae infection.

Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products

BackgroundThe yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilise its metabolism as a basis for bio-based production.ResultsTo illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L−1 2-PE from minimal medium.ConclusionsThe strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.

Evaluation of Basic Fermentation Parameters and Effective Combinations of Predominant Yeasts from Traditional Starter Materials of Indigenous Communities from Northeast India

In this study, yeasts inherent in traditional starter materials of four indigenous communities from northeast India were characterized. These included Saccharomyces and non-Saccharomyces types representing several genera and species such as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Candida glabrata and Candida tropicalis. The yeasts were cultured in synthetic minimal media (2% glucose and 2% starch), both individually and in combinations. The propensity of growth of individual isolates in glucose appeared as W. anomalus > C. tropicalis > C. glabrata > S. cerevisiae. In starch, the propensity appeared as C. tropicalis > W. anomalus > C. glabrata > S. cerevisiae. The findings were incongruent when isolates were co-cultured in dual combinations in glucose and starch. In glucose, W. anomalus could not prosper with C. tropicalis, which otherwise was reversed in starch. C. tropicalis dominated all the co-cultures in starch followed by W. anomalus, S. cerevisiae and C. glabrata. In glucose, the order appeared as W. anomalus > C. tropicalis > S. cerevisiae > C. glabrata. S. cerevisiae could not thrive in competition with C. tropicalis and appeared dominant over C. glabrata in both glucose and starch. W. anomalus was dominant over S. cerevisiae and C. glabrata, while C. tropicalis outnumbered C. glabrata in both carbon sources. The fermentation efficiency was highest when S. cerevisiae and C. glabrata were co-cultured together, in both starch and glucose. This study suggests an advantage in co-culturing selective indigenous yeasts as consortia to yield a productive fermentation output that could have commercial benefit but additional strains of each species still needed to be investigated.

Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives

Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called “magic mushrooms”. Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.

Genetic and phenotypic characteristics of <i>Klebsiella michiganensis</i> isolates

The aim of the study was to identify an optimal research target for detection of Klebsiella michiganensis isolates, determine their genetic and phenotypic characteristics necessary for identification. Here, we examined 11 Klebsiella oxytoca strains, lacking (atypical, negative) a marker 5-aminosalicylate decarboxylase (detected by the chromogenic reaction by 5-aminosalicylic acid) unique for the genus Klebsiella bacteria. They were selected for genetic analysis subsequent to a phenotypic characterization of K. oxytoca clinical isolates, collected in within 2015–2018 period in medical institutions in St. Petersburg. Two K. oxytoca and two Raoultella ornithinolytica clinical strains displaying typical properties were used as a control. The presence of 5-aminosalicylate decarboxylase was detected by the chromogenic reaction with the “Klebsiella 5-ASK CHROME C” nutrient medium (Pasteur Institute, St. Petersburg). Substrate utilization as the sole carbon source was detected on a solid minimal synthetic medium added with 2 g/L substrate during incubation for 72 hours at 37°C. Biochemical bacteria features were studied by the microvolume method with the “Rapid-Entero” test system (Pasteur Institute, St. Petersburg). Genetic strain characterization was performed by estimating 16S rRNA, gyrA, rpoB by using a routine PCR with primer sequences described before. Two rpoB gene fragments with a total length 834 bp, 16S rRNA gene fragment — 387 bp, and gyrA gene fragment — 441 bp were amplified followed by their sequencing by Singer on an ABI 3130 automatic capillary sequencer (Applied Biosystems, USA) and subsequently determined similarity levels. Amplification pattern for pehX gene PCR fragments was performed by using a method described elsewhere with two primer pairs flanking fragment AD with a 513 bp length and 344 bp CD-long motifs. While examining 11 clinical bacterial strains identified earlier as Klebsiella oxytoca, lacking (atypical, negative) a 5-aminosalicylate decarboxylase (detected by the chromogenic reaction by 5-aminosalicylic acid) unique for the genus Klebsiella, molecular techniques identified 9 K. michiganensis strains and 1 strain highly homologous to Klebsiella kielensis based on the rpoB gene nucleotide sequence, confirming its high informative value. We used the methods for estimating a similarity level for sequenced fragments of 16S rRNA genes (fragment length 387 bp), gyrA gene (fragment length 441 bp), rpoB gene (rpoB-b and rpoB-e with a total fragments length 834 bp), and the analysis of marker amplicon patterns for pehX gene (AD, CD). It was shown that for the 4 K. oxytoca strains, 99–100% similarity to K. michiganensis was identified for all fragments in the sequenced genes. Moreover, similarity of all 9 strains detected with K. michiganensis was revealed only in the rpoB gene, hereby allowing to recommend it as the most informative approach. The pehX gene encoding polygalacturonase was verified by PCR in the majority of K. michiganensis strains, pointing that this approach is not rational for their identification (distinguish with K. oxytoca). The most informative for the phenotypic identification of K. michiganensis are were assays characterized by a common profile for the majority of strains: lack of 5-aminosalicylate decarboxylase, lack of utilized histamine, dulcitol, tricarballylic acid; positive for indole production, as well as D-melezitose and putrescine utilization.

Hydrolytic enzyme production of endophytic fungi isolated from soybean (Glycine max)

This current approach aimed to isolate endophytic fungi from soybean (Glycine max L.) and to assess for their ability to produce hydrolytic extracellular enzymes such as carboxymethyl cellulase (CMC-ase), xylanase and amylase in order to evaluate their ecological role within the host plant. Ten endophytic fungi were isolated from soybean (roots, stems, and leaves). All isolated endophytes (100%) exhibited CMC-ase, xylanase and amylase activities using the minimal synthetic medium (MSM) for enzyme production supplemented with 2% agar in presence of the appropriate carbon source including carboxymethyl cellulose (CMC) for CMC-ase, xylan for xylanase and starch for amylase). The endophytic isolates showed growth stimulation on CMC and xylan media and were hyperactive in the presence of these substrates more than glucose. On the other hand, starch was investigated as the lower preferable substrate for these endophytes. These relevant results endeavored to explore endophytic fungi and to investigate their production of industrial microbial enzymes.

Multiple pathways for the formation of the γ-glutamyl peptides γ-glutamyl-valine and γ- glutamyl-valyl-glycine in Saccharomyces cerevisiae.

The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p)2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p)2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.

Bacillus subtilis TE3: A promising biological control agent against Bipolaris sorokiniana, the causal agent of spot blotch in wheat (Triticum turgidum L. subsp. durum)

Bipolaris sorokiniana, the causal agent of wheat spot blotch, is an emerging phytopathogen in the Yaqui Valley, Sonora, Mexico. Currently, the unique method to control spot blotch on that region is employing synthetic fungicides with environmental and human health issues. Thus, the aim of this work was to identify and characterize native wheat-associated bacteria in order to find promising biological control strains that can be used as a sustainable alternative to control spot blotch. The identification of antagonistic strains was carried out by confrontation assay against B. sorokiniana. Antagonistic strains to B. sorokiniana were taxonomically classified by 16S rRNA gene sequencing, and characterized on important biological traits: (i) inhibition of the target phytopathogen, (ii) growth rate, and (iii) cytotoxic activity. Out 195 bacterial strains tested, 14 exhibited antagonistic activity against B. sorokiniana at different degree (1.6–8.0 mm of inhibition zone). The Antagonistic strains were taxonomically identified as members of Bacillus (eight strains), Paenibacillus (one strain), Pseudomonas (two strains), Achromobacter (one strain), Delftia (one strain), and Stenotrophomonas (one strain). The strain TE3, identified as Bacillus subtilis, showed promising biological control traits: (i) broad spectrum inhibition against B. sorokiniana, (ii) growth capacity in synthetic minimal medium, and (iii) non-cytotoxic activity to erythrocyte (γ-hemolysis). The application of cell suspension or cell-free culture filtrate of B. subtilis TE3 under highly conducive conditions (28 °C and 100% HR) for wheat spot blotch, resulted in a significant reduction of that disease. In conclusion, B. subtilis TE3 and its antifungal metabolites are promising effective treatment to control the causal agent of the wheat spot blotch.

Generation of hydrogen sulfide from sulfur assimilation in Escherichia coli.

Many organisms produce endogenous hydrogen sulfide (H2S) as a by-product of protein, peptide, or L-cysteine degradation. Recent reports concerning mammalian cells have demonstrated that H2S acts as a signaling molecule playing important roles in various biological processes. In contrast to mammals, bacterial H2S signaling remains unclear. In this work, we demonstrate that Escherichia coli generates H2S through the assimilation of inorganic sulfur, without L-cysteine degradation. Comparison of phenotypes and genomes between laboratory E. coli K-12 strains revealed a major contribution of CRP (a protein that controls the expression of numerous genes involved in glycolysis) to H2S generation. We found that H2S was produced by cells growing in a synthetic minimal medium containing thiosulfate as a sole inorganic sulfur source, but not in a medium only containing sulfate. Furthermore, E. coli generated H2S in a CRP-dependent manner as a response to glucose starvation. These results indicate that CRP plays a key role in the generation of H2S coupled to thiosulfate assimilation, whose molecular mechanisms remains to be elucidated. Here, we propose a potential biological role of the H2S as a signaling mediator for a cross-talk between carbon and sulfur metabolism in E. coli.

Hyperthermophilic Archaeon Thermococcus kodakarensis Utilizes a Four-Step Pathway for NAD+ Salvage through Nicotinamide Deamination.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.