Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

Structural and spectroscopic characterization of RufO indicates a new biological role in rufomycin biosynthesis.

Rufomycins constitute a class of cyclic heptapeptides isolated from actinomycetes. They are secondary metabolites that show promising treatment against Mycobacterium tuberculosis infections by inhibiting a novel drug target. Several nonproteinogenic amino acids are integrated into rufomycins, including a conserved 3-nitro-tyrosine. RufO, a cytochrome P450 (CYP)-like enzyme, was proposed to catalyze the formation of 3-nitro-tyrosine in the presence of O2 and NO. To define its biological function, the interaction between RufO and the proposed substrate tyrosine is investigated using various spectroscopic methods that are sensitive to the structural change of a heme center. However, a low- to high-spin state transition and a dramatic increase in the redox potential that are commonly found in CYPs upon ligand binding have not been observed. Furthermore, a 1.89-Å crystal structure of RufO shows that the enzyme has flexible surface regions, a wide-open substrate access tunnel, and the heme center is largely exposed to solvent. Comparison with a closely related nitrating CYP reveals a spacious and hydrophobic distal pocket in RufO, which is incapable of stabilizing a free amino acid. Molecular docking validates the experimental data and proposes a possible substrate. Collectively, our results disfavor tyrosine as the substrate of RufO and point to the possibility that the nitration occurs during or after the assembly of the peptides. This study indicates a new function of the unique nitrating enzyme and provides insights into the biosynthesis of nonribosomal peptides.

Biosynthesis of Tasikamides via Pathway Coupling and Diazonium-Mediated Hydrazone Formation.

Naturally occurring hydrazones are rare despite the ubiquitous usage of synthetic hydrazones in the preparation of organic compounds and functional materials. In this study, we discovered a family of novel microbial metabolites (tasikamides) that share a unique cyclic pentapeptide scaffold. Surprisingly, tasikamides A-C (1-3) contain a hydrazone group (C═N─N) that joins the cyclic peptide scaffold to an alkyl 5-hydroxylanthranilate (AHA) moiety. We discovered that the biosynthesis of 1-3 requires two discrete gene clusters, with one encoding a nonribosomal peptide synthetase (NRPS) pathway for assembling the cyclic peptide scaffold and another encoding the AHA-synthesizing pathway. The AHA gene cluster encodes three ancillary enzymes that catalyze the diazotization of AHA to yield an aryl diazonium species (diazo-AHA). The electrophilic diazo-AHA undergoes nonenzymatic Japp-Klingemann coupling with a ß-keto aldehyde-containing cyclic peptide precursor to furnish the hydrazone group and yield 1-3. The studies together unraveled a novel mechanism whereby specialized metabolites are formed by the coupling of two biosynthetic pathways via an unprecedented in vivo Japp-Klingemann reaction. The findings raise the prospect of exploiting the arylamine-diazotizing enzymes (AAD) for the in vivo synthesis of aryl compounds and modification of biological macromolecules.

Promoter exchange of the cryptic nonribosomal peptide synthetase gene for oligopeptide production in Aspergillus oryzae.

Oligopeptides with functional activities are of current interest in the nutraceutical and medical sectors. The development of the biosynthetic process of oligopeptides through a nonribosomal peptide synthetase (NRPS) system has become more challenging. To develop a production platform for nonribosomal peptides (NRPs), reprogramming of transcriptional regulation of the acv gene encoded ACV synthetase (ACVS) was implemented in Aspergillus oryzae using the CRISPR-Cas9 system. Awakening silent acv expression was successfully achieved by promoter substitution. Among the three exchanged promoters, AoPgpdA, AoPtef1, and PtPtoxA, the replacement of the native promoter with AoPgpdA led to the highest ACV production in A. oryzae. However, the ACV production of the AoPGpdA strain was also dependent on the medium composition, in which urea was the best nitrogen source, and a C:N ratio of 20:1 was optimal for tripeptide production. In addition to cell growth, magnesium ions are an essential element for ACV production and might participate in ACVS activity. It was also found that ACV was the growth-associated product of the engineered strain that might be a result of constitutive transcriptional control by the AoPgpdA promoter. This study offers a potential strategy for nonribosomal ACV production using the fungal system, which is applicable for redesigning bioactive oligopeptides with industrial relevance.

Actinomadura violacea sp. nov., a madurastatin A1-producing strain isolated from lichen in Thailand.

An actinomycete strain, LCR2-06T, isolated from a lichen sample on rock collected from Chiang Rai Province (Pong Phra Bat Waterfall), Thailand, was characterized using a polyphasic approach. The strain grew at 25-45 °C, pH 6-11 and on International Streptomyces Project 2 agar plate with 5 % (w/v) NaCl. It contained meso-diaminopimelic acid as the diamino acid in whole-cell hydrolysates. Rhamnose, ribose, xylose, madurose, glucose and galactose were detected as whole-cell sugar hydrolysates. Mycolic acids were absent. The N-acyl type of muramic acid was acetyl. The strain contained C16 : 0, TBSA 10-methyl C18 : 0 and 2-hydroxy C16 : 0 as the predominant fatty acids and MK-9(H6), MK-9(H4) and MK-9(H8) as the major menaquinones. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol and unidentified phospholipid. The draft genome of strain LCR2-06T was closely related to Actinomadura barringtoniae TBRC 7225T (99.2 %), Actinomadura nitritigenes NBRC 15918T (98.8 %), Actinomadura montaniterrae TISTR 2400T (98.5 %) and Actinomadura physcomitrii JCM 33455T (97.9 %). The draft genome of LCR2-06T was 11.1 Mb with 10 588 coding sequences with an average G+C content of 72.7 mol%. Results of genomic analysis revealed that the ANIb and ANIm values between strain LCR2-06T and A. montaniterrae TISTR 2400T were 90.0 and 92.0 %, respectively. The digital DNA-DNA hybridization value was 43.9 % in comparison with the draft genome of A. montaniterrae TISTR 2400T. The strain produced an antibacterial compound active against Bacillus subtilis ATCC 6633 and Kocuria rhizophila ATCC 9341. The results of taxonomic analysis suggested that strain LCR2-06T represented a novel species of the genus Actinomadura for which the name Actinomadura violacea sp. nov. is proposed. The type strain is LCR2-06T (=JCM 33065T=KCTC 49547T=NBRC 114810T=LMG 32136T=TISTR 2935T).

Biosynthesis of a New Fusaoctaxin Virulence Factor in Fusarium graminearum Relies on a Distinct Path To Form a Guanidinoacetyl Starter Unit Priming Nonribosomal Octapeptidyl Assembly.

Fusarium graminearum is a pathogenic fungus causing huge economic losses worldwide via crop infection leading to yield reduction and grain contamination. The process through which the fungal invasion occurs remains poorly understood. We recently characterized fusaoctaxin A in F. graminearum, where this octapeptide virulence factor results from an assembly line encoded in fg3_54, a gene cluster proved to be involved in fungal pathogenicity and host adaptation. Focusing on genes in this cluster that are related to fungal invasiveness but not to the biosynthesis of fusaoctaxin A, we here report the identification and characterization of fusaoctaxin B, a new octapeptide virulence factor with comparable activity in wheat infection. Fusaoctaxin B differs from fusaoctaxin A at the N-terminus by possessing a guanidinoacetic acid (GAA) unit, formation of which depends on the combined activities of the protein products of fgm1-3. Fgm1 is a cytochrome P450 protein that oxygenates l-Arg to 4(R)-hydroxyl-l-Arg in a regio- and stereoselective manner. Then, Cß-Cγ bond cleavage proceeds in the presence of Fgm3, a pyridoxal-5'-phosphate-dependent lyase, giving guanidinoacetaldehyde and l-Ala. Rather than being directly oxidized to GAA, the guanidine-containing aldehyde undergoes spontaneous cyclization and subsequent enzymatic dehydrogenation to provide glycociamidine, which is linearized by Fgm2, a metallo-dependent amidohydrolase. The GAA path in F. graminearum is distinct from that previously known to involve l-Arg:l-Gly aminidotransferase activity. To provide this nonproteinogenic starter unit that primes nonribosomal octapeptidyl assembly, F. graminearum employs new chemistry to process l-Arg through inert C-H bond activation, selective C-C bond cleavage, cyclization-based alcohol dehydrogenation, and amidohydrolysis-associated linearization.

Identification of Novel Rotihibin Analogues in Streptomyces scabies, Including Discovery of Its Biosynthetic Gene Cluster.

Streptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II. IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters.

Functional genomics study of Pseudomonas putida to determine traits associated with avoidance of a myxobacterial predator.

Predation contributes to the structure and diversity of microbial communities. Predatory myxobacteria are ubiquitous to a variety of microbial habitats and capably consume a broad diversity of microbial prey. Predator-prey experiments utilizing myxobacteria have provided details into predatory mechanisms and features that facilitate consumption of prey. However, prey resistance to myxobacterial predation remains underexplored, and prey resistances have been observed exclusively from predator-prey experiments that included the model myxobacterium Myxococcus xanthus. Utilizing a predator-prey pairing that instead included the myxobacterium, Cystobacter ferrugineus, with Pseudomonas putida as prey, we observed surviving phenotypes capable of eluding predation. Comparative transcriptomics between P. putida unexposed to C. ferrugineus and the survivor phenotype suggested that increased expression of efflux pumps, genes associated with mucoid conversion, and various membrane features contribute to predator avoidance. Unique features observed from the survivor phenotype when compared to the parent P. putida include small colony variation, efflux-mediated antibiotic resistance, phenazine-1-carboxylic acid production, and increased mucoid conversion. These results demonstrate the utility of myxobacterial predator-prey models and provide insight into prey resistances in response to predatory stress that might contribute to the phenotypic diversity and structure of bacterial communities.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

Enzymatic Synthesis of γ-Glutamylvalylglycine Using Bacterial γ-Glutamyltranspeptidase.

γ-Glutamylvalylglycine is known as a potent "kokumi" substance that increases the thickness, continuity, and mouthfulness of the taste of food. In this study, γ-glutamylvalylglycine was enzymatically synthesized from glutamine and valylglycine through the transpeptidation reaction of bacterial γ-glutamyltranspeptidase. The reaction conditions decided for the production of γ-glutamylvalylglycine are 20 mM glutamine, 100 mM valylglycine, 30 mU/mL γ-glutamyltranspeptidase, and 0.856 M (5%) NaCl at pH 8 and 37 °C, and 18.6 mM γ-glutamylvalylglycine was synthesized. Metal cations, Na+ and Mg2+, improved the transpeptidation reaction of γ-glutamyltranspeptidase, and the production of γ-glutamylvalylglycine increased. Moreover, by feeding a suitable amount of glutamine every 5 h, the maximum production of γ-glutamylvalylglycine increased 1.7 times after 20 h of incubation.

Antifungal mechanism of Bacillus amyloliquefaciens strain GKT04 against Fusarium wilt revealed using genomic and transcriptomic analyses.

The application of endophytic bacteria, particularly members of the genus Bacillus, offers a promising strategy for the biocontrol of plant fungal diseases, owing to their sustainability and ecological safety. Although multiple secondary metabolites that demonstrate antifungal capacity have been identified in diverse endophytic bacteria, the regulatory mechanisms of their biosynthesis remain largely unknown. To elucidate this, we sequenced the entire genome of Bacillus amyloliquefaciens GKT04, a strain isolated from banana root, which showed high inhibitory activity against Fusarium oxysporum f. sp. cubense race 4 (FOC4). The GKT04 genome consists of a circular chromosome and a circular plasmid, which harbors 4,087 protein-coding genes and 113 RNA genes. Eight gene clusters that could potentially encode antifungal components were identified. We further applied RNA-Seq analysis to survey genome-wide changes in the gene expression of strain GKT04 during its inhibition of FOC4. In total, 575 upregulated and 242 downregulated genes enriched in several amino acid and carbohydrate metabolism pathways were identified. Specifically, gene clusters associated with difficidin, bacillibactin, and bacilysin were significantly upregulated, and their gene regulatory networks were constructed. Our work thereby provides insights into the genomic features and gene expression patterns of this B. amyloliquefaciens strain, which presents an excellent potential for the biocontrol of Fusarium wilt.

Malonate utilization by Pseudomonas aeruginosa affects quorum-sensing and virulence and leads to formation of mineralized biofilm-like structures.

Pseudomonas aeruginosa is an opportunistic pathogen that uses malonate among its many carbon sources. We recently reported that, when grown in blood from trauma patients, P. aeruginosa expression of malonate utilization genes was upregulated. In this study, we explored the role of malonate utilization and its contribution to P. aeruginosa virulence. We grew P. aeruginosa strain PA14 in M9 minimal medium containing malonate (MM9) or glycerol (GM9) as a sole carbon source and assessed the effect of the growth on quorum sensing, virulence factors, and antibiotic resistance. Growth of PA14 in MM9, compared to GM9, reduced the production of elastases, rhamnolipids, and pyoverdine; enhanced the production of pyocyanin and catalase; and increased its sensitivity to norfloxacin. Growth in MM9 decreased extracellular levels of N-acylhomoserine lactone autoinducers, an effect likely associated with increased pH of the culture medium; but had little effect on extracellular levels of PQS. At 18 hr of growth in MM9, PA14 formed biofilm-like structures or aggregates that were associated with biomineralization, which was related to increased pH of the culture medium. These results suggest that malonate significantly impacts P. aeruginosa pathogenesis by influencing the quorum sensing systems, the production of virulence factors, biofilm formation, and antibiotic resistance.

Integrated Omics Strategy Reveals Cyclic Lipopeptides Empedopeptins from Massilia sp. YMA4 and Their Biosynthetic Pathway.

Empedopeptins-eight amino acid cyclic lipopeptides-are calcium-dependent antibiotics that act against Gram-positive bacteria such as Staphylococcus aureus by inhibiting cell wall biosynthesis. However, to date, the biosynthetic mechanism of the empedopeptins has not been well identified. Through comparative genomics and metabolomics analysis, we identified empedopeptin and its new analogs from a marine bacterium, Massilia sp. YMA4. We then unveiled the empedopeptin biosynthetic gene cluster. The core nonribosomal peptide gene null-mutant strains (ΔempC, ΔempD, and ΔempE) could not produce empedopeptin, while dioxygenase gene null-mutant strains (ΔempA and ΔempB) produced several unique empedopeptin analogs. However, the antibiotic activity of ΔempA and ΔempB was significantly reduced compared with the wild-type, demonstrating that the hydroxylated amino acid residues of empedopeptin and its analogs are important to their antibiotic activity. Furthermore, we found seven bacterial strains that could produce empedopeptin-like cyclic lipopeptides using a genome mining approach. In summary, this study demonstrated that an integrated omics strategy can facilitate the discovery of potential bioactive metabolites from microbial sources without further isolation and purification.

Identification of Active Site Residues of the Siderophore Synthesis Enzyme PvdF and Evidence for Interaction of PvdF with a Substrate-Providing Enzyme.

The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.

Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Glutathione Synthesis in Cancer Cells.

Tripeptide GSH is associated not only with the control and maintenance of redox cell homeostasis, but also with the processes of detoxification, proliferation, cell differentiation, and regulation of cell death. Disruptions in GSH synthesis and changes in the GSH/GSSG ratio are common for many pathological conditions, including malignant neoplasms. Numerous data indicate the importance of GSH and the GSH/GSSG ratio in the regulation of tumor cell viability, in the initiation of tumor development, progression, and drug resistance. However, control of the mechanism of GSH synthesis in malignant tumors remains poorly understood. This review discusses the features of GSH synthesis and its regulation in tumor cells. The role of GSH in the mechanisms of apoptosis, necroptosis, ferroptosis, and autophagy is considered.

Mining New Plipastatins and Increasing the Total Yield Using CRISPR/Cas9 in Genome-Modified Bacillus subtilis 1A751.

CRISPR/Cas9 is one of the robust and effective gene manipulation tools which has been widely applied in various organisms. In this study, the plipastatin gene cluster was successfully expressed in genome-modified Bacillus subtilis 1A751 by disrupting the surfactin operon (srf) through CRISPR/Cas9 technology. The presumed plipastatin biosynthetic pathway was proposed based on the analysis of its biosynthetic gene cluster. Two new plipastatins were identified by a combination of ultra-high performance liquid chromatography-coupled electron spray ionization-tandem mass spectrometry and gas chromatography-mass spectrometry analyses, together with nine known plipastatins or their derivatives. The yield of plipastatin was as high as 1600 mg/L which is the highest reported to date. Antimicrobial experiments revealed that its methanolic extracts exhibited powerful inhibitory effects on the growth of the tested pathogens and fungi. The results from this investigation highlight the remarkable utility of CRISPR/Cas9 in mining new plipastatins and increasing the total plipastatin yield, providing a new pipeline for the industrial application of plipastatin.

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A.

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish.

In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.