Poly-γ-glutamic Acid Synthesis, Gene Regulation, Phylogenetic Relationships, and Role in Fermentation.
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ABSTRACT:
SUBMITTER: Hsueh YH
PROVIDER: S-EPMC5751247 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMolasses is a wildly used feedstock for fermentation, but it also poses a severe wastewater-disposal problem worldwide. Recently, the wastewater from yeast molasses fermentation is being processed into fulvic acid (FA) powder as a fertilizer for crops, but it consequently induces a problem of soil acidification after being directly applied into soil. In this study, the low-cost FA powder was bioconverted into a value-added product of γ-PGA by a glutamate-independent producer of Bacillus velezensis GJ11.ResultsFA power could partially substitute the high-cost substrates such as sodium glutamate and citrate sodium for producing γ-PGA. With FA powder in the fermentation medium, the amount of sodium glutamate and citrate sodium used for producing γ-PGA were both decreased around one-third. Moreover, FA powder could completely substitute Mg2+, Mn2+, Ca2+, and Fe3+ in the fermentation medium for producing γ-PGA. In the optimized medium with FA powder, the γ-PGA was produced at 42.55 g/L with a productivity of 1.15 g/(L·h), while only 2.87 g/L was produced in the medium without FA powder. Hydrolyzed γ-PGA could trigger induced systemic resistance (ISR), e.g., H2O2 accumulation and callose deposition, against the pathogen's infection in plants. Further investigations found that the ISR triggered by γ-PGA hydrolysates was dependent on the ethylene (ET) signaling and nonexpressor of pathogenesis-related proteins 1 (NPR1).ConclusionsTo our knowledge, this is the first report to use the industry waste, FA powder, as a sustainable substrate for microbial synthesis of γ-PGA. This bioprocess can not only develop a new way to use FA powder as a cheap feedstock for producing γ-PGA, but also help to reduce pollution from the wastewater of yeast molasses fermentation.
| S-EPMC7594462 | biostudies-literature
Project description:The main forms of poly-γ-glutamic acid (γ-PGA) applied in agriculture include agricultural γ-PGA and γ-PGA super absorbent polymer (SAP). Laboratory experiments were conducted with a check treatment CK (no γ-PGA added) and two different forms of γ-PGA added to sandy loam soil (T and TM stand for γ-PGA and γ-PGA SAP) at four different soil mass ratios (0.05% (1), 0.10% (2), 0.15% (3) and 0.20% (4)) to determine their effects on sandy loam soil hydro-physical properties. Both of them could reduce the cumulative infiltration of soil water. The total available water (TAW) which the soil water content (SWC) from field water capacity (FC) to permanent wilting point (PWP) after γ-PGA added into sandy loam soil had no significant different compared with CK, and the TAW was highest at the treatment of γ-PGA with 0.10% addition amount into sandy loam soil. However, the TAW of sandy loam soil increased dramatically with the γ-PGA SAP addition amount increasing. TM3 had the highest soil water absorption among the treatments with γ-PGA SAP. The T1 to T4 treatments with γ-PGA addition slightly prolonged retention time (RT) when SWC varied from FC to PWP compared with CK. For γ-PGA SAP addition treatments, the time for SWC varied from FC to PWP was 1.48 times (TM1), 1.88 times (TM2), 2.01 times (TM3) and 2.87 times (TM4) longer than that of CK, respectively. The results of this study will provide further information for the use of these materials in agricultural application.
| S-EPMC7983855 | biostudies-literature
Project description:A nanoformulation composed of curdlan, a linear polysaccharide of 1,3-β-linked d-glucose units, hydrogen bonded to poly(γ -glutamic acid) (PGA), was developed to stimulate macrophage. Curdlan/PGA nanoparticles (C-NP) are formulated by physically blending curdlan (0.2 mg mL-1 in 0.4 m NaOH) with PGA (0.8 mg mL-1 ). Forster resonance energy transfer (FRET) analysis demonstrates a heterospecies interpolymer complex formed between curdlan and PGA. The 1 H-NMR spectra display significant peak broadening as well as downfield chemical shifts of the hydroxyl proton resonances of curdlan, indicating potential intermolecular hydrogen bonding interactions. In addition, the cross peaks in 1 H-1 H 2D-NOESY suggest intermolecular associations between the OH-2/OH-4 hydroxyl groups of curdlan and the carboxylic-/amide-groups of PGA via hydrogen bonding. Intracellular uptake of C-NP occurs over time in human monocyte-derived macrophage (MDM). Furthermore, C-NP nanoparticles dose-dependently increase gene expression for TNF-α, IL-6, and IL-8 at 24 h in MDM. C-NP nanoparticles also stimulate the release of IL-lβ, MCP-1, TNF-α, IL-8, IL-12p70, IL-17, IL-18, and IL-23 from MDM. Overall, this is the first demonstration of a simplistic nanoformulation formed by hydrogen bonding between curdlan and PGA that modulates cytokine gene expression and release of cytokines from MDM.
| S-EPMC7839312 | biostudies-literature
Project description:BackgroundPoly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production.ResultsIn this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain.ConclusionsWe proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.
| S-EPMC5440981 | biostudies-literature
Project description:Poly-γ-glutamic acid (γ-PGA) production is commonly achieved using glycerol, citrate, and L-glutamic acid as substrates. The constitutive expression of the γ-PGA synthetase enabled γ-PGA production with Bacillus subtilis from glucose only. The precursors for γ-PGA synthesis, D- and L-glutamate, are ubiquitous metabolites. Hence, the metabolic flux toward γ-PGA directly depends on the concentration and activity of the synthetase and thereby on its expression. To identify pathway bottlenecks and important metabolites that are highly correlated with γ-PGA production from glucose, we engineered B. subtilis strains with varying γ-PGA synthesis rates. To alter the rate of γ-PGA synthesis, the expression level was controlled by two approaches: (1) Using promoter variants from the constitutive promoter P veg and (2) Varying induction strength of the xylose inducible promoter P xyl . The variation in the metabolism caused by γ-PGA production was investigated using metabolome analysis. The xylose-induction strategy revealed that the γ-PGA production rate increased the total fluxes through metabolism indicating a driven by demand adaption of the metabolism. Metabolic bottlenecks during γ-PGA from glucose were identified by generation of a model that correlates γ-PGA production rate with intracellular metabolite levels. The generated model indicates the correlation of certain metabolites such as phosphoenolpyruvate with γ-PGA production. The identified metabolites are targets for strain improvement to achieve high level γ-PGA production from glucose.
| S-EPMC7002566 | biostudies-literature
Project description:Poly-gamma-glutamic acid (PGA) is a promising bio-based polymer that shares many functions with poly (acrylic acid) and its derivatives. Thus, technologies for efficient production and molecular size control of PGA are required to expand the application of this useful biopolymer. In Bacillus strains, PGA is synthesized by the PgsBCA protein complex, which is encoded by the pgsBCA gene operon, otherwise is known as ywsC and ywtAB operons and/or capBCA operon. Hence, we investigated responsible components of the PgsBCA complex in B. subtilis for over-production of PGA. In particular, we constructed genomic pgsBCA gene-deletion mutants of B. subtilis. And also, we assembled high copy-number plasmids harboring ?A-dependent promoter, leading to high-level expression of all combinations of pgsBCA, pgsBC, pgsBA, pgsCA, pgsB, pgsC, and/or pgsA genes. Subsequently, PGA production of the transformed B. subtilis mutant was determined in batch fermentation using medium supplemented with L-glutamate. PGA production by the transformants introduced with pgsBC genes (lacking the genomic pgsBCA genes) was 26.0?±?3.0 g L-1, and the enantiomeric ratio of D- and L-glutamic acid (D/L-ratio) in the produced PGA was 5/95. In contrast, D/L-ratio of produced PGA by the transformants introduced with pgsBCA genes (control strains) was 75/25. In conclusion, B. subtilis without pgsA gene could over-produce PGA with an L-rich enantiomeric ratio.
| S-EPMC6029982 | biostudies-literature
Project description:Poly-γ-glutamic acid (γ-PGA) is a promising microbial polymer with potential applications in industry, agriculture and medicine. The use of high γ-PGA-producing strains is an effective approach to improve productivity of γ-PGA. In this study, we developed a mutant, F3-178, from Bacillus subtilis GXA-28 using genome shuffling. The morphological characteristics of F3-178 and GXA-28 were not identical. Compared with GXA-28 (18.4 ± 0.8 g l-1 ), the yield of γ-PGA was 1.9-fold higher in F3-178 (34.3 ± 1.2 g l-1 ). Results from batch fermentation in 3.7 l fermenter showed that F3-178 was satisfactory for industrial production of γ-PGA. Metabolic studies suggested that the higher γ-PGA yield in F3-178 could be attributed to increased intracellular flux and uptake of extracellular glutamate. Real-time PCR indicated that mRNA level of pgsB in F3-178 was 18.8-fold higher than in GXA-28, suggesting the higher yield might be related to the overexpression of genes involved in γ-PGA production. This study demonstrated that genome shuffling can be used for rapid improvement of γ-PGA strains, and the possible mechanism for the improved phenotype was also explored at the metabolic and transcriptional levels.
| S-EPMC5072198 | biostudies-literature
Project description:Zinc and copper are essential micronutrients that serve as a cofactors for numerous enzymes. However, when present at elevated concentrations, zinc and copper are highly toxic to bacteria. To combat the effects of zinc and copper excess, bacteria have evolved a wide array of defense mechanisms. Here, we show that the Gram-positive soil bacterium, Bacillus subtilis, produces the extracellular polymeric substance, poly-gamma-glutamate (γ-PGA) as a protective mechanism in response to zinc and copper excess. Furthermore, we provide evidence that zinc and copper dependent γ-PGA production is independent of the DegS-DegQ two-component regulatory system and likely occurs at a posttranscriptional level through the small protein, PgsE. These data provide new insight into bacterial metal resistance mechanisms and contribute to our understanding of the regulation of bacterial γ-PGA biosynthesis. IMPORTANCE Zinc and copper are potent antimicrobial compounds. As such, bacteria have evolved a diverse range of tools to prevent metal intoxication. Here, we show that the Gram-positive model organism, Bacillus subtilis, produces poly-gamma-glutamic acid (γ-PGA) as a protective mechanism against zinc and copper intoxication and that zinc and copper dependent γ-PGA production occurs by a yet undefined mechanism independent of known γ-PGA regulation pathways.
| S-EPMC9045300 | biostudies-literature
Project description:The safety of nanomaterials, a crucial consideration for clinical translation, is enhanced by using building blocks that are biologically nontoxic. Here, we used poly(γ-glutamic acid) (γ-PGA) and dopamine as building blocks of polymeric nanomaterials for carrying hydrophobic anticancer drugs. The introduction of phenylalanine onto γ-PGA enabled the resulting amphiphilic derivative of γ-PGA acid to self-assemble in the presence of the anticancer drug paclitaxel (PTX) to form PTX-encapsulated micelles. The surfaces of PTX-loaded micelles were then coated with polymerized dopamine (PDA). The PDA-coated, amphiphilic γ-PGA-based micelles (AM) carrying PTX (PDA/AM/P) exerted near-infrared-responsive photothermal effects. Near-infrared irradiation of cancer cells treated with PDA/AM/P nanoparticles produced a greater anticancer effect than that observed in other treatment groups, indicating a synergistic effect. Intravenous administration of PDA/AM/P completely ablated tumors and prevented their recurrence. Notably, the in vivo safety profile of PDA/AM/P nanoparticles allowed PTX to be delivered at a 3.6-fold higher dose than was possible with PTX solubilized in surfactant, and circumvented the side effects of the surfactant. These results support the multifunctional potential of PDA/AM for the delivery of various hydrophobic drugs and imaging dyes for safe translation of nanomaterials into the clinic.
| S-EPMC6543094 | biostudies-literature
Project description:We report the synthesis and characterization of high-molecular weight poly(l-glutamic acid) based brush polymers. Utilizing a combination of ring-opening metathesis polymerization of norbornene based monomers and ring-opening polymerization of ?-benzyl-l-glutamate N-carboxyanhydride, high-molecular weight ?-benzyl protected poly(l-glutamic acid) brush polymers are synthesized. Controlled and complete deprotection of the benzyl groups using trimethylsilyl iodide resulted in poly(l-glutamic acid) based brush polymers with molecular weights up to 3.6 MDa, which may potentially be used to prepare size-controlled unimolecular polymeric nanomedicine for drug delivery applications. Camptothecin brush poly(l-glutamic acid) conjugates were prepared and their stability, drug release kinetics, and in vitro toxicity were studied.
| S-EPMC6716790 | biostudies-literature