ABSTRACT
Safe and eco-friendly preservatives are crucial to preventing food spoilage and illnesses, as foodborne diseases caused by pathogens result in approximately 600 million cases of illness and 420,000 deaths annually. ε-Poly-L-lysine (ε-PL) is a novel food preservative widely used in many countries. However, its commercial application has been hindered by high costs and low production. In this study, ε-PL's biosynthetic capacity was enhanced in Streptomyces albulus WG608 through metabolic engineering guided by multi-omics techniques. Based on transcriptome and metabolome data, differentially expressed genes (fold change >2 or <0.5; p < 0.05) and differentially expressed metabolites (fold change >1.2 or <0.8) were separately subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The integrative analysis of transcriptome, metabolome, and overexpression revealed the essential roles of isocitrate lyase, succinate dehydrogenase, flavoprotein subunit, diaminopimelate dehydrogenase, polyphosphate kinase, and polyP:AMP phosphotransferase in ε-PL biosynthesis. Subsequently, a strain with enhanced ATP supply, L-lysine supply, and ε-PL synthetase expression was constructed to improve its production. Finally, the resulting strain, S. albulus WME10, achieved an ε-PL production rate of 77.16 g/L in a 5 L bioreactor, which is the highest reported ε-PL production to date. These results suggest that the integrative analysis of the transcriptome and metabolome can facilitate the identification of key pathways and genetic elements affecting ε-PL synthesis, guiding further metabolic engineering and thus significantly enhancing ε-PL production. The method presented in this study could be applicable to other valuable natural antibacterial agents.
Subject(s)
Metabolic Engineering , Polylysine , Streptomyces , Streptomyces/metabolism , Streptomyces/genetics , Metabolic Engineering/methods , Polylysine/biosynthesis , Polylysine/metabolism , Metabolome , Transcriptome , Metabolomics/methods , MultiomicsABSTRACT
Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL25-35) and ε-poly-l-ß-lysine consisting of l-ß-lysine residues (ε-PßL4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL5-14-FAM, with HeLa cells. Unlike ε-PαL25-35-FAM, ε-PαL5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 µM) was required for the internalization events. ε-PαL5-14 has a chain length almost equal to that of the membrane permeable ε-PßL4-13, which can enter cells at low concentrations. Considering that the basicity of the ß-amino group is higher than that of α-amino acid at physiological pH, ε-PßL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PßL would be more advantageous for cellular internalization of cargo proteins than ε-PαL25-35.
Subject(s)
Cell-Penetrating Peptides , Endocytosis , Polylysine , Humans , HeLa Cells , Polylysine/chemistry , Polylysine/metabolism , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/metabolism , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Polyelectrolytes/chemistry , Click ChemistryABSTRACT
Human norovirus (HuNoV) is an enteric infectious pathogen belonging to the Caliciviridae family that causes occasional epidemics. Circulating alcohol-tolerant viral particles that are readily transmitted via food-borne routes significantly contribute to the global burden of HuNoV-induced gastroenteritis. Moreover, contact with enzymes secreted by other microorganisms in the environment can impact the infectivity of viruses. Hence, understanding the circulation dynamics of Caliciviridae is critical to mitigating epidemics. Accordingly, in this study, we screened whether environmentally abundant secretase components, particularly proteases, affect Caliciviridae infectivity. Results showed that combining Bacillaceae serine proteases with epsilon-poly-L-lysine (EPL) produced by Streptomyces-a natural antimicrobial-elicited anti-Caliciviridae properties, including against the epidemic HuNoV GII.4_Sydney_2012 strain. In vitro and in vivo biochemical and virological analyses revealed that EPL has two unique synergistic viral inactivation functions. First, it maintains an optimal pH to promote viral surface conformational changes to the protease-sensitive structure. Subsequently, it inhibits viral RNA genome release via partial protease digestion at the P2 and S domains in the VP1 capsid. This study provides new insights regarding the high-dimensional environmental interactions between bacteria and Caliciviridae, while promoting the development of protease-based anti-viral disinfectants.
Subject(s)
Bacillaceae , Polylysine , Serine Proteases , Streptomyces , Streptomyces/enzymology , Polylysine/pharmacology , Polylysine/chemistry , Polylysine/metabolism , Serine Proteases/metabolism , Bacillaceae/enzymology , RNA, Viral/genetics , RNA, Viral/metabolism , Humans , Genome, Viral , Animals , Norovirus/drug effects , Norovirus/genetics , Virus Inactivation/drug effects , Caliciviridae/genetics , Antiviral Agents/pharmacologyABSTRACT
ε-Poly-l-lysine (ε-PL), a natural food preservative with various advantages, is primarily produced by Streptomyces. It has attracted considerable attentions for the outstanding antibacterial activity, safety, heat stability, water solubility and other remarkable properties. In this study, a food-grade recombinant Bacillus subtilis was constructed for the biocatalysis of ε-PL. Firstly, the d-alanine racemase gene (alrA) was deleted from the genome of Bacillus subtilis 168 to construct an auxotrophic B. subtilis 168 (alrA-). Based on the shuttle plasmid pMA5, a food-grade plasmid pMA5a was constructed by replacing the genes of kanamycin resistance (Kanr) and ampicillin resistance (Ampr) with alrA and the gene encoding α-peptide of ß-galactosidase (lacZα), respectively. Subsequently, codon-optimized ε-PL synthase gene (pls) and P-pls were ligated into pMA5a and transformed in E. coli DH5α and expressed in B. subtilis 168 (alrA-). Finally, the whole-cell biocatalysis conditions for ε-PL production by B. subtilis 168 (alrA-)/pMA5a-pls were optimized, and the optimal conditions were 30°C, pH 4, l-lysine concentration of 0.6â¯g/L, bacterial concentration of 15â¯% (w/v) and a catalytic time of 7â¯h. The ε-PL production reached a maximum of 0.33 ± 0.03â¯g/L. The product was verified to be ε-PL by HPLC and tricine-SDS-PAGE. The information obtained in this study shows critical reference for the food-grade heterologous expression of ε-PL.
Subject(s)
Bacillus subtilis , Biocatalysis , Polylysine , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacillus subtilis/enzymology , Polylysine/metabolism , Polylysine/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Plasmids/genetics , Escherichia coli/genetics , Escherichia coli/metabolismABSTRACT
Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.
Subject(s)
Nanoparticles , Urinary Bladder Neoplasms , Mice , Animals , Humans , Urinary Bladder/pathology , Polylysine/metabolism , CRISPR-Cas Systems/genetics , Urinary Bladder Neoplasms/therapy , Urinary Bladder Neoplasms/drug therapy , Genetic TherapyABSTRACT
The purpose of the study was to investigate the mechanism of inactivation of Serratia liquefaciens by different treatments, namely corona discharge plasma (CDP), ε-polylysine (ε-PL), and corona discharge plasma combined with ε-polylysine (CDP plus ε-PL). The results showed that the combined treatment of CDP and ε-PL exhibited significant antibacterial effects. The total number of colonies of S. liquefaciens dropped by 0.49 log CFU/mL following 4 min of CDP treatment, 4MIC ε-PL treatment for 6 h alone decreased the amounts of colonies by 2.11 log CFU/mL, and 6 h of treatment with 4MIC ε-PL after the bacterium was treated with CDP could decrease the number of colonies by 6.77 log CFU/mL. Scanning electron microscopy images showed that the combined treatment of CDP and ε-PL caused the most serious damage to the cell morphology. Electrical conductivity, nucleic acid, and PI staining indicated that the combined treatment dramatically enhanced the permeability of the cell membrane. In addition, the combined treatment led to a significant decrease in SOD and POD enzyme activities in S. liquefaciens, which prevented energy metabolism. Finally, the determination of free and intracellular ε-PL concentrations confirmed that the treatment of CDP could cause the bacteria to bind more ε-PL and exert more significant bacterial inhibition. Therefore, CDP and ε-PL had a synergistic effect in the inhibition of S. liquefaciens.
Subject(s)
Polylysine , Serratia liquefaciens , Polylysine/pharmacology , Polylysine/metabolism , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Cell Membrane/metabolism , Microscopy, Electron, ScanningABSTRACT
Streptomyces albulus NBRC14147 produces a homopoly(amino acid), ε-poly-L-lysine (ε-PL). Due to its antibiotic activity, thermostability, biodegradability, and non-toxicity to humans, ε-PL is used as a food preservative. In this study, homology searches of diaminopimelate (DAP) pathway genes (dapB and dapE), in an S. albulus genome database, were shown to encode predicted enzymes using dapB or dapE in Escherichia coli strain complementation assays. We observed that dapB and dapE transcriptional levels were weak during ε-PL production stages. Therefore, we strengthened this expression using an ermE constitutive promoter. Engineered strains generated faster growth and ε-PL production rates when compared with the control strain. Moreover, maximum ε-PL yields in S. albulus, where dapB was constitutively expressed, were approximately 14% higher when compared with the control strain. These findings showed that enhanced lysine biosynthetic gene expression generated faster and higher ε-PL production levels.
Subject(s)
Polylysine , Streptomyces , Humans , Fermentation , Gene Expression , Polylysine/genetics , Polylysine/metabolism , Streptomyces/metabolism , Diaminopimelic Acid/metabolismABSTRACT
BACKGROUND: The aim of this study was to construct crosslinked polylysine-hyaluronic acid microspheres (pl-HAM) ladened with gingival mesenchymal stem cells (GMSCs) and explore its biologic behavior in soft tissue regeneration. METHODS: The effects of the crosslinked pl-HAM on the biocompatibility and the recruitment of L-929 cells and GMSCs were detected in vitro. Moreover, the regeneration of subcutaneous collagen tissue, angiogenesis and the endogenous stem cells recruitment were investigated in vivo. We also detected the cell developing capability of pl-HAMs. RESULTS: The crosslinked pl-HAMs appeared to be completely spherical-shaped particles and had good biocompatibility. L-929 cells and GMSCs grew around the pl-HAMs and increased gradually. Cell migration experiments showed that pl-HAMs combined with GMSCs could promote the migration of vascular endothelial cells significantly. Meanwhile, the green fluorescent protein-GMSCs in the pl-HAM group still remain in the soft tissue regeneration area 2 weeks after surgery. The results of in vivo studies showed that denser collagen deposition and more angiogenesis-related indicator CD31 expression in the pl-HAMs+ GMSCs + GeL group compared with the pl-HAMs + GeL group. Immunofluorescence showed that CD44, CD90, CD73 co-staining positive cells surrounded the microspheres in both pl-HAMs + GeL group and pl-HAM + GMSCs + GeL group. CONCLUSIONS: The crosslinked pl-HAM ladened with GMSCs system could provide a suitable microenvironment for collagen tissue regeneration, angiogenesis and endogenous stem cells recruitment, which may be an alternative to autogenous soft tissue grafts for minimally invasive treatments for periodontal soft tissue defects in the future.
Subject(s)
Mesenchymal Stem Cells , Polylysine , Polylysine/metabolism , Polylysine/pharmacology , Hyaluronic Acid/pharmacology , Microspheres , Endothelial Cells , Angiogenesis , Cell Differentiation , Gingiva/metabolism , Stem Cells , Collagen/metabolism , Tissue EngineeringABSTRACT
The blood-brain barrier (BBB) is a protective cellular anatomical layer with a dynamic micro-environment, tightly regulating the transport of materials across it. To achieve in-vivo characteristics, an in-vitro BBB model requires the constituent cell types to be layered in an appropriate order. A cost-effective in-vitro BBB model is desired to facilitate central nervous system (CNS) drug penetration studies. Enhanced integrity of tight junctions observed during the in-vitro BBB establishment and post-experiment is essential in these models. We successfully developed an in-vitro BBB model mimicking the in-vivo cell composition and a distinct order of seeding primary human brain cells. Unlike other in-vitro BBB models, our work avoids the need for pre-coated plates for cell adhesion and provides better cell visualization during the procedure. We found that using bovine collagen-I coating, followed by bovine fibronectin coating and poly-L-lysine coating, yields better adhesion and layering of cells on the transwell membrane compared to earlier reported use of collagen and poly-L-lysine only. Our results indicated better cell visibility and imaging with the polyester transwell membrane as well as point to a higher and more stable Trans Endothelial Electrical Resistance values in this plate. In addition, we found that the addition of zinc induced higher claudin 5 expressions in neuronal cells. Dolutegravir, a drug used in the treatment of HIV, is known to appear in moderate concentrations in the CNS. Thus, dolutegravir was used to assess the functionality of the final model and cells. Using primary cells and an in-house coating strategy substantially reduces costs and provides superior imaging of cells and their tight junction protein expression. Our 4-cell-based BBB model is a suitable experimental model for the drug screening process.
Subject(s)
Blood-Brain Barrier , Polylysine , Animals , Cattle , Humans , Blood-Brain Barrier/physiology , Cell Line , Polylysine/metabolism , Polylysine/pharmacology , Endothelial Cells , Microscopy, ConfocalABSTRACT
Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.
Subject(s)
Heparin , Mesenchymal Stem Cells , Humans , Heparin/pharmacology , Heparin/metabolism , Polylysine/pharmacology , Polylysine/chemistry , Polylysine/metabolism , Interferon-gamma/pharmacology , Interferon-gamma/metabolism , Osteogenesis , Cell DifferentiationABSTRACT
ε-poly-L-lysine (ε-PL) is the main secondary metabolite of Streptomyces albulus, and it is widely used in the food industry. Polylysine synthetase (Pls) is the last enzyme in the ε-PL biosynthetic pathway. Our previous study revealed that Pls overexpressed in S. albulus CICC11022 result in the efficient production of ε-PL. In this study, a Pls gene knockout strain was initially constructed. Then, genomic, transcriptomic and metabolomic approaches were integrated to study the effects of the high expression and knockout of Pls on the gene expression and metabolite synthesis of S. albulus. The high expression of Pls resulted in 598 significantly differentially expressed genes (DEGs) and 425 known differential metabolites, whereas the inactivation of Pls resulted in 868 significant DEGs and 374 known differential metabolites. The expressions of 8 and 35 genes were negatively and positively associated with the Pls expression, respectively. Subsequently, the influence mechanism of the high expression and inactivation of Pls on the ε-PL biosynthetic pathway was elucidated. Twelve metabolites with 30% decreased yield in the high-expression strain of Pls but 30% increased production in the Pls knockout strain were identified. These results demonstrate the influence of Pls on the metabolism of S. albulus. The present work can provide the theoretical basis for improving the production capacity of ε-PL by means of metabolic engineering or developing bioactive substances derived from S. albulus.
Subject(s)
Polylysine , Streptomyces , Polylysine/genetics , Polylysine/metabolism , Transcriptome , Ligases/genetics , Ligases/metabolism , Ligases/pharmacology , Streptomyces/metabolism , FermentationABSTRACT
ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.
Subject(s)
Polylysine , Transcriptome , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Anti-Bacterial Agents , Butanols , Carbon , Citrate (si)-Synthase/metabolism , Diaminopimelic Acid/metabolism , Ethanol , Fermentation , Hazardous Substances , Metabolome , NAD/metabolism , Polylysine/metabolism , Sigma Factor/metabolism , Tricarboxylic AcidsABSTRACT
The effects of ε-polylysine (ε-PL) at different concentrations (0.005 %, 0.010 %, 0.020 %, and 0.030 %) on the structure and gelling behavior of pork myofibrillar protein (MP) under oxidative stress were explored. The incorporation of ε-PL significantly restrained oxidation-induced sulfhydryl and solubility losses (up to 9.72 % and 41.9 %, respectively) as well as protein crosslinking and aggregation. Compared with the oxidized control, ε-PL at low concentrations (0.005 % - 0.020 %) promoted further unfolding and destabilization of MP, while 0.030 % ε-PL led to refolding of MP and enhanced its thermal stability. The ε-PL-induced physicochemical changes favored the formation of a finer and more homogeneous three-dimensional network structure, therefore obviously enhancing the strength and water-holding capacity (WHC) of thermally induced oxidized MP gels, with the ε-PL at 0.020 % showed the greatest enhancement. This work revealed for the first time that ε-PL can significantly ameliorate the oxidation stability and gel-forming ability of meat proteins.
Subject(s)
Myofibrils , Polylysine , Gels/chemistry , Meat Proteins/analysis , Meat Proteins/metabolism , Muscle Proteins/chemistry , Myofibrils/chemistry , Oxidative Stress , Polylysine/metabolism , Polylysine/pharmacology , Water/chemistryABSTRACT
Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.
Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Histones/metabolism , Humans , Leukocyte Elastase/metabolism , Peptidyl-Dipeptidase A/metabolism , Polylysine/metabolism , Protein Binding , SARS-CoV-2 , Salivary Proteins and Peptides/metabolism , Salivary Proteins and Peptides/pharmacology , Spike Glycoprotein, CoronavirusABSTRACT
The antimicrobial activity of ε-poly-l-lysine (EPL) has been documented, but its antifungal activity on yeast is not well defined and its mechanism of action has been vaguely explained. Our studies revealed that on both, Candida albicans and Saccharomyces cerevisiae, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) were 250 µg·mL-1; EPL produced a K+ and Ca2+ efflux, and at higher concentrations also an efflux of material absorbing at 260 nm, small peptides, and phosphate is produced, along with the inhibition of fermentation and extracellular acidification and respiration. Moreover, growth was inhibited, reactive oxygen species (ROS) production increased, and cell viability decreased. The polycation also produced plasma membrane potential hyperpolarization. The effects were dependent both on the cell quantity and polycation concentration, as well as the media used. The plasma membrane disruption was confirmed by TEM and PI staining.
Subject(s)
Antifungal Agents , Candida albicans , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Candida albicans/metabolism , Microbial Sensitivity Tests , Polylysine/metabolism , Polylysine/pharmacology , Saccharomyces cerevisiae/metabolismABSTRACT
Epsilon-poly-L-lysine (ε-PL) is an unusual biopolymer composed of L-lysine produced by several microorganisms, especially by the genus Streptomyces. Due to its excellent antimicrobial activity, good water solubility, high safety, and biodegradable nature, ε-PL with a GRAS status has been widely used in food and pharmaceutical industries. In the past years, studies have focused on the biotechnological production of É-PL, the biosynthetic mechanism of microbial É-PL, and its application. To provide new perspectives from recent advances, the review introduced the methods for the isolation of É-PL producing strains and the biosynthetic mechanism of microbial É-PL. We summarized the strategies for the improvement of É-PL producing strains, including physical and chemical mutagenesis, ribosome engineering and gene engineering, and compared the different metabolic regulation strategies for improving É-PL production, including medium optimization, nutrient supply, pH control, and dissolved oxygen control. Then, the downstream purification methods of É-PL and its recent applications in food and medicine industries were introduced. Finally, we also proposed the potential challenges and the perspectives for the production of ε-PL.
Subject(s)
Polylysine , Streptomyces , Biopolymers/metabolism , Biotechnology/methods , Culture Media/metabolism , Polylysine/chemistry , Polylysine/metabolism , Streptomyces/genetics , Streptomyces/metabolismABSTRACT
Cryopreservation of human erythrocytes via suitable cryoprotectants is essential for transfusion during emergencies, but the conventional glycerolization method requires a tedious thawing-deglycerolization process. Alternatively, trehalose, a nonreducing disaccharide, has gained much attention as a biocompatible cryoprotectant due to its nature in living organisms capable of surviving extreme cold and desiccation. In this work, cryopreservation of human erythrocytes was realized through high intracellular trehalose enhanced by benzyl alcohol at 4 °C with membrane stabilization of maltotriose-grafted ε-poly(L-lysine). Intracellular trehalose could reach 94.2 ± 12.1 mM with slight impacts on morphology and cell functions, and the post-storage cryosurvival of human erythrocytes could achieve 96.2 ± 3.4% via membrane protection by the glycopeptide. It has been demonstrated that the functional glycopeptide performed as an extracellular cryoprotectant accompanied by high intracellular trehalose for synergistic cryopreservation of human erythrocytes in the biocompatible glycerol-free conditions. This two-step approach involving augmentation of intracellular trehalose at a hypothermic temperature and membrane stabilization of the functional glycopeptide could be an alternative way for human cell cryopreservation.
Subject(s)
Polylysine , Trehalose , Cryopreservation/methods , Cryoprotective Agents/chemistry , Cryoprotective Agents/metabolism , Cryoprotective Agents/pharmacology , Erythrocytes , Glycopeptides/metabolism , Humans , Polylysine/metabolism , Polylysine/pharmacology , Trehalose/chemistry , TrisaccharidesABSTRACT
Ferrocene-based polymers as redox mediators are considered versatile and important in the study of glucose biosensors. Poly-L-lysine (PLL), as a cationic polymer, possesses good properties including biocompatibility, biodegradation and water solubility. In this work, PLL was modified with ferrocene carboxylate in a very simple way by activating the carboxyl group of Fc, which reacted with the amino groups of the polymer. The resulting product was analysed by FTIR. Performance as a redox mediator (Fc-PLL) with the enzyme glucose oxidase was tested by cyclic voltammetry and showed an increase in the oxidation current in the presence of glucose in PBS pH 7.4. Additionally, performance as a biosensor was evaluated by amperometry and gave a linear range of 0-10 mM, a limit of detection of 23 µM, a sensitivity of 6.55 µA/cm2 mM and high selectivity. To evaluate the charged regions of Fc-PLL/GOx on the electrode surface, analysis by scanning electrochemical microscopy showed remarkable activity. The Fc-PLL redox polymer as a glucose biosensor has been well accepted as this kind of material, and the results showed remarkable activity as an electron transfer mediator between the redox polymer and the GOx enzyme.
Subject(s)
Biosensing Techniques , Glucose , Biosensing Techniques/methods , Electrodes , Enzymes, Immobilized/chemistry , Glucose/analysis , Glucose Oxidase/chemistry , Metallocenes , Oxidation-Reduction , Polylysine/metabolism , Polymers/chemistryABSTRACT
Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNALys or Val-tRNALys reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.
Subject(s)
Peptide Biosynthesis , Polylysine , RNA, Messenger , RNA, Transfer , Ribosomes , Peptides/metabolism , Polylysine/metabolism , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Transfer, Lys/metabolism , Ribosomes/metabolismABSTRACT
BACKGROUND: AdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-L-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces. RESULTS: In this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomyces albulus NK660 by heterologously expressing adpA from S. neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S. albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S. albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S. albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF-MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S. albulus. CONCLUSIONS: Here, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.