Microbial Cell Factories最新文献

Conventional methods for measuring antibacterial activity, such as disk-diffusion assays, have limitations in quantitative reliability and require long incubation times making them unsuitable for high-throughput applications. To address these limitations, we automated a turbidity-based assay using readily available equipment and Bayesian data analysis, enabling accurate and precise antibacterial quantification from high-throughput experiments. In this study, we demonstrate the method applied to lysostaphin, a potent anti-staphylococcal agent and promising candidate for therapeutic applications. The turbidity assay monitors optical density changes upon lysostaphin-induced lysis of a susceptible Staphylococcus strain. We validated the use of autoclaved Staphylococcus carnosus TM300 as suitable indicator strain and optimized assay conditions for dynamic range of 0.63-10 mg L^-1 lysostaphin. Our integrated approach provides a robust, scalable, and reproducible platform for quantifying active lysostaphin, paving the way for its application in high-throughput screening and process development. We believe that the approach is adaptable to other turbidity-based assays, such as those assessing endolysin activity.

Background: Interest in microbial biosurfactants has increased due to the rising demand for environmentally friendly and sustainable surfactants. Waste frying oil provides a renewable and low-cost feedstock for their production. This study aimed to isolate, characterize, and evaluate the antibacterial mechanism of a biosurfactant synthesized by Achromobacter xylosoxidans PX106473 using waste frying oil as an economical carbon source.

Results: Achromobacter xylosoxidans PX106473 produced a biosurfactant with significant activity, including an emulsification index (E24%) of 66.7% against kerosene and substantial oil displacement and hemolytic activities. According to the results of thin-layer chromatography (TLC), the produced biosurfactant contained lipids and amino acids. Fourier transform infrared spectroscopy (FT-IR) results revealed the presence of an N-H group, aliphatic hydrocarbons, and amide peaks, which suggest a lipid-peptide linkage, providing further evidence for its putative lipopeptide nature. Hexadecanoic acid, with an area percentage of 76.44, was the dominant component of the lipopeptide based on gas chromatography-mass spectrometry (GC-MS) results. The produced biosurfactant demonstrated good inhibitory activity against E. coli and S. aureus. These biological findings were further supported by in silico assays; molecular docking studies showed that hexadecanoic acid binds stably to key bacterial proteins from E. coli (DNA gyrase B, -6.4 kcal/mol) and S. aureus (PBP2a, -3.9 kcal/mol), indicating a potential dual-target mechanism.

Conclusion: Achromobacter xylosoxidans efficiently produced a putative lipopeptide biosurfactant from waste frying oil with strong emulsifying and antibacterial properties, providing an economical and sustainable solution with potential in various environmental and pharmaceutical applications.

Background: The growing threat of antibiotic resistance underscores the urgent need for novel therapeutic agents. Antimicrobial peptides (AMPs), such as human defensins, are promising candidates due to their broad-spectrum activities, but their widespread application is severely hindered by high production costs and limited stability. Among these, human neutrophil peptide-2 (HNP2), a member of the α-defensin family, is a particularly potent AMP that kills microorganisms by disrupting their membrane integrity. The eukaryotic microalga Chlamydomonas reinhardtii (C. reinhardtii) has emerged as a low-cost and efficient bioreactor for exogenous protein production, yet its potential for expressing human defensins remains largely unexplored. This study aimed to express a tandem trimer of the HNP2 mature peptide (3×HNP2) in C. reinhardtii and to characterize its stability, safety, and antibacterial functions.

Results: In this study, a gene encoding 3×HNP2 was successfully expressed in the C. reinhardtii strain CC-5325, yielding a fusion protein of approximately 22 kDa. The recombinant protein's expression remained stable for over five months of continuous subculturing. Obtained via fermentation technology and affinity purification, the purified 3×HNP2 demonstrated potent antibacterial activity against the Gram-negative pathogens Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The protein exhibited high thermal stability (up to 90 °C), broad pH tolerance (pH 2-10), and resistance to degradation by proteinase K and papain. Mechanistic investigations using propidium iodide (PI) staining and scanning electron microscopy (SEM) confirmed that 3×HNP2 acts by disrupting bacterial membrane integrity. Furthermore, qRT-PCR analysis revealed that 3×HNP2 significantly downregulated the expression of key virulence-associated genes (lecA, phzA2, csgA, and rpoS). Biosafety assays showed that the peptide had minimal hemolytic activity and low cytotoxicity against mammalian cell lines.

Conclusions: This work establishes a green platform for producing a functional human defensin in C. reinhardtii. It demonstrates that algae-derived 3×HNP2 is a stable, safe, and effective antimicrobial agent, suggesting its potential as a candidate for further development in antimicrobial therapeutics or food safety applications.

Background: The growing consumer preference for natural and sustainable products has heightened interest in biopigments across pharmaceutical, cosmetic, and food industries. In this study, we investigate endophytic fungi as a viable and eco-friendly source for the production of bioactive natural pigments.

Methods and results: A promising strain, Aspergillus westerdijkiae 17P, was isolated from Betula pendula and assessed for its pigment-producing potential and associated bioactivities. The biomass extract was fractionated, and the resulting components were evaluated for antimicrobial, antioxidant, anticancer, neuroprotective, and peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist activities. Among the fractions, 17P2 exhibited broad-spectrum antimicrobial effects, notable antioxidant activity (83% DPPH radical scavenging at 1000 mg/mL), and cytotoxicity against MCF-7 and HepG2 cancer cell lines, with IC₅₀ values of 250 mg/mL. Isothermal titration calorimetry (ITC) demonstrated strong binding affinities of 17P2 to acetylcholinesterase (Kd = 1.63 µM) and butyrylcholinesterase (Kd = 0.03 µM), indicating potential anti-Alzheimer's properties. Additionally, significant interactions with monoamine oxidase A and PPAR-γ suggest possible antidepressant and antidiabetic applications. Four major pigment fractions (17P1-17P4) were purified and structurally characterized using UHPLC-MS and NMR, revealing key metabolites such as aluminium and iron aspergillic acid complexes, penicillic acid, and preussin. Notably, gamma irradiation at 2000 Gy significantly enhanced the red, yellow, and orange pigments yield compared to the non-irradiated control cultures.

Conclusions: Collectively, these findings position A. westerdijkiae 17P as a valuable and versatile biotechnological resource for the sustainable production of multifunctional fungal pigments with potential industrial and therapeutic applications.