ABSTRACT The emergence of antibiotic‐resistant pathogens, particularly methicillin‐resistant Staphylococcus aureus (MRSA), represents a major public health threat, with limited treatment options. Bacteriophage‐derived endolysins and their engineered chimeric derivatives have attracted attention as alternative antibacterial agents due to their rapid bacteriolytic activity, target specificity, and low resistance propensity. ASEnd was constructed by fusing the enzymatically active LYZ2 domain from S. aureus phage φMR11 with the SH3b‐type cell wall–binding domain from phage K via a flexible (G4S) 3 linker. Structural integrity was assessed in silico using Ramachandran plot analysis, ERRAT, Verify3D, and ProSA‐web. The recombinant protein was expressed in Escherichia coli BL21 (DE3) and purified by immobilized metal affinity chromatography. Antibacterial activity was evaluated against MRSA, methicillin‐susceptible S. aureus (MSSA), and other bacterial strains using disk diffusion, turbidity reduction, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. pH tolerance, storage stability, and cytotoxicity toward HEK‐293 cells were also examined. ASEnd was obtained as a soluble 34.7 kDa protein with favorable structural metrics (>99% of residues in favored Ramachandran regions). The protein exhibited potent activity against S. aureus , producing inhibition zones up to 19 mm and rapidly reducing turbidity at low concentrations. ASEnd demonstrated MIC and MBC values of 1.56 and 3.125 µg/mL, respectively, representing a 16‐fold improvement over LYZ2. The enzyme retained activity between pH 6 and 8, but declined significantly below pH 5, and gradual loss was observed during storage at 4°C (21%, 42%, and 78% reductions after 1, 7, and 14 days), with complete inactivation after 3 weeks. ASEnd showed no cytotoxicity toward HEK‐293 cells and displayed selective activity against MRSA and MSSA, with limited cross‐reactivity against Streptococcus pyogenes (2.4‐fold lower) and no effect on E. coli . These findings identify ASEnd as a rationally designed chimeric endolysin with enhanced potency and selectivity against S. aureus and a favorable safety profile. Further stability optimization, clinical isolate testing, and in vivo validation are required to establish its therapeutic potential.
ABSTRACT Pichia pastoris has emerged as a highly efficient heterologous protein expression system with significant potential for secretory production. However, its secretion efficiency is influenced by multiple factors, among which intracellular redox homeostasis plays a critical role. In this study, differential EGFP‐secreting strains (GS115‐High/Low) were constructed via genomic integration of site‐directed mutations at N ‐glycosylation sites in the α‐mating factor signal peptide of P. pastoris . Notably, the expression of the superoxide dismutase ( SOD1 ) gene was significantly upregulated in the high‐secretion strain GS115‐High. To further investigate the regulatory role of SOD1 in protein secretion, the SOD1 gene was overexpressed in both high‐ and low‐secretion strains. Experimental results demonstrated that SOD1 overexpression markedly enhanced EGFP secretion in the high‐secretion strain ( p ≤ 0.05), but had no significant effect on the low‐secretion strain. Transcriptomic analysis revealed that amino acid metabolism pathways and oxidoreductase activity were significantly enhanced in the high‐secretion strain, suggesting a synergistic optimization of secretion efficiency through metabolic resource reallocation and antioxidant capacity. This study demonstrates that glycosylation modification and SOD1‐mediated redox regulation both contribute to enhanced EGFP secretion, providing a basis for a “glycosylation‐antioxidant” dual‐track optimization strategy for the rational design of industrial P. pastoris strains and high‐efficiency protein production.
ABSTRACT Dunaliella species are among the most significant biological sources of carotenoids, notably beta‐carotene. Given that chemical synthesis offers a more cost‐effective alternative to biotechnological cultivation, optimizing production strategies is essential for economic viability. This study investigated the optimization of bioproduct synthesis in a Brazilian strain of Dunaliella salina by evaluating the effects of high photon flux density (PFD) and temperature under low‐salinity conditions (1 mol L −1 NaCl) to facilitate downstream processing. Batch cultivations were conducted at 26°C across a PFD gradient ranging from 100 to 2000 µmol photons m −2 s −1 , with additional cultivations performed at 30°C under maximum irradiance. The results demonstrated that increasing PFD to 2000 µmol photons m −2 s −1 significantly enhanced metabolic performance, yielding a 25% increase in biomass concentration, a 62% rise in biomass productivity, and an 82% surge in total carotenoid production. Furthermore, carbohydrate content increased by 61%, whereas lipid levels remained stable, exhibiting a fatty acid profile conducive to biodiesel production. The temperature variation up to 30°C had no effect on biomass concentration or biomass composition, affecting only the fatty acid composition. These findings underscore the robust potential of this strain for the large‐scale biotechnological production of carotenoids and other high‐value compounds within a biorefinery framework.
Algal biorefineries provide a platform for producing fuels, biodegradable materials, and higher-value co-products from the same biomass resource. This review examines integrated pathways that connect microalgal and macroalgal cultivation with biodiesel production, hydrothermal liquefaction (HTL), anaerobic digestion (AD), polysaccharide extraction, and bioplastic manufacture. Microalgal lipid contents of 40%-70% of dry biomass can support biodiesel production, whereas hydrothermal processing of wet biomass has produced bio-crude yields of 30-50 wt% with energy densities of approximately 35-40 MJ/kg. Macroalgal polysaccharides, including alginate, agar, and carrageenan, can be processed into films, blends, and composites with reported tensile strengths of 18-45 MPa. However, the environmental and economic performance of these routes depends on cultivation configuration, dewatering requirements, product recovery, allocation method, and system boundary. Published techno-economic estimates place stand-alone algal biodiesel production at approximately USD 4-7 gal-1. In contrast, integrated scenarios can approach USD 2.50 gal-1 when favorable biomass productivity and co-product credits are assumed. Machine-learning (ML) methods have mainly been applied to laboratory data analysis and process prediction, while evidence of commercial real-time control remains limited. The review identifies mass-balance reporting, long-duration outdoor cultivation, standardized biodegradation testing, and harmonized techno-economic and life-cycle assessment as priorities for scale-up.
The rapid expansion of the global textile industry, driven by the fast-fashion paradigm, which is characterized by low-cost, short-lived garments made from inexpensive synthetic fibers, has intensified environmental pressures through increased waste generation, resource depletion, microplastic pollution, and greenhouse gas emissions. Conventional polyethylene terephthalate (PET) recycling remains limited by high energy demands, harsh processing conditions, and the generation of low-value products. This review examines emerging upcycling strategies targeting PET-derived monomers, including terephthalic acid (TPA) and ethylene glycol, with particular emphasis on converting TPA into high-value chemicals, functional materials, and specialty polymers. By integrating biological and materials engineering approaches, these strategies offer a transformative pathway for converting textile waste into value-added products, thereby supporting the development of a circular and sustainable PET economy. Unlike previous surveys, this work focuses on the upcycling of TPA, providing a critical evaluation of how advanced protein engineering is driving improvements in enzymatic depolymerization and bioconversion pathways toward high-value products, thereby filling a key gap in existing research, which is predominantly centered on primary recycling approaches.
