Optimizing enzyme properties to enhance dihydroxyacetone production via methylglyoxal biosensor development.

BACKGROUND: Dihydroxyacetone (DHA) stands as a crucial chemical material extensively utilized in the cosmetics industry. DHA production through the dephosphorylation of dihydroxyacetone phosphate, an intermediate product of the glycolysis pathway in Escherichia coli, presents a prospective alternative for industrial production. However, insights into the pivotal enzyme, dihydroxyacetone phosphate dephosphorylase (HdpA), remain limited for informed engineering. Consequently, the development of an efficient tool for high-throughput screening of HdpA hypermutants becomes imperative. RESULTS: This study introduces a methylglyoxal biosensor, based on the formaldehyde-responding regulator FrmR, for the selection of HdpA. Initial modifications involved the insertion of the FrmR binding site upstream of the -35 region and into the spacer region between the -10 and -35 regions of the constitutive promoter J23110. Although the hybrid promoter retained constitutive expression, expression of FrmR led to complete repression. The addition of 350 µM methylglyoxal promptly alleviated FrmR inhibition, enhancing promoter activity by more than 40-fold. The methylglyoxal biosensor system exhibited a gradual increase in fluorescence intensity with methylglyoxal concentrations ranging from 10 to 500 µM. Notably, the biosensor system responded to methylglyoxal spontaneously converted from added DHA, facilitating the separation of DHA producing and non-producing strains through flow cytometry sorting. Subsequently, the methylglyoxal biosensor was successfully applied to screen a library of HdpA mutants, identifying two strains harboring specific mutants 267G > T and D110G/G151C that showed improved DHA production by 68% and 114%, respectively. Expressing of these two HdpA mutants directly in a DHA-producing strain also increased DHA production from 1.45 to 1.92 and 2.29 g/L, respectively, demonstrating the enhanced enzyme properties of the HdpA mutants. CONCLUSIONS: The methylglyoxal biosensor offers a novel strategy for constructing genetically encoded biosensors and serves as a robust platform for indirectly determining DHA levels by responding to methylglyoxal. This property enables efficiently screening of HdpA hypermutants to enhance DHA production.

Stabilizing oil-oil interfaces with mixed-shell polymeric nanoparticles prepared via PISA and the grafting combination.

The preparation of mixed-shell polymeric nanoparticles (MSPNs) and their stabilized non-aqueous Pickering emulsions was described in this study. Poly(methyl methacrylate)-poly(4-vinylpyridine) (PMMA-P4VP) diblock copolymer nanoparticles with different morphologies including spheres, worms and vesicles were first prepared via reversible addition-fragmentation chain transfer-based polymerization induced self-assembly in toluene. C18 alkyl chains were subsequently grafted onto the surfaces of the as-prepared PMMA-P4VP nanoparticles, affording C18/PMMA-P4VP MSPNs with P4VP blocks as the core and C18/PMMA chains as the mixed shells. MSPNs were utilized as Pickering emulsifiers to prepare non-aqueous Pickering emulsions, where [Bmim][PF6] and toluene oils were selected. Two kinds of different Pickering emulsions, [Bmim][PF6]-in-toluene and toluene-in-[Bmim][PF6], could be formed, depending on the initial location of MSPNs. However, neither of them could be generated when PMMA-P4VP diblock copolymer nanoparticles were adopted as Pickering emulsifiers, indicating MSPNs were better than diblock copolymer nanoparticle precursors in the aspect of stabilizing oil-oil interfaces. The formation mechanisms of different kinds of Pickering emulsions were unraveled in this study.

High cell-density fermentation, expression and purification of bacteriophage lysin TSPphg, a thermostable antimicrobial protein from extremophilic Thermus bacteriophage TSP4.

Recently, high cell-density (HCD) cultivation has become an important tool for production of many microbial products. However, to the best of our knowledge, no study regarding HCD fermentation, overproduction and purification of thermostable bacteriophage lysin has been reported. Here, by employing a glucose-limited fed-batch strategy, we performed high density fermentation of the host Escherichia coli BL21(DE3) cells, compared the efficiency of high pressure homogenization, ultrasonication and thermolysis in bacterial cell disruption after HCD cultivation, and purified TSPphg, a thermostable lysin derived from extremophilic bacteriophage TSP4. On the 20-L scale, the overproduction level of TSPphg was up to 67.8 ± 0.7%. In total, we obtained a broth titer of 3322.8 ± 26 mg/L TSPphg with a purity of 95.5 ± 0.7% from a bacterial cell mass of 86.3 ± 4.9 g/L after 26 h of fermentation. The overall productivity of TSPphg was 127.8 ± 1 mg/L/h. Additionally, the antimicrobial activity of purified TSPphg against both Gram-negative (Escherichia coli O157) and Gram-positive (Staphylococcus aureus) pathogenic bacteria was further confirmed by scanning electron microscope analysis. Summarily, for the first time, we have established a relatively stable and efficient HCD cultivation and purification process for recovery of thermostable lysins from extremophilic Thermus bacteriophages. Our results provide insights into the strategies for time-saving and cost-effective production of antimicrobial proteins to replace or supplement antibiotics in the current age of mounting antibiotic resistance.

Stimuli-responsive antioxidant Pickering emulsions stabilized by functionalized cellulose nanocrystals.

Stimuli-responsive antioxidant Pickering emulsions play crucial role in many industrial areas. This study demonstrated for the first time oil-in-water Pickering emulsions with outstanding antioxidation and responsive demulsification stabilized by functionalized cellulose nanocrystals (CNCs). Dialdehyde cellulose nanocrystals (DACs) were first prepared through the oxidation of CNCs with periodate, followed by the grafting of p-aminophenols (PAPs) onto their surfaces through Schiff base reaction, affording PAP grafted DACs (DAC-g-PAP) via dynamic covalent linkage. The degree of the oxidation (DO) of DACs had a significant effect on the yield of the targeting DAC-g-PAP nanoparticles. High DO (≥40 %) potentially led to the degradation of DACs during the grafting of PAP. The introduced PAP endowed DACs with excellent radical scavenging capability, thereby providing antioxidant properties while improving the hydrophobicity. DAC-g-PAP nanoparticles were then applied as Pickering emulsifiers to prepare oil-in-water Pickering emulsions. The resultant Pickering emulsions indicated exceptional antioxidant and pH-responsiveness together with good freezing-thaw stability. The structures of DAC-g-PAP nanoparticles were thoroughly characterized in this study.

Family function and anxiety among junior school students during the COVID-19 pandemic: a moderated mediation model.

Background: The prevalence of anxiety among adolescents is relatively high during an epidemic. Studies have reported that family function and perceived stress are important factors affecting adolescents' anxiety. However, only few studies have explored the factors influencing the relationship between family function and anxiety. Therefore, this study explored the mediating and moderating mechanisms underlying this relationship among junior school student during the COVID-19 pandemic. Methods: 745 junior school students completed family function, perceived stress, and anxiety questionnaires. Results: (1) The junior school students that were left-behind tended to show lower family function (t = -4.21, p < 0.001), greater perceived stress (t = 2.72, p < 0.01), and higher anxiety (t = 4.24, p < 0.001), (2) Family function in junior school students was negatively associated with anxiety (r = -0.35, p < 0.001); perceived stress mediated the relationship between family function and anxiety (p < 0.001), and (3) Whether the student was left-behind (LB) moderated the link between family function and anxiety (ß = -0.16, t = -3.33, p < 0.001) and between family function and perceived stress (ß = -0.22, t = -2.61, p < 0.001). Conclusion: These findings suggest a negative association between family function and anxiety. Knowledge of the mediating role of perceived stress and moderating role of being left-behind may help prevent and improve anxiety among junior school students during the COVID-19 pandemic.

TSPphg Lysin from the Extremophilic Thermus Bacteriophage TSP4 as a Potential Antimicrobial Agent against Both Gram-Negative and Gram-Positive Pathogenic Bacteria.

New strategies against antibiotic-resistant bacterial pathogens are urgently needed but are not within reach. Here, we present in vitro and in vivo antimicrobial activity of TSPphg, a novel phage lysin identified from extremophilic Thermus phage TSP4 by sequencing its whole genome. By breaking down the bacterial cells, TSPphg is able to cause bacteria destruction and has shown bactericidal activity against both Gram-negative and Gram-positive pathogenic bacteria, especially antibiotic-resistant strains of Klebsiella pneumoniae, in which the complete elimination and highest reduction in bacterial counts by greater than 6 logs were observed upon 50 µg/mL TSPphg treatment at 37 °C for 1 h. A murine skin infection model further confirmed the in vivo efficacy of TSPphg in removing a highly dangerous and multidrug-resistant Staphylococcus aureus from skin damage and in accelerating wound closure. Together, our findings may offer a therapeutic alternative to help fight bacterial infections in the current age of mounting antibiotic resistance, and to shed light on bacteriophage-based strategies to develop novel anti-infectives.