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Biosynthetic Conversion of Ag? to highly Stable Ag? Nanoparticles by Wild Type and Cell Wall Deficient Strains of Chlamydomonas reinhardtii.

ABSTRACT: In the current study, two different strains of the green, freshwater microalga Chlamydomonas reinhardtii bioreduced Ag? to silver nanoparticles (AgNPs), which have applications in biosensors, biomaterials, and therapeutic and diagnostic tools. The bioreduction takes place in cell cultures of C. reinhardtii at ambient temperature and atmospheric pressure, thus eliminating the need for specialized equipment, harmful reducing agents or the generation of toxic byproducts. In addition to the visual changes in the cell culture, the production of AgNPs was confirmed by the characteristic surface plasmon resonance (SPR) band in the range of 415?425 nm using UV-Vis spectrophotometry and further evolution of the SPR peaks were studied by comparing the peak intensity at maximum absorbance over time. X-ray diffraction (XRD) determined that the NPs were Ag?. Micrographs from transmission electron microscopy (TEM) revealed that 97 ± 2% AgNPs were <10 nm in diameter. Ag? to AgNP conversion was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The AgNPs were stable over time in the cell culture media, acetone, NaCl and reagent alcohol solutions. This was verified by a negligible change in the features of the SPR band after t > 300 days of storage at 4 °C.

SUBMITTER: Rahman A

PROVIDER: S-EPMC6337529 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:An intact cell wall is critical for cellular interactions with the environment and protecting the cell from environmental challenges. Signaling mechanisms are necessary to monitor cell wall integrity and to regulate cell wall production and remodeling during growth and division cycles. The green alga, Chlamydomonas, has a proteinaceous cell wall of defined structure that is readily removed by gametolysin (g-lysin), a metalloprotease released during sexual mating. Naked cells treated with g-lysin induce the mRNA accumulation of >100 cell wall-related genes within an hour, offering a system to study signaling and regulatory mechanisms for de novo cell wall assembly. Combining quantitative RT-PCR and luciferase reporter assays to probe transcript accumulation and promoter activity, we revealed that up to 500-fold upregulation of cell wall-related genes was driven at least partly by transcriptional activation upon g-lysin treatment. To investigate how naked cells trigger this rapid transcriptional activation, we tested whether osmotic stress and cell wall integrity are involved in this process. Under a constant hypotonic condition, comparable levels of cell wall-gene activation were observed by g-lysin treatment. In contrast, cells in an iso- or hypertonic condition showed up to 80% reduction in the g-lysin-induced gene activation, suggesting that osmotic stress is required for full-scale responses to g-lysin treatment. To test whether mechanical perturbation of cell walls is involved, we isolated and examined a new set of cell wall mutants with defective or little cell walls. All cell wall mutants examined showed a constitutive upregulation of cell wall-related genes at a level that is only achieved by treatment with g-lysin in wild-type cells. Our study suggests a cell wall integrity monitoring mechanism that senses both osmotic stress and mechanical defects of cell walls and regulates cell wall-gene expression in Chlamydomonas, which may relate to cell wall integrity signaling mechanisms in other organisms.

Project description:The green microalga Chlamydomonas reinhardtii is emerging as a promising cell biofactory for secreted recombinant protein (RP) production. In recent years, the generation of the broadly used cell wall-deficient mutant strain UVM4 has allowed for a drastic increase in secreted RP yields. However, purification of secreted RPs from the extracellular space of C. reinhardtii strain UVM4 is challenging. Previous studies suggest that secreted RPs are trapped in a matrix of cell wall protein aggregates populating the secretome of strain UVM4, making it difficult to isolate and purify the RPs. To better understand the nature and behaviour of these extracellular protein aggregates, we analysed and compared the extracellular proteome of the strain UVM4 to its cell-walled ancestor, C. reinhardtii strain 137c. When grown under the same conditions, strain UVM4 produced a unique extracellular proteomic profile, including a higher abundance of secreted cell wall glycoproteins. Further characterization of high molecular weight extracellular protein aggregates in strain UVM4 revealed that they are largely comprised of pherophorins, a specific class of cell wall glycoproteins. Our results offer important new insights into the extracellular space of strain UVM4, including strain-specific secreted cell wall proteins and the composition of the aggregates possibly related to impaired RP purification. The discovery of pherophorins as a major component of extracellular protein aggregates will inform future strategies to remove or prevent aggregate formation, enhance purification of secreted RPs, and improve yields of recombinant biopharmaceuticals in this emerging cell biofactory. KEY POINTS: • Extracellular protein aggregates hinder purification of recombinant proteins in C. reinhardtii • Unassembled cell wall pherophorins are major components of extracellular protein aggregates • Known aggregate composition informs future strategies for recombinant protein purification.

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Biosynthetic Conversion of Ag? to highly Stable Ag? Nanoparticles by Wild Type and Cell Wall Deficient Strains of Chlamydomonas reinhardtii.

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