Acutely induced cell mortality in the unicellular green alga Chlamydomonas reinhardtii (Chlorophyceae) following exposure to acrylic resin nanoparticles.

Nanoparticles have unique properties that make them attractive for use in industrial and medical technology industries but can also be harmful to living organisms, making an understanding of their molecular mechanisms of action essential. We examined the effect of three different sized poly(isobutyl-cyanoacrylate) nanoparticles (iBCA-NPs) on the unicellular green alga Chlamydomonas reinhardtii. We found that exposure to iBCA-NPs immediately caused C. reinhardtii to display abnormal swimming behaviors. Furthermore, after one hour, most of the cells had stopped swimming and 10%-30% of cells were stained with trypan blue, suggesting that these cells had severely impaired plasma membranes. Observation of the cyto-ultrastructure showed that the cell walls had been severely damaged and that many iBCA-NPs were located in the space between the cell wall and plasma membrane, as well as inside the cytosol in some cases. A comparison of three strains of C. reinhardtii with different cell wall conditions further showed that the cell mortality ratio increased more rapidly in the absence of a cell wall. Interestingly, cell mortality over time was essentially identical regardless of iBCA-NP size if the total surface area was the same. Furthermore, direct observation of the trails of iBCA-NPs indicated that the first trigger was their contact with the cell wall, which is most likely accompanied by the inactivation or removal of adsorbed proteins from the cell wall surface. Cell mortality was accompanied by the overproduction of reactive oxygen species, which was detected more readily in cells grown under constant light rather than in the dark.

UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems.

KEY MESSAGE: In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory. Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.

Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains.

Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type. In this study, we evaluated the possibility of a new mutant strain, met1, which contains a tag in the maintenance type methyltransferase gene that is expected to play a key role in the maintenance of transcriptional gene silencing. The versatile usefulness of the UVM4 strain to express heterologous genes was also analyzed. We failed to overexpress CrSSL3 cDNA, which is the codon-adjusted squalene synthase-like gene originated from Botryococcus braunii, using the common expression cassette in the wild-type CC-1690 and UVM4 strains. However, we succeeded in isolating western blot-positive transformants through the combinational use of the UVM4 strain and ble2A expression system of which expression cassette bears a fused ORF of the target gene and the antibiotic resistance gene ble via the foot-and-mouth disease virus (FMDV) self-cleaving 2A sequence. It is noteworthy that even with this system, huge deviations in the accumulated protein levels were still observed among the UVM4 transformants.

Construction of individual, fused, and co-expressed proteins of endoglucanase and ß-glucosidase for hydrolyzing sugarcane bagasse.

At least a combination of endoglucanase (EglII) and ß-glucosidase (BglZ) is required for hydrolyzing crystalline cellulose. To understand the catalytic efficiency of combination enzymes for converting biomass to sugars, EglII and BglZ were constructed in the form of individual, fused as well as co-expression proteins, and their activities for hydrolyzing sugarcane bagasse were evaluated. The genes, eglII isolated from Bacillus amyloliquefaciens PSM3.1 earlier and bglZ from B. amyloliquefaciens ABBD, were expressed extracellularly in Bacillus megaterium MS941. EglII exhibited both exoglucanase and endoglucanase activities, and BglZ belonging to the glycoside hydrolase 1 family (GH 1) showed ß-glucosidase activity. A combination of EglII and BglZ showed activity on substrates Avicel, CMC and sugarcane bagasse. Specifically for hydrolyzing sugarcane bagasse, fused protein (fus-EglII+BglZ), co-expression protein (coex-BglZ+EglII), and mixed-individual protein (mix-EglII+BglZ) produced cellobiose as the main product, along with a small amount of glucose. The amount of reducing sugars released from the hydrolyzing bleached sugarcane bagasse (BSB) using fus-EglII+BglZ and mix-EglII+BglZ was 2.7- and 4.2-fold higher, respectively, than steamed sugarcane bagasse (SSB), indicating the synergetic enzymes worked better on treated sugarcane bagasse. Compared with fus-EglII+BglZ and mix-EglII+BglZ, coex-BglZ+EglII released more mol reducing sugars from SSB, indicating the enzymes were potential for biomass conversion. Additionally, coex-BglZ+EglII acted on BSB 2.5-fold faster than fus-EglII+BglZ. Thus, coex-bglZ+eglII expression system was the best choice to produce enzymes for hydrolyzing sugarcane baggase.

ß-D-xylosidase from Geobacillus thermoleovorans IT-08: biochemical characterization and bioinformatics of the enzyme.

The gene encoding a thermostable ß-D-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6-8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k cat/K M) of 0.0048 ± 0.0010 s(-1) mM(-1) on p-nitrophenyl-ß-D-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel ß-strands) and catalytic module (residues 157 to 604 forming five-bladed ß-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.

Screening, gene sequencing and characterising of lipase for methanolysis of crude palm oil.

Staphylococcus sp. WL1 lipase (LipFWS) was investigated for methanolysis of crude palm oil (CPO) at moderate temperatures. Experiments were conducted in the following order: searching for the suitable bacterium for producing lipase from activated sludge, sequencing lipase gene, identifying lipase activity, then synthesising CPO biodiesel using the enzyme. From bacterial screening, one isolated specimen which consistently showed the highest extracellular lipase activity was identified as Staphylococcus sp. WL1 possessing lipFWS (lipase gene of 2,244 bp). The LipFWS deduced was a protein of 747 amino acid residues containing an α/ß hydrolase core domain with predicted triad catalytic residues to be Ser474, His704 and Asp665. Optimal conditions for the LipFWS activity were found to be at 55 °C and pH 7.0 (in phosphate buffer but not in Tris buffer). The lipase had a K(M) of 0.75 mM and a V(max) of 0.33 mMmin(-1) on p-nitrophenyl palmitate substrate. The lyophilised crude LipFWS performed as good as the commonly used catalyst potassium hydroxide for methanolysis of CPO. ESI-IT-MS spectra indicated that the CPO was converted into biodiesel, suggesting that free LipFWS is a worthy alternative for CPO biodiesel synthesis.