Advances in algal lipid metabolism and their use to improve oil content.

Microalgae are eukaryotic photosynthetic micro-organisms that convert CO2 into carbohydrates, lipids, and other valuable metabolites. They are considered promising chassis for the production of various bioproducts, including fatty acid-derived biofuels. However, algae-based biofuels are not yet commercially available, mainly because of their low yields and high production cost. Optimizing strains to improve lipid productivity using the principles of synthetic biology should help move forward. This necessitates developments in the following areas: (1) identification of molecular bricks (enzymes, transcription factors, regulatory proteins etc.); (2) development of genetic tools; and (3) availability of high-throughput phenotyping methods. Here, we highlight the most recent developments in some of these areas and provide examples of the use of genome editing tools to improve oil content.

Overexpression of a MYB1 Transcription Factor Enhances Triacylglycerol and Starch Accumulation and Biomass Production in the Green Microalga Chlamydomonas reinhardtii.

Microalgae are promising platforms for biofuel production. Transcription factors (TFs) are emerging as key regulators of lipid metabolism for biofuel production in microalgae. We previously identified a novel TF MYB1, which mediates lipid accumulation in the green microalga Chlamydomonas under nitrogen depletion. However, the function of MYB1 on lipid metabolism in microalgae under standard growth conditions remains poorly understood. Here, we examined the effects of MYB1 overexpression (MYB1-OE) on lipid metabolism and physiological changes in Chlamydomonas. Under standard growth conditions, MYB1-OE transformants accumulated 1.9 to 3.2-fold more triacylglycerols (TAGs) than that in the parental line (PL), and total fatty acids (FAs) also significantly increased. Moreover, saturated FA (C16:0) was enriched in TAGs and total FAs in MYB1-OE transformants. Notably, starch and protein content and biomass production also significantly increased in MYB1-OE transformants compared with that in PL. Furthermore, RT-qPCR results showed that the expressions of key genes involved in TAG, FA, and starch biosynthesis were upregulated. In addition, MYB1-OE transformants showed higher biomass production without a compromised cell growth rate and photosynthetic activity. Overall, our results indicate that MYB1 overexpression not only enhanced lipid content but also improved starch and protein content and biomass production under standard growth conditions. TF MYB1 engineering is a promising genetic engineering tool for biofuel production in microalgae.

Enhanced triacyclglycerols and starch synthesis in Chlamydomonas stimulated by the engineered biodegradable nanoparticles.

Microalgae are promising feedstock for renewable fuels. The accumulation of oils in microalgae can be enhanced by nanoparticle exposure. However, the nanoparticles employed in previous studies are mostly non-biodegradable, which hinders nanoparticles developing as promising approach for biofuel production. We recently reported the engineered resin nanoparticles (iBCA-NPs), which were found to be biodegradable in this study. When the cells of green microalga Chlamydomonas reinhardtii were exposed to the iBCA-NPs for 1 h, the cellular triacyclglycerols (TAG) and starch contents increased by 520% and 60% than that in the control. The TAG production improved by 1.8-fold compared to the control without compromised starch production. Additionally, the content of total fatty acids increased by 1.3-fold than that in control. Furthermore, we found that the iBCA-NPs addition resulted in increased cellular reactive oxygen species (ROS) content and upregulated the activities of antioxidant enzymes. The relative expressions of the key genes involved in TAG and starch biosynthesis were also upregulated. Overall, our results showed that short exposure of the iBCA-NPs dramatically enhances TAG and starch accumulation in Chlamydomonas, which probably resulted from prompt upregulated expression of the key genes in lipid and starch metabolic pathways that were triggered by over-accumulated ROS. This study reported a useful approach to enhance energy-rich reserve accumulation in microalgae. KEY POINTS: 1. The first attempt to increase oil and starch in microalgae by biodegradable NPs. 2. The biodegradability of iBCA-NPs by the BOD test was more than 50% after 28 days. 3. The iBCA-NPs induce more energy reserves than that of previously reported NPs.

[Dose-effect relationship of DMSO and Tween 80 influencing the growth and viability of murine bone marrow-derived cells in vitro].

This study was purpose to examine the effect of dimethyl sulfoxide (DMSO) and Tween 80 on the growth and viability of stromal cells (BMSC), colony-forming units for granulocytes and macrophages (CFU-GM) and bone marrow endothelial cell line (BMEC) from murine bone marrow in vitro, and to analyze the concentration-effect relationship. The colony yields of colony-forming units fibroblastic (CFU-F) and CFU-GM were assessed in the murine bone marrow cell cultures at various concentrations of DMSO or Tween 80 and in the control groups. The MTT assay and trypan blue exclusion were used to determine the cell viability and percentage of survival in BMSC and BMEC cultures with or without either of these organic solvents. The results showed that the colony yields of both CFU-F and CFU-GM were decreased significantly (p<0.05 or <0.01) at the concentrations (v/v final) of 2% DMSO or 0.005%-0.01% Tween 80 respectively, as compared with control. The cell viability and percentage of survival of BMSC and BMEC cultures were significantly reduced (p<0.05 or <0.01) at 0.5%-1.0% DMSO or 0.002%-0.005% Tween 80, as compared with control. With the increase of volume fractions of these solvents, the decreased percentages of corresponding measurements were increased by degrees. It is concluded that when the concentration of DMSO or Tween 80 goes to a certain level in cell culture medium, either of the organic solvents has an inhibitory action or/and cytotoxicity on the growth and viability of BMSCs, CFU-GM and BMECs. The growth inhibition and cytotoxic response are more significant at higher concentrations of these solvents.

[Determination of residual monomers in polymer water treatment chemicals using high performance liquid chromatography].

A method was developed for the determination of residual monomers in polymer water treatment chemicals (sodium of polyepoxysuccinic acid (PESA), acrylic acid/maleic anhydride copolymer (AA/MA), polyacrylic acid (PAA), sodium of hydrolyzed polymaleic acid (HPMA), and maleic anhydride/acrylic acid/methyl acrylate copolymer (MA/AA/MAc) using high performance liquid chromatography. The separation was performed on a ZORBAX 300SB-C18 column (5 microm, 150 mm x 4.6 mm). The mobile phase was 0.01 mol/L KH2PO4 (pH 2.3, adjusted by 5% H3PO4)-methanol (95 : 5, v/v) with a flow rate of 0.6 mL/min. The detection wavelength was set at 210 nm and the column temperature was 30 degrees C. The sample was diluted by the mobile phase and filtrated for analysis. The residual monomers of maleic acid, fumaric acid and acrylic acid were completely separated and determined in 10 min, and the limits of detection were 0.5, 0.5, and 0.2 mg/L, respectively. The average recoveries were 98.9% - 103.7% with the relative standard deviations of 1.09% - 1.69%. The correlation coefficients for the linear equations were 0.999 6 - 0.999 9. These results demonstrate that the proposed method is simple, sensitive and reliable for the determination of the residual monomers in five polymer water treatment chemicals.