Screening of astaxanthin-overproducing Xanthophyllomyces dendrorhous strains via iterative ARTP mutagenesis and cell sorting by flow cytometry.

AIMS: The astaxanthin-producing yeast Xanthophyllomyces dendrorhous is widely used in aquaculture. Due to the production of carotenoid, this yeast shows visible color; however, high-throughput approaches for identification of astaxanthin-overproducing strains remain rare. METHODS AND RESULTS: This study verified an effective approach to identify astaxanthin-overproducing mutants of X. dendrorhous by flow cytometry (FCM) and cell sorting. First, the mutant libraries were generated by atmospheric and room-temperature plasma (ARTP) mutagenesis. Second, a highly direct correlation between the concentrations of intracellular astaxanthin and the levels of emitting fluorescence was constructed by testing a variety of astaxanthin-contained populations via FCM and cell sorting. Third, iterative cell sorting efficiently improves the identification of astaxanthin-overproducing strains. Finally, two mutants producing 4.96 mg astaxanthin g-1 DCW (dry cell weight) and 5.30 mg astaxanthin g-1 DCW were obtained, which were 25.3% and 33.8% higher than that of the original strain, respectively. CONCLUSIONS: This study demonstrated that iterative ARTP mutagenesis along with cell sorting by FCM is effective for identifying astaxanthin-overproduction strains.

A redescription of the rare eucyclopine copepod Eucyclops productus Kiefer, 1939 (Multicrustacea: Copepoda: Cyclopoida: Cyclopidae) and a ke to Eucyclops subgenera and species of China and adjacent areas.

The rare freshwater copepod Eucyclops (Speratocyclops) productus Kiefer, 1939 from Lake Mandongco, Tibet Autonomous Region, China, is redescribed. This species was originally found and described from a pond south of Chushul village, and many of the details did not meet modern standards for describing the species. In this paper, the morphological characteristics of this species are described in detail and compared with other members of the subgenus Speratocyclops species recorded in China. The key features of the species are: 1) long caudal rami, 2) the coxal seta of the fourth pair of legs armed with long hairs in the proximal part and short denticles in the distal part, 3) the inner spine of the fifth pair of legs long and strong. An identification key to 22 species of Eucyclops known from China and adjacent areas is also provided.

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one-hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two-hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild-type strain did not largely modify the carotenoid profile, indicating translational and/or post-translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild-type strain and the XdWC2-disrupted strain apparently improved the production of monocyclic carotenoid 3-hydroxy-3', 4'-didehydro-ß, ψ-carotene-4-one (HDCO) rather than ß-carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over-producing strains.

Comprehensive Analysis of CRISPR-Cas9 Editing Outcomes in Yeast Xanthophyllomyces dendrorhous.

DNA repair after Cas9 cutting can result in deletions/insertions, genomic rearrangements, and rare nucleotide substitutions. However, most work has only focused on deletions/insertions resulting from repair after CRISPR-Cas9 action. Here, we comprehensively analyzed the editing outcomes induced by CRISPR-Cas9 treatment in yeast Xanthophyllomyces dendrorhous by Sanger and Illumina sequencing and identified diverse DNA repair patterns, including DNA deletions, interchromosomal translocations, and on-target nucleotide substitutions (point mutations). Some deletions were observed repeatedly, and others, especially large deletions, varied in size. Genome sequencing and structural variation analysis showed that the interchromosomal translocations happened between Cas9 target sites and the endogenous ADH4 promoter. In contrast to previous studies, analysis revealed that the on-target point mutations were not random. Importantly, these point mutations showed strong sequence dependence that is not consistent with previous work in Hela cells, where CRISPR-mediated substitutions were found to lack sequence dependence and conversion preferences. Finally, we found that the non-homologous end joining components Ku70, Ku80, Mre11, or RAD50, and the overlapping roles of non-essential DNA polymerases were necessary for the production of both point mutations and deletions. This work expands our knowledge of CRISPR-Cas9 mediated DNA repair.

Metabolome and transcriptome profiling provide insights into green apple peel reveals light- and UV-B-responsive pathway in anthocyanins accumulation.

BACKGROUND: In nature, green apple are associated with the accumulation of chlorophyll, while red apple varieties are associated with anthocyanins accumulation. Notably, in this study, the green skin color apple variety 'white winter pearmain' treated with ultraviolet-B (UV-B) exhibited red skins and marked anthocyanin accumulation, while visible light could not. But there are few reports on the biosynthesis difference of anthocyanins in green apple by visible light and UV-B-treatment. Here, we explored the difference of metabolites and genes expression level in green apple by transcriptomic and metabolic. RESULTS: The metabolic analysis revealed that there were 152 and 178 significantly changed metabolites in the visible light and UV-B-treated green apple, respectively, compared to the control, and flavone, flavonol, and anthocyanin were the most significantly increased; and transcriptomic analysis showed that 37,110 and 37,709 differentially expressed genes, including 382 and 475 transcription factors (TFs) were detected in light and UV-B-treatment fruit, respectively. Quantitative reverse transcription PCR (qRT-PCR) results confirmed changes in the expression levels of genes encoding metabolites involved in the flavonoid synthesis pathways. The flavonoid metabolic flux in the UV-B treatment increased the accumulation of cyanidin 3-glucoside and cyanidin 3, 5-diglucoside compared to under the light-treatment. Furthermore, we performed qRT-PCR analysis of anthocyanin biosynthesis genes and predicted the gene of MD00G1134400 (a UDP glucose-flavonoid 3-0-glucosyltransferase) may be a candidate gene for anthocyanins accumulation and highly expressed in UV-B-treatment fruit. Expression profiles of several transcription factors of the families MYB, bHLH, NAC were highly correlated with the content of the anthocyanin. CONCLUSIONS: The composition and contents of anthocyanins in green apple in UV-B-treatment very greatly. A series of metabolites and candidate genes were revealed through combined analysis of metabolome and transcriptome. These results provide an important data for dissecting candidate genes and molecular basis governing green apple color formation in response to visible light and UV-B light.

The severe toxicity of CuO nanoparticles to the photosynthesis of the prokaryotic algae Arthrospira sp.

This research first verified that prokaryotic algae are more sensitive to toxicity of CuO nanoparticles (CuO NPs) than eukaryotic algae and that CuO NPs damaged photosynthesis of prokaryotic algae (Arthrospira sp.) but had no effect on respiration. The Cu2+ released by CuO NPs caused a bending deformation of the thylakoid, which was an important cause of the decline in photosynthetic capacity. In addition, the D1 protein was the most susceptible site to CuO NPs. The degradation of D1 protein reduced photosynthetic electron transport, which enhanced the excess excitation energy to cause the accumulation of reactive oxygen species (ROS) to further result in oxidative stress on algae. Dissolved organic matter (DOM) increased the toxicity of CuO NPs to photosynthesis of Arthrospira sp. The damage of photosynthesis caused by CuO NPs is an important reason why CuO NPs have a serious toxicity to algae.

Cadmium-induced genome-wide DNA methylation changes in growth and oxidative metabolism in Drosophila melanogaster.

BACKGROUND: Cadmium (Cd)-containing chemicals can cause serious damage to biological systems. In animals and plants, Cd exposure can lead to metabolic disorders or death. However, for the most part the effects of Cd on specific biological processes are not known. DNA methylation is an important mechanism for the regulation of gene expression. In this study we examined the effects of Cd exposure on global DNA methylation in a living organism by whole-genome bisulfite sequencing (WGBS) using Drosophila melanogaster as model. RESULTS: A total of 71 differentially methylated regions and 63 differentially methylated genes (DMGs) were identified by WGBS. A total of 39 genes were demethylated in the Cd treatment group but not in the control group, whereas 24 showed increased methylation in the former relative to the latter. In most cases, demethylation activated gene expression: genes such as Cdc42 and Mekk1 were upregulated as a result of demethylation. There were 37 DMGs that overlapped with differentially expressed genes from the digital expression library including baz, Act5C, and ss, which are associated with development, reproduction, and energy metabolism. CONCLUSIONS: DNA methylation actively regulates the physiological response to heavy metal stress in Drosophila in part via activation of apoptosis.

Mechanism of long-term toxicity of CuO NPs to microalgae.

Little is known regarding the detailed mechanism of CuO NPs' toxicity to microalgal primary metabolism pathway. Photosynthesis and respiration are the most important primary metabolism and the main sources of production of reactive oxygen species (ROS), but the effect of CuO NPs on both of them has not been systematically studied to date. Our research demonstrated that long-term treatment with CuO NPs significantly inhibited activities of photosynthesis and respiration in microalgae, and the photosynthesis was more sensitive to the toxicity of CuO NPs than respiration. CuO NPs could be absorbed by microalgae and be converted into Cu2O NPs concentrated in chloroplast. The internalized Cu, regardless of whether the exposure was Cu2+ or CuO NPs had the same capacity to damage chloroplast structure. The result also shows that the oxygen-evolving complex (OEC) in the photosynthetic electron transport chain was the most sensitive site to CuO NPs and Cu2+-treated microalgae had the same damage site as that of CuO NPs, which may be related to the Mn cluster that is dissociated by Cu ions released from CuO NPs. The damage of OEC inhibited photosynthetic electron transport to increase excess excited energy, which caused the accumulation of ROS in chloroplast. The accumulation of ROS damaged the structure of cell membrane and aggravated the PSII photoinhibition, further decreasing the efficiency of light energy utilization. In conclusion, the Cu ionic toxicity of photosynthetic apparatus by CuO NPs resulted in the carbon starvation and the accumulation of ROS to inhibit the growth of microalgae.

Dangguishaoyao-San attenuates LPS-induced neuroinflammation via the TLRs/NF-κB signaling pathway.

INTRODUCTION: Dangguishaoyao-San (DSS) is composed of six traditional Chinese medicines, including Angelica sinensis, Paeoniae radix, Rhizoma Ligusticum, Poria cocos, Rhizoma Atractylodis Macrocephalae, and Rhizoma Alismatis. DSS has been reported to be effective in alleviating the symptoms of Alzheimer's disease (AD). The aim of this study was to investigate the mechanism of action of DSS in vitro using lipopolysaccharide (LPS)-stimulated BV-2 microglia cells. MATERIALS AND METHODS: BV-2 cells were pretreated with 0.58-1.16 mg/mL of DSS for 2 h and then treated with 1 µg/mL LPS for 24 h. Cell viability was determined by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels were measured by Western blots. Inflammatory factors were detected by enzyme-linked immunosorbent assays (ELISAs). The mRNA levels of inflammatory factors were analyzed by quantitative real-time PCR (qRT-PCR). RESULTS: DSS treatment at concentrations of 0.58-1.16 mg/mL resulted in no significant cytotoxicity. DSS attenuated the release of pro-inflammatory factors, such as interleukin-1ß (IL-1ß), iNOS and tumor necrosis factor-α (TNF-α) in LPS-induced BV-2 cells. DSS attenuated the mRNA expression of pro-inflammatory cytokines, TLR2, and TLR4 and decreased TLR4 and TLR protein levels as well as the phosphorylation of IκB in LPS-induced BV-2 cells. DSS also down-regulated the nuclear translocation of p65. CONCLUSION: This study demonstrated that DSS has a protective effect on neuroinflammation in LPS-induced BV-2 microglia cells through the TLRs/NF-κB signaling pathway.