A strategy to reduce the byproduct glucose by simultaneously producing levan and single cell oil using an engineered Yarrowia lipolytica strain displaying levansucrase on the surface.

Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

Transcriptome and Metabolome Integration Reveals the Impact of Fungal Elicitors on Triterpene Accumulation in Sanghuangporus sanghuang.

Sanghuangporus sanghuang is a large wood-decaying mushroom highly valued in traditional Chinese medicine due to its medicinal properties, including hypoglycemic, antioxidant, antitumor, and antibacterial properties effects. Its key bioactive compounds include flavonoids and triterpenoids. Specific fungal genes can be selectively induced by fungal elicitors. To investigate the effect of fungal polysaccharides derived from Perenniporia tenuis mycelia on the metabolites of S. sanghuang, we conducted metabolic and transcriptional profiling with and without elicitor treatment (ET and WET, respectively). Correlation analysis showed significant differences in triterpenoid biosynthesis between the ET and WET groups. In addition, the structural genes associated with triterpenoids and their metabolites in both groups were verified using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Through metabolite screening, three triterpenoids were identified: betulinol, betulinic acid, and 2-hydroxyoleanolic acid. Excitation treatment increased the level of betulinic acid by 2.62-fold and 2-hydroxyoleanolic acid by 114.67-fold compared to WET. The qRT-PCR results of the four genes expressed in secondary metabolic pathways, defense gene activation, and signal transduction showed significant variation between the ET and WET groups. Overall, our study suggests that the fungal elicitor induced the aggregation of pentacyclic triterpenoid secondary metabolites in S. sanghuang.

A laser-induced graphene electrochemical immunosensor for label-free CEA monitoring in serum.

The cost-effective construction of self-designed conductive graphene patterns is crucial to the fabrication of graphene-based electrochemical devices. Here, a label-free carcinoembryonic antigen (CEA) electrochemical immunosensor is developed based on the surface engineering of a laser-induced graphene (LIG)/Au electrode. The LIG electrode was produced with a smart and inexpensive 450 nm semiconductor laser through three electrode patterns under ambient conditions. Then the LIG/Au electrode was organized by conformal anchoring of Au nanoparticles (NPs) on the LIG work area using chloroauric acid as the precursor. Good electrochemical activity with improved conductivity of the LIG/Au electrode was obtained under optimized conditions of laser intensity, carving depth, and chlorogenic acid dosage, to name a few. The LIG/Au electrode was carbonylated based on Au-S∼COOH using 11-mercaptoundecanoic acid (MUA). The antibody was covalently bound on the work area to form a label-free immunosensor. The constructed immunosensor shows high sensitivity with a good response in the range of low concentrations from 0.01 to 100 ng mL-1, low detection limit (5.0 pg mL-1), high selectivity compared with some possible interference, and can be applied in a bovine serum solution without the need of sample labeling and pretreatment. Moreover, the immunosensor is mechanically flexible with minimal change in signal output after bending at different angles. It shows an easy and green electrode preparation method that combines 3D porous structures of graphene, uniform immobilization of Au NPs, binder-free, easy covalent binding of an antibody, and good mechanical properties. Hence, the present method has great potential for applications involving electrochemical biosensors.

Circular RNA expression profiles alter significantly after intracerebral hemorrhage in rats.

Circular RNAs (circRNAs) are a class of covalently closed non-coding RNAs, and aberrant alteration of their expression patterns is studied in numerous diseases. This study aimed to investigate whether intracerebral hemorrhage (ICH) affected circRNA expression profiles in the rat brain. Adult male Sprague-Dawley rats were subjected to intrastriatal injection of autologous artery blood to establish the ICH model. The cerebral cortex around hematoma was collected to perform circRNA microarray at 6 h, 12 h and 24 h. Quantitative reverse transcription-PCR (qRT-PCR) was used to validate the results. Bioinformatic methods were applied to predict ceRNA network and perform enrichment analyses for parent genes at three time points and target mRNAs. 111, 1145, 1751 up-regulated and 47, 732, 1329 down-regulated circRNAs were detected in the cerebral cortex of rats at 6 h, 12 h and 24 h after ICH compared with sham group. Most were from exonic regions. 93 were up-regulated and 20 were down-regulated at all three time points. Microarray results of 3 circRNAs were confirmed via qRT-PCR. GO and KEGG analyses for parent genes showed transition from protein complex assembly, cell-cell adhesion and cAMP signaling pathway at 6 h to intracellular signal transduction, protein phosphorylation and glutamatergic synapse at 12 h and 24 h. A circRNA-miRNA-mRNA network was successfully predicted. Enrichment analyses of targeted mRNAs indicated transcriptional regulations and pathways including Rap1, Ras, MAPK, PI3K-Akt, TNF and Wnt signaling and pathways in cancer. This was the first study to demonstrate that ICH significantly altered the expression of circRNAs with promising targets for therapeutic intervention.

Simultaneous production of single cell oil and fumaric acid by a newly isolated yeast Aureobasidium pullulans var. aubasidani DH177.

Microbial oils can be used for biodiesel production and fumaric acid (FA) is widely used in the food and chemical industries. In this study, the production of lipids and FA by Aureobasidium pullulans var. aubasidani DH177 was investigated. A high initial carbon/nitrogen ratio in the medium promoted the accumulation of lipids and FA. When the medium contained 12.0% glucose and 0.2% NH4NO3, the yeast strain DH177 accumulated 64.7% (w/w) oil in its cells, 22.4 g/l cell biomass and 32.3 g/l FA in a 5-L batch fermentation. The maximum yields of oil and FA were 0.12 g/g and 0.27 g/g of consumed sugar, respectively. The compositions of the produced fatty acids were C14:0 (0.6%), C16:0 (24.9%), C16:1 (4.4%), C18:0 (2.1%), C18:1 (57.6%), and C18:2 (10.2%). Biodiesel obtained from the extracted oil burned well. This study provides the pioneering utilization of the yeast strain DH177 for the integrated production of oil and FA.

Single Cell Oil Production from Hydrolysates of Inulin by a Newly Isolated Yeast Papiliotrema laurentii AM113 for Biodiesel Making.

Microbial oils are among the most attractive alternative feedstocks for biodiesel production. In this study, a newly isolated yeast strain, AM113 of Papiliotrema laurentii, was identified as a potential lipid producer, which could accumulate a large amount of intracellular lipids from hydrolysates of inulin. P. laurentii AM113 was able to produce 54.6% (w/w) of intracellular oil in its cells and 18.2 g/l of dry cell mass in a fed-batch fermentation. The yields of lipid and biomass were 0.14 and 0.25 g per gram of consumed sugar, respectively. The lipid productivity was 0.092 g of oil per hour. Compositions of the fatty acids produced were C14:0 (0.9%), C16:0 (10.8%), C16:1 (9.7%), C18:0 (6.5%), C18:1 (60.3%), and C18:2 (11.8%). Biodiesel obtained from the extracted lipids could be burnt well. This study not only provides a promising candidate for single cell oil production, but will also probably facilitate more efficient biodiesel production.

Comparative transcriptome analysis reveals multiple functions for Mhy1p in lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica.

Yarrowia lipolytica is considered as a promising microbial cell factory for bio-oil production due to its ability to accumulate a large amount of lipid. However, the regulation of lipid metabolism in this oleaginous yeast is elusive. In this study, the MHY1 gene was disrupted, and 43.1% (w/w) intracellular oil based on cell dry weight was obtained from the disruptant M-MHY1, while only 30.2% (w/w) lipid based on cell dry weight was obtained from the reference strain. RNA-seq was then performed to analyze transcriptional changes during lipid biosynthesis after MHY1 gene inactivation. The expression of 1597 genes, accounting for 24.7% of annotated Y. lipolytica genes, changed significantly in the disruptant M-MHY1 during lipid biosynthesis. Differential gene expression analysis indicated that Mhy1p performs multiple functions and participates in a wide variety of biological processes, including lipid, amino acid and nitrogen metabolism. Notably, data analysis revealed increased carbon flux through lipid biosynthesis following MHY1 gene inactivation, accompanied by decreased carbon flux through amino acid biosynthesis. Moreover, Mhy1p regulates the cell cycle, and the cell cycle rate was enhanced in the disruptant M-MHY1. These results suggest that Mhy1p plays critical regulatory roles in diverse aspects of various biological processes, especially in lipid biosynthesis, amino acid and nitrogen metabolism and cell cycle. Our dataset appears to elucidate the crucial role of Mhy1p in lipid biosynthesis and serves as a resource for exploring physiological dimorphic growth in Y. lipolytica.

Geographic distribution of cadmium and its interaction with the microbial community in the Longjiang River: risk evaluation after a shocking pollution accident.

A shocking Longjiang River cadmium pollution accident occurred in 2012, the effects of which on microbial communities remain unclear. Alkaline precipitation technology was applied for remediation, but concerns rose about the stability of this technology. To understand the geographic distribution of cadmium and its correlation with microbes, in this study, 39 water samples and 39 sludge samples from this river and 2 soil samples from the nearby farmland were collected for chemical and microbial analyses. The Cd concentrations of all water samples were lower than 0.005 mg/L and reached the quality standards for Chinese surface water. A ranking of sludge samples based on Cd contents showed sewage outfall > dosing sites > farmland, all of which were higher than the quality standard for soil. Alkaline precipitation technology was effective for Cd precipitation. Cd was unstable; it was constantly dissolving and being released from the sludge. The Cd content of each phase was mainly influenced by the total Cd content. Over 40,000 effective sequences were detected in each sample, and a total of 59,833 OTUs and 1,273 genera were found using Illumina MiSeq sequencing. Two phyla and 39 genera were notably positively correlated with the Cd distribution, while the cases of 10 phyla and 6 genera were the opposite.

High-Level Production of Exopolysaccharides by a Cosmic Radiation-Induced Mutant M270 of the Maitake Medicinal Mushroom, Grifola frondosa (Agaricomycetes).

A new Grifola frondosa mutant, M270, was successfully isolated for high production of exopolysaccharides (EPSs) using cosmic radiation-induced mutagenesis. We found that the mutant M270 had a clearer and thicker EPS layer (~10 µm) adhering to mycelia than those of its parent strain 265 after Congo red staining. In the 20-L batch fermentation for M270, 10.3 g/L of EPS and 17.9 g/L of dry mycelia biomass were obtained after 204 hours of fermentation. Furthermore, a main water-soluble fraction (EP1) in the EPS was purified from M270 and then confirmed to be heteroglycan-protein complex with 91% (w/w) total carbohydrates and 9% (w/w) total proteins. Four kinds of monosaccharide-D-mannose, D-glucosamine, D-glucose, and D-xylose-were detected in EP1 with a molar ratio of 17.6:1.8:100:2.5. The molecular mass of the main component in EP1 was 8.9 kDa. The EPS from M270 significantly inhibited the growth of sarcoma 180 solid tumors in mice. This G. frondosa M270 mutant could serve as a better candidate strain for polysaccharide production.

[The protective effects of insulin on cardiocyte apoptosis challenged by burn serum].

OBJECTIVE: To investigate the protective effects of insulin on burn serum-challenged cardiocyte apoptosis and its mechanism. METHODS: Burn-serum challenged cardiocytes were pretreated with insulin and inhibitors to pathway SB203580 and LY294002. The expression of cardiac myofilament proteins cleaved-caspase-3, Bax and phosphorylation nuclear factor-ΚB inhibitive factor α (p-IΚBα) were examined by Western blotting. The mRNA expression of tumor necrosis factor-α (TNF-α) was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR). Apoptosis of cardiocyte was observed after Hoechst 33258 staining. Further blocking experiments were used to investigate the cytoprotective pathway of insulin. RESULTS: Insulin could significantly decrease the expression of cleaved-caspase-3 (2.22 ± 0.30 vs. 4.84 ± 0.74, P < 0.01), Bax (1.33 ± 0.35 vs. 3.74 ± 0.65, P < 0.01), p-IΚBα (1.43 ± 0.62 vs. 3.62 ± 0.74, P < 0.01), TNF-α (0.72 ± 0.27 vs. 2.02 ± 0.63, P < 0.01) and the cardiocyte apoptosis rate [(9.4 ± 3.4)% vs. (19.1 ± 5.6)%, P < 0.01] in cardiocytes challenged by burn serum. Further blocking experiments showed that LY294002, phosphatidylinositol-3-kinase (PI3K)/Akt activation inhibitor, could mitigate the protective effects of insulin. Meanwhile, SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38MAPK) pathway, was able to inhibit cardiocyte injury challenged by burn serum, and it was as effective as insulin. CONCLUSION: For cardiocytes challenged by burn serum, insulin may decrease inflammatory cytokine expression and apoptosis via regulating PI3K/Akt and p38MAPK pathway.