Saccharomyces cerevisiae cellular engineering for the production of FAME biodiesel.

The unsustainable and widespread utilization of fossil fuels continues to drive the rapid depletion of global supplies. Biodiesel has emerged as one of the most promising alternatives to conventional diesel, leading to growing research interest in its production. Microbes can facilitate the de novo synthesis of a type of biodiesel in the form of fatty acid methyl esters (FAMEs). In this study, Saccharomyces cerevisiae metabolic activity was engineered to facilitate enhanced FAME production. Initially, free fatty acid concentrations were increased by deleting two acetyl-CoA synthetase genes (FAA1, FAA4) and an acyl-CoA oxidase gene (POX1). Intracellular S-adenosylmethionine (SAM) levels were then enhanced via the deletion of an adenosine kinase gene (ADO1) and the overexpression of a SAM synthetase gene (SAM2). Lastly, the S. cerevisiae strain overproducing free fatty acids and SAM were manipulated to express a plasmid encoding the Drosophila melanogaster Juvenile Hormone Acid O-Methyltransferase (DmJHAMT). Using this combination of engineering approaches, a FAME concentration of 5.79 ± 0.56 mg/L was achieved using these cells in the context of shaking flask fermentation. To the best of our knowledge, this is the first detailed study of FAME production in S. cerevisiae. These results will provide a valuable basis for future efforts to engineer S. cerevisiae strains for highly efficient production of biodiesel.

CDs-g-C3N4-oleaginous yeast hybrid system: Microbial lipid synthesis and fermentation residual reutilization.

The utilization of solar energy and fast-growing heterotrophic microbes for biofuel production has been recognized as a promising approach to achieve carbon neutrality and address energy crisis. In this work, we synthesized different kinds of photocatalysts based on graphitic carbon nitride (g-C3N4). We found that carbon dots modified-graphitic carbon nitride (CDs-g-C3N4) showed the highest photocatalytic activity. Subsequently, we developed a photocatalyst-microbe hybrid (PMH) system by combining CDs-g-C3N4 with an oleaginous yeast strain, Cutaneotrichosporon dermatis ZZ-46. Under visible light irradiation, the lipid yield of this PMH system reached 1.70 g/L at 120 h, representing a 36 % increase compared to the control. The photocatalytic reaction-induced ROS and the reductive photogenerated electrons facilitated ZZ-46 cells to synthesize more lipids. Furthermore, the fermentation residual of this PMH system was reutilized to prepare biochar via pyrolysis. The biochar generated at 550 °C (BC-550) demonstrated exceptional adsorption capabilities, particularly with a 57 % adsorption rate for methylene blue (MB), and maintained its perfect adsorption efficacy even after five regeneration cycles. These results offer promising avenues for addressing energy shortages and environmental contamination.

A Report on the Use and Safety of Nonsteroidal Anti-inflammatory Drugs among 22,553 Children from Sample Hospitals in Shanghai.

Context: The safety of medication for pediatric patients has always been a concern, and non steroidal anti-inflammatory drugs (NSAIDs) are one of the essential and commonly used drugs in children. Therefore, it is necessary to conduct a study on the efficacy and safety of NSAIDs in pediatric patients. Objective: To study the use and safety of nonsteroidal anti-inflammatory drugs (NSAIDs) among 22 553 pediatric patients from 14 hospitals in Shanghai. Methods: We collected the clinical data of 22 553 pediatric patients who received NSAIDs during their stay in 14 hospitals in Shanghai from January 2005 to May 2011, which were then retrospectively analyzed. The use of nimesulide, paracetamol, and ibuprofen was observed among these children. The age and gender distribution, discharge status, length of hospital stay, and types of diseases treated with NSAIDs were analyzed. The relationship between death and length of hospital stay was assessed. The safety of NSAIDs in these children was discussed. Results: The response rate of nimesulide and ibuprofen was 71.23% and 73.12%, respectively. There was no significant difference in response rate between the two drugs (P > .05). The response rate of paracetamol was the lowest among the three drugs (59.67%, P < .05). The average length of hospital stay was significantly longer in children receiving paracetamol than in those receiving nimesulide. The average length of hospital stay was significantly longer in children receiving nimesulide than in those receiving ibuprofen (P < .05). The diseases treated with nimesulide were less diverse than those treated with ibuprofen and paracetamol. To be specific, bronchopneumonia was predominant among all the diseases treated with nimesulide. Although bronchopneumonia was also the most common among all the diseases treated with ibuprofen and paracetamol, the diseases treated with these two drugs were more diverse. The incidence of abnormal liver function among children receiving nimesulide was significantly lower than in those receiving ibuprofen and paracetamol (P < .05). There was no significant difference in the incidence of abnormal liver function caused by paracetamol and ibuprofen (P > .05). Conclusion: Nimesulide and ibuprofen achieved a generally higher response rate than paracetamol among the surveyed children from Shanghai. Although bronchopneumonia was the most common diagnosis among all children treated with NSAIDs, the diagnoses were less diverse in those treated with nimesulide. The length of hospital stay was the shortest among children receiving ibuprofen, while the response rate of paracetamol was the lowest. The incidence of abnormal liver function was the lowest in children receiving nimesulide. All of the three NSAIDs might induce liver function impairment, but the risk was not significantly different between them. This study also has some limitations, such as limited drug types and regional limitations. In summary, Nimesulide is a highly effective and safe non steroidal anti-inflammatory drug that can meet the clinical medication needs of pediatric patients. Future research is contemplating the clinical benefits of Nimesulide in treating more diagnostic types besides pediatric bronchopneumonia, in order to investigate its greater medicinal value.

Microbial lipid synthesis based on visible light-driven oxygen doped-graphitic carbon nitride /oleaginous yeast hybrid system.

The use of solar energy and heterotrophic microbes to synthesize microbial lipids is a promising strategy to solve energy crisis and reduce CO2 emissions. In this study, a photocatalyst, oxygen-doped graphitic carbon nitride (O-g-C3N4), was synthesized and combined with an oleaginous yeast strain, Cutaneotrichosporon dermatis ZZ-46, to construct a photocatalyst-microbe hybrid (PMH) system. Under illumination, the lipid yield of the PMH system reached 1.61 g/L after 96 h (87 % higher than that of control). NADPH/NADP+ ratio of ZZ-46 cells in the PMH system increased. Metabolomics results revealed that glutathione generation was increased, and the fatty acid decomposition pathway in ZZ-46 cells was inhibited in the PMH system. This study provides a new approach for the synthesis of microbial lipids based on solar energy and heterotrophic microbes.

Isolation and stereospecific synthesis of indole alkaloids with lipid-lowering effects from the marine-derived fungus Colletotrichum gloeosporioides BB4.

Seven undescribed compounds, colletotrichindoles A-E, colletotrichaniline A, and colletotrichdiol A, as well as three known compounds, (-)-isoalternatine A, (+)-alternatine A and 3-hydroxybutan-2-yl 2-phenylacetate were isolated from the marine-derived fungus Colletotrichu gloeosporioides BB4. The racemic mixtures colletotrichindole A，colletotrichindole C, and colletotrichdiol A were further separated by chiral chromatography to give three pairs of enantiomers (10S,11R,13S)/(10R,11S,13R)-colletotrichindole A, (10R,11R,13S)/(10S,11S,13R)-colletotrichindole C, and (9S,10S)/(9R,10R)-colletotrichdiol A, respectively. The chemical structures of seven undescribed compounds and the known compounds, (-)-isoalternatine A, and (+)-alternatine A were determined using a combination of NMR, MS, X-ray diffraction, ECD calculations, and/or chemical synthesis. All possible enantiomers of colletotrichindoles A-E were synthesized and used to determine the absolute configurations of the natural products by comparing their spectroscopic data and HPLC retention times on a chiral column. In addition, the X-ray crystal structures of the known compounds (-)-isoalternatine A and (+)-alternatine A were also obtained to confirm their absolute configurations. (10S,11R,13S)-Colletotrichindole A, colletotrichindole B, and (+)-alternatine A significantly reduced triglyceride levels in 3T3-L1 cells with EC50 values of 5.8, 9.0, and 1.3 µM, respectively.

The gut microbiota-brain axis: Role of the gut microbial metabolites of dietary food in obesity.

Obesity, a social epidemic disease, threatens human health and affects economic stability worldwide. With the emergence of knowledge implicating the human gut microbiome, a complex relationship between the enteric nervous system, the gut microbiota and the central nervous system has become the focus of research attention. Which allows for the microbiota to influence the metabolism of an organism significantly. To date, data from animal and human studies provide a theoretical basis, that is food-derived natural compounds can work on anti-obesity via the microbiota-gut-brain axis (MGBA). Thus, we systematically summarize the role of the major metabolites in dietary metabolism and the potential for combating obesity and metabolism disorder. Simultaneously, the review examines the sufficient evidence surrounding the MGBA as a regulator of obesity. It will provide a new clue for developing the potential of gut-microbiota-targeted strategies and lay a good foundation for its operation for food intervention in anti-obesity.

Draft Genome Assembly and Annotation for Cutaneotrichosporon dermatis NICC30027, an Oleaginous Yeast Capable of Simultaneous Glucose and Xylose Assimilation.

The identification of oleaginous yeast species capable of simultaneously utilizing xylose and glucose as substrates to generate value-added biological products is an area of key economic interest. We have previously demonstrated that the Cutaneotrichosporon dermatis NICC30027 yeast strain is capable of simultaneously assimilating both xylose and glucose, resulting in considerable lipid accumulation. However, as no high-quality genome sequencing data or associated annotations for this strain are available at present, it remains challenging to study the metabolic mechanisms underlying this phenotype. Herein, we report a 39,305,439 bp draft genome assembly for C. dermatis NICC30027 comprised of 37 scaffolds, with 60.15% GC content. Within this genome, we identified 524 tRNAs, 142 sRNAs, 53 miRNAs, 28 snRNAs, and eight rRNA clusters. Moreover, repeat sequences totaling 1,032,129 bp in length were identified (2.63% of the genome), as were 14,238 unigenes that were 1,789.35 bp in length on average (64.82% of the genome). The NCBI non-redundant protein sequences (NR) database was employed to successfully annotate 11,795 of these unigenes, while 3,621 and 11,902 were annotated with the Swiss-Prot and TrEMBL databases, respectively. Unigenes were additionally subjected to pathway enrichment analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups of proteins (COG), Clusters of orthologous groups for eukaryotic complete genomes (KOG), and Non-supervised Orthologous Groups (eggNOG) databases. Together, these results provide a foundation for future studies aimed at clarifying the mechanistic basis for the ability of C. dermatis NICC30027 to simultaneously utilize glucose and xylose to synthesize lipids.

An important link between the gut microbiota and the circadian rhythm: imply for treatments of circadian rhythm sleep disorder.

Currently, gut microbiota living in the gastrointestinal tract, plays an important role in regulating host's sleep and circadian rhythms. As a tool, gut microbiota has great potential for treating circadian disturbance and circadian insomnia. However, the relationship between gut microbiota and circadian rhythms is still unclear, and the mechanism of action has still been the focus of microbiome research. Therefore, this article summarizes the current evidences associating gut microbiota with factors that impact host circadian rhythms neurology sleep disorder. Moreover, we discuss the changes to these systems in sleep disorder and the potential mechanism of intestinal microbiota in regulating circadian rhythms neurology sleep disorder via microbial metabolites. Meanwhile, based on the role of intestinal flora, it is provided a novel insight into circadian related insomnia and will be benefit the dietary treatment of circadian disturbance and the circadian related insomnia.

Halophilic archaea and their extracellular polymeric compounds in the treatment of high salt wastewater containing phenol.

Phenol is one of the major organic pollutants in high salt industrial wastewaters. The biological treatment of such waste using microorganisms is considered to be a cost-effective and eco-friendly method. However, in this process, salt tolerance of microorganisms is one of the main limiting factors. Halophilic microorganisms, especially halophilic archaea are thought to be appropriate for such treatment. To develop a novel effective biological method for high salt phenol wastewater treatment, the influence of phenol in high salt phenol wastewater on halophilic archaea and their extracellular polymeric substances (EPS) should be investigated. In the present study, using phenol enrichment method, 75 halophilic archaeal strains were isolated from Wuyongbulake salt lake sediment sample. The majority of the identified strains were phenol-tolerant. Six strains with high phenol tolerance were chosen, and the phenol scavenging effect was observed in the microbial suspension, supernatant, and EPS. It was noticed that the phenol degradation rate of suspensions of both strains 869-1, and 121-1 in salt water exhibited the highest rates of 83.7%, while the supernatant of strain 869-1 reached the highest rate of 78.2%. When combined with the comprehensive analysis of the artificial wastewater simulation experiment, it was discovered that in the artificial wastewater containing phenol, the phenol degradation rate of suspension of strain A387 exhibited the highest rates of 55.74% both, and supernatant of strain 630-3 reached the highest rate of 62.3%. The EPS produced by strains A00135, 558-1, 869-1, 121-1 and A387 removed 100% phenol within 96 h, and the phenol removal efficiency of EPS produced by 869-1 reached 56.1% under an artificial wastewater simulation experiment with high salt (15%NaCl) condition. The present study suggests that halophilic archaea and their EPS play an important role in phenol degradation. This approach could be potentially used for industrial high-salt wastewater treatment.

Haloterrigena gelatinilytica sp. nov., a new extremely halophilic archaeon isolated from salt-lake.

Two extremely halophilic strains, designated SYSU A558-1T and SYSU A121-1, were isolated from a saline sediment sample collected from Aiding salt-lake, China. Cells of strains SYSU A558-1T and SYSU A121-1 were Gram-stain-negative, coccoid, and non-motile. The strains were aerobic and grew at NaCl concentration of 10-30% (optimum, 20-22%), at 20-55 °C (optimum, 37-42 °C) and at pH 6.5-8.5 (optimum, 7.0-8.0). Cells lysed in distilled water. The polar lipids were phosphatidyl choline, phosphatidylglycerol phosphate methyl ester, disulfated diglycosyl diether-1 and unidentified glycolipid. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the two strains SYSU A558-1T and SYSU A121-1 were closely related to the membranes of the genus Haloterrigena. Phylogenetic and phylogenomic trees of strains SYSU A558-1T and SYSU A121-1 demonstrated a robust clade with Haloterrigena turkmenica, Haloterrigena salifodinae and Haloterrigena salina. The genomic DNA G + C content of strains SYSU A558-1T and SYSU A121-1 were 65.8 and 65.0%, respectively. Phenotypic, phylogenetic, chemotaxonomic and genome analysis suggested that the two strains SYSU A558-1T and SYSU A121-1 represent a novel species of the genus Haloterrigena, for which the name Haloterrigena gelatinilytica sp. nov. is proposed. The type strain is SYSU A558-1T (= KCTC 4259T = CGMCC 1.15953T).

Effect of Dihydropyridine Enrichment in the Microstructure of the Palisade Layer on the Stability of Fat Nano-emulsions.

Relationship between the stability of fat nano-emulsions and the incorporated drug at the molecular level are rarely known. Herein, fat nano-emulsions containing dihydropyridine drugs were prepared and the microstructure of their palisade layers were investigated.The prepared 1.0 mg/mL nimodipine nano-emulsion was found to contain 65.50% drug in the palisade layer. The increasing drug concentration led to a decrease-increase-decrease trend in centrifugal stability constant, particle size and proton nuclear magnetic resonance (1H NMR) signal intensity of the lecithin trimethyl ammonium group in the nimodipine and felodipine nano-emulsions. The 1H NMR spectra of test solutions including nano-emulsions suggest that increasing drugs penetrated into the palisade layer, resulting in the lecithin arrangement from loose to tight, and then from monolayer to bilayer. Nimodipine and felodipine nano-emulsions showed two valley values at concentrations of 0.15 and 0.75 mg/mL, and 0.30 and 0.90 mg/mL respectively, which indicated that the nano-emulsion has two more stable states corresponding to the tightly arranged mono- and bi-palisade layer. These two concentrations are positively correlated with lipophilicity of nimodipine and felodipine. Further, nimodipine liposomes were prepared to validate the effect of drugs on the arrangement of lecithin in the palisade layer. 1H NMR characterizations of the liposomes showed a similar profile to that of nano-emulsions. These results demonstrated that the increasing drug concentration could cause a rearrangement of lecithin in the palisade layer, thus affecting emulsion stability.

CYP2C19 is involved in the effect of Wuzhi tablet (Schisandra sphenanthera extract) and its constituents on the pharmacokinetics of intravenous voriconazole.

The co-administration of voriconazole (VCZ) and Wuzhi tablet (WZ) is frequently prescribed for solid organ transplantation patients in China. However, the pharmacokinetic interactions between VCZ and WZ as well as its bioactive constituents, such as schisandrin A and schisandrol B, remain unknown. Therefore, the effects of WZ and the two lignans on the metabolism of VCZ and the potential role of cytochromeP450 (CYP450), especially cytochrome P450 2C19 (CYP2C19), were investigated. The results showed that WZ extensively inhibited the activities of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Noteworthy, 2.5 mg/mL WZ almost completely inhibited the activity of 2C19, and the inhibition ratio reached 78.6±3% and 63.5±4.6% for schisandrin A and schisandrol B at concentrations 100 µM, respectively. In addition, rats were treated with a single or consecutive 14 day oral dose of WZ (250 mg/kg), schisandrol B (10 mg/kg) and schisandrin A (10 mg/ kg). In rats treated with WZ, the AUC0-∞ value for intravenous VCZ dosing was increased by 80.2% (single dose, p < 0.05) and 66.4% (dosage for 14 day, p < 0.05) and the Cmax was increased by 10.5% (p < 0.05) and (20.6%, p < 0.05), respectively, much greater than that when VCZ (28 mg/kg) was given alone. Unexpectedly, the AUC and Cmax values after schisandrol B and schisandrin A treatment were significantly increased. However, the mRNA expression of liver CYP2C19 and the protein expression of liver CYP2C19 were surprisingly increased after treatment with WZ, schisandrol B and schisandrin A in rats. Therefore, attention should be paid to when WZ and VCZ are administered concomitantly, as dosage adjustment might become necessary. Further clinical study is warranted to validate the interaction between WZ and VCZ.

LNK deficiency decreases obesity-induced insulin resistance by regulating GLUT4 through the PI3K-Akt-AS160 pathway in adipose tissue.

In recent years, LNK, an adapter protein, has been found to be associated with metabolic diseases, including hypertension and diabetes. We found that the expression of LNK in human adipose tissue was positively correlated with serum glucose and insulin in obese people. We examined the role of LNK in insulin resistance and systemic energy metabolism using LNK-deficient mice (LNK-/-). With consumption of a high-fat diet, wild type (WT) mice accumulated more intrahepatic triglyceride, higher serum triglyceride (TG), free fatty acid (FFA) and high sensitivity C-reactive protein (hsCRP) compared with LNK-/- mice. However, there was no significant difference between LNK-/- and WT mice under normal chow diet. Meanwhile, glucose transporter 4 (GLUT4) expression in adipose tissue and insulin-stimulated glucose uptake in adipocytes were increased in LNK-/- mice. LNK-/- adipose tissue showed activated reactivity for IRS1/PI3K/Akt/AS160 signaling, and administration of a PI3K inhibitor impaired glucose uptake. In conclusion, LNK plays a pivotal role in adipose glucose transport by regulating insulin-mediated IRS1/PI3K/Akt/AS160 signaling.

Comparative Glucose and Xylose Coutilization Efficiencies of Soil-Isolated Yeast Strains Identify Cutaneotrichosporon dermatis as a Potential Producer of Lipid.

Glucose and xylose are the major hydrolysates of lignocellulose, and therefore, it is of great implication to identify the microbes involved in simultaneous utilization of glucose and xylose. In this study, the strain ZZ-46 isolated from the soil of Nanyang, China, could simultaneously assimilate glucose and xylose efficiently to produce lipid. Upon cultivation with a 2:1 glucose/xylose mixture as the carbon source for 144 h, the cell biomass, lipid concentration, lipid content, and lipid yield of ZZ-46 reached 19.85 ± 0.39 g/L, 9.53 ± 0.60 g/L, 48.05 ± 3.51%, and 0.142 ± 0.003 g/g sugar, respectively. Moreover, C16 and C18 fatty acids were the main constituents of lipid produced by ZZ-46. In addition, ZZ-46 was identified as Cutaneotrichosporon dermatis by the morphology features and phylogenetic analyses. The strain ZZ-46 would have good perspective in practical application for converting lignocellulose into microbial lipid.

Paeoniflorin exerts neuroprotective effects in a transgenic mouse model of Alzheimer's disease via activation of adenosine A1 receptor.

Alzheimer's disease (AD) is the most common cause of dementia, characterised by advanced cognitive and memory deterioration with no effective treatments available. Previous in vitro and in vivo studies suggest that paeoniflorin (PF), a major bioactive constituent of Radix Paeoniae, might possess anti-dementia properties; however, the underlying mechanism remains unclear. The aim of the current study was to determine the therapeutic effects of PF in a transgenic mouse model of AD and to identify its mechanism. Transgenic mice with five familial AD mutations (5XFAD) were used in this study. We showed that 28 days of PF (5 mg/kg, ip) treatment significantly decreased the escape latency and path length in the Morris water maze test and increased the alternation rate in the T-maze test, compared to the vehicle treatment group. In addition, PF treatment significantly alleviated amyloid ß plaque burden, inhibited astrocyte activation, and decreased IL-1ß and TNF-α expression in the brain of 5XFAD mice. However, the anti-cognitive deficits, anti-amyloidogenic, and anti-inflammatory effects of PF were abolished by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.3 mg/kg), an adenosine A1 receptor (A1R) antagonist. In conclusion, our results suggest that PF might act as a potential therapeutic agent for AD via activation of adenosine A1R.

Recognition of PDL1/L2 by different induced-fit mechanisms of PD1: a comparative study of molecular dynamics simulations.

Recent clinical data has shown that some cancers choose to express PDL2 compared to PDL1. Therefore, a detailed and comparative study of the dynamic binding mechanism between PD1/PDL1 and PD1/PDL2 can guide drug design towards PD1. Herein, long time-scale classical molecular dynamics simulation, binding free energy calculation, energy decomposition and homology modeling for PD1/PDL2 were used to shed light on the differences in the binding mechanisms of the PD1/PDL1 and PD1/PDL2 complexes. On one hand, our results reveal a different binding mechanism of PD1 binding to PDL1 and PDL2, which is mainly attributed to the induced-fit from different proteins, that is, the C'D loop of PD1 is essential for PD1/PDL1, while the CD loop of PDL2 is critical for PD1/PDL2. Particularly, the "enclosed" conformation of PDL2 leads to a higher affinity between PD1-PDL2 in comparison to the affinity between PD1-PDL1. For PD1/PDL1, the key residues of N66, Y68, Q75, T76, K78, D85, I126, L128, A132, I134 and E136 are the dominant residues for stabilizing the protein-protein interaction (PPI). For PD1/PDL2, the key residues are mainly concentrated in the FG loop, including N33/Q75/L128/A132/Q133/I134/K135. These findings provide a comprehensive understanding of the distinctive binding kinetics and thermodynamic features, which will contribute meaningfully for the design of peptides and small molecule inhibitors to selectively break the PPI interfaces of PD1/PDL1 and PD1/PDL2.

Molecular basis for feedback inhibition of tyrosine-regulated 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) is responsible for the biosynthesis of essential aromatic compounds in microorganisms and plants. It plays a crucial role in the regulation of the carbon flow into the shikimate pathway. Until now, the crystal structures and regulatory mechanisms of dimeric DAHPS enzymes from type Iα subclass have not been reported. Here, we reported dimeric structures of the tyrosine-regulated DAHPS from Escherichia coli, both in its apo form and complex with the inhibitor tyrosine at 2.5 and 2.0 Šresolutions, respectively. DAHPS(Tyr) has a typical (ß/α)8 TIM barrel, which is decorated with an N-terminal extension and an antiparallel ß sheet, ß6a/ß6b. Inhibitor tyrosine binds at a cavity formed by residues of helices α3, α4, strands ß6a, ß6b and the adjacent loops, and directly interacts with residues P148, Q152, S181, I213 and N8*. Although the small angle X-ray scattering profiles from DAHPS(Tyr) with and without tyrosine shows that tyrosine binding leaves most of DAHPS(Tyr) structures unaffected. The comparison of the liganded and unliganded crystal structures reveals that conformational changes of residues P148, Q152 and I213 initiate a transmission pathway to propagate the allosteric signal from the tyrosine-binding site to the active site, which is different from DAHPS(Phe), a phenylalanine-regulated isozyme from E. coli. In addition, mutations of five tyrosine-binding residues P148, Q152, S181, I213 and N8* leads to tyrosine-resistant DAHPS(Tyr) enzymes. These findings provide a new insight into the regulatory mechanism of DAHPS enzymes and a basis for further engineering studies.

Efficient CRISPR-Cas9 mediated multiplex genome editing in yeasts.

BACKGROUND: The thermotolerant methylotrophic yeast Ogataea polymorpha has been regarded as an important organism for basic research and biotechnological applications. It is generally recognized as an efficient and safe cell factory in fermentative productions of chemicals, biofuels and other bio-products. However, it is difficult to genetically engineer for the deficiency of an efficient and versatile genome editing technology. RESULTS: In this study, we developed a CRISPR-Cas9-assisted multiplex genome editing (CMGE) approach including multiplex genes knock-outs, multi-locus (ML) and multi-copy (MC) integration methods in yeasts. Based on CMGE, various genome modifications, including gene deletion, integration, and precise point mutation, were performed in O. polymorpha. Using the CMGE-ML integration method, three genes TAL from Herpetosiphon aurantiacus, 4CL from Arabidopsis thaliana and STS from Vitis vinifera of resveratrol biosynthetic pathway were simultaneously integrated at three different loci, firstly achieving the biosynthesis of resveratrol in O. polymorpha. Using the CMGE-MC method, ∼ 10 copies of the fusion expression cassette P ScTEF1 -TAL-P ScTPI1 -4CL-P ScTEF2 -STS were integrated into the genome. Resveratrol production was increased ~ 20 fold compared to the one copy integrant and reached 97.23 ± 4.84 mg/L. Moreover, the biosynthesis of human serum albumin and cadaverine were achieved in O. polymorpha using CMGE-MC to integrate genes HSA and cadA, respectively. In addition, the CMGE-MC method was successfully developed in Saccharomyces cerevisiae. CONCLUSIONS: An efficient and versatile multiplex genome editing method was developed in yeasts. The method would provide an efficient toolkit for genetic engineering and synthetic biology researches of O. polymorpha and other yeast species.

Hypouricemic and Nephroprotective Effects of an Active Fraction from Polyrhachis Vicina Roger On Potassium Oxonate-Induced Hyperuricemia in Rats.

BACKGROUND/AIMS: The objective of this study is to evaluate the hypouricemic and nephroprotective effects of an active fraction from Polyrhachis vicina Roger (AFPR) in potassium oxonate-induced hyperuricemic rats. METHODS: Hyperuricemia was induced by potassium oxonate in male rats. AFPR was orally administered to hyperuricemic rats for 12 consecutive weeks. Serum, liver and kidney samples were collected for effects and mechanism analysis. The levels of serum uric acid (SUA) were measured by the phosphotungstic acid method, xanthine oxidase (XOD) activity in the hepatic and serum samples were measured by ultraviolet spectrophotometry, serum levels of interleukin-1 (IL-1ß), interleukin-1 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by ELISA, the levels of serum creatinine (SCr), blood urea nitrogen (BUN), super oxide dismutase (SOD) and malondialdehyde (MDA) in serum were determined by colorimetric method. Protein expression of renal URAT1, GLUT9, and OAT1 were analyzed by Western blot. RESULTS: AFPR significantly decreased the levels of SUA, serum and hepatic XOD, SCr, BUN, and MDA as well as increased SOD. In addition, AFPR treatment significantly reduced the levels of proinflammatory cytokines in serum, including IL-1ß, IL-6 and TNF-α. Moreover, we found the significant decrease in protein expression of URAT1 and GLUT9, and the significant increase in protein expression of OAT1 in the kidney in AFPR treated groups compared to the model groups of hyperuricemia. CONCLUSION: These findings suggest that AFPR has anti-hyperuricemic activity attributed to the inhibition of uric acid generation in the liver and probably to the enhancement of urate excretion in the kidney, and possess nephroprotective effect in hyperuricemic rats due to its anti-inflammatory and antioxidant activities.

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.