The Property of a Key Amino Acid Determines the Function of Farnesyl Pyrophosphate Synthase in Sporobolomyces pararoseus NGR.

Farnesyl pyrophosphate synthase (FPPS) catalyzes the synthesis of C15 farnesyl diphosphate (FPP) from C5 dimethylallyl diphosphate (DMAPP) and two or three C5 isopentenyl diphosphates (IPPs). FPP is an important precursor for the synthesis of isoprenoids and is involved in multiple metabolic pathways. Here, farnesyl pyrophosphate synthase from Sporobolomyces pararoseus NGR (SpFPPS) was isolated and expressed by the prokaryotic expression system. The SpFPPS full-length genomic DNA and cDNA are 1566 bp and 1053 bp, respectively. This gene encodes a 350-amino acid protein with a predicted molecular mass of 40.33 kDa and a molecular weight of 58.03 kDa (40.33 kDa + 17.7 kDa), as detected by SDS-PAGE. The function of SpFPPS was identified by induction, purification, protein concentration and in vitro enzymatic activity experiments. Structural analysis showed that Y90 was essential for chain termination and changing the substrate scope. Site-directed mutation of Y90 to the smaller side-chain amino acids alanine (A) and lysine (K) showed in vitro that wt-SpFPPS catalyzed the condensation of the substrate DMAPP or geranyl diphosphate (GPP) with IPP at apparent saturation to synthesize FPP as the sole product and that the mutant protein SpFPPS-Y90A synthesized FPP and C20 geranylgeranyl diphosphate (GGPP), while SpFPPS-Y90K hydrolyzed the substrate GGPP. Our results showed that FPPS in S. pararoseus encodes the SpFPPS protein and that the amino acid substitution at Y90 changed the distribution of SpFPPS-catalyzed products. This provides a baseline for potentially regulating SpFPPS downstream products and improving the carotenoid biosynthesis pathway.

Creation of gastroenteric anastomosis through natural orifice in rats by magnetic compression technique.

BACKGROUND: Being one of the core techniques of magnetic surgery, magnetic compression technique (MCT) has been used for digestive tract anastomosis reconstruction in experimental studies. This study verified the feasibility of gastroenteric anastomosis through natural orifice using MCT in rats. METHODS: The parent and daughter magnets were designed and manufactured for oral and anal insertion in 20 Sprague-Dawley rats. After anesthesia, the parent magnet was inserted into the colon spleen area through the anus, and the daughter magnet was inserted into the stomach through the mouth. Then the two magnets were positioned to attract each other and bind together. The position of the two magnets was monitored using X-ray. The time required for the formation of the anastomosis and expulsion of the magnets were recorded. 2 weeks later, the animal was sacrificed and the anastomotic specimen was obtained which was observed under naked eye and microscope. RESULTS: The gastroenteric anastomosis was successfully performed via natural orifices in 18 out of 20 rats. The mean time to construct the anastomosis was 3.78 ± 0.88 min. X-ray examination showed that the magnets were in the appropriate position in 17 rats. The magnets were excreted in 9.47 ± 1.62 days after surgery. The gross and microscopic examination of the specimen showed that the anastomoses were patent and the mucosa at the anastomotic was smooth. The mean bursting pressure of the anastomosis was 136.94 ± 6.79 mmHg. CONCLUSION: It is feasible to perform gastroenteric anastomosis through natural orifices by MCT.

[Neuroprotective effect of cinnamaldehyde in MPTP-induced mouse model of subacute Parkinson's disease].

This paper aims to explore the neuroprotective effect of cinnamaldehyde(CA) in mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced subacute Parkinson's disease(PD) and the mechanism. To be specific, male C57 BL/6 mice(n=72, SPF) were randomized into control group, model group, positive control(madopar 0.1 mg·g~(-1)) group, and low-dose, me-dium-dose, and high-dose CA groups(0.15, 0.30, 0.60 mg·g~(-1)). MPTP(intraperitoneal injection, 0.03 mg·g~(-1), once a day for 5 days) was used to induce subacute PD in mice except for the control group. The administration began from the day of modeling and lasted 19 days. On the 0 th, 12 th, and 19 th day, the open field test, pole test, and rotarod test were carried out. After the tests, the mice were killed and brains were separated. In addition, the organ index was measured. The number of cells in substantia nigra(SN) in the midbrain of MPTP-induced PD model mice was detected based on hematoxylin and eosin(HE) staining. The levels of tyrosine hydroxylase(TH)-and α-synuclein(α-Syn)-positive cells in SN were determined by immunohistochemical staining, and the protein levels of TH and α-Syn in SN by Western blot. The results showed that the MPTP-stimulated mice had abnormal behaviors such as erect hair, arched back, rigidity of the tail, slow movement, and tremor, decreased number of crossings and rearing, increased frequency of urination and defecation, longer time of pole climbing, and shorter time of staying on the rotating rod. In addition, the mice showed obvious damage of neurons in the SN and reduced neuron cells in irregular arrangement with some shrinking. In addition, the average optical density of TH in SN decreased and that of α-Syn increased. All these suggested the successful modeling. CA displayed obvious therapeutic effect on the PD mice, as manifested by the increased number of crossings and rearing, decreased frequency of urination and defecation, shorter time of climbing pole, longer time of staying on the rotating rod, and more neuron cells in the SN with a few pykno-tic cells. Moreover, CA significantly alleviated the decrease of TH and the overexpression of α-Syn in SN. As a result, the MPTP-induced injury of dopaminergic neurons was alleviated. The performance of 0.3 mg·g~(-1) CA was the best. This study is expected to lay a scientific basis for the development of CA products.

A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations.

Currently, the most widely used strategies for molecular cloning are sticky-end ligation-based cloning, TA cloning, blunt-end ligation-based cloning and ligase-independent cloning. In this study we have developed a novel mini-vector pANY1 which can simultaneously meet the requirements of all these cloning strategies. In addition, the selection of appropriate restriction digestion sites is difficult in some cases because of the presence of internal sites. In this study, an annealing of PCR products (APP)-based sticky-end cloning strategy was introduced to avoid this issue. Additionally, false positives occur during molecular cloning, which increases the workload of isolating positive clones. The plasmid pANY1 contains a ccdB cassette between multiple cloning sites, which efficiently avoids these false positives. Therefore, this mini-vector should serve as a useful tool with wide applications in biosciences, agriculture, food technologies, etc.

Western diet feeding influences gut microbiota profiles in apoE knockout mice.

BACKGROUND: Gut microbiota plays an important role in many metabolic diseases such as diabetes and atherosclerosis. Apolipoprotein E (apoE) knock-out (KO) mice are frequently used for the study of hyperlipidemia and atherosclerosis. However, it is unknown whether apoE KO mice have altered gut microbiota when challenged with a Western diet. METHODS: In the current study, we assessed the gut microbiota profiling of apoE KO mice and compared with wild-type mice fed either a normal chow or Western diet for 12 weeks using 16S pyrosequencing. RESULTS: On a western diet, the gut microbiota diversity was significantly decreased in apoE KO mice compared with wild type (WT) mice. Firmicutes and Erysipelotrichaceae were significantly increased in WT mice but Erysipelotrichaceae was unchanged in apoE KO mice on a Western diet. The weighted UniFrac principal coordinate analysis exhibited clear separation between WT and apoE KO mice on the first vector (58.6%) with significant changes of two dominant phyla (Bacteroidetes and Firmicutes) and seven dominant families (Porphyromonadaceae, Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, Helicobacteraceae, Erysipelotrichaceae and Veillonellaceae). Lachnospiraceae was significantly enriched in apoE KO mice on a Western diet. In addition, Lachnospiraceae and Ruminococcaceae were positively correlated with relative atherosclerosis lesion size in apoE KO. CONCLUSIONS: Collectively, our study showed that there are marked changes in the gut microbiota of apoE KO mice, particularly challenged with a Western diet and these alterations may be possibly associated with atherosclerosis.

A silica sands-based method for faithful analysis of microbial communities and DNA isolation from a wide range of species.

A silica sands-based method has been developed to isolate high quality genomic DNAs from cells of animals, plants and microorganisms, such as Hemisalanx prognathus, Spinacia oleracea, Pichia pastoris, Bacillus licheniformis and Escherichia coli. To the best of our knowledge, no DNA isolation method has so wide application until now. In addition, this method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16s rDNA sequencing. As a result, the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit, indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities.

Dietary Cocoa Powder Improves Hyperlipidemia and Reduces Atherosclerosis in apoE Deficient Mice through the Inhibition of Hepatic Endoplasmic Reticulum Stress.

Cocoa powder is rich in flavonoids, which have many beneficial effects on human health, including antioxidative and anti-inflammatory effects. The aim of our study was to investigate whether the intake of cocoa powder has any influence on hyperlipidemia and atherosclerosis and examine the underlying molecular mechanisms. We fed apoE knockout mice a Western diet supplemented with either 0.2% (low group) or 2% (high group) cocoa powder for 12 weeks. The groups fed dietary cocoa powder showed a significant reduction in both plasma cholesterol levels and aortic atherosclerosis compared to the control group. Analysis of mRNA profiling of aortic atherosclerotic lesions revealed that the expression of several genes related to apoptosis, lipid metabolism, and inflammation was significantly reduced, while the antiapoptotic gene Bcl2 was significantly increased in the cocoa powder group compared to the control. RT-PCR analysis along with Western blotting revealed that a diet containing cocoa powder inhibited the expression of hepatic endoplasmic reticulum stress. These data suggest that cocoa powder intake improves hyperlipidemia and atherosclerosis, and such beneficial effects are possibly mediated through the suppression of hepatic endoplasmic reticulum stress.

Isolation and Analysis of Salt Response of Lactobacillusplantarum FS5-5 from Dajiang.

From 15 samples of dajiang collected in northeast of China, three salt resistant lactic acid bacteria were isolated and identified as Lactobacillusplantarum through physiological studies and 16S rDNA sequence alignment. L. plantarum FS5-5 showed better growth in an environment with 12 % (w/v) NaCl than the other two strains. The expression of proteins extracted from L.plantarum FS5-5 cultured in de Man, Rogosa, and Sharp (MRS) containing 0, 3, 6 and 9 % (w/v) NaCl was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results showed that 42 kinds of proteins were identified, which could be divided into three groups: 27 kinds of proteins related to protein synthesis and degradation, six kinds of proteins related to carbohydrate metabolism and energy metabolism, nine proteins related to nucleic acid metabolism. Overexpression of these proteins imply that a series of changes have occurred in the process of protein synthesis and degradation, carbohydrate metabolism, energy metabolism and nucleic acid metabolism after L.plantarum FS5-5 exposed to salt stress. All these proteins may have effects on the salt-tolerant characteristics of the L.plantarum FS5-5.

A simple and convenient sticky/blunt-end ligation method for fusion gene construction.

Here we present a simple and convenient sticky/blunt-end ligation method for fusion gene construction. The fusion gene is constructed by seamless ligation of 5'-end phosphorylated blunt ends instead of by overlap extension PCR (OE-PCR). Therefore, the challenge of amplifying large DNA fragments (e.g., the large bifunctional enzyme gene constructed by fusion of two monofunctional enzyme genes) by PCR can be avoided. In addition, synthesis of the inner primers for OE-PCR is not necessary, indicating that this method should be especially convenient for construction of fusion genes with various combinations of multiple fragments (e.g., chimeric gene libraries, fusion gene libraries). As a modification of the commonly used fusion gene construction technique, this method may find a wide range of applications in bioscience and biotechnology.

A modified version of the digestion-ligation cloning method for more efficient molecular cloning.

Here we describe a modified version of the digestion-ligation approach for efficient molecular cloning. In comparison with the original method, the modified method has the additional steps of gel purification and a second ligation after the first ligation of the linearized vector and DNA insert. During this process, the efficiency and reproducibility could be significantly improved for both stick-end cloning and blunt-end cloning. As an improvement of the very important molecular cloning technique, this method may find a wide range of applications in bioscience and biotechnology.

A method suitable for DNA extraction from humus-rich soil.

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

Progress in Gene Editing and Metabolic Regulation of Saccharomyces cerevisiae with CRISPR/Cas9 Tools.

The CRISPR/Cas9 systems have been developed as tools for genetic engineering and metabolic engineering in various organisms. In this review, various aspects of CRISPR/Cas9 in Saccharomyces cerevisiae, from basic principles to practical applications, have been summarized. First, a comprehensive review has been conducted on the history of CRISPR/Cas9, successful cases of gene disruptions, and efficiencies of multiple DNA fragment insertions. Such advanced systems have accelerated the development of microbial engineering by reducing time and labor, and have enhanced the understanding of molecular genetics. Furthermore, the research progress of the CRISPR/Cas9-based systems in the production of high-value-added chemicals and the improvement of stress tolerance in S. cerevisiae have been summarized, which should have an important reference value for genetic and synthetic biology studies based on S. cerevisiae.

Characterization of a novel multifunctional ß-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea.

Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

Research progress of engineering microbial cell factories for pigment production.

Pigments are widely used in people's daily life, such as food additives, cosmetics, pharmaceuticals, textiles, etc. In recent years, the natural pigments produced by microorganisms have attracted increased attention because these processes cannot be affected by seasons like the plant extraction methods, and can also avoid the environmental pollution problems caused by chemical synthesis. Synthetic biology and metabolic engineering have been used to construct and optimize metabolic pathways for production of natural pigments in cellular factories. Building microbial cell factories for synthesis of natural pigments has many advantages, including well-defined genetic background of the strains, high-density and rapid culture of cells, etc. Until now, the technical means about engineering microbial cell factories for pigment production and metabolic regulation processes have not been systematically analyzed and summarized. Therefore, the studies about construction, modification and regulation of synthetic pathways for microbial synthesis of pigments in recent years have been reviewed, aiming to provide an up-to-date summary of engineering strategies for microbial synthesis of natural pigments including carotenoids, melanins, riboflavins, azomycetes and quinones. This review should provide new ideas for further improving microbial production of natural pigments in the future.

An experimental study of magnetic compression technique for ureterovesical anastomosis in rabbits.

This study aimed to explore the feasibility of the magnetic compression technique (MCT) for ureterovesical anastomosis in a rabbit model with ureteral obstruction. The distal ureteral obstruction model using female New Zealand rabbits was induced by ligating the distal end of the right ureter with silk thread for four weeks. A pair of cylindrical NdFeB magnets (daughter magnet and parent magnet) with a hole in the center was used for the ureterovesical anastomosis. The daughter magnet and the parent magnet were respectively placed close to the obstruction site through the dilated proximal ureter and urethra, and then the daughter-parent magnets pair was attracted together automatically. Postoperative X-rays were taken to confirm the position of the magnets. The anastomotic stoma specimens were obtained two weeks postoperatively, and the anastomotic stoma formation was observed by the naked eye and histological staining. The operation time and the anastomotic burst pressure were measured. The ureter was significantly dilated in the fourth week after ligation, which satisfied the placement of the daughter magnet. The ureterovesical magnet placements were successfully performed in ten experimental rabbits, with an operation time of 36.5 ± 6.09 min. The parent and daughter magnets attracted each other well and were subsequently removed through the urethra two weeks postoperatively, resulting in the establishment of ureterovesical anastomosis. The anastomotic burst pressure was 147.5 ± 14.59 mmHg. Gross specimens and histological examination of the anastomotic stoma showed that the anastomotic stoma healed well. MCT is feasible and simple for ureterovesical anastomosis.

The strategy of cell extract based metal organic frameworks (CE-MOF) for improved enzyme characteristics.

A convenient cell extract based metal organic frameworks (CE-MOF) strategy was used to produce self-assembled hybrid microparticles of enzymes with improved characteristics. It was shown that many metal ions and enzymes could be used to construct catalytically active CE-MOF microparticles. As a proof-of-principle study, the ß-xylosidase BH3683 was used to prepare FeSO4-CE-MOF-BH3683 microparticles to explore the factors influencing preparation of the microparticles. As a result, DNA, RNA, polysaccharides and proteins were found to play important roles in the formation of the microparticles and affected enzyme activities through interaction with enzyme molecules. Compared with the free BH3683, the optimum temperature of FeSO4-CE-MOF-BH3683 increased 5 °C, and the relative activity at 70 °C increased two times. Moreover, FeSO4-CE-MOF-BH3683 have stronger tolerance to different concentrations of various organic solvents and high-concentration xylose than the free BH3683, and the CE-MOF microparticles prepared by BH3683 and xylanase XynII could catalyze high-concentration xylan more efficiently than their free counterparts. In addition, FeSO4-CE-MOF-BH3683 exhibited about 40 % of its initial activity after reused for 10 times, showing satisfactory reusability. To sum up, this strategy might have wide application potential in the fields of biocatalysis, biofuel production, fertilizer industry, etc.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

Biochemical characterization of an engineered bifunctional xylanase/feruloyl esterase and its synergistic effects with cellulase on lignocellulose hydrolysis.

Herein, the xylanase and feruloyl esterase domains of the xylanase/feruloyl esterase bifunctional enzyme (Xyn-Fae) from Prevotella ruminicola 23 were identified using N- and C-terminal truncation mutagenesis. In addition, a novel and more efficient xylanase/feruloyl esterase bifunctional enzyme XynII-Fae was constructed, and its synergistic action with a commercial cellulase for lignocellulose hydrolysis was studied. When 40% cellulase was replaced by XynII-Fae, the production of reducing sugars increased by 65% than that with the cellulase alone, and the conversions of xylan and glucan were increased by 125.1% and 54.3%, respectively. When 80% cellulase was substituted by XynII-Fae, up to 43.5 µg/mL ferulic acid and 418.7 µg/mL acetic acid were obtained. The XynII-Fae could also accelerate the hydrolysis of wheat straw and sugarcane bagasse with commercial cellulase. These results indicated that the synergistic action of XynII-Fae with cellulase could dramatically improve the hydrolysis efficiency of lignocellulose, showing the great potential for industrial applications.

Establishment of Yan-Zhang's staging of digestive tract magnetic compression anastomosis in a rat model.

Magnetic compression anastomosis, also known as magnamosis, is a safe and feasible method for digestive tract anastomosis. However, the pathological process involved in magnamosis of the digestive tract has not been investigated. This study aimed to establish the stages of digestive tract magnamosis in a rat model. Eighty-four Sprague-Dawley albino rats (200-250 g) were randomly divided into 14 groups (n = 6 per group). All rats underwent colonic magnamosis. Starting from postoperative day (POD) 1, one group of rats was sacrificed every other day to obtain the specimens. Burst pressure at the anastomotic site of each specimen was examined. Gross and histological examination of the anastomotic site was performed to establish the stages of the digestive tract magnamosis. Colonic magnamosis was successfully performed in all rats and the mean anastomosis time was 5.62 ± 0.91 min. The postoperative survival rate was 100%. The lowest anastomotic burst pressure was 78.33 ± 3.44 mmHg on POD3. The anastomotic burst pressure gradually increased and stabilized on POD21. Macroscopic and histological examination showed that the anastomotic mucosal and serosal layer did not heal on POD1. The serosal layer of the anastomosis healed by adhesion on POD3, and the mucosal layer began to heal on POD3-11 and was established by POD21. According to the anastomotic bursting pressure, digestive tract magnamosis can be staged into the magnetic maintenance, fragile, strengthening, and stable phases, which on histology correspond to the serosal adhesion formation, serosal healing, mucosal healing, and stereotyping, respectively.

Novel biocatalytic strategy of levan: His-ELP-intein-tagged protein purification and biomimetic mineralization.

Here a versatile fusion tag composed of His-tag, intein, and elastin-like polypeptide (ELP) tag was prepared for the first time to be fused with levansucrase SacB to construct a recombinant His-ELP-intein-SacB (HEIS) protein to realize nonchromatographic purification of SacB. The efficient biomimetic mineralization of CaHPO4 and HEIS-based hybrid-hydrangea (CaHPO4-HEIS-HH) with good reusability, excellent storage stability and 254.3% improved relative levan yield was prepared with the biomimetic mineralization method. Additionally, the CaHPO4-HEIS-HH showed outstanding operation activity when catalyzing sucrose in solution and up to 75% sucrose conversion rate in fruit juices. The mechanism of biomimetic mineralization was analyzed to show that the HEIS protein might serve as a "binder" to assemble the nanoflakes during biomimetic mineralization. The CaHPO4-HEIS-HH was applicable for efficient production of the levan-type prebiotic polysaccharides, and this approach should be highly valuable for nonchromatographic purification and convenient preparation of various encapsulated enzymes for more efficient catalysis.