Hypoglycemic property of soy isoflavones from hypocotyl in Goto-Kakizaki diabetic rats.

The present study was carried out to investigate the hypoglycemic effect of soy isoflavones from hypocotyl in GK diabetic rats. A single administration and long-term administration tests were conducted in GK diabetic rats to test the hypoglycemic effect of soy isoflavones. At the end of long-term administration trial, blood protein, cholesterol, triglyceride, glycosylated serum protein, C-reactive protein, insulin, aminotransferase, lipid peroxide, interleukin-6, tumor necrosis factor-α were estimated. Inhibition of soy isoflavones against α-amylase and α-glucosidase, as well as on glucose uptake into brush border membrane vesicles or Caco-2 cells were determined in vitro. In single administration experiment, soy isoflavones reduced postprandial blood glucose levels in GK rats. In long-term administration, hypoglycemic effect of soy isoflavones was first observed at week 12 and maintained till week 16. A significant reduction in fasting blood glucose, C-reactive protein, and lipid peroxide was noted at week 16. However, there was no significant treatment effect on blood insulin. Furthermore, soy isoflavone administration resulted in significant decreases in glycosylated serum protein, tumor necrosis factor-α, and interleukin-6. Other biochemical parameters, such as protein, cholesterol, triglyceride and aminotransferases were not modified, however. The results in vitro showed that soy isoflavones showed a potent inhibitory effect on intestinal α-glucosidase, but not on pancreatic α-amylase. Soy isoflavones also decreased glucose transport potency into brush border membrane vesicles or Caco-2 cells. It is concluded that soy isoflavones from hypocotyl, performs hypoglycemic function in GK rats with type 2 diabetes, maybe via suppression of carbohydrate digestion and glucose uptake in small intestine.

"Perfect" designer chromosome V and behavior of a ring derivative.

Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis. We designed and de novo synthesized 536,024-base pair chromosome synV in the "Build-A-Genome China" course. We corrected an initial isolate of synV to perfectly match the designed sequence using integrative cotransformation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated editing in 22 steps; synV strains exhibit high fitness under a variety of culture conditions, compared with that of wild-type V strains. A ring synV derivative was constructed, which is fully functional in Saccharomyces cerevisiae under all conditions tested and exhibits lower spore viability during meiosis. Ring synV chromosome can extends Sc2.0 design principles and provides a model with which to study genomic rearrangement, ring chromosome evolution, and human ring chromosome disorders.

Enhanced Bioconversion of Cellobiose by Industrial Saccharomyces cerevisiae Used for Cellulose Utilization.

Cellobiose accumulation and the compromised temperature for yeast fermentation are the main limiting factors of enzymatic hydrolysis process during simultaneous saccharification and fermentation (SSF). In this study, genes encoding cellobiose transporter and ß-glucosidase were introduced into an industrial Saccharomyces cerevisiae strain, and evolution engineering was carried out to improve the cellobiose utilization of the engineered yeast strain. The evolved strain exhibited significantly higher cellobiose consumption rate (2.8-fold) and ethanol productivity (4.9-fold) compared with its parent strain. Besides, the evolved strain showed a high cellobiose consumption rate of 3.67 g/L/h at 34°C and 3.04 g/L/h at 38°C. Moreover, little cellobiose was accumulated during SSF of Avicel using the evolved strain at 38°C, and the ethanol yield from Avicel increased by 23% from 0.34 to 0.42 g ethanol/g cellulose. Overexpression of the genes encoding cellobiose transporter and ß-glucosidase accelerated cellobiose utilization, and the improvement depended on the strain background. The results proved that fast cellobiose utilization enhanced ethanol production by reducing cellobiose accumulation during SSF at high temperature.

Deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) induces simultaneous utilization of xylose and glucose in xylose-utilizing Saccharomyces cerevisiae.

Simultaneous co-utilization of xylose and glucose is a key issue in engineering microbes for cellulosic ethanol production. We coupled xylose utilization with glucose metabolism by deletion of D-ribulose-5-phosphate 3-epimerase (RPE1) through pentose phosphate pathway flux. Simultaneous utilization of xylose and glucose then occurred in the engineered Saccharomyces cerevisiae strain with the xylose utilization pathway. Xylose consumption occurred at the beginning of glucose consumption by the engineered yeast without RPE1 in a mixed sugar fermentation. About 3.2 g xylose l(-1) was utilized simultaneously with consumption of 40.2 g glucose l(-1) under O2-limited conditions. In addition, an approximate ratio (~1:10) for xylose and glucose consumption was observed in the fermentation with different sugar concentration by the engineered strain without RPE1. Simultaneous utilization of xylose is realized by the coupling of glucose metabolism and xylose utilization through RPE1 deletion in xylose-utilizing S. cerevisiae.

Optimization of CDT-1 and XYL1 expression for balanced co-production of ethanol and xylitol from cellobiose and xylose by engineered Saccharomyces cerevisiae.

Production of ethanol and xylitol from lignocellulosic hydrolysates is an alternative to the traditional production of ethanol in utilizing biomass. However, the conversion efficiency of xylose to xylitol is restricted by glucose repression, causing a low xylitol titer. To this end, we cloned genes CDT-1 (encoding a cellodextrin transporter) and gh1-1 (encoding an intracellular ß-glucosidase) from Neurospora crassa and XYL1 (encoding a xylose reductase that converts xylose into xylitol) from Scheffersomyces stipitis into Saccharomyces cerevisiae, enabling simultaneous production of ethanol and xylitol from a mixture of cellobiose and xylose (main components of lignocellulosic hydrolysates). We further optimized the expression levels of CDT-1 and XYL1 by manipulating their promoters and copy-numbers, and constructed an engineered S. cerevisiae strain (carrying one copy of PGK1p-CDT1 and two copies of TDH3p-XYL1), which showed an 85.7% increase in xylitol production from the mixture of cellobiose and xylose than that from the mixture of glucose and xylose. Thus, we achieved a balanced co-fermentation of cellobiose (0.165 g/L/h) and xylose (0.162 g/L/h) at similar rates to co-produce ethanol (0.36 g/g) and xylitol (1.00 g/g).

Balance of XYL1 and XYL2 expression in different yeast chassis for improved xylose fermentation.

Reducing xylitol formation is necessary in engineering xylose utilization in recombinant Saccharomyces cerevisiae for ethanol production through xylose reductase/xylitol dehydrogenase pathway. To balance the expression of XYL1 and mutant XYL2 encoding xylose reductase (XR) and NADP(+)-dependent xylitol dehydrogenase (XDH), respectively, we utilized a strategy combining chassis selection and direct fine-tuning of XYL1 and XYL2 expression in this study. A XYL1 gene under the control of various promoters of ADH1, truncated ADH1 and PGK1, and a mutated XYL2 with different copy numbers were constructed into different xylose-utilizing modules, which were then expressed in two yeast chassises W303a and L2612. The strategy enabled an improved L2612-derived recombinant strain with XYL1 controlled by promoter PGK1 and with two copies of XYL2. The strain exhibited a 21.3% lower xylitol yield and a 40.0% higher ethanol yield. The results demonstrate the feasibility of the combinatorial strategy for construction of an efficient xylose-fermenting S. cerevisiae.

Purification, characterization, and cloning of fibrinolytic metalloprotease from Pleurotus ostreatus mycelia.

A fibrinolytic protease (PoFE) was purified from the cultured mycelia of the edible oyster mushroom Pleurotus ostreatus, using a combination of various chromatographies. The purification protocol resulted in an 876-fold purification of the enzyme, with a final yield of 6.5%. The apparent molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE, fibrin-zymography, and size exclusion using FPLC. The optimal reaction pH value and temperature were pH 6.5 and 35 degrees C, respectively. PoFE effectively hydrolyzed fibrinogen, preferentially digesting the A alpha-chain and the B beta-chain over the gamma-chain. Enzyme activity was enhanced by the addition of Ca2+, Zn2+, and Mg2+ ions. Furthermore, PoFE activity was potently inhibited by EDTA, and it was found to exhibit a higher specificity for the chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 19 amino acid residues of the N-terminal sequence were ALRKGGAAALNIYSVGFTS, which is extremely similar to the metalloprotease purified from the fruiting body of P. ostreatus. In addition, we cloned the PoFE protein, encoding gene, and its nucleotide sequence was determined. The cDNA of cloned PoFE is 867 nucleotides long and consists of an open reading frame encoding 288 amino acid residues. Its cDNA showed a high degree of homology with PoMEP from P. ostreatus fruiting body. The mycelia of P. ostreatus may thus represent a potential source of new therapeutic agents to treat thrombosis.

Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea.

A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 degrees C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chain over the Bbeta- and r-chains. Enzyme activity was inhibited by Cu(2+) and Co(2+), but enhanced by the addition of Ca(2+) and Mg(2+) ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.