查看更多>>摘要：Heparosan as an acidic polysaccharide is mainly applied for heparin biosynthesis and drug delivery. Escherichia coli Nissle 1917 (EcN) naturally synthesizes and secrets heparosan as its capsular polysaccharides. In this study, we described the metabolic engineering of EcN to enhance heparosan production by optimizing the biosynthesis of precursors UDP-GlcA and UDP-GlcNAc and the expression of heparosan synthase. The orthologs of heparosan synthetic pathway enzymes from five species were expressed and comparatively investigated. bsGalU and ecKfiD for UDP-GlcA and ecGlmM for UDP-GlcNAc were introduced into EcN and the production of heparosan was increased from 0.15 g/L to 0.34 g/L, 0.39 g/L and 0.37 g/L, respectively. Combinational overexpression of bsGalU, ecKfiD and ecGlmM improved heparosan production to 0.80 g/L in flask cultures. After further upregulation of the endogenous heparosan synthases KfiAC, the titer of heparosan was improved to 1.29 g/L. Meanwhile, pathway engineering also led to the fluctuation of molecular weights between 312.39 and 410.84 kDa. Eventually, the engineered strain EC048 with overexpression of bsGalU, ecKfiD, ecGlmM and KfiAC produced 11.50 g/L heparosan in 3-L fed-batch fermentor, demonstrating EcN as a good microbial chassis is applicable for engineering an efficient heparosan cell factory.
查看更多>>摘要：Protein stability is crucial in enzymatic catalysis. To improve the efficiency in the searching for thermostablizing mutations, we applied a sequence consensus approach focusing on dimeric interface residues of ketoreductase ChKRED20. The strategy returned a success rate of 43%, revealing 9 beneficial mutations from 21 candidates with improved kinetic or thermodynamic stability. Several combinatorial mutants were then constructed, and mutant M8K displayed the highest thermostability, with a melting temperature (Tm) of 89 C and a half inactivation temperature (T-50) of 93.4 C, both of over 35 C increase compared to the wild-type. M8K could remain stable for at least 7 days at its optimal reaction temperature of 55 C. Its inactivation half-life (t(1/2)) was 110 min at 90 C, while the wild-type was 18.6 min at 60 C. The results were interpreted in the context of structural and molecular dynamic simulation analysis, which revealed the addition of intramolecular interactions, decreased conformational flexibility and increased compactness, all in agreement with the observed effect.
查看更多>>摘要：Basidiomycetes produce various sesquiterpenoids and their relevance for pharmaceutical and agricultural applications and understanding their biosynthetic machinery to produce these secondary metabolites have attracted significant interest. Because sesquiterpene synthases (STSs) and cytochrome P450 monooxygenases (P450s) play pivotal roles in the production of sesquiterpenoids, functional characterization of these enzymes is fundamentally essential. In this study, we found 11 possible STSs from the white-rot basidiomycete Phanerochaete chrysosporium (PcSTSs) and isolated nine of these as full-length cDNAs encoding a mature open reading frame. Using the isolated cDNAs, we performed heterologous expression of PcSTSs in Saccharomyces cerevisiae. Meta-bolic studies revealed that seven of the PcSTSs produce a series of sesquiterpene scaffolds, including (E)-alpha-bisabolene. Furthermore, we constructed a co-expression system of (E)-alpha-bisabolene synthase and P450 from P. chrysosporium (PcCYP). Semi-comprehensive screening using 120 isoforms of PcCYPs resulted in the identification of CYP5158A1 and CYP5144C8, two P450s capable of decorating (E)-alpha-bisabolene.
查看更多>>摘要：L-aspartate-alpha-decarboxylase (PanD) is an essential enzyme catalysing the decarboxylation of L-aspartate to beta-alanine in organisms. To perform the catalytic functions, PanD pro-proteins need to be self-cleaved to form two subunits: active alpha-subunit and beta-subunit. However, the processes of self-cleavage have diverged in different organisms for unknown reasons. To reveal the possible divergence mechanisms, the molecular evolution, selection pressures and site-directed mutagenesis of the panD gene family were explored in this study. The evolution analysis revealed that the panD genes in bacteria have diverged into three clades: Class I, Class II and Class III. Furthermore, 9 positive selection sites (A13, T14, V23, L32, V44, N49, L55, L78, and V85 in BsupanD) were detected. As shown by SDS-PAGE assay and catalytic activity determination in the mutants of BsupanD and EcoPanD, three of those sites (T14, V44 and V85) affect the PanD activities and are involved in the divergence of panD self-cleavage, while the other 6 sites only influenced the enzymatic activities of PanD. Furthermore, the structure analysis indicated that the structural mechanisms of the 9 sites affecting the catalysis were various. In all, three sites contributing to the divergence of PanD self-cleavage were revealed, and the results also provide foundation for the industrial application of PanD in beta-alanine synthesis.
查看更多>>摘要：We selected Bacillus licheniformis NY1505 by screening a strain capable of producing alpha-glucosidase inhibitors in both aerobic and anaerobic environments in vitro and spore formation. To confirm whether this strain proliferates in the intestine and produces alpha-glucosidase inhibitor, the spores of this strain were administered to mice orally. As the results, it was confirmed that 107 cells and about 300 units of alpha-glucosidase inhibitor per 1 g feces were excreted in the feces after three weeks of administration as spores. And after two weeks of stopping administration, Bacillus licheniformis NY1505 in the intestine are cleared. This means that Bacillus licheniformis NY1505 steadily proliferated in the intestine and produced alpha-glucosidase inhibitors and excreted in the feces. Also, it has an advantage in its use as it can easily eliminate Bacillus licheniformis NY1505 from the intestine. This method of ingesting only microorganisms is a more efficient and new method than the existing method of administering an alpha-glucosidase inhibitor that consumes a large amount of purified product. This method shows a process in which microorganisms capable of proliferating in the intestine directly produce and supply specific secondary metabolites in the intestine.
查看更多>>摘要：Protein aggregation can affect the stability and function of proteins, and may lead to developing diseases, but reports on the in vivo effect of aggregates are scarce. In the current study, the effect of phenylalanine (Phe) and indole presence was first investigated on the structure and stability of human lysozyme (HLZ) and its aggregation under in vitro condition. Tm measurements, circular dichroism and spectrofluorimetric spectra, as well as and transmission electron microscopy (TEM) were performed in this stage. In the next step, pathogenicity of HLZ amorphous aggregates formed in presence or absence of the additives was investigated in vivo, by subcutaneous injection to adult male Wistar rats. Resulting inflamed tissues were studied by hematoxylin and eosin (HE), Congo red and Sudan black staining. Serum levels of liver enzymes (Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST)), specific inflammatory cytokines (Tumor Necrosis Factor Alpha (TNF-alpha) and Interleukin 6 (IL-6)) as well as glucose, cholesterol, and triglyceride levels were measured. Amorphous aggregates of HLZ caused inflammation and affected the number of fat cells, macrophages, cytokines, liver enzymes and glucose. Indole, that increases amorphous aggregates amount as shown with CD, fluorescence, and TEM experiments, leads into more severe inflammation. In presence of Phe, (which stabilizes HLZ structure) a markedly milder inflammatory state is observed in histological results and no increase could be detected in the inflammation-related parameters. In conclusion, amorphous aggregates of HLZ may be pathogenic in vivo, and presence of anti-aggregation compounds (such as Phe) can be effective in diminishing their deleterious manifestations.
Tran, Vy Ha NguyenPerna, ValentinaMikkelsen, Maria DalgaardNguyen, Thuan Thi...
9页
查看更多>>摘要：Endo-fucoidanases, including EC 3.2.1.211 endo-alpha-1,3-L-fucanase and EC 3.2.1.212 endo-alpha-1,4-L-fucanase activities, catalyze depolymerization of fucoidans - a group of bioactive, sulfated fucosyl-polysaccharides found primarily in brown macroalgae (brown seaweeds). Quantitative assessment of endo-fucoidanase activity is critical for characterizing endo-fucoidanase kinetics and for comparing the action of different endo-fucoidanases on different types of fucoidans. However, the current state-of-the-art endo-fucoidanase assay consists of a qualitative assessment based on Carbohydrate-Polyacrylamide Gel Electrophoresis. Here, we report a new quantitative endo-fucoidanase assay based on real time spectral evolution profiling of changes in substrate and product during endo-fucoidanase action using Fourier Transform InfraRed spectroscopy (FTIR) combined with Parallel Factor Analysis (PARAFAC). The FTIR-PARAFAC assay was validated by monitoring the reaction progress of three different microbial endo-fucoidanase enzymes, FcnA Delta 229, FFA2 and Fhf1 Delta 470, on two different fucoidan substrates. The substrates were purified from the brown macroalgae Fucus evanescens and Fucus vesiculosus, respectively. The evolution profiling showed that the strongest spectral change of the fucoidans during enzymatic depolymerization occurred in the spectral range 1220-1260 cm(-1), but the profiles differed depending on the substrate and the enzyme used. Spectral changes within 1220-1260 cm(-1) are in agreement with the enzymatic depolymerization inducing signature changes in the mid-infrared absorption of sulfated fucosyls as sulfate ester bonds and C-O stretching vibrations absorb in this spectral region. Based on the data obtained, we also introduce an activity unit for endo-fucoidanases: One endo-fucoidanase Unit, Uf, is the amount of enzyme able to catalyze a change in the FTIR-PARAFAC score by 0.01 during 498 s of reaction (8.3 min) on 20 g/L pure fucoidan from F. evanescens at 42 degrees C, pH 7.4, 100 mM NaCl and 10 mM CaCl2. This new quantitative endo-fucoidanase assay can pave the way for better kinetic characterizations as well as novel explorations of endo-fucoidanases.
查看更多>>摘要：The alpha-L-rhamnosidase BtRha from Bacteroides thetaiotao VPI-5482 is a specific enzyme that selectively hydrolyzes the alpha-1,2 glycosidic bond between rhamnose and rhamnose, allowing the bioconversion of epimedin C to icariin. In this study, BtRha was molecularly modified using B-factor-saturation mutagenesis strategy and the introduction of disulfide bonds, resulting in a mutant with significantly improved catalytic efficiency, S592C, and two thermally stable mutants, E39W and E39W-S592C. The results showed that the half-lives of E39W and E39WS592C at 55 C were 10.4 and 9.4-fold higher, respectively, than that of the original enzyme, The mutant S592C showed a 63.3% reduction in Km value and a 163.6% increase in catalytic efficiency (kcat/Km value), which improved the ability to hydrolyze epimedin C to icariin effectively. In addition, high-level expression of alpha-L-rhamnosidase mutant S592C was established. With 0.1 mM IPTG as an inducer, induction temperature of 32 C, induction pH of 7.0 and induction OD600 of 50, the maximum activity of mutant S592C reached 182.0 U/ mL in terrific broth medium after 22 h. This is the highest enzyme activity of alpha-L-rhamnosidase which can convert epimedin C to icariin to date. All the results provide a specific and cost-effective alpha-L-rhamnosidase mutant, which will raise its potential interest for the food and pharmaceutical applications.
查看更多>>摘要：Minor flavonoids, such as isoquercitin, icariin and icariside II, have desired bioactivity and bioavailability. To enhance the production efficiency on converting rutin and epimedin C into minor flavonoids, a novel GH family 78 alpha-L-rhamnosidase PodoRha from the Paenibacillus odorifer was cloned and heterologous over-expressed in E. coli BL21(DE3). PodoRha exhibited a high catalytic ability in cleaving outer alpha-1, 2-rhamnopyranosyl moieties of epimedin C and alpha-1, 6-rhamnopyranosyl of rutin. It displayed the highest activity at 45 ? and pH 6.5, and has great thermostability at 40 ?. Under the optimal transformation condition (40 ?, pH 6.5, 1 h incubation), 10 g/L of Rutin was 99.86% transformed into 6.95 g/L of isoquercitin by PodoRha with a crude productivity of 6.95 g.L-1.h(-1); 1.964 g/L icariin was detected after the epimedin C of the total Epimedium flavonoids (10 g/L) were 99.98% converted by PodoRha. Moreover, by using a two-stage conversion system with a crude icariside II productivity of 2.081 g.L-1.h(-1), 5 g/L epimedin C was 99.85% converted into 3.122 g/L of icariside II with 1 h incubation under pH 6.5 at 40 ? after addition of 5.5 U/mL PodoRha; then with 30 min incubation under pH 6.5 at 90 ? after addition of 0.8 U/mL IagBgl1.
查看更多>>摘要：The naturally occurring and mutated promoters inserted into expression plasmids or Escherichia coli chromosome are essential for recombinant protein production and metabolic engineering. Analyzing their activities and screening the promoter libraries require the simple and easy-to-use reporter. Here, we developed a novel and efficient approach to detect the promoter activity, based on E. coli cell growth inhibited by overexpression of bacteriophage phi X174 gene E product (LyE), but recovered by pre-overexpression of Bacillus subtilis MraY (BsMraY). Under the conditional LyE construct expression in the absence or the presence of the BsMraY, activities of promoters including the reported P-T7/lac, P-tac, P-BAD, P-rha, P-hucR, P-prpB, P-cum, the wild type and engineered Ptet for leaky and induced expression, the PthrC for auto-induction, and the P-ms for constitutive expression were assayed. In one-plasmid coexpression system, the P-BAD promoter activity detected using the reporter gene was related to the insertion site. The constructed LyE toxic effects were correlated with toxin expression levels, as determined by the split green fluorescent protein reconstitution. Microscopic analysis showed that cells lysis occurred by the LyE induced with arabinose. Taken together, the toxin reporter construct is a convenient and cost-effective tool to examine the promoter activity in E. coli.
NSTL
Elsevier