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Zhu Q.,East China University of Science and Technology | Chen X.,East China University of Science and Technology | Wu J.,East China University of Science and Technology | Zhou Y.,East China University of Science and Technology | And 6 more authors.
European Food Research and Technology | Year: 2017

Chlorella vulgaris: is a nutritional food with high protein content. Thus, 43 protein sequences from C. vulgaris were in silico gastrointestinal digested with the aid of BIOPEP. A peptide library of 468 di- and tri-peptides was built from the produced peptides. Six peptides, AAR, VPA, VPW, IPL, IPR, and PPL, were selected for DPP-IV inhibitory assay based on their sequence feature with Pro or Ala at the second N-terminal site. VPA, VPW, IPL, and IPR had potency of DPP-IV inhibition. VPW and IPR with the same N-terminal and second N-terminal sites as those of diprotin A (IPI) and diprotin B (VPL) achieved relative inhibitory IC50 value of 6.4 and 6.9 versus IPI. These peptides were further demonstrated gastrointestinal stable in vitro and could also inhibit the DPP-IV in mouse serum. Molecular docking illustrated the inhibitory mechanism of VPW and IPR. Both VPW and IPR binding with DPP-IV through hydrogen bonds, van Edward Mars interactions, and hydrophobic interactions. However, VPW formed more stable interaction with DPP-IV. The results suggested that C. vulgaris proteins would be a good source for DPP-IV inhibitory peptides. © 2017 Springer-Verlag Berlin Heidelberg


Yin K.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | Wang Q.,Shanghai Collaborative Innovation Center for Biomanufacturing | Xiao J.,East China University of Science and Technology | And 3 more authors.
FEMS Microbiology Letters | Year: 2017

As a leading pathogen, Edwardsiella piscicida can cause hemorrhagic septicemia in fish and gastro-intestinal infections in humans. The two-component regulatory system EsrA-EsrB plays essential roles in pathogenesis through the type III and type VI secretion systems, and hemolysin production in E. piscicida. It is unclear whether other virulence- or stress response-associated genes are regulated by EsrA-EsrB. In this study, the proteomes of wild-type E. piscicida EIB202 and esrB mutant strains were compared to reveal EsrB regulon components after growth in Luria-Bertani broth (LB). Overall, the expression levels of nine genes exhibited significant changes, and five of them required the presence of EsrB, while others exhibited higher levels in the esrB mutant. The diverse functions of these proteins were identified, including amino acid metabolism, oxidative stress defense and energy production. Interestingly, superoxidase dismutase and thiol peroxidase were the most significantly down-regulated by EsrB. Furthermore, other reported reactive oxygen species (ROS) resistance-related genes were also down-regulated by EsrB as revealed by quantitative real-time. Compared with the wild-type and the complement strain esrB+, ΔesrB displayed a significantly enhanced ROS resistance. These results demonstrated that EsrB plays important roles in the ROS resistance pathway in E. piscicida grown in LB conditions. © FEMS 2016. All rights reserved.


Yin Y.,East China University of Science and Technology | Cai M.,East China University of Science and Technology | Zhou X.,East China University of Science and Technology | Li Z.,Shanghai JiaoTong University | And 2 more authors.
Applied Microbiology and Biotechnology | Year: 2016

The knowledge of biosynthesis gene clusters, production improving methods, and bioactivity mechanisms is very important for the development of filamentous fungi metabolites. Metabolic engineering and heterologous expression methods can be applied to improve desired metabolite production, when their biosynthesis pathways have been revealed. And, stable supplement is a necessary basis of bioactivity mechanism discovery and following clinical trial. Aspergillus terreus is an outstanding producer of many bioactive agents, and a large part of them are polyketides. In this review, we took polyketides from A. terreus as examples, focusing on 13 polyketide synthase (PKS) genes in A. terreus NIH 2624 genome. The biosynthesis pathways of nine PKS genes have been reported, and their downstream metabolites are lovastatin, terreic acid, terrein, geodin, terretonin, citreoviridin, and asperfuranone, respectively. Among them, lovastatin is a well-known hypolipidemic agent. Terreic acid, terrein, citreoviridin, and asperfuranone show good bioactivities, especially anticancer activities. On the other hand, geodin and terretonin are mycotoxins. So, biosynthesis gene cluster information is important for the production or elimination of them. We also predicted three possible gene clusters that contain four PKS genes by homologous gene alignment with other Aspergillus strains. We think that this is an effective way to mine secondary metabolic gene clusters. © 2016, Springer-Verlag Berlin Heidelberg.


Ma X.,East China University of Science and Technology | Xiao Y.,East China University of Science and Technology | Xu H.,Collaborative Innovation Center for Petrochemical New Materials | Lei K.,East China University of Science and Technology | And 2 more authors.
Materials Science and Engineering C | Year: 2016

Drug-eluting stents with biodegradable polymers as reservoirs have shown great potential in the application of interventional therapy due to their capability of local drug delivery. Herein, poly(l-lactide-co-ϵ-caprolactone) (PLCL) with three different compositions as carriers for ciprofloxacin lactate (CIP) was coated on ureteral stents by the dipping method. To simulate a body environment, degradation behavior of PLCL as both the bulk film and the stent coating was evaluated in artificial urine (AU, pH 6.20) respectively at 37 °C for 120 days by tracing their weight/Mn loss, water absorption and surface morphologies. Furthermore, the release profile of the eluting drug CIP on each stent exhibited a three-stage pattern, which was greatly affected by the degradation behavior of PLCL except for the burst stage. Interestingly, the degradation results on both macroscopic and molecular level indicated that the release mechanism at stage I was mainly controlled by chain scission instead of the weight loss or morphological changes of the coatings. While for stage II, the release profile was dominated by erosion resulting from the hydrolysis reaction autocatalyzed by acidic degradation residues. In addition, ciprofloxacin-loaded coatings displayed a significant bacterial resistance against E. coli and S. aureus without obvious cytotoxicity to Human foreskin fibroblasts (HFFs). Our results suggested that PLCL copolymers with tunable degradation rate as carriers for ciprofloxacin lactate could be used as a promising long-term antibacterial coating for ureteral stents. © 2016 Elsevier B.V. All rights reserved.


Zhang J.,Shanghai Key Laboratory of Advanced Polymeric Materials | Xiao Y.,Shanghai Key Laboratory of Advanced Polymeric Materials | Xiao Y.,Shanghai Collaborative Innovation Center for Biomanufacturing | Xu H.,Collaborative Innovation Center for Petrochemical New Materials | And 3 more authors.
Polymer Chemistry | Year: 2016

The introduction of reactive groups such as -NH2, -COOH etc. onto a poly(ϵ-caprolactone) (PCL) backbone was necessary for further modification but a well-controlled approach remains a challenge for synthetic chemistry. Carboxyl functionalized PCL was typically prepared via three steps involving the synthesis of the corresponding monomer with a carboxyl-protecting group, polymerization and the removal of the protection. Except for obtaining purified monomers and a decent polymerization, the most critical step in carboxyl PCL synthesis was the deprotection from the degradable main chain. Therefore, electronic effects and steric hindrance of the protecting group were taken into account with the aim for controllable polymerization and feasible deprotection. Substituents including -CH3, H and NO2 with discriminative electronegativity on the para position of the benzyl protecting group have been selected to investigate their behavior in monomer preparation, polymerization and deprotection, respectively. It turned out that the electron donating group (-CH3) displayed the highest selectivity in the monomer preparation, excellent control over the polymerization degree and the most efficient removal of the protecting groups without degradation of the backbone. In addition, the reactivity of the pendant carboxyl groups on PCL was demonstrated by amidation with 4-amino-2,2,6,6-tetramethylpiperidinyloxy (4-amino-TEMPO). Our results also provide guidance information on preparing well-defined biodegradable polymers with pendant reactive groups such as polypeptides, expanding the library of novel biomaterials. © 2016 The Royal Society of Chemistry.


Wei C.,East China University of Science and Technology | Zhang Y.,East China University of Science and Technology | Xu H.,Collaborative Innovation Center for Petrochemical New Materials | Xu Y.,East China University of Science and Technology | And 2 more authors.
Journal of Materials Chemistry B | Year: 2016

Well-defined diselenide-centered biodegradable tri-block copolymers methoxyl poly(ethylene glycol)-b-poly(ϵ-caprolactone)-b-methoxyl poly(ethylene glycol) (mPEG-PCL-Se)2 were precisely synthesized by the combination of ring opening polymerization using di(1-hydroxyethylene) diselenide as a new initiator and a facile coupling reaction. The amphiphilic block copolymers enabled the formation of self-assembled micelles which revealed an excellent reductive response to glutathione (GSH) due to the unique reduction-responsive cleavage of the diselenide bond. Such GSH response ensured an enhanced release of anticancer drugs (DOX) from the micelles in simulative tumor microenvironments; moreover, the drug release could be changed to some extent through fine-tuning the chemical composition of the copolymers. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements confirmed that the DOX-loaded micelles could be efficiently taken up by oral squamous carcinoma (HN30) cells and DOX was released into the nuclei of cancer cells following 4 h of incubation. The cell viability assays showed the diselenide-containing polymers were nontoxic up to a tested concentration (400 μg mL-1), while the DOX-loaded micelles exhibited an evident inhibition toward HN30 cells. Therefore, the reduction-labile biodegradable (mPEG-PCL-Se)2 may offer an alternative platform for tumor-targeting therapy. © The Royal Society of Chemistry 2016.


Chu T.,East China University of Science and Technology | Huang Y.,East China University of Science and Technology | Hou M.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | And 5 more authors.
Applied and Environmental Microbiology | Year: 2015

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter ParaBAD, into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo-triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo, the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines. © 2015, American Society for Microbiology.


Zhang X.,East China University of Science and Technology | He H.,East China University of Science and Technology | Yin Y.,East China University of Science and Technology | Zhou W.,East China University of Science and Technology | And 4 more authors.
Journal of Biotechnology | Year: 2016

Light, as an important environmental signal, generally brings about a broad regulation in fungal metabolism. In this work, we aim to explore the light-responded metabolic rules so as to further develop a feasible and effective light regulation strategy for production of anticancer polyketide 1403C by marine fungus Halorosellinia sp.. Light derived production enhancement of polyketides was first found in shake flask. To further understand this well working black box, light-responded cell growth, polyketides biosynthesis, metabolic behaviors (enzymes activities and organic acids levels) and mycelia morphology were then investigated in 5-L bioreactor. By comparing cultures under constant irradiation and dark conditions, the entire bioprocess was divided into two phases. During 0-60. h, light presumably stimulated relevant metabolism to generate sufficient energy, NADPH and carbon skeleton, particularly malonyl-CoA, which was favorable for mycelia growth and polyketides accumulation. After 60. h, light did harm to biomass and polyketides production. Consequently, a light-dark shift strategy was proposed and verified in 5-L bioreactor. It led to a maximal 1403C production of 1.67. g/L, which was 24% and 74% higher than those obtained under constant irradiation and dark conditions, respectively. © 2016 Elsevier B.V.


Chu T.,East China University of Science and Technology | Ni C.,East China University of Science and Technology | Zhang L.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | And 5 more authors.
Microbial Cell Factories | Year: 2015

Background: Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers. Results: In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS=67.0%) or E. tarda EIB202 (RPS=72.3%). Conclusions: Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts. © Chu et al.


Huang M.,East China University of Science and Technology | Gao Y.,East China University of Science and Technology | Zhou X.,East China University of Science and Technology | Zhang Y.,East China University of Science and Technology | And 2 more authors.
Bioprocess and Biosystems Engineering | Year: 2016

Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris. © 2016 Springer-Verlag Berlin Heidelberg

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