Key Laboratory of Industrial Biotechnology

Wuxi, China

Key Laboratory of Industrial Biotechnology

Wuxi, China
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Zhou S.,Key Laboratory of Industrial Biotechnology | Ding R.,Key Laboratory of Industrial Biotechnology | Chen J.,Key Laboratory of Industrial Biotechnology | Chen J.,Jiangnan University | And 3 more authors.
ACS Synthetic Biology | Year: 2017

A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from E. coli were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the PBAD promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (PssrA-UTRrpsT and PdnaKJ-UTRrpsT) and two cascade PUTRs (PUTRssrA-PUTRinfC-rplT and PUTRalsRBACE-PUTRinfC-rplT) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the PBAD promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products. © 2017 American Chemical Society.

Tang C.-D.,Key Laboratory of Industrial Biotechnology | Wang J.-Q.,Key Laboratory of Industrial Biotechnology | Wu M.-C.,Jiangnan University
Journal of Agricultural and Food Chemistry | Year: 2012

A cDNA fragment of the Anman5A, a gene that encodes an acidophilic β-mannanase of Aspergillus niger LW-1 (abbreviated as AnMan5A), was cloned and functionally expressed in Pichia pastoris. Homology alignment of amino acid sequences verified that the AnMan5A belongs to the glycoside hydrolase (GH) family 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assay demonstrated that the recombinant AnMan5A (reAnMan5A), a N-glycosylated protein with an apparent molecular weight of 52.0 kDa, was secreted into the medium. The highest reAnMan5A activity expressed by one P. pastoris transformant, labeled as GSAnMan4-12, reached 29.0 units/mL. The purified reAnMan5A displayed the highest activity at pH 3.5 and 70 °C. It was stable at a pH range of 3.0-7.0 and at a temperature of 60 °C or below. Its activity was not significantly affected by an array of metal ions and ethylenediaminetetraacetic acid (EDTA). The Km and Vmax of the reAnMan5A, toward locust bean gum, were 1.10 mg/mL and 266.7 units/mg, respectively. © 2011 American Chemical Society.

Xu J.Z.,Key Laboratory of Industrial Biotechnology | Zhang J.L.,OriGene Biotechnology Co. | Guo Y.F.,Key Laboratory of Industrial Biotechnology | Jia Q.D.,Key Laboratory of Industrial Biotechnology | Zhang W.G.,Key Laboratory of Industrial Biotechnology
Preparative Biochemistry and Biotechnology | Year: 2014

Fructose-1,6-bisphosphatase (FBPase), which is mainly used to supply NADPH, has an important role in increasing L-lysine production by Corynebacterium glutamicum. However, C. glutamicum FBPase is negatively regulated at the metabolic level. Strains that overexpressed Escherichia coli fructose-1,6-bisphosphatase in C. glutamicum were constructed, and the effects of heterologous FBPase on cell growth and L-lysine production during growth on glucose, fructose, and sucrose were evaluated. The heterologous fructose-1,6-bisphosphatase is insensitive to fructose 1-phosphate and fructose 2,6-bisphosphate, whereas the homologous fructose-1,6-bisphosphatase is inhibited by fructose 1-phosphate and fructose 2,6-bisphosphate. The relative enzyme activity of heterologous fructose-1,6-bisphosphatase is 90.8% and 89.1% during supplement with 3 mM fructose 1-phosphate and fructose 2,6-bisphosphate, respectively. Phosphoenolpyruvate is an activator of heterologous fructose-1,6-bisphosphatase, whereas the homologous fructose-1,6-bisphosphatase is very sensitive to phosphoenolpyruvate. Overexpression of the heterologous fbp in wild-type C. glutamicum has no effect on L-lysine production, but fructose-1,6-bisphosphatase activities are increased 9- to 13-fold. Overexpression of the heterologous fructose-1,6-bisphosphatase increases L-lysine production in C. glutamicum lysC T311I by 57.3% on fructose, 48.7% on sucrose, and 43% on glucose. The dry cell weight (DCW) and maximal specific growth rate (μ) are increased by overexpression of heterologous fbp. A "funnel-cask" diagram is first proposed to explain the synergy between precursors supply and NADPH supply. These results lay a definite theoretical foundation for breeding high L-lysine producers via molecular target. © 2014 Copyright Taylor and Francis Group, LLC.

Ni Y.,Key Laboratory of Industrial Biotechnology | Wang Y.,Key Laboratory of Industrial Biotechnology | Sun Z.,Key Laboratory of Industrial Biotechnology
Applied Biochemistry and Biotechnology | Year: 2012

Clostridium acetobutylicum strains used in most Chinese ABE (acetone-butanol- ethanol) plants favorably ferment starchy materials like corn, cassava, etc., rather than sugar materials. This is one major problem of ABE industry in China and significantly limits the exploitation of cheap waste sugar materials. In this work, cane molasses were utilized as substrate in ABE production by Clostridium saccharobutylicum DSM 13864. Under optimum conditions, total solvent of 19.80 g/L (13.40 g/L butanol) was reached after 72 h of fermentation in an Erlenmeyer flask. In a 5-L bioreactor, total solvent of 17.88 g/L was attained after 36 h of fermentation, and the productivity and yield were 0.50 g/L/h and 0.33 g ABE/g sugar consumption, respectively. To further enhance the productivity, a two-stage semicontinuous fermentation process was steadily operated for over 8 days (205 h, 26 cycles) with average productivity (stage II) of 1.05 g/L/h and cell concentration (stage I) of 7.43 OD 660, respectively. The average batch fermentation time (stage I and II) was reduced to 21?25 h with average solvent of 15.27 g/L. This study provides valuable process data for the development of industrial ABE fermentation process using cane molasses as substrate. © Springer Science+Business Media, LLC 2012.

Wang Z.,Key Laboratory of Industrial Biotechnology | Wang Y.,Key Laboratory of Industrial Biotechnology | Zhang D.,Key Laboratory of Industrial Biotechnology | Li J.,Key Laboratory of Industrial Biotechnology | And 5 more authors.
Bioresource Technology | Year: 2010

Alkaline polygalacturonate lyase (PGL) production by Pichia pastoris GS115 was used as a model to study the mechanism and strategy for enhancing heterologous protein production. In order to enhance cell viability and volumetric recombinant protein productivity, sorbitol, which had been confirmed to be a non-repressive carbon source, was added together with methanol during the induction phase. The resultant PGL activity was up to 1593 U mL-1, which was enhanced 1.85-fold compared to the control (863 U mL-1) cultured with sorbitol added at a constant rate of 3.6 g h-1 L-1 after an induction period of 100 h. Further results revealed that an appropriate sorbitol co-feeding strategy not only decreased the cell mortality to 8.8% (the control is about 23.1%) in the end of fermentation, but also reduced the proteolytic degradation of PGL. © 2009 Elsevier Ltd. All rights reserved.

Ding Y.,Jiangnan University | Ding Y.,Key Laboratory of Industrial Biotechnology | Cai Y.,Key Laboratory of Industrial Biotechnology | Cai Y.,Jiangnan University
Biopolymers | Year: 2013

The conformational dynamics of xylanase A from Streptomyces lividans (Sl-XlnA) were studied using Molecular Dynamics (MD) simulation to identify the thermally sensitive regions. Sl-XlnA begins to unfold at loop4 and this unfolding expands to the loops near the N-terminus. The high flexibility of loop6 during the 300 K simulation is related to its function. The intense movements of the 310-helices also affect the structural stability. The interaction between the α4β5-loop and the neighboring α5β6-loop plays a crucial role in stabilizing the region from the α4β5-loop to α6. The most thermally sensitive region is from β3 to loop4. The high mobility of the long loop4 easily transfers to the adjacent β4 and α4 and causes β4 and α4 to fluctuate. And, salt bridges ASP124-ARG79, ASP200-ARG159, and ASP231-LYS166 formed a "clamp" to stabilize the region including α4, β4, β5, β6, and β7. © 2013 Wiley Periodicals, Inc.

Gao X.,Key Laboratory of Industrial Biotechnology | Cui W.,Key Laboratory of Industrial Biotechnology | Ding N.,Key Laboratory of Industrial Biotechnology | Liu Z.,Key Laboratory of Industrial Biotechnology | And 2 more authors.
Prion | Year: 2013

Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387 and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward LeupNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wildtype BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry. © 2013 Landes Bioscience.

Chen J.,Jiangnan University | Ding Y.,Jiangnan University | Ding Y.,Key Laboratory of Industrial Biotechnology | Xu W.,Jiangnan University
Journal of Biological Systems | Year: 2013

Metabolic networks are useful representations of the metabolic capabilities of cells. A comparison of metabolic networks across species is essential to better understand how evolutionary pressures shape these networks. By comparing the set of reactions that are expected to occur in an organism with the set of reactions in reference metabolic pathways, it is possible to infer the main metabolic functions of an organism. In this paper, the metabolic networks of the mesophilic archaeon Methanosarcina acetivorans and the thermophilic archaeon Methanopyrus kandleri have been reconstructed based on the KEGG LIGAND database, followed by four topological statistical analyses of the nodes in the two networks to compare their metabolic networks. The values of average degree and characteristic path length are very small but clustering coefficient is relatively large. The results show that the complete metabolic networks of M. acetivorans and M. kandleri possessed small-world network properties. Then we used Girvan-Newman modular algorithm to identify hub modules and compared hub modules with non-hub modules, respectively. The results show that M. kandleri metabolic network has a better modular organization than the M. acetivorans network. M. acetivorans includes 39 modules, 25 modules of them are independent, and 15 modules are functionally pure. On the other hand, M. kandleri includes 30 modules. Among them, there are 20 independent modules, and 14 of them are functionally pure. These results further indicated that the present approach for identifying modules yields modules that have biologically significant functions. We also identified hub modules of the metabolic networks and found that these hub modules are carbohydrate metabolism and amino acid metabolism. The conclusions obtained from such studies provide a broad overview of the similarities and differences between organism's metabolic networks. These will be very helpful for further research on thermostability of methanogens. © 2013 World Scientific Publishing Company.

Liu J.,University of Georgia | Liu J.,Key Laboratory of Industrial Biotechnology | Zeng L.,University of Georgia | Zeng L.,Qingdao Agricultural University | And 3 more authors.
Soil Research | Year: 2013

Soil water repellency (SWR) caused by organic coatings on soil particles can lead to serious loss in crop production and turfgrass quality. In laboratory experiments, we tested the novel concept of direct application of enzymes to alleviate SWR. In a biofuel research project on fungal pre-treatment of switchgrass (Panicum virgatum L.) for improved saccharification, enzymatic co-products (mainly laccase mixed with other trace enzymes) were produced based on fermentation periods of 18, 36, 54, and 72 days. We characterised enzyme activities of the 18-72-day crude enzyme extracts (CEE) and applied undiluted or diluted solutions (dilutions of 5-, 10-, and 100-fold) to eight air-dried, SWR soils from several golf courses. These soils exhibited water drop penetration times (WDPT) of 345-7439s (i.e. moderately to very strongly hydrophobic) and all showed a large decrease in SWR to WDPT <60s after application of undiluted CEE and various dilutions of CEE for 3 days with a 1:1 soil:solution ratio (10g air-dried soil and 10mL CEE solution). The observed decrease in WDPT was positively related to increased enzyme activity level for each soil in an exponential or logarithmic relationship. Most of the improvement in SWR was observed within 1 day. Enzyme activity was maintained to varying degrees in the soil solution for up to 5 days. These preliminary results suggest that it may be feasible to use direct enzyme application from CEE, as a biomass fermentation byproduct, for remediation of hydrophobic soils, which could also offer a cost benefit for biomass fermentation. © CSIRO 2013.

Li Z.,Jiangnan University | Li B.,Jiangnan University | Gu Z.,Jiangnan University | Du G.,Jiangnan University | And 5 more authors.
Carbohydrate Research | Year: 2010

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca2+, Ba2+, and Zn2+ in a concentration-dependent manner, while it is dramatically inhibited by Hg2+. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation. © 2010.

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