SynBio Research Platform

Tianjin, China

SynBio Research Platform

Tianjin, China
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Sun T.,Tianjin University | Sun T.,SynBio Research Platform | Chen L.,Tianjin University | Chen L.,SynBio Research Platform | And 2 more authors.
Journal of Proteome Research | Year: 2017

Bacterial small RNAs (sRNAs) and two-component systems (TCSs) were two vital regulatory mechanisms employed by microorganisms to respond to environmental changes and stresses. As a promising "autotrophic cell factory", photosynthetic cyanobacteria have attracted a lot of attention these years. Although most studies focused on studying the roles of sRNAs or TCS regulators in stress response in photosynthetic cyanobacteria, limited work has elucidated their potential crosstalk. Our previous work has identified a negative sRNA regulator CoaR and a positive response regulator Slr1037 both related to 1-butanol stress regulation in Synechocystis sp. PCC6803. In this work, the potential crosstalk between CoaR and Slr1307 (i.e., the coregulated genes mediated by CoaR and Slr1037) was identified and validated through quantitative proteomics and quantitative real-time PCR (qRT-PCR), respectively. The results showed that the sensitive phenotype to 1-butanol of Δslr1037 could be rescued by suppressing coaR in Δslr1037, probably due to the fact that some target genes of Slr1037 could be reactivated by repression of CoaR. Twenty-eight coregulated proteins mediated by CoaR and Slr1037 were found through quantitative proteomics, and 10 of the annotated proteins were validated via qRT-PCR. This study proved the existence of crosstalk between sRNAs and response regulators and provided new insights into the coregulation of biofuel resistance in cyanobacteria. © 2017 American Chemical Society.

Sun T.,Tianjin University | Sun T.,SynBio Research Platform | Pei G.,Tianjin University | Pei G.,SynBio Research Platform | And 5 more authors.
Biotechnology for Biofuels | Year: 2017

Background: Microbial small RNAs (sRNAs) have been proposed as valuable regulatory elements for optimizing cellular metabolism for industrial purposes. However, little information is currently available on functional relevance of sRNAs to biofuels tolerance in cyanobacteria. Results: Here, we described the identification and functional characterization of a novel 124 nt sRNA Ncl1460 involved in tolerance to biofuel 1-butanol in Synechocystis sp. PCC 6803. The expression of Ncl1460 was verified by blotting assay and its length was determined through 3′ RACE. Further analysis showed that Ncl1460 was a negative regulator of slr0847 (coaD) and slr0848 operon responsible for coenzyme A (CoA) synthesis possibly via promoter-directed transcriptional silencing mechanisms which has been widely discovered in eukaryote; thus Ncl1460 was designated as CoaR (CoA Biosynthesis Regulatory sRNA). The possible interaction between CoaR and target genes was suggested by CoA quantification and green fluorescent protein assays. Finally, a quantitative proteomics analysis showed that CoaR regulated tolerance to 1-butanol possibly by down-regulating CoA biosynthesis, resulting in a decrease of fatty acid metabolism and energy metabolism. Conclusions: As the first reported sRNA involved CoA synthesis and 1-butanol tolerance in cyanobacteria, this study provides not only novel insights in regulating mechanisms of essential pathways in cyanobacteria, but also valuable target for biofuels tolerance and productivity modifications. © 2017 The Author(s).

Yang B.,Nankai University | Yang B.,Key Laboratory of Molecular Microbiology and Technology | Feng L.,Nankai University | Feng L.,Key Laboratory of Molecular Microbiology and Technology | And 7 more authors.
Nature Communications | Year: 2015

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. Here we identify a virulence-regulating pathway in which the biotin protein ligase BirA signals to the global regulator Fur, which in turn activates LEE (locus of enterocyte effacement) genes to promote EHEC adherence in the low-biotin large intestine. LEE genes are repressed in the high-biotin small intestine, thus preventing adherence and ensuring selective colonization of the large intestine. The presence of this pathway in all nine EHEC serotypes tested indicates that it is an important evolutionary strategy for EHEC. The pathway is incomplete in closely related small-intestinal enteropathogenic E. coli due to the lack of the Fur response to BirA. Mice fed with a biotin-rich diet show significantly reduced EHEC adherence, indicating that biotin might be useful to prevent EHEC infection in humans. © 2015 Macmillan Publishers Limited. All rights reserved.

Peng C.,Tianjin University | Luo H.,Tianjin University | Zhang X.,Tianjin University | Gao F.,Tianjin University | Gao F.,SynBio Research Platform
Frontiers in Microbiology | Year: 2015

DNA replication, one of the central events in the cell cycle, is the basis of biological inheritance. In order to be duplicated, a DNA double helix must be opened at defined sites, which are called DNA replication origins (ORIs). Unlike in bacteria, where replication initiates from a single replication origin, multiple origins are utilized in the eukaryotic genomes. Among them, the ORIs in budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have been best characterized. In recent years, advances in DNA microarray and next-generation sequencing technologies have increased the number of yeast species involved in ORIs research dramatically. The ORIs in some non-conventional yeast species such as Kluyveromyces lactis and Pichia pastoris have also been genome-widely identified. Relevant databases of replication origins in yeast were constructed, then the comparative genomic analysis can be carried out. Here, we review several experimental approaches that have been used to map replication origins in yeast and some of the available web resources related to yeast ORIs. We also discuss the sequence characteristics and chromosome structures of ORIs in the four yeast species, which can be utilized to improve yeast replication origins prediction. © 2015 Peng, Luo, Zhang and Gao.

Luo H.,Tianjin University | Zhang C.-T.,Tianjin University | Gao F.,Tianjin University | Gao F.,SynBio Research Platform
Frontiers in Microbiology | Year: 2014

DNA replication is one of the most basic processes in all three domains of cellular life. With the advent of the post-genomic era, the increasing number of complete archaeal genomes has created an opportunity for exploration of the molecular mechanisms for initiating cellular DNA replication by in vivo experiments as well as in silico analysis. However, the location of replication origins (oriCs) in many sequenced archaeal genomes remains unknown. We present a web-based tool Ori-Finder 2 to predict oriCs in the archaeal genomes automatically, based on the integrated method comprising the analysis of base composition asymmetry using the Z-curve method, the distribution of origin recognition boxes identified by FIMO tool, and the occurrence of genes frequently close to oriCs. The web server is also able to analyze the unannotated genome sequences by integrating with gene prediction pipelines and BLAST software for gene identification and function annotation. The result of the predicted oriCs is displayed as an HTML table, which offers an intuitive way to browse the result in graphical and tabular form. The software presented here is accurate for the genomes with single oriC, but it does not necessarily find all the origins of replication for the genomes with multiple oriCs. Ori-Finder 2 aims to become a useful platform for the identification and analysis of oriCs in the archaeal genomes, which would provide insight into the replication mechanisms in archaea. © 2014 Luo, Zhang and Gao.

Qi Z.,Tianjin University | Qi Z.,SynBio Research Platform | Pei G.,Tianjin University | Pei G.,SynBio Research Platform | And 4 more authors.
Scientific Reports | Year: 2014

Microbial syntrophic metabolism has been well accepted as the heart of how methanogenic and other anaerobic microbial communities function. In this work, we applied a single-cell RT-qPCR approach to reveal gene-expression heterogeneity in a model syntrophic system of Desulfovibrio vulgaris and Methanosarcina barkeri, as compared with the D. vulgaris monoculture. Using the optimized primers and single-cell analytical protocol, we quantitatively determine gene-expression levels of 6 selected target genes in each of the 120 single cells of D. vulgaris isolated from its monoculture and dual-culture with M. barkeri. The results demonstrated very significant cell-to-cell gene-expression heterogeneity for the selected D. vulgaris genes in both the monoculture and the syntrophic dual-culture. Interestingly, no obvious increase in gene-expression heterogeneity for the selected genes was observed for the syntrophic dual-culture when compared with its monoculture, although the community structure and cell-cell interactions have become more complicated in the syntrophic dual-culture. In addition, the single-cell RT-qPCR analysis also provided further evidence that the gene cluster (DVU0148-DVU0150) may be involved syntrophic metabolism between D. vulgaris and M. barkeri. Finally, the study validated that single-cell RT-qPCR analysis could be a valuable tool in deciphering gene functions and metabolism in mixed-cultured microbial communities.

Wang Y.,Tianjin University | Wang Y.,SynBio Research Platform | Chen L.,Tianjin University | Chen L.,SynBio Research Platform | And 2 more authors.
Biotechnology for Biofuels | Year: 2016

Background: 3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. In our previous study, the biosynthetic pathway of 3-HP was constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803, which led to 3-HP production directly from CO2 at a level of 837.18 mg L−1 (348.8 mg/g dry cell weight). As the production and accumulation of 3-HP in cells affect cellular metabolism, a better understanding of cellular responses to 3-HP synthesized internally in Synechocystis will be important for further increasing 3-HP productivity in cyanobacterial chassis. Results: Using a engineered 3-HP-producing SM strain, in this study, the cellular responses to 3-HP internally produced were first determined using a quantitative iTRAQ-LC-MS/MS proteomics approach and a LC-MS-based targeted metabolomics. A total of 2264 unique proteins were identified, which represented about 63 % of all predicted protein in Synechocystis in the proteomic analysis; meanwhile intracellular abundance of 24 key metabolites was determined by a comparative metabolomic analysis of the 3-HP-producing strain SM and wild type. Among all identified proteins, 204 proteins were found up-regulated and 123 proteins were found down-regulated, respectively. The proteins related to oxidative phosphorylation, photosynthesis, ribosome, central carbon metabolism, two-component systems and ABC-type transporters were up-regulated, along with the abundance of 14 metabolites related to central metabolism. The results suggested that the supply of ATP and NADPH was increased significantly, and the precursor malonyl-CoA and acetyl-CoA may also be supplemented when 3-HP was produced at a high level in Synechocystis. Confirmation of proteomic and metabolomic results with RT-qPCR and gene-overexpression strains of selected genes was also conducted, and the overexpression of three transporter genes putatively involved in cobalt/nickel, manganese and phosphate transporting (i.e., sll0385, sll1598 and sll0679) could lead to an increased 3-HP production in Synechocystis. Conclusions: The integrative analysis of up-regulated proteome and metabolome data showed that to ensure the high-efficient production of 3-HP and the normal growth of Synechocystis, multiple aspects of cells metabolism including energy, reducing power supply, central carbon metabolism, the stress responses and protein synthesis were enhanced in Synechocystis. The study provides an important basis for further engineering cyanobacteria for high 3-HP production. © 2016 The Author(s).

Lin F.,Tianjin University | Guo X.,Tianjin University | Lu W.,Tianjin University | Lu W.,SynBio Research Platform
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2015

Ginsenosides are the major pharmacological components in ginseng. Microorganisms from a ginseng field were isolated to identify transformation of ginsenosides. Based on HPLC and LC–MS analysis, strain LFJ1403 showed strong activities to transform ginsenoside Rb1 to Rd as the sole product. Phylogenetic analysis of 18S rDNA indicated that LFJ1403 belonged to Aspergillus versicolor. Through comparing four systems of transforming Rb1 to Rd, strain LFJ1403 was found to secrete ginsenoside-converting enzymes in the spore production phase of plate culture. This result suggested that the enzyme could be directly obtained from the plate. The spore suspension, which contained the exocrine enzyme, was easy to prepare and efficient for biotransformation of ginsenoside Rb1 to Rd. Further study showed that the maximum bioconversion rate was 96 % (w/w) in shake flasks when a spore suspension system was used with optimized biotransformation conditions. Scale-up of this system to 2L resulted in an 85 % conversion rate. The ginsenoside Rb1 converting enzyme was separated by gradient HPLC with Q-Sepharose column, and its β-glucosidase activity and Rb1-converting ability was assayed by the 4-Nitrophenyl-β-d-glucopyranoside (PNPG) method and HPLC with C18 column, respectively. We obtained 130 U ml−1 enzymatic activity with the purified β-glucosidase. This is the first report on efficiently converting ginsenoside using extracellular enzyme directly from the fungus spore production phase of solid culture. © 2015, Springer International Publishing Switzerland.

Wang G.,Tianjin University | Wang G.,SynBio Research Platform | Huang D.,Nankai University | Li Y.,Tianjin University | And 5 more authors.
Bioresource Technology | Year: 2015

In this work, wheat bran (WB) was utilized as feedstock to synthesize fumaric acid by Rhizopus oryzae. Firstly, the pretreatment process of WB by dilute sulfuric acid hydrolysis undertaken at 100. °C for 30. min offered the best performance for fumaric acid production. Subsequently, through optimizing the seed culture medium, a suitable morphology (0.55. mm pellets diameter) of R. oryzae was obtained. Furthermore, a metabolic-based approach was developed to profile the differences of intracellular metabolites concentration of R. oryzae between xylose (the abundant sugar in wheat bran hydrolysate (WBH)) and glucose metabolism. The xylitol, sedoheptulose 7-phosphate, ribulose 5-phosphate, glucose 6-phosphate, proline and serine were responsible for fumaric acid biosynthesis limitation in xylose fermentation. Consequently, regulation strategies were proposed, leading to a 149% increase in titer (up to 15.4. g/L). Finally, by combinatorial regulation strategies the highest production was 20.2. g/L from WBH, 477% higher than that of initial medium. © 2014 Elsevier Ltd.

Liang D.-M.,Tianjin University | Liang D.-M.,Key Laboratory of Systems Bioengineering | Liang D.-M.,SynBio Research Platform | Liu J.-H.,Tianjin University | And 14 more authors.
Chemical Society Reviews | Year: 2015

Glycosylation reactions mainly catalyzed by glycosyltransferases (Gts) occur almost everywhere in the biosphere, and always play crucial roles in vital processes. In order to understand the full potential of Gts, the chemical and structural glycosylation mechanisms are systematically summarized in this review, including some new outlooks in inverting/retaining mechanisms and the overview of GT-C superfamily proteins as a novel Gt fold. Some special features of glycosylation and the evolutionary studies on Gts are also discussed to help us better understand the function and application potential of Gts. Natural product (NP) glycosylation and related Gts which play important roles in new drug development are emphasized in this paper. The recent advances in the glycosylation pattern (particularly the rare C- and S-glycosylation), reversibility, iterative catalysis and protein auxiliary of NP Gts are all summed up comprehensively. This review also presents the application of NP Gts and associated studies on synthetic biology, which may further broaden the mind and bring wider application prospects. © The Royal Society of Chemistry.

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