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Tianjin, China

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.

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.

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.

Li Y.,Tianjin University | Li Y.,SynBio Research Platform | Lin Z.,Tianjin University | Lin Z.,SynBio Research Platform | And 11 more authors.
Metabolic Engineering | Year: 2015

Engineering cellular metabolism for improved production of valuable chemicals requires extensive modulation of bacterial genome to explore complex genetic spaces. Here, we report the development of a CRISPR-Cas9 based method for iterative genome editing and metabolic engineering of Escherichia coli. This system enables us to introduce various types of genomic modifications with near 100% editing efficiency and to introduce three mutations simultaneously. We also found that cells with intact mismatch repair system had reduced chance to escape CRISPR mediated cleavage and yielded increased editing efficiency. To demonstrate its potential, we used our method to integrate the β-carotene synthetic pathway into the genome and to optimize the methylerythritol-phosphate (MEP) pathway and central metabolic pathways for β-carotene overproduction. We collectively tested 33 genomic modifications and constructed more than 100 genetic variants for combinatorially exploring the metabolic landscape. Our best producer contained15 targeted mutations and produced 2.0. g/L β-carotene in fed-batch fermentation. © 2015 International Metabolic Engineering Society.

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