Abstract:

Abstract:[Objective] The objective of this research was to compare CRISRP-Cas9 system and mazF-mediated method for large deletions in Saccharomyces cerevisiae.[Methods] We made a 26.5 kb deletion from YKL072W to YKL061W using the foresaid two methods. The two methods were analyzed from the perspective of transformation rate and accuracy of deletion.[Results] There were five colonies appeared on the plate in average using CRISRP-Cas9 system and all of these were correct. And 100 colonies were observed using the mazF-mediated method and the accuracy was 93%, a little bit lower than CRISPR-Cas9 system.[Conclusion] Both methods are good to make large deletion in Saccharomyces cerevisiae. The CRISRP-Cas9 system has a high accuracy and easy to use, the mazF-mediated method is stable and does not need the PAM sequence.

Abstract:[Objective] To detect the clustered regularly interspaced short palindromic repeats (CRISPR) in Vibrio parahaemolyticus, and analyze its structural diversities in different sources of V. parahaemolyticus strains.[Methods] The primers of convincing CRISPR structure CRISPR-1 were designed by using the sequence according to CRISPR database, while the primers of questionable CRISPR structure CRISPR-2 were designed according to the literature. PCR approach was used to detect the CRISPR locus in all 79 strains, and all CRISPR sequences were analyzed using CRISPR Finder. Furthermore, the structural diversities of CRISPR in different sources of V. parahaemolyticus were analyzed using bioinformatics methods.[Results] The positive rate of CRISPR-1 was 92.41% and of CRISPR-2 96.20%. The strains possessed with these two locus accounted for 89.87% of the total strains, and only one strain did not contain any locus. There was no difference between the repeats of CRISPR-1 and CRISPR-2 from different sources of V. parahaemolyticus, whereas the spacers of those two locus in clinical strains had more variations than those in environmental strains. Therefore, two CRISPR locus formed 8 spectral patterns (numbers A-H) according to the variations of spacers. Except type F, the patterns A-E, G only were found in the clinical strains. And the type H containing none of locus was only found in environmental strains.[Conclusion] CRISPR commonly existed in V. parahaemolyticus. There were differences in the structure of CRISPR between environmental and clinical strains.

Abstract:Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) is a kind of genetic structure containing interval repeat sequences and is widespread in bacteria and archaea genome. CRISPR/CAS system mediated by RNA can provide bacteria with a specific immune protection mechanism to resist the invasion of the virus or phage. Through the transformation of CRISPR/CAS system Ⅱ, it has become another efficient technology for targeted gene modification after the zinc finger nuclease (ZFNs) and Tate nuclease (TALENs), which is of flexibility, efficiency, cheapness and easy-operating. At present, CRISPR/Cas technology has been applied to many organisms such as microorganism, mammalian cells, fruit flies, rice and some research achievements in genetic modification have been obtained. The structure, classification, molecular mechanism of genome editing, application prospect in industrial microorganism and problems of CRISPR/Cas system are reviewed in this paper.

Abstract:CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technology, established in 2013, guided by special RNA and initiated by endonuclease, has been developed into the third generation of genome editing technology, which is widely used in genome editing among varieties of species including animals, plants, bacteria and fungi with high efficiency and specificity. This review briefly introduces the characteristics and the application of three genome editing technologies based on endonucleases, describes the composes and mechanism of CRISPR/Cas9 mediated genome editing, summarizes the application of this tool in Saccharomyces cerevisiae and filamentous fungi in genome editing. In the other hand, we put forward the protocol about using CRISPR/Cas9 technology in filamentous fungi, such as the design of sgRNA cassette, the optimization of Cas9 cassette, the screening of resistance marker, the selection of receptor, and so on. Furthermore, some suggestions were given about the problems which often encounter in practical application, such as off-target effects, the addition of Cas9 nuclear localization signal, the selection of promoter, and multi-gene editing, to provide a theoretical and technological references for the beginners in filamentous fungus gene editing field.

Abstract:The discovery of CRISPR-Cas system breaks certain theory which adaptive immunity had been long time considered as unique characteristics of eukaryotic organism. CRISPR-Cas, a new adaptive immunity system widespread in bacteria and archaea, protects the host from invasion of exogenous nucleic acids by capturing and using the Cas protein and crRNA to resisting it when invading again. In recent years, CRISPR-Cas system has aroused extensive attention and exploration. From microbial perspective, this article introduces a brief overview on classification, mechanism and application which have achieved significant breakthrough to provide valuable reference for further research on CRISPR-Cas system and its application.

Abstract:Heredity and variation embody the beauty and mystery of life. In the process of continuous exploration, researchers have established genetics that is full of rigorous systematic and scientific methods. It is generally known that gene editing tools are indispensable in the study of genetics. CRISPR/Cas discovered in recent years as a new gene editing toolbox, has many characteristics, such as strong specificity and target ability, and become a hot spot of research and development. Here, we review the latest progress of CRISPR/Cas system including diversity, molecular mechanism and application.

Abstract:[Objective] The purpose of this study is to study the acetylation of PhaE, one subunit of polyhydroxyalkanoate (PHA) synthase (PhaEC), and its function in PHA biosynthesis in Haloferax mediterranei.[Methods] PHA granules were collected by sucrose density gradient centrifugation. The acetylated sites of PhaE were identified by LC-MS/MS. The acetylated lysine (K) sites were mutated to arginine (R, mimics deacetylation) or glutamine (Q, mimics acetylation), and the mutated gene was knocked into the chromosome on its original location through pop-in/pop-out method. Taking wild-type strain as control, the cell growth, ability of glucose consumption, and the PHA accumulation were detected in different mutated strains. The relative abundance of PhaE on the PHA granule was also analyzed by Western blot in wild-type and mutated strains.[Results] Two acetylated sites (K105 and K170) were identified on PhaE. Six different mutant (K105R, K170R, K105Q, K170Q, K105R/K170R and K105Q/K170Q) strains were constructed. The fermentation experiments showed that none of the single-site mutation could affect the cell growth and PHA synthesis, and no obvious effect was observed when the two sites were simultaneously mutated to Q. However, the PHA accumulation significantly decreased when these two acetylated sites were mutated to R at the same time. Interestingly, Western blotting showed that abundance of PhaE on the PHA granules almost decreased to 58% of the wild type.[Conclusion] The deacetylation of PhaE decreased the PHA synthesis from glucose metabolism, which was likely resulted from the inefficient interaction between the deacetylated PhaE and the PHA granules (or PhaC on the PHA granules), thus led to reduction of the activity of PhaEC synthase for PHA biosynthesis.

Abstract:[Objective] MCP2923 is a putative of Comamonas testosteroni CNB-1. The objective of this study was to experimentally characterize MCP2923 for chemotactic response.[Methods] Using swimming plate assay, we determined chemotaxis towards 35 aromatic compounds and 9 Tricarboxylic Acid Cycle intermediates. Agrose-in-plug was used to screen aromatic chemoattractants that might bind to MCP2923 directly. To study the ligand of MCP2923, Isothermal Titration Calorimetry experiment was done to 11 chemoattractants.[Results] Swimming plate assay showed that CNB-1 responded to 12 aromatic compounds and 9 Tricarboxylic Acid Cycle intermediates that were defined as strong, medium and weak chemoattractants. Knockout MCP2923 gene reduced chemotactic responses to these chemoattractants. Complemented with MCP2923 gene to CNB-1△20, chemotaxis toward 15 chemoattractants was restored. Deletion of the ligand binding domain of MCP2923, chemotaxis failed to complement. Isothermal Titration Calorimetry experiment showed no response to the tested 11 compounds, including protocatechuic acid and 4-hydroxybenzoic acid.[Conclusion] MCP2923 mediates chemotaxis towards both aromatic compounds and Tricarboxylic Acid Cycle cycle intermediates by CNB-1. The ligand binding domain of MCP2923 is necessary for triggering chemotaxis toward these chemoattractants.

Abstract:To accommodate the variable environments, bacterial cells often need to take swift changes on metabolism while keep their genomic sequence unchanged. Thus, epigenetic regulation becomes an important way for bacteria because no genomic alteration is required. As one of the DNA modifications, DNA methylation is the most well-known epigenetic tools among all the organisms. Here in this review, we gave a comprehensive introduction of two orphan methyltransferases Dam (DNA adenine methyltransferase) and CcrM (Cell cycle-regulated methyltransferase), as examples of epigenetic regulation in prokaryotes. We majorly talked about their role in bacterial DNA replication initiation, DNA mismatch repair, regulation of gene expression, virulence and phase variation. Moreover, we also discussed the trends in this field based on a newly-generated technology Chromosome Conformation Capture (3C) and a novel DNA phosphorothioate modification.

Abstract:Protein post-translational modifications, as an important step in protein maturation, participate in the regulation of diverse protein biological functions. Methylation is one of the modifications and plays multiple roles in cellular processes, ranging from eukaryotes to prokaryotes. Protein methylation occurs on various amino acid residues, such as lysine, arginine, glutamine and others. This review summarized the progress in protein methylation, including both eukaryote and prokaryote, as well as histone and non-histone proteins.

Abstract:Toxin-Antitoxin Systems (TA) are prevalent in mobile genetic elements and chromosomes of prokaryotes. They have been classified into six different types based on the nature of the antitoxin and the way of activating the toxin. TA systems have been shown to play roles in the formation of dormancy and persister cell formation, and they have also been shown to participate in other important physiological processes such as programmed cell death, biofilm formation and general stresses responses. In the last three decades, the cellular targets and the regulation of TA systems have been revealed in both commensal and pathogenic bacteria; however, they have been largely unexplored in environmental microbes. In addition, most studies have focused on Type Ⅱ TA systems, and more efforts need to be devoted to other types of TA systems. This review focused on recent discoveries of novel TA systems, their novel targets, the interaction among different TA systems, and the regulatory roles of the antitoxins. Moreover, we discussed the potential future directions for research in the TA field as well as their applications in biotechnology and in medicine.

Abstract:[Objective] The aim of this study was to study the diversity of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.[Methods] Ion exchange chromatography, genome sequencing and heterologous expression were used to study the diversity of lipolytic enzymes in Stenotrophomonas maltophilia OUC_Est10.[Results] Stenotrophomonas maltophilia OUC_Est10 could secret a wide range of lipolytic enzymes (lipases and esterases) as revealed by ion exchange chromatography. The complete genome is of 4668743 bp in length, with an average GC content of 66.25%. Genome annotation indicated the presence of 33 candidate genes whose products possess the predicted lipolytic enzyme activities. Analysis of catalytic features was carried out by expressing five putative lipolytic enzyme genes, and lipolytic enzymes in OUC_Est10 had different catalytic properties.[Conclusion] We proved that Stenotrophomonas maltophilia OUC_Est10 was a good candidate to produce diverse lipolytic enzymes, with potential applications in various fields.

Abstract:Traditional bioassay-guided natural product discovery has led to re-isolation of a large quantity of known compounds, and thus dramatically stunted the process of drug development. Large-scale genome sequencing revealed the existence of numerous cryptic biosynthetic gene clusters in microbes, which have attracted increasing attention in the research field of natural product discovery. In this review, we summarized the strategies for activation of microbial cryptic gene clusters at pathway-specific and pleiotropic levels, and discussed trends in structural identification technologies used for genome-guided compound mining. Activation of cryptic gene clusters will open new era for effective discovery of novel bioactive natural products.

Abstract:Red/ET recombineering is an in vivo homologous recombination-based genetic engineering method used primarily in Escherichia coli by using short homology arms (40-50 bp), based on the expression of either redα/redβ from the Red operon of λ phage or the analogous recE/recT from Rac prophage that are located in the E. coli chromosome. It can rapidly, efficiently and accurately modify and manipulate genomic and episome DNA. In this review, we introduce the progress of Red/ET recombineering in E. coli and other bacteria, and its application in microbial genome mining, especially in the field of heterologous expression of microbial biosynthetic gene clusters.