Diversity and exploration of bioactive marine actinomycetes in the Bay of Bengal of the Puducherry coast of India

The present study was designed to investigate the Puducherry coast of the Bay of Bengal, India for the diversity of bioactive actinomycetes. A total of 50 actinomycete strains were isolated from the marine sediments and most of the strains were belongs to Streptomyces. These strains were identified by means of morphological physiological, biochemical and cultural characteristics. The isolates were subjected to shake flask fermentation and the secondary metabolites were extracted with ethyl acetate and screened for cytotoxicity, hemolytic activity and antimicrobial activity against selected bacterial and fungal pathogens. The cytotoxic activity was evaluated using HeLa cell lines by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay, hemolytic activity on mouse erythrocytes and the antifungal activity was evaluated by MTT cytotoxic assay against Aspergillus niger, Aspergillus fumigatus and Candida albicans. The antibacterial activity was studied against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. The cytotoxicity and antimicrobial activity of secondary metabolite was found to be concentration dependent and nearly 24% of isolates showed significant antimicrobial, hemolytic and cytotoxic activity. The results of our study indicate the diversity and bioactive potential of marine actinomycetes isolated in the Puducherry coast.     

Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering

Production of secondary metabolites is a process influenced by several physico-chemical factors including nutrient supply, oxygenation, temperature and pH. These factors have been traditionally controlled and optimized in industrial fermentations in order to enhance metabolite production. In addition, traditional mutagenesis programs have been used by the pharmaceutical industry for strain and production yield improvement. In the last years, the development of recombinant DNA technology has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathways. These efforts are usually focused in redirecting precursor metabolic fluxes, deregulation of biosynthetic pathways and overexpression of specific enzymes involved in metabolic bottlenecks. In addition, efforts have been made for the heterologous expression of biosynthetic gene clusters in other organisms, looking not only for an increase of production levels but also to speed the process by using rapidly growing and easy to manipulate organisms compared to the producing organism. In this review, we will focus on these genetic approaches as applied to bioactive secondary metabolites produced by actinomycetes.     

Targeting the Bacillus subtilis genome: an efficient and clean method for gene disruption

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented β cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne β site-specific recombinase is transferred into the background. Protein β catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术.该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化....     

Mechanisms of resistance to self-produced toxic secondary metabolites in plants

Plants produce a variety of secondary metabolites to protect themselves from pathogens and herbivores and/or to influence the growth of neighbouring plants. Some of these metabolites are toxic to the producing cells when their target sites are present in the producing organisms. Therefore, a specific self-resistance mechanism must exist in these plants. Self-resistance mechanisms, including extracellular excretion, vacuolar sequestration, vesicle transport, extracellular biosynthesis, and accumulation of the metabolite in a non-toxic form, have been proposed thus far. Recently, a new mechanism involving mutation of the target protein of the toxic metabolite has been elucidated. We review here the mechanisms that plants use to prevent self-toxicity from the following representative compounds: cannabinoids, flavonoids, diterpene sclareol, alkaloids, benzoxazinones, phenylpropanoids, cyanogenic glycosides, and glucosinolates.     

A role for alternative end-joining factors in homologous recombination and genome editing in Chinese hamster ovary cells

CRISPR technologies greatly foster genome editing in mammalian cells through site-directed DNA double strand breaks (DSBs). However, precise editing outcomes, as mediated by homologous recombination (HR) repair, are typically infrequent and outnumbered by undesired genome alterations. By using knockdown and overexpression studies in Chinese hamster ovary (CHO) cells as well as characterizing repaired DNA junctions, we found that efficient HR-mediated genome editing depends on alternative end-joining (alt-EJ) DNA repair activities, a family of incompletely characterized DNA repair pathways traditionally considered to oppose HR. This dependency was influenced by the CRISPR nuclease type and the DSB-to-mutation distance, but not by the DNA sequence surrounding the DSBs or reporter cell line. We also identified elevated Mre11 and Pari, and low Rad51 expression levels as the most rate-limiting factors for HR in CHO cells. Counteracting these three bottlenecks improved precise genome editing by up to 75%. Altogether, our study provides novel insights into the complex interplay of alt-EJ and HR repair pathways, highlighting their relevance for developing improved genome editing strategies.     

基因组挖掘技术在海洋放线菌天然产物研究开发中的应用及展望

利用微生物的基因组信息预测其合成特定天然产物的潜能, 进而进行新化合物分离纯化和结构鉴定的基因组挖掘技术, 已经成为国内外研究的热点, 并在多种细菌和真菌的天然产物发现中得到成功应用。本文综述了基因组挖掘技术的最新进展, 包括生物信息分析和结构预测、基因组指导的天然产物的发现、沉默基因的激活和异源表达技术等, 以及我国学者开发的转录组挖掘技术, 并重点综述了影像质谱技术在基因组挖掘中的应用。目前对海洋放线菌进行基因组挖掘的研究还比较少, 而基因组挖掘技术的发展, 将极大地促进对海洋放线菌天然产物的发现和鉴定。未来除了充分挖掘可培养微生物的基因组, 对未培养微生物宏基因组的挖掘将进一步深入。此外, 除了开发利用基因组中合成天然产物的结构基因和调节基因, 还应该充分开发利用其他不同的遗传元件, 包括不同转录活性和响应不同环境条件和信号的启动子, 以及具有调节作用的RNA等。     

ABC transporters involved in the transport of plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, polyphenols, quinones and many further compounds having combined structures of those groups. Physiological roles of those metabolites for plants are still under investigation, but they play, at least in part, important functions as protectants for plant bodies against herbivores and pathogens, as well as from physical stresses like ultraviolet light and heat. In order to accomplish these functions, biosyntheses and accumulation of secondary metabolites are highly regulated in a temporal and spatial manner in plant organs, where they can appropriately accumulate. In this mini-review, I introduce the mechanism of accumulation and membrane transport of these metabolites, in particular, focusing on ATP-binding cassette transporters involved.     

Sampling techniques and comparative extraction procedures for quantitative determination of intra- and extracellular metabolites in filamentous fungi

Two methods for rapid sampling and three procedures for extraction of metabolites from the filamentous fungus Monascus ruber were compared. It is shown that arrest of metabolism by either dropping the mycelial cultures in liquid nitrogen or by spraying them on a 60% solution of methanol kept at −40°C followed by rapid centrifugation at −10°C were equally effective. Metabolites were extracted from mycelia using different procedures including acid and alkaline treatments, permeabilization by cold chloroform and extraction by boiling buffered ethanol, to demonstrate that the latter method gave the best results both in terms of recovery and stability of metabolites. In addition, this method is very simple to handle and allows the use of very low amounts (i.e. 10–20 mg dry mass) of cellular material since the removal of ethanol by evaporation after extraction results in a concentration step of metabolites.     

Rational engineering strategies for achieving high-yield,high-quality and high-stability of natural product production in actinomycetes

Actinomycetes are recognized as excellent producers of microbial natural products, which have a wide range of applications, especially in medicine, agriculture and stockbreeding. The three main indexes of industrialization (titer, purity and stability) must be taken into overall consideration in the manufacturing process of natural products. Over the past decades, synthetic biology techniques have expedited the development of industrially competitive strains with excellent performances. Here, we summarize various rational engineering strategies for upgrading the performance of industrial actinomycetes, which include enhancing the yield of natural products, eliminating the by-products and improving the genetic stability of engineered strains. Furthermore, the current challenges and future perspectives for optimizing the industrial strains more systematically through combinatorial engineering strategies are also discussed.     

Serine recombinases as tools for genome engineering

The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ?C31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ?C31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.     

Genetic recombination pathways and their application for genome modification of human embryonic stem cells

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens.     

Trends in the search for bioactive microbial metabolites

Summary Bioactive microbial metabolites are attracting increasing attention as useful agents for medicine, veterinary medicine, agriculture, and as unique biochemical tools. A review of the current trends in the discovery-of new metabolites shows that the number of active compounds with non-antibiotic type of activity has increased, resulting in an expansion of the variety of bioactivity of microbial metabolites. Factors that contribute to the increased rate of discovery include: development of new methods for activity measurement, exploitation of novel groups of microorganisms as sources of active compounds, new directions for chemical modification, and incorporation of newer knowledge of biotechnology into screening systems. To exemplify this, typical screening methods, and chemical and biological properties of several bioactive compounds obtained by these methods are discussed.     

植物次生代谢基因簇研究进展

类似于原核生物的操纵子,在真核生物（如酵母、真菌、昆虫等）基因组中也出现了彼此功能相关的非同源基因成簇存在的现象。这些基因形成基因簇,可参与多种次生代谢途径。近年来,植物中也发现了越来越多的参与次生代谢产物合成的基因簇,它们已成为植物生物学研究的热点。本文总结并分析了植物中已鉴定的次生代谢基因簇。这些基因簇存在于玉米（Zea mays L.）、水稻（Oryza sativa L.）、拟南芥（Arabidopsis thaliana（L.） Heynh.）、番茄（Solanum lycopersicum L.）等植物的基因组中,分别参与合成苯并噁唑嗪酮类、萜类和生物碱类等次生代谢产物。本文通过解析这些基因簇的组成及结构特点,对其特征进行总结,探讨了基因簇形成的分子机理及其调控机制,对植物次生代谢基因簇在合成生物学及代谢工程学中的研究方向和应用前景进行了展望。     

Evolutionary relics dominate the small number of secondary metabolism genes in the hemibiotrophic fungus Dothistroma septosporum

Fungal secondary metabolites have important functions for the fungi that produce them, such as roles in virulence and competition. The hemibiotrophic pine needle pathogen Dothistroma septosporum has one of the lowest complements of secondary metabolite (SM) backbone genes of plant pathogenic fungi, indicating that this fungus produces a limited range of SMs. Amongst these SMs is dothistromin, a well-characterised polyketide toxin and virulence factor that is required for expansion of disease lesions in Dothistroma needle blight disease. Dothistromin genes are dispersed across six loci on one chromosome, rather than being clustered as for most SM genes. We explored other D. septosporum SM genes to determine if they are associated with gene clusters, and to predict what their likely products and functions might be. Of nine functional SM backbone genes in the D. septosporum genome, only four were expressed under a range of in planta and in culture conditions, one of which was the dothistromin PKS backbone gene. Of the other three expressed genes, gene knockout studies suggested that DsPks1 and DsPks2 are not required for virulence and attempts to determine a functional squalestatin-like SM product for DsPks2 were not successful. However preliminary evidence suggested that DsNps3, the only SM backbone gene to be most highly expressed in the early stage of disease, appears to be a virulence factor. Thus, despite the small number of SM backbone genes in D. septosporum, most of them appear to be poorly expressed or dispensable for virulence in planta. This work contributes to a growing body of evidence that many fungal secondary metabolite gene clusters might be non-functional and may be evolutionary relics.     

Organization of an Arabidopsis thaliana gene cluster on chromosome 4 including the RPS2 gene, in the Brassica nigra genome

 Genetic and physical maps, consisting of a large number of DNA markers for Arabidopsis thaliana chromosomes, represent excellent tools to determine the organization of related genomes such as those of Brassica. In this paper we report the chromosomal localization and physical analysis by pulsed-field gel electrophoresis (PFGE) of a well-defined gene complex of A. thaliana in the Brassica nigra genome (B genome n=8). This complex is approximately 30 kb in length in A. thaliana and contains a cluster of six genes including ABI1 (ABA-responsive), RPS2 (resistance against Pseudomonas syringae, a bacterial disease), CK1 (casein kinase I), NAP (nucleosome-assembly protein), X9 and X14 (both of unknown function). The Arabidopsis chromosomal complex was found to be duplicated and conserved in gene number at different levels in the Brassica genome. Linkage group B1 had the most-conserved arrangement carrying all six genes tightly linked. Group B4 had an almost complete complex except for the absence of RPS2. Other partial complexes of fewer members were found on three other chromosomes. Our studies demonstrate that by this approach it is possible to identify ancestrally related chromosome segments in a complex and duplicated genome, such as the genome of B. nigra, permitting one to draw conclusions as to its origin and evolution. Received: 11 July 1997 / Accepted: 9 October 1997     

Recent advances in developing molecular tools for targeted genome engineering of mammalian cells

Various biological molecules naturally existing in diversified species including fungi, bacteria, and bacteriophage have functionalities for DNA binding and processing. The biological molecules have been recently actively engineered for use in customized genome editing of mammalian cells as the molecule-encoding DNA sequence information and the underlying mechanisms how the molecules work are unveiled. Excitingly, multiple novel methods based on the newly constructed artificial molecular tools have enabled modifications of specific endogenous genetic elements in the genome context at efficiencies that are much higher than that of the conventional homologous recombination based methods. This minireview introduces the most recently spotlighted molecular genome engineering tools with their key features and ongoing modifications for better performance. Such ongoing efforts have mainly focused on the removal of the inherent DNA sequence recognition rigidity from the original molecular platforms, the addition of newly tailored targeting functions into the engineered molecules, and the enhancement of their targeting specificity. Effective targeted genome engineering of mammalian cells will enable not only sophisticated genetic studies in the context of the genome, but also widely-applicable universal therapeutics based on the pinpointing and correction of the disease-causing genetic elements within the genome in the near future. [BMB Reports 2015; 48(1): 6-12]     

Relocation of genes generates non-conserved chromosomal segments in Fusarium graminearum that show distinct and co-regulated gene expression patterns

Background

Genome comparisons between closely related species often show non-conserved regions across chromosomes. Some of them are located in specific regions of chromosomes and some are even confined to one or more entire chromosomes. The origin and biological relevance of these non-conserved regions are still largely unknown. Here we used the genome of Fusarium graminearum to elucidate the significance of non-conserved regions.

Results

The genome of F. graminearum harbours thirteen non-conserved regions dispersed over all of the four chromosomes. Using RNA-Seq data from the mycelium of F. graminearum, we found weakly expressed regions on all of the four chromosomes that exactly matched with non-conserved regions. Comparison of gene expression between two different developmental stages (conidia and mycelium) showed that the expression of genes in conserved regions is stable, while gene expression in non-conserved regions is much more influenced by developmental stage. In addition, genes involved in the production of secondary metabolites and secreted proteins are enriched in non-conserved regions, suggesting that these regions could also be important for adaptations to new environments, including adaptation to new hosts. Finally, we found evidence that non-conserved regions are generated by sequestration of genes from multiple locations. Gene relocations may lead to clustering of genes with similar expression patterns or similar biological functions, which was clearly exemplified by the PKS2 gene cluster.

Conclusions

Our results showed that chromosomes can be functionally divided into conserved and non-conserved regions, and both could have specific and distinct roles in genome evolution and regulation of gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-191) contains supplementary material, which is available to authorized users.