Linking chemistry and genetics in the growing cyanobactin natural products family.

Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ~30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins.

Utilization of real-time PCR to detect Rangifer Cornu contamination in Cervi Parvum Cornu.

Cervi parvum cornu (CPC) is a well-known ethnopharmacological source, whereas Rangifer cornu (RC) is not considered to be a major source. CPC is distributed in sliced form. Addition of RC to CPC has become an issue in CPC distribution because the appearance of sliced RC is not different from sliced CPC. Therefore, a real-time polymerase chain reaction (PCR) method was developed in this study to detect contaminating RC in CPC. The C-VIC and R-FAM primer/probe sets were designed to specifically amplify CPC and RC DNA, respectively. The specificities and sensitivities of real-time PCR using two primer/probe sets and the applicability of the real-time PCR to powder mixtures, which involved mixtures of powdered CPC and powdered RC in diverse ratios, were evaluated. Real-time PCR using C-VIC and R-FAM primer/probe sets specifically and sensitively amplified both CPC and RC DNA. Furthermore, real-time RCR sensitively detected RC DNA in the powder mixtures of CPC and RC. These results indicate that this real-time PCR method using two primer/probe sets is sufficiently applicable for the detection of contaminant RC in CPC.

Integrating carbon-halogen bond formation into medicinal plant metabolism.

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Bacterial biosurfactants, and their role in microbial enhanced oil recovery (MEOR).

Surfactants are chemically synthesized surface-active compounds widely used for large number of applications in various industries. During last few years there is increase demand of biological surface-active compounds or biosurfactants which are produced by large number of microorganisms as they exert biodegradability, low toxicity and widespread application compared to chemical surfactants. They can be used as emulsifiers, de-emulsifiers, wetting agents, spreading agents, foaming agents, functional food ingredients and detergents. Various experiments at laboratory scale on sand-pack columns and field trials have successfully indicated effectiveness of biosurfactants in microbial enhanced oil recovery (MEOR).

The use of mushroom-forming fungi for the production of N-glycosylated therapeutic proteins.

The market for N-glycosylated therapeutic proteins represents multi-billion dollars in sales and is growing more than 10% each year. This requires cost-effective production platforms that display correct and homogeneous N-glycosylation. Based on recent results, we propose to use mushroom-forming basidiomycetes for the production of N-glycosylated therapeutic proteins.

New natural product biosynthetic chemistry discovered by genome mining.

Analyses of plant and microbial genome sequences have revealed many genes and gene clusters encoding proteins similar to those known to be involved in the biosynthesis of structurally-complex natural products. Many of these genes and gene clusters do not direct the production of known metabolites of the organism in which they are found. Others represent novel gene clusters that direct the biosynthesis of known natural products. This article highlights several examples of new biosynthetic chemistry discovered in the course of investigating such gene clusters.

[Strategies on the construction of high-quality microbial natural product library--a review].

The microbial secondary metabolites are always the main source of the natural drugs. The historical paradigm of the deep ocean as a biological 'desert' has shifted to one of a 'rainforest' owing to the isolation of many novel microbes and their associated bioactive compounds. A high quality microbial and its natural product library are crucial for successful drug and other screenings. However, how to build up the library efficiently is still faced with many bottlenecks. To overcome the difficulties and limitations, we reviewed the following strategies: (1) diversifying microbial sources and dereplication; (2) diversifying gene sources and dereplication; (3) diversifying microbial metabolite sources and dereplication; (4) novel methods and technologies for bioactive secondary metabolites, especially the high-throughput synergy screening for multi-target drugs. Bioactive compounds isolated using the above chemical microbiology strategies have not only shown importance in biotechnological and pharmaceutical applications but also increased our understanding of the diversity of microbe, ecosystem functions and the exploitable biology.

Phytochemistry and pharmacogenomics of natural products derived from traditional Chinese medicine and Chinese materia medica with activity against tumor cells.

The cure from cancer is still not a reality for all patients, which is mainly due to the limitations of chemotherapy (e.g., drug resistance and toxicity). Apart from the high-throughput screening of synthetic chemical libraries, natural products represent attractive alternatives for drug development. We have done a systematic bioactivity-based screening of natural products derived from medicinal plants used in traditional Chinese medicine. Plant extracts with growth-inhibitory activity against tumor cells have been fractionated by chromatographic techniques. We have isolated the bioactive compounds and elucidated the chemical structures by nuclear magnetic resonance and mass spectrometry. By this strategy, we identified 25-O-acetyl-23,24-dihydro-cucurbitacin F as a cytotoxic constituent of Quisqualis indica. Another promising compound identified by this approach was miltirone from Salvia miltiorrhiza. The IC50 values for miltirone of 60 National Cancer Institute cell lines were associated with the microarray-based expression of 9,706 genes. By COMPARE and hierarchical cluster analyses, candidate genes were identified, which significantly predicted sensitivity or resistance of cell lines to miltirone.

Drug discovery from natural products.

Natural product compounds are the source of numerous therapeutic agents. Recent progress to discover drugs from natural product sources has resulted in compounds that are being developed to treat cancer, resistant bacteria and viruses and immunosuppressive disorders. Many of these compounds were discovered by applying recent advances in understanding the genetics of secondary metabolism in actinomycetes, exploring the marine environment and applying new screening technologies. In many instances, the discovery of a novel natural product serves as a tool to better understand targets and pathways in the disease process. This review describes recent progress in drug discovery from natural sources including several examples of compounds that inhibit novel drug targets.

Establishment of in vitro test system for the evaluation of the estrogenic activities of natural products.

In order to evaluate estrogenic compounds in natural products, an in vitro detection system was established. For this system, the human breast cancer cell line MCF7 was stably transfected using an estrogen responsive chloramphenicol acetyltransferase (CAT) reporter plasmid yielding MCF7/pDsCAT-ERE119-Ad2MLP cells. To test the estrogenic responsiveness of this in vitro assay system, MCF7/pDsCAT-ERE119-Ad2MLP cells were treated with various concentrations of 17beta-estradiol. Treatments of 10(-8) to 10(-12) M 17beta-estradiol revealed significant concentration dependent estrogenic activities compared with ethanol. We used in vitro assay system to detect estrogenic effects in Puerariae radix and Ginseng radix Rubra extracts. Treatment of 500 and 50 microg/ml of Puerariae radix extracts increased the transcriptional activity approximately 4- and 1.5-fold, respectively, compared with the ethanol treatment. Treatment of 500, 50, and 5 microg/ml of Ginseng radix Rubra extracts increased the transcriptional activity approximately 3.2-, 2.7-, and 1.4-fold, respectively, compared with the ethanol treatment. These observations suggest that Puerariae radix and Ginseng radix Rubra extracts have effective estrogenic actions and that they could be developed as estrogenic supplements.

Diversifying microbial natural products for drug discovery.

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.