Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora

Here we report associations between secondary metabolite production and phylogenetically distinct but closely related marine actinomycete species belonging to the genus Salinispora. The pattern emerged in a study that included global collection sites, and it indicates that secondary metabolite production can be a species-specific, phenotypic trait associated with broadly distributed bacterial populations. Associations between actinomycete phylotype and chemotype revealed an effective, diversity-based approach to natural product discovery that contradicts the conventional wisdom that secondary metabolite production is strain specific. The structural diversity of the metabolites observed, coupled with gene probing and phylogenetic analyses, implicates lateral gene transfer as a source of the biosynthetic genes responsible for compound production. These results conform to a model of selection-driven pathway fixation occurring subsequent to gene acquisition and provide a rare example in which demonstrable physiological traits have been correlated to the fine-scale phylogenetic architecture of an environmental bacterial community.     

Genetic manipulation of secondary metabolite biosynthesis for improved production in Streptomyces and other actinomycetes

Journal of Industrial Microbiology & Biotechnology - Actinomycetes continue to be important sources for the discovery of secondary metabolites for applications in human medicine, animal health,...     

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.     

Engineering secondary metabolite production in plants

Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.     

Tetrodotoxin production of actinomycetes isolated from marine sediment

Ten actinomycetes isolated from various marine sediments were investigated for the production of tetrodotoxin (TTX). Tissue culture assay, high performance liquid chromatography and gas chromatography-mass spectrometry confirmed that the nine strains produced TTX. Actinomycetes may be responsible for TTX accumulation in marine environments.     

Synthetic biology of secondary metabolite biosynthesis in actinomycetes: Engineering precursor supply as a way to optimize antibiotic production

Actinomycetes are a rich source for the synthesis of medically and technically useful natural products. The genes encoding the enzymes for their biosynthesis are normally organized in gene clusters, which include also the information for resistance (in the case of antibacterial compounds), regulation, and transport. This facilitates the manipulation of such pathways by molecular genetic techniques. Recent advances in DNA sequencing and analytical chemistry revealed that not only new strains isolated from yet unexplored habitats, but also already known strains possess a large potential for the synthesis of novel compounds. Synthetic Biology now offers a new perspective to exploit this potential further by generating novel pathways, and thereby novel products, by combining different biosynthetic steps originating from different bacteria. The supply of precursors, which are subsequently incorporated into the final product, is often already organized in a modular manner in nature and may directly be exploited for Synthetic Biology. Here we report examples for the synthesis of building blocks and possibilities to modify and optimize antibiotic biosynthesis, exemplary for the synthesis of the manipulation of the synthesis of the glycopeptide antibiotic balhimycin.     

UV-B辐射增强对陆地植物次生代谢的影响

平流层臭氧的减薄已导致地表中波紫外辐射(UV-B,280～320nm)增强,由于UV-B能被许多生物大分子如蛋白质和核酸吸收并引起分子构象的变化,因此可对植物的各方面产生影响。本文将近年来特别是近5年的UV-B辐射增强对植物次生代谢物影响的研究工作进行了综述。主要包括：UV-B辐射增强对植物紫外吸收物的影响和可能的机制;环境因子的复合作用对植物紫外吸收物的影响和可能的机制;UV-B辐射增强对次生代谢物影响的生态学意义。并对该领域未来的研究作了展望。     

Regulation of secondary metabolite production in filamentous ascomycetes

Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.     

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.     

Genetic potential for secondary metabolite production in stromatolite communities

The cyanobacterial communities associated with stromatolites surviving in extreme habitats are a potentially rich source of bioactive secondary metabolites. We screened for the potential for production of bioactive metabolites in diverse species of cyanobacteria isolated from stromatolites in Hamelin Pool, Shark Bay, Australia. Using degenerate primer sets, putative peptide synthetase and polyketide synthase genes were detected from strains of Symploca, Leptolyngybya, Microcoleus, Pleuorocapsa, and Plectonema sp. Sequence analysis indicates the enzymes encoded by these genes may be responsible for the production of different secondary metabolites, such as hepatotoxins and antibiotics. Computer modelling was also conducted to predict the putative amino acid recognised by the unknown adenylation domain in the NRPS sequences. Mass spectral analysis also allowed the putative identification of the cyclic peptides cyanopeptolin S and 21-bromo-oscillatoxin A in two of the isolates. This is the first time evidence of secondary metabolite production has been shown in stromatolite-associated microorganisms.     

Evolution of secondary metabolite genes in three closely related marine actinomycete species

The marine actinomycete genus Salinispora is composed of three closely related species. These bacteria are a rich source of secondary metabolites, which are produced in species-specific patterns. This study examines the distribution and phylogenetic relationships of genes involved in the biosynthesis of secondary metabolites in the salinosporamide and staurosporine classes, which have been reported for S. tropica and S. arenicola, respectively. The focus is on "Salinispora pacifica," the most recently discovered and phylogenetically diverse member of the genus. Of 61 S. pacifica strains examined, 15 tested positive for a ketosynthase (KS) domain linked to the biosynthesis of salinosporamide K, a new compound in the salinosporamide series. Compound production was confirmed in two strains, and the domain phylogeny supports vertical inheritance from a common ancestor shared with S. tropica, which produces related compounds in the salinosporamide series. There was no evidence for interspecies recombination among salA KS sequences, providing further support for the geographic isolation of these two salinosporamide-producing lineages. In addition, staurosporine production is reported for the first time for S. pacifica, with 24 of 61 strains testing positive for staD, a key gene involved in the biosynthesis of this compound. High levels of recombination were observed between staD alleles in S. pacifica and the cooccurring yet more distantly related S. arenicola, which produces a similar series of staurosporines. The distributions and phylogenies of the biosynthetic genes examined provide insight into the complex processes driving the evolution of secondary metabolism among closely related bacterial species.     

Intracellular ATP levels affect secondary metabolite production in Streptomyces spp

The addition of extracellular ATP (exATP) to four Streptomyces strains had similar effects: low exATP levels stimulated antibiotic production and high levels reduced it. Compared with antibiotic production, the concentrations of intracellular ATP (inATP) in the tested strains were opposite, which suggests a role of inATP in regulating secondary metabolite production. Under inactivation of the polyphosphate kinase gene (ppk) in Streptomyces lividans, we observed the same results: when the inATP level in the mutant strain was lower than in the parent strain, more antibiotic was produced. Combining all the results, a strong inverse relationship between [inATP] and the secondary metabolite production is suggested by this study.     

Repression of secondary metabolite production by exogenous cAMP in Monascus

The cAMP signal pathway controls various biological functions, including secondary metabolism of filamentous fungi. We found that exogenous cAMP represses the production of lovastatin, red pigments, and citrinin in Monascus. Interestingly, a mutant MK-1 with increased lovastatin and red pigments production was not influenced by cAMP on these productions, indicating that cAMP signaling might be lacking in MK-1.     

The role of polyketides in secondary metabolite production by penicillia

The participation of polyketides in the biogenesis of natural products has long been bolstered by chemical analogies. Many isotopic tracer studies have validated the acetate-polymalonate route, via presumptive extended poly-β-carbonyl intermediates, to a variety of fungal metabolites. Though implicit as antibiotic precursors, the ephemeral polyketides have not been isolated, nor perhaps with the exception of acetoacetate, can oligoketides become incorporated intact into secondary metabolites. However, a prototypical oligoketides in its stable lactone form, methyltriacetic lactone (3, 6-dimethyl-l-hydroxy-2-pyrone), has been obtained from the tropolone-producing mold P. Stipitatum. A convenient synthesis of this metabolite, by methylation of triacetic lactone followed by partition chromatographic separation of the resultant positional isomers, has been devised. In an experiment with ¹⁴C-formate, it was shown that the hypothetical, enzyme-bound polyketide precursor to methyltriacetic lactone is probably involved in stipitatie arid formation, and that the origin of the “extra” methyl or methyl-derived carbons of both substances arises from the identical “C₁” pool. Radioactive tracer experiments concerning the biogenesis of pulvilloric acid, a fairly unstable antibiotic substance produced by P. Pulvillorum, showed that its exocyclic carboxyl is formed following initial methyl transfer, whereas the ring system of the molecule is essentially acetate-polymalonate derived. In order to test the hypothesis that methyl-branched C₁₄ polyketide precursors to pulvilloric acid exist and may become integrated into the fatty acid multienzyme complex, presumptive fatty acid congeners to pulvilloric acid such as. 1-methylmyristie, 4-methyllauric, or 2-methyllauric acids were sought. These substances were, however, absent from the mycelial fatty acid spectrum, as well as from the fatty acid moieties of a crystalline glyceridc mixture obtained from the beer. Alternative approaches to the detection or isolation of polyketides are discussed.     

Engineering the plant cell factory for secondary metabolite production

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study. This revised version was published online in August 2006 with corrections to the Cover Date.