Hybrid isoprenoid secondary metabolite production in terrestrial and marine actinomycetes

Terpenoids are among the most ubiquitous and diverse secondary metabolites observed in nature. Although actinomycete bacteria are one of the primary sources of microbially derived secondary metabolites, they rarely produce compounds in this biosynthetic class. The terpenoid secondary metabolites that have been discovered from actinomycetes are often in the form of biosynthetic hybrids called hybrid isoprenoids (HIs). HIs include significant structural diversity and biological activity and thus are important targets for natural product discovery. Recent screening of marine actinomycetes has led to the discovery of a new lineage that is enriched in the production of biologically active HI secondary metabolites. These strains represent a promising resource for natural product discovery and provide unique opportunities to study the evolutionary history and ecological functions of an unusual group of secondary metabolites.     

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.     

An <Emphasis Type="Italic">N</Emphasis>-acyl homolog of mycothiol is produced in marine actinomycetes

Marine actinomycetes have generated much recent interest as a potentially valuable source of novel antibiotics. Like terrestrial actinomycetes the marine actinomycetes are shown here to produce mycothiol as their protective thiol. However, a novel thiol, U25, was produced by MAR2 strain CNQ703 upon progression into stationary phase when secondary metabolite production occurred and became the dominant thiol. MSH and U25 were maintained in a reduced state during early stationary phase, but become significantly oxidized after 10 days in culture. Isolation and structural analysis of the monobromobimane derivative identified U25 as a homolog of mycothiol in which the acetyl group attached to the nitrogen of cysteine is replaced by a propionyl residue. This N-propionyl-desacetyl-mycothiol was present in 13 of the 17 strains of marine actinomycetes examined, including five strains of Salinispora and representatives of the MAR2, MAR3, MAR4 and MAR6 groups. Mycothiol and its precursor, the pseudodisaccharide 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol, were found in all strains. High levels of mycothiol S-conjugate amidase activity, a key enzyme in mycothiol-dependent detoxification, were found in most strains. The results demonstrate that major thiol/disulfide changes accompany secondary metabolite production and suggest that mycothiol-dependent detoxification is important at this developmental stage.     

Chemical and molecular characterization of Phomopsis and Cytospora-like endophytes from different host plants in Brazil

Phomopsis and related taxa comprise important endophytic and plant pathogenic species, and are known for the production of a diverse array of secondary metabolites. Species concepts within this group based on morphological characters and assumed host specificity do not reflect phylogenetic affinities. Additional phenotypic characters, such as profiles of secondary metabolites, are needed for practical species recognition. We investigated 36 strains of Phomopsis spp. and Cytospora-like fungi, obtained as endophytes of different host plants in Brazil, using metabolite profiling based on HPLC-UV/liquid chromatography -mass spectrometry (LC-MS) combined with cluster analysis of the results. Strains were also subjected to phylogenetic analyses based on internal transcribed spacer (ITS) rDNA. Six chemotypes were identified. Chemotypes 1-5 contained Phomopsis strains, while Cytospora-like strains formed the chemotype 6. Strains of chemotype 1 typically produced alternariols, altenusin, altenuene, cytosporones, and dothiorelones. Alternariol and seven unknown compounds were consistently produced by strains of chemotype 2. Members of chemotypes 3-5 produced poor metabolite profiles containing few chemical markers. Cytospora-like endophytes (chemotype 6) produced a characteristic set of metabolites including cytosporones and dothiorelones. Bayesian and Maximum Parsimony (MP) trees classified strains of each chemotype into single phylogenetic lineages or closely related groups. Strains of chemotypes 1 and 2 formed a monophyletic group along with Diaporthe neotheicola. The remaining Phomopsis strains formed monophyletic (chemotype 4) or polyphyletic (chemotypes 3 and 5) lineages inside a large and well supported clade. Cytospora-like strains formed a monophyletic lineage located at an intermediary position between Diaporthe/Phomopsis and Valsa/Cytospora clades. The combined results show that the production of secondary metabolites by Phomopsis and related Diaporthales may be species-specific, giving support to the use of metabolite profiling and chemical classification for phenotypic recognition and delimitation of species.     

植物次生代谢途径的遗传操作

植物次生代谢产物种类极其丰富,是人类的宝贵资源,这些产物及其合成途径相关酶具有空间特异性分布的特征。植物次生代谢途径的调控是个复杂的过程,受代谢产物水平、多酶复合物相互作用等多种因素的影响。通过遗传操作改造代谢过程,调控产物在植物体内的含量,是一条切实可行和具有广阔发展空间的途径。目前,改造植物次生代谢途径可以采取单基因操作和多基因操作两种策略进行。     

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.     

Biodiversity of Actinomycetes Associated with Caribbean Sponges and Their Potential for Natural Product Discovery

Marine actinomycetes provide a rich source of structurally unique and bioactive secondary metabolites. Numerous genera of marine actinomycetes have been isolated from marine sediments as well as several sponge species. In this study, 16 different species of Caribbean sponges were collected from four different locations in the coastal waters off Puerto Rico in order to examine diversity and bioactive metabolite production of marine actinomycetes in Caribbean sponges. Sediments were also collected from each location, in order to compare actinomycete communities between these two types of samples. A total of 180 actinomycetes were isolated and identified based on 16S rRNA gene analysis. Phylogenetic analysis revealed the presence of at least 14 new phylotypes belonging to the genera Micromonospora, Verruscosispora, Streptomyces, Salinospora, Solwaraspora, Microbacterium and Cellulosimicrobium. Seventy-eight of the isolates (19 from sediments and 59 from sponges) shared 100 % sequence identity with Micromonospora sp. R1. Despite having identical 16S rRNA sequences, the bioactivity of extracts and subsequent fractions generated from the fermentation of both sponge- and sediment-derived isolates identical to Micromonospora sp. R1 varied greatly, with a marked increase in antibiotic metabolite production in those isolates derived from sponges. These results indicate that the chemical profiles of isolates with high 16S rRNA sequence homology to known strains can be diverse and dependent on the source of isolation. In addition, seven previously reported dihydroquinones produced by five different Streptomyces strains have been purified and characterized from one Streptomyces sp. strain isolated in this study from the Caribbean sponge Agelas sceptrum.     

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

Background  

Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.     

Mining novel biosynthetic machineries of secondary metabolites from actinobacteria

ABSTRACT

Secondary metabolites produced by actinobacteria have diverse structures and important biological activities, making them a useful source of drug development. Diversity of the secondary metabolites indicates that the actinobacteria exploit various chemical reactions to construct a structural diversity. Thus, studying the biosynthetic machinery of these metabolites should result in discovery of various enzymes catalyzing interesting and useful reactions. This review summarizes our recent studies on the biosynthesis of secondary metabolites from actinobacteria, including the biosynthesis of nonproteinogenic amino acids used as building blocks of nonribosomal peptides, the type II polyketide synthase catalyzing polyene scaffold, the nitrous acid biosynthetic pathway involved in secondary metabolite biosynthesis and unique cytochrome P450 catalyzing nitrene transfer. These findings expand the knowledge of secondary metabolite biosynthesis machinery and provide useful tools for future bioengineering.     

Chemical and physiological characterization of taxa in theFusarium sambucinum complex

Forty-one isolates ofFusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains ofF. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remainingF. sambucinum strains produced T-2 toxin, TB1 and TB2.Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB.Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.     

Intra-clade metabolomic profiling of MAR4 Streptomyces from the Macaronesia Atlantic region reveals a source of anti-biofilm metabolites

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC–MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 ( 1 ) against S. aureus was confirmed.     

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.     

Marine actinomycetes as a source of novel secondary metabolites

A set of 600 actinomycetes strains which were isolated from marine sediments from various sites in the Pacific and Atlantic Oceans were screened for the production of bioactive secondary metabolites. Marine streptomycete strains were found to be producers of well known chemically diverse antibiotics isolated from terrestrial streptomycetes, as in the case of marine Micromonospora strains. New marine members of the rare genus Verrucosispora seem to be a promising source for novel bioactive secondary metabolites as shown in the case of the abyssomicin producing strain AB-18-032.     

中国丝状真菌次级代谢分子调控研究进展

在目前已知的具有生物活性的微生物次级代谢物中约有50%是由丝状真菌产生的,其中包括人们所熟知的青霉素、环孢菌素A以及洛伐他汀等。鉴于丝状真菌次级代谢物在农业、医药和工业上的重要价值,它们的生物合成及其分子调控一直备受关注。丝状真菌次级代谢物的生物合成是一个复杂的过程,一般涉及多步酶学反应,该过程往往受到不同水平的调控。深入了解丝状真菌次级代谢的分子调控机制,可以为其产量的提高、新骨架化合物的发掘以及隐性次级代谢物的激活奠定重要的理论基础。本文以丝状真菌次级代谢分子调控为主线,重点介绍近40年来我国科研工作者在该领域取得的研究进展,并对这一领域未来的发展进行展望。     

Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.     

Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genes dhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.     

Heterologous production of clavulanic acid intermediates in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028.