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....     

Marine actinomycetes as an emerging resource for the drug development pipelines

Many representatives of the order Actinomycetales are prolific producers of thousands of biologically active secondary metabolites. Actinomycetes from terrestrial sources have been studied and screened since the 1950s, yielding many important anti-infective and anti-cancer drugs. However, frequent re-discovery of the same compounds in terrestrial actinomycetes have made them less attractive for screening programs in the recent years. At the same time, actinomycetes isolated from the marine environment currently receive considerable attention due to the structural diversity and unique biological activities of their secondary metabolites. This review highlights achievements and challenges in the isolation of marine actinomycetes, some examples of bioactive metabolites identified by conventional screening, and presents new developments in the field of genome mining and heterologous expression of biosynthetic gene clusters leading to the discovery of novel compounds.     

Impact of the first <Emphasis Type="Italic">Streptomyces</Emphasis> genome sequence on the discovery and production of bioactive substances

An important addition to the field of bacterial genomics is the recent publication of the complete genome sequence of Streptomyces coelicolor. This strain has been for some decades the model organism for streptomycetes and other filamentous actinomycetes, Gram-positive bacteria highly valuable for their ability to produce thousands of bioactive metabolites, many of which have found important applications in medicine and agriculture. We discuss here the impacts that the S. coelicolor genome sequence is likely to have on the production of bioactive metabolites by current industrial strains, on the possible development of future superhost(s) for the production of valuable drugs, and on the search for new bioactive substances from microbial sources.     

培菌昆虫相关放线菌的次级代谢产物研究进展

昆虫共生微生物是一种特殊的微生物资源,其中放线菌在昆虫肠道、体表和巢穴中广泛分布。近年来,人们从培菌昆虫来源的放线菌中分离得到多种新型化合物,可以选择性抑制菌圃的致病真菌,部分还对植物致病真菌、昆虫致病真菌、人类病原菌和癌细胞有抑制活性。因此,研究培菌昆虫相关微生物不仅有助于了解宿主与微生物的共生机制,还能发掘新的活性物质,用于生物农药、生物医药的开发。本文对培菌昆虫来源放线菌次级代谢产物的研究进展进行了综述。     

Marine actinomycetes: An ongoing source of novel bioactive metabolites

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds. There are more than 22,000 known microbial secondary metabolites, 70% of which are produced by actinomycetes, 20% from fungi, 7% from Bacillus spp. and 1–2% by other bacteria. Among the actinomycetes, streptomycetes group are considered economically important because out of the approximately more than 10,000 known antibiotics, 50–55% are produced by this genus. The ecological role of actinomycetes in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate marine strains for search and discovery of new drugs. The search for and discovery of rare and new actinomycetes is of significant interest to drug discovery due to a growing need for the development of new and potent therapeutic agents. Modern molecular technologies are adding strength to the target-directed search for detection and isolation of bioactive actinomycetes, and continued development of improved cultivation methods and molecular technologies for accessing the marine environment promises to provide access to this significant new source of chemical diversity with novel/rare actinomycetes including new species of previously reported actinomycetes.     

红树林放线菌研究进展

红树林是生长在热带和亚热带海岸潮间带的一种独特植物群落,构成陆地与海洋间的过渡,具有水分高、盐分高、耐缺氧等特征的特殊生态系统及很高的生物多样性,是筛选和发现放线菌新物种和药用生物活性次级代谢产物的宝贵资源。在物种鉴定方面,放线菌分类学是目前红树林放线菌物种鉴定的重要手段。本文综述了红树林放线菌物种鉴定主要方法、红树林放线菌生态环境和物种分布特征、生物学活性及其次级代谢产物等,并展望了该领域研究与发展的重要问题与趋势。     

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.     

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.     

放线菌与枯草芽孢杆菌的共培养及其对活性次生代谢产物的影响

为探讨共培养对放线菌产生活性次生代谢产物的影响,结合抗菌活性测定及HPLC-PDA分析,研究了22株放线菌的单培养及其与枯草芽孢杆菌的共培养发酵代谢产物的差异,并选取抗菌活性较强的链霉菌FXJ2.014进一步研究其代谢产物。发现FXJ2.014、FXJ1.296、AS4.1252三株菌与枯草芽孢杆菌共培养时产生其在相同条件下单培养时没有的物质,其中链霉菌FXJ2.014单培养时主要产生醌霉素A,共培养时产物中增加了醌霉素结构类似物FXJ2.014-HB。进一步的抗菌、抗肿瘤活性测定结果表明,两者的生物活性有较显著的差异,且FXJ2.014-HB对多种肿瘤细胞系的抑制活性普遍弱于高毒性的醌霉素A,为有潜力的细胞毒性较小的抗生素。共培养是一条很有希望的发掘放线菌活性次生代谢产物的新途径。     

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.     

植物内生放线菌及其生理活性物质研究进展

植物内生放线菌是一类具有巨大开发潜力的新微生物资源。目前从许多活体植物组织内已分离到种类众多的植物内生放线菌,已有的研究表明植物内生放线菌能产生许多重要的生理活性物质,如抗生素类物质、植物生长促进剂、植物生长抑制剂以及具有新特性的酶类。植物内生放线菌在农业生产、医药新药的筛选上显示出广阔的应用前景。     

Discovery of novel metabolites from marine actinomycetes

Recent findings from culture-dependent and culture-independent methods have demonstrated that indigenous marine actinomycetes exist in the oceans and are widely distributed in different marine ecosystems. There is tremendous diversity and novelty among the marine actinomycetes present in marine environments. Progress has been made to isolate novel actinomycetes from samples collected at different marine environments and habitats. These marine actinomycetes produce different types of new secondary metabolites. Many of these metabolites possess biological activities and have the potential to be developed as therapeutic agents. Marine actinomycetes are a prolific but underexploited source for the discovery of novel secondary metabolites.     

放线菌次级代谢产物研究进展

随着耐药细菌和新型病毒的不断出现，癌症的发病率和死亡率持续上升，迫使人们不断寻找新的化合物来治疗疾病。放线菌次级代谢产物结构新颖，作用独特，具有抗菌、杀虫、抗肿瘤、免疫抑制等活性，广泛应用于医疗、农业、食品等领域，深入挖掘放线菌资源来开发新型抗生素潜力巨大。然而从自然界分离的放线菌生产目标化合物的能力较弱，这直接影响其工业应用，增加其生产成本，因此构建目标化合物高产菌株显得尤为重要。本文以此为出发点，从放线菌新药资源挖掘和放线菌产抗能力提高两个方面对近年来的研究情况进行概述，为放线菌资源开发提供参考。     

鼎湖山国家自然保护区林下土壤微生物新结构生物活性代谢产物的研究进展

鼎湖山国家自然保护区特殊的地理位置和气候条件造就了独特的生态环境,孕育了丰富的植物和微生物资源。为了挖掘其中的微生物资源,对近年来鼎湖山林下土壤来源的真菌和放线菌次生代谢物研究进行了综述。     

Physiology and nutritional aspects of actinomycetes: an overview

A review is presented of the nutritional requirements and physiology of actinomycetes, with special emphasis on: (a) selective isolation of unusual forms; (b) colonial morphogenesis and induction of sporulation; (c) biosynthesis of secondary metabolites of value, with particular reference to antibiotics and industrial enzymes. The future potential of actinomycetes as major contributors of useful bioactive metabolites is also indicated.     

Fungal endophytes and bioprospecting

Horizontally transmitted fungal endophytes are an ecological group of fungi, mostly belonging to the Ascomycota, that reside in the aerial tissues and roots of plants without inducing any visual symptoms of their presence. These fungi appear to have a capacity to produce an array of secondary metabolites exhibiting a variety of biological activity. Although the ability of fungi to produce unique bioactive metabolites is well known, endophytes have not been exploited, perhaps because we are only beginning to understand their distribution and biology. This review emphasizes the need to routinely include endophytic fungi in the screening of organisms for bioactive metabolites and novel drugs; it also underscores the need to use information obtained concerning fungal secondary metabolite production from other groups of fungi for a targeted screening approach.     

Phylogenetic characterization of culturable actinomycetes associated with the mucus of the coral Acropora digitifera from Gulf of Mannar

The marine environment is a virtually untapped source of novel actinomycete diversity and its metabolites. Investigating the diversity of actinomycetes in other marine macroorganisms, like seaweeds and sponges, have resulted in isolation of novel bioactive metabolites. Actinomycetes diversity associated with corals and their produced metabolites have not yet been explored. Hence, in this study we attempted to characterize the culturable actinomycetes population associated with the coral Acropora digitifera. Actinomycetes were isolated from the mucus of the coral wherein the actinomycetes count was much higher when compared with the surrounding seawater and sediment. Actinobacteria-specific 16S rRNA gene primers were used for identifying the isolates at the molecular level in addition to biochemical tests. Amplified ribosomal DNA restriction analysis using three restriction enzymes revealed several polymorphic groups within the isolates. Sequencing and blast analysis of the isolates revealed that some isolates had only 96.7% similarity with its nearest match in GenBank indicating that they may be novel isolates at the species level. The isolated actinomycetes exhibited good antibacterial activity against various human pathogens. This study offers for the first time a prelude about the unexplored culturable actinomycetes diversity associated with a scleractinian coral and their bioactive capabilities.     

Pathway Engineering in Secondary Metabolite-Producing Actinomycetes

Abstract

Actinomycetes represent the microbial group richest in production of variable secondary metabolites. These mostly bioactive molecules are the end products of complex multistep biosynthetic pathways. Recent progress in the molecular genetics and biochemistry of the biosynthetic capacities of actinomycetes enables first attempts to redesign these pathways in a directed fashion. However, in contrast to several examples of designed biochemical improvement of primary metabolic processes in microorganisms, none of the products or strains derived from pathway engineering in actinomycetes discussed herein have reached pilot or production scale. The main reasons for this slow progress are the complicated pathways themselves, their complex regulation during the actinomycete cell cycle, and their uniqueness, as most pathways and products are specific for a strain rather than for a given species or larger taxonomic group. However, the modular use of a minimum of very similar enzymes and their conversion of similar intermediates to form the building blocks for the production of a maximum of divergent end products gives hope for the future application of these genetic models for the redesign of complex pathways for modified or new natural products. Several strategies that can be followed to reach this aim are discussed, mainly for the variable 6-deoxyhexose metabolism as an ubiquitously applicable example.     

Bioactive compounds from marine actinomycetes

Actinomycetes are one of the most efficient groups of secondary metabolite producers and are very important from an industrial point of view. Among its various genera, Streptomyces, Saccharopolyspora, Amycolatopsis, Micromonospora and Actinoplanes are the major producers of commercially important biomolecules. Several species have been isolated and screened from the soil in the past decades. Consequently the chance of isolating a novel actinomycete strain from a terrestrial habitat, which would produce new biologically active metabolites, has reduced. The most relevant reason for discovering novel secondary metabolites is to circumvent the problem of resistant pathogens, which are no longer susceptible to the currently used drugs. Existence of actinomycetes has been reported in the hitherto untapped marine ecosystem. Marine actinomycetes are efficient producers of new secondary metabolites that show a range of biological activities including antibacterial, antifungal, anticancer, insecticidal and enzyme inhibition. Bioactive compounds from marine actinomycetes possess distinct chemical structures that may form the basis for synthesis of new drugs that could be used to combat resistant pathogens.     

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.