<Emphasis Type="Italic">Streptomyces</Emphasis> and <Emphasis Type="Italic">Saccharopolyspora</Emphasis> hosts for heterologous expression of secondary metabolite gene clusters

Natural products discovery from actinomycetes has been on the decline in recent years, and has suffered from a lack of innovative ways to discover new secondary metabolites within a background of the thousands of known compounds. Recent advances in whole genome sequencing have revealed that actinomycetes with large genomes encode multiple secondary metabolite pathways, most of which remain cryptic. One approach to address the expression of cryptic pathways is to first identify novel pathways by bioinformatics, then clone and express them in well-characterized hosts with known secondary metabolomes. This process should eliminate the tedious dereplication process that has hampered natural products discovery. Several laboratory and industrial production strains have been used for heterologous production of secondary metabolite pathways. This review discusses the results of these studies, and the pros and cons of using various Streptomyces and one Saccharopolyspora strain for heterologous expression. This information should provide an experimental basis to help researchers choose hosts for current application and future development to express heterologous secondary metabolite pathways in yields sufficient for rapid scale-up, biological testing, and commercial production.     

PCR screening reveals considerable unexploited biosynthetic potential of ansamycins and a mysterious family of AHBA‐containing natural products in actinomycetes

Aims

Ansamycins are a family of macrolactams that are synthesized by type I polyketide synthase (PKS) using 3‐amino‐5‐hydroxybenzoic acid (AHBA) as the starter unit. Most members of the family have strong antimicrobial, antifungal, anticancer and/or antiviral activities. We aimed to discover new ansamycins and/or other AHBA‐containing natural products from actinobacteria.

Methods and Results

Through PCR screening of AHBA synthase gene, we identified 26 AHBA synthase gene–positive strains from 206 plant‐associated actinomycetes (five positives) and 688 marine‐derived actinomycetes (21 positives), representing a positive ratio of 2·4–3·1%. Twenty‐five ansamycins, including eight new compounds, were isolated from six AHBA synthase gene–positive strains through TLC‐guided fractionations followed by repeated column chromatography. To gain information about those potential ansamycin gene clusters whose products were unknown, seven strains with phylogenetically divergent AHBA synthase genes were subjected to fosmid library construction. Of the seven gene clusters we obtained, three show characteristics for typical ansamycin gene clusters, and other four, from Micromonospora spp., appear to lack the amide synthase gene, which is unusual for ansamycin biosynthesis. The gene composition of these four gene clusters suggests that they are involved in the biosynthesis of a new family of hybrid PK‐NRP compounds containing AHBA substructure.

Conclusions

PCR screening of AHBA synthase is an efficient approach to discover novel ansamycins and other AHBA‐containing natural products.

Significance and Impact of the Study

This work demonstrates that the AHBA‐based screening method is a useful approach for discovering novel ansamycins and other AHBA‐containing natural products from new microbial resources.     

放线菌来源活性天然产物发现研究进展

放线菌(Actinomycetes)是抗生素等活性天然产物的重要来源，在临床治疗病原菌感染等重大疾病方面发挥着重要作用。由于抗生素滥用等原因导致临床上出现的以耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)等为代表的耐药病菌的种类和数量急剧增加，对人类生存造成了重大威胁。结合现代生物技术进展，加大放线菌来源新抗生素的开发力度刻不容缓。本文对新时期放线菌来源抗生素的发现现状、基于生理操作和基因操作的放线菌来源天然产物挖掘的技术方法等进行综述，以期对放线菌来源活性天然产物的发现提供借鉴。     

Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products

Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture‐dependent and ‐independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR‐43 exhibited cytotoxic activity against cancer cell lines. JBIR‐34 and ‐35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.     

Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers

Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.     

Red Soils Harbor Diverse Culturable Actinomycetes That Are Promising Sources of Novel Secondary Metabolites

Red soils, which are widely distributed in tropical and subtropical regions of southern China, are characterized by low organic carbon, high content of iron oxides, and acidity and, hence, are likely to be ideal habitats for acidophilic actinomycetes. However, the diversity and biosynthetic potential of actinomycetes in such habitats are underexplored. Here, a total of 600 actinomycete strains were isolated from red soils collected in Jiangxi Province in southeast China. 16S rRNA gene sequence analysis revealed a high diversity of the isolates, which were distributed into 26 genera, 10 families, and 7 orders within the class Actinobacteria; these taxa contained at least 49 phylotypes that are likely to represent new species within 15 genera. The isolates showed good physiological potentials for biosynthesis and biocontrol. Chemical screening of 107 semirandomly selected isolates spanning 20 genera revealed the presence of at least 193 secondary metabolites from 52 isolates, of which 125 compounds from 39 isolates of 12 genera were putatively novel. Macrolides, polyethers, diketopiperazines, and siderophores accounted for most of the known compounds. The structures of six novel compounds were elucidated, two of which had a unique skeleton and represented characteristic secondary metabolites of a putative novel Streptomyces phylotype. These results demonstrate that red soils are rich reservoirs for diverse culturable actinomycetes, notably members of the families Streptomycetaceae, Pseudonocardiaceae, and Streptosporangiaceae, with the capacity to synthesize novel bioactive compounds.     

拟孢囊菌属放线菌的研究进展

拟孢囊菌属(Kibdelosporangium)是一个经典的丝状放线菌类群。自Shearer等于1986年建立该属以来,不断有新的菌株从不同生境中被纯培养分离得到,目前已有效发表8个种、2个亚种。拟孢囊菌属作为稀有放线菌属,是获得新型抗生素类次生代谢产物的重要资源。本文结合我们分离到的一株该属菌株的功能研究、基因组分析和相关文献资料,系统综述了拟孢囊菌属放线菌的建立、分类学特征、属内种的分布、活性次级代谢产物的发现以及其他功能应用和开发前景,以期为该属其他新分离菌株的分类鉴定、新颖次级代谢产物的发现、功能基因资源的挖掘与开发提供借鉴。     

Stress-driven discovery of a cryptic antibiotic produced by Streptomyces sp. WU20 from Kueishantao hydrothermal vent with an integrated metabolomics strategy

Marine hydrothermal microorganisms respond rapidly to the changes in the concentrations and availability of metals within hydrothermal vent microbial habitats which are strongly influenced by elevated levels of heavy metals. Most hydrothermal vent actinomycetes possess a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites. Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known compounds. Here, we report the identification of a novel antibiotic produced by Streptomyces sp. WU20 isolated from the metal-rich hydrothermal vents in Taiwan Kueishantao, following a strategy based on metal induction of silent genes combined with metabolomics analytical methods. HPLC-guided isolation by tracking the target peak resulted in the characterization of the novel compound 1 with antimicrobial activity against Bacillus subtilis. The stress metabolite 1 induced by nickel is structurally totally different compared with the normally produced compounds. This study underlines the applicability of metal induction combined with metabolic analytical techniques in accelerating the exploration of novel antibiotics and other medically relevant natural products.

     

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.     

Protoplast Mutation and Genome Shuffling Induce the Endophytic Fungus Tubercularia sp. TF5 to Produce New Compounds

Tubercularia sp. TF5 is an endophytic fungal strain isolated from the medicinal plant Taxus mairei. Previously, taxol has been detected in the fermentation products of this strain. However, it lost the capability of producing taxol after long-term laboratory culture. Herein, we tried to reactivate the production of taxol by protoplast mutations and genome shuffling. The protoplasts of Tub. sp. TF5 were prepared from its mycelia, and mutated by UV and NTG. The mutant strains regenerated from the mutated protoplasts were selected and classified into four groups on the basis of their phenotypes, the profile of their metabolites analyzed by TLC, MS, and bioassay data. Then, genome shuffling was subsequently carried out with eight mutant strains, with two representatives from each protoplast mutant group, and genome shuffling mutant strains were obtained and screened using the same screening procedure. Although taxol has not been detected in any mutant, two important mutants, M-741 and G-444 were selected for metabolites isolation and determination due to their phenotypes, and differences in TLC analysis result from TF5 and other mutants. Three new sesquiterpenoids, namely tuberculariols A–C (1–3), and a known dihydroisocoumarin (4) were obtained from M-741. Eighteen novel compounds were isolated from G-444, including five new sesquiterpenoids (5-9), two new dihydroisocoumarins (10, 11), one new tetralone (12), together with 10 known compounds (13–20, 1, and 2). The compounds isolated from the M-741 and G-444 were different in structure types and substitutions from those of TF5 (15, 21–29). The results showed, for the first time, that protoplast mutations and genome shuffling are efficient approaches to mining natural products from endophytic fungi. Understanding the mechanisms of unlocking the biosynthesis of new metabolites will facilitate the manipulation of the secondary metabolism in fungi.     

抗菌活性放线菌的界面微滴移液互作分离筛选方法

[背景]放线菌是天然产物的宝库,目前应用于临床的天然抗生素有70％来源于放线菌的次级代谢产物.随着细菌对传统抗生素耐药问题的日趋严重,如何从自然生境中高效筛选新型活性放线菌资源并发现新型抗生素成为当前微生物学者面临的重要挑战.通过传统方法筛选活性放线菌不仅费时费力、试剂耗材消耗量大,并且筛选通量非常有限,难以对自然样品...     

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.     

Biodiscovery from rare actinomycetes: an eco-taxonomical perspective

Microbial natural products, in particular, the ones produced by the members of the order Actinomycetales, will continue to represent an important route to the discovery of novel classes of bioactive compounds. As a result, the search for and discovery of lesser-known and/or novel actinomycetes is of significant interest to the industry due to a growing need for the development of new and potent therapeutic agents, mainly against drug resistant bacteria. Current advancements in genomics and metagenomics are adding strength to the target-directed search for detection and isolation of bioactive actinomycetes. New discoveries, however, will only stem from a sound understanding and interpretation of knowledge derived from conventional studies conducted since the discovery of streptomycin, on the ecology, taxonomy, physiology and metabolism of actinomycetes, and from a combination of this knowledge with currently available and continuously advancing molecular tools. Such a powerful information platform will then inevitably reveal the whereabouts, taxonomical and chemical identities of previously undetected bioactive actinomycetes including novel species of streptomycetes as potential producers of novel drug candidates.     

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.     

西沙海藻共附生放线菌的分离鉴定及其抗菌活性评价

【目的】为了探究南海海藻共附生放线菌资源的多样性及潜在的应用价值,对中国西沙群岛来源的海藻进行共附生放线菌的分离鉴定与抗菌活性筛选。【方法】利用稀释涂布平板法,采用2种不同分离培养基对不同采样位点的6种海藻进行放线菌分离;通过16S rRNA基因序列分析、构建系统发育树对分离的放线菌进行鉴定;用打孔法对无乳链球菌(Streptococcus agalactiae)等10种敏感细菌进行抗菌活性筛选;对筛选得到的目标活性菌株HZ014进行全基因组测序,通过AntiSMASH在线工具分析其次级代谢产物生物合成基因簇,预测其产生新型活性物质的潜力。【结果】从6种海藻中分离得到36株共附生放线菌,基于16S rRNA基因序列比对和系统发育分析,鉴定结果为链霉菌属(Streptomyces) 2株、红球菌属(Rhodococcus) 2株、诺卡氏菌属(Nocardia)3株、小单孢菌属(Micromonospora) 5株和盐孢菌属(Salinispora) 24株;抗菌活性筛选结果表明,36株共附生放线菌发酵粗提物对至少1种敏感细菌表现出一定的抑制作用,不同菌株发酵粗提物的抗菌活性存在明显差异,...     

Mycolic acid-containing bacteria induce natural-product biosynthesis in Streptomyces species

Natural products produced by microorganisms are important starting compounds for drug discovery. Secondary metabolites, including antibiotics, have been isolated from different Streptomyces species. The production of these metabolites depends on the culture conditions. Therefore, the development of a new culture method can facilitate the discovery of new natural products. Here, we show that mycolic acid-containing bacteria can influence the biosynthesis of cryptic natural products in Streptomyces species. The production of red pigment by Streptomyces lividans TK23 was induced by coculture with Tsukamurella pulmonis TP-B0596, which is a mycolic acid-containing bacterium. Only living cells induced this pigment production, which was not mediated by any substances. T. pulmonis could induce natural-product synthesis in other Streptomyces strains too: it altered natural-product biosynthesis in 88.4% of the Streptomyces strains isolated from soil. The other mycolic acid-containing bacteria, Rhodococcus erythropolis and Corynebacterium glutamicum, altered biosynthesis in 87.5 and 90.2% of the Streptomyces strains, respectively. The coculture broth of T. pulmonis and Streptomyces endus S-522 contained a novel antibiotic, which we named alchivemycin A. We concluded that the mycolic acid localized in the outer cell layer of the inducer bacterium influences secondary metabolism in Streptomyces, and this activity is a result of the direct interaction between the mycolic acid-containing bacteria and Streptomyces. We used these results to develop a new coculture method, called the combined-culture method, which facilitates the screening of natural products.     

Rare actinomycetes: a potential storehouse for novel antibiotics

New antimicrobial agents are desperately needed to combat the increasing number of antibiotic resistant strains of pathogenic microorganisms. Natural products remain the most propitious source of novel antibiotics. It is widely accepted that actinobacteria are prolific producers of natural bioactive compounds. We argue that the likelihood of discovering a new compound having a novel chemical structure can be increased with intensive efforts in isolating and screening rare genera of microorganisms. Screening rare actinomycetes and their previously under-represented genera from unexplored environments in natural product screening collections is one way of achieving this. Rare actinomycetes are usually regarded as the actinomycete strains whose isolation frequency is much lower than that of the streptomycete strains isolated by conventional methods. Many natural environments are still either unexplored or under-explored and thus, can be considered as a prolific resource for the isolation of less exploited microorganisms. More and different ecological niches need to be studied as sources of a greater diversity of novel microorganisms. In this review, we wish to update our understanding of the potential of the rare actinomycetes by focusing on the ways and means of enhancing their bio-discovery potential.     

Rare actinomycetes: a potential storehouse for novel antibiotics

New antimicrobial agents are desperately needed to combat the increasing number of antibiotic resistant strains of pathogenic microorganisms. Natural products remain the most propitious source of novel antibiotics. It is widely accepted that actinobacteria are prolific producers of natural bioactive compounds. We argue that the likelihood of discovering a new compound having a novel chemical structure can be increased with intensive efforts in isolating and screening rare genera of microorganisms. Screening rare actinomycetes and their previously under-represented genera from unexplored environments in natural product screening collections is one way of achieving this. Rare actinomycetes are usually regarded as the actinomycete strains whose isolation frequency is much lower than that of the streptomycete strains isolated by conventional methods. Many natural environments are still either unexplored or under-explored and thus, can be considered as a prolific resource for the isolation of less exploited microorganisms. More and different ecological niches need to be studied as sources of a greater diversity of novel microorganisms. In this review, we wish to update our understanding of the potential of the rare actinomycetes by focusing on the ways and means of enhancing their bio-discovery potential.     

西沙石珊瑚共附生放线菌的分离培养及抗菌活性评价

【背景】珊瑚礁生态系统是海洋中一类极其重要的生态系统,健康珊瑚礁中丰富的共附生放线菌群体是珊瑚抵御各种致病菌的重要防线,因此,这类放线菌是寻找抗菌活性分子的重要资源,其药用潜力巨大。【目的】从西沙石珊瑚样品中分离共附生放线菌,并从中筛选具有良好抗菌活性的菌株。【方法】通过稀释涂布法分离珊瑚共附生放线菌,并根据16S rRNA基因序列构建系统发育树进行菌种鉴定;通过平板对峙法对放线菌进行抗菌活性筛选并确定目标菌株;将目标菌株涂布于不同氯化钠浓度的ISP2固体培养基上培养,测试其盐度耐受能力;通过平板对峙法对该菌株发酵产物的热稳定性和光稳定性进行测试;采用NanoPore和Illumina方法完成目标活性放线菌全基因组测序,并通过antiSMASH在线分析预测其次级代谢产物生物合成基因簇及其结构类型。【结果】从6份西沙石珊瑚样品中分离得到104株可培养放线菌,根据菌落形态和分离来源去重后对其中27株放线菌进行16S rRNA基因序列测序,通过序列比对和系统发育树分析将菌株初步鉴定为盐孢菌属(Salinispora)(25株)、链霉菌属(Streptomyces)(1株)和戈登菌属(Gord...     

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.