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,难以对自然样品中的复杂微生物群落进行整体全面的解析。【目的】提出一种基于多孔板液滴阵列培养的新策略,可高通量筛选抗菌活性放线菌。分析模式放线菌在微液滴中的培养特征与筛选条件,为进一步建立基于液滴阵列技术的超高通量活性放线菌筛选平台奠定基础。【方法】采用界面移液技术,将传统的多孔板高通量筛选体系微缩至1μL水平,在油相填充的多孔板(96孔板)中生成微升培养液滴阵列,每个微孔液滴中封装一个放线菌孢子或菌丝团。经过短期培养,放线菌在微滴中完成菌丝分化与次级代谢产物的分泌。这时,通过第二步界面移液技术与液滴融合加入带有荧光标记的指示菌,通过全菌拮抗筛选定位活性目标菌株,并将活性谱转化为量化的荧光数值。【结果】通过对模式放线菌的测试发现,放线菌菌丝可以在微液滴中达到最佳培养状态,并积累足够的生物质与代谢物,对荧光指示菌有明显的抑制作用。【结论】通过建立上述基于微孔板液滴阵列的高通量筛选技术,能从单细胞水平快速筛选出具有抑菌活性的菌株,显著节约了筛选成本并提高了筛选通量,为新型活性天然产物的发现提供了一种新的简单有效的筛选方法。     

CRISPR/Cas9在放线菌合成生物学研究中的应用和发展

放线菌是活性天然产物和抗生素药物的重要来源。利用合成生物学高效地开发其中丰富的天然产物资源,将为加速新药开发奠定坚实的基础。CRISPR/Cas9作为一种多功能基因编辑系统,因其便捷高效而被广泛应用于真核生物的遗传操作。但在原核生物尤其是放线菌中的应用仍处于起步阶段,机遇和挑战并存。本综述总结了目前CRISPR/Cas9系统在放线菌基因编辑和调控,以及活性天然产物的产量提升、生物合成机制解析和资源开发等方面的研究进展。同时,也对该系统在应用中面临的包括重组修复效率低,以及靶向切割效率不足等关键挑战进行了分析,并提出了相应的优化解决方法。随着CRISPR/Cas9在放线菌应用中的不断完善和发展,将极大地推动放线菌的合成生物学研究,促进其中天然产物资源的有效挖掘和应用开发。     

微生物中抗耐药菌活性天然产物的研究进展

近年来,细菌耐药性已成为全球性问题。微生物作为抗生素的重要来源,在发掘抗耐药菌新型抗生素的研究中承担了重要角色,许多微生物来源天然化合物展现了显著的抗耐药菌活性。本文主要综述了近十年发现的微生物源抗耐药菌天然化合物,并概括了抗耐药菌活性天然产物的筛选方法,以期对抗耐药菌新型抗生素的发现做铺垫。     

新型angucycline/angucyclinone类天然产物的研究进展 (2010–2020)

Angucycline/angucyclinone类天然产物是Ⅱ型聚酮类化合物中一个庞大的家族,其产生菌广泛存在于自然界中。由于这类化合物具有丰富的生物活性和独特的化学结构,吸引了众多科学家的浓厚兴趣。随着合成生物学的发展和特殊环境来源放线菌资源的开发,不断有新骨架的angucycline/angucyclinone类天然产物被发现,极大地丰富了这一家族天然产物的结构多样性。本文对2010–2020年期间,利用改变培养条件、基因改造、基因组挖掘、活性导向、特殊环境来源放线菌培养等不同策略从放线菌中所发现的新型angucycline/angucyclinone类化合物进行综述,并对合成生物学方法在这类化合物的发现和开发中的作用进行了讨论。     

可培养海洋放线菌生物多样性的研究进展

海洋放线菌是新药开发和天然活性产物的重要来源,海洋放线菌的生物多样性是代谢产物功能多样性的基础,因此研究可培养放线菌的生物多样性具有重要的意义。综述了近年来可培养的海洋放线菌生物多样性的研究进展,尤其是海绵共附生放线菌、深海放线菌和海洋固有放线菌的研究进展,对可培养的海洋放线菌的分离培养方法,包括样品处理、培养基的选择等进行了重点介绍,并对未培养海洋放线菌的分离培养进行了探讨,强调了建立区域性海洋放线菌菌种及基因资源库的重要性。     

海洋微生物抗肿瘤天然产物研究进展

近年来,海洋微生物已成为抗肿瘤天然产物研究的热点,目前从海洋微生物中发现了大量新的抗肿瘤天然产物。文章综述了近几年从海洋微生物(海洋放线菌、真菌和细菌)中分离得到的抗肿瘤活性天然产物的研究进展。     

昆虫共生放线菌多样性、功能及其活性次生代谢产物研究进展

作为一类具有重要经济价值的微生物资源,已发现的放线菌所产的生物活性物质占到所有微生物生物活性物质的70%,其中包括抗生素、免疫抑制剂和酶抑制剂等。从昆虫体内分离放线菌将会是发现新菌种和新化合物的重要渠道。本文主要阐述了昆虫共生放线菌的多样性及功能,总结了昆虫共生放线菌的活性次生代谢产物的种类及其应用进展情况,最后对昆虫共生放线菌代谢活性物质研究与开发中存在的问题提出了对策。     

新方法新技术与新型抗生素发现

日趋严峻的细菌耐药性问题给“抗生素传奇时代”带来了严重威胁, 传统的抗菌药物开发手段已很难应对, 因此急需新思路来寻找新型抗生素。近年来, 一些新方法新技术如新型筛选模型的构建、基因组挖掘技术以及组合生物合成等给天然产物的筛选引发了革命性的变革, 也为放线菌来源的新型抗生素的发现带来了新的希望。本文将对这些新技术及其在新型抗生素研发中的应用进展进行阐述。     

小单胞菌属次级代谢产物及其生物活性研究进展

小单胞菌属(Micromonospora)为稀有放线菌,广泛分布在土壤、海洋和动植物中,其所产代谢产物不仅具有抗菌、抗肿瘤、抗HIV等多种生物活性,而且化学结构新颖多样。本文从化学结构分类、生物活性等方面对近几年已报道的小单胞菌属来源的重要天然产物做了简要综述,以期为小单胞菌天然产物的开发和应用奠定基础。     

A guide to successful bioprospecting: informed by actinobacterial systematics

New structurally diverse natural products are discovered when novel screening procedures are introduced or when high quality biological materials from new sources are examined in existing screens, hence it is important to foster these two aspects of novelty in drug discovery programmes. Amongst prokaryotes, actinomycetes, notably streptomycetes, remain a rich source of new natural products though it has become increasingly difficult to find such metabolites from common actinomycetes as screening ‘old friends’ leads to the costly rediscovery of known compounds. The bioprospecting strategy which is the subject of this review is based upon the premise that new secondary metabolites can be found by screening relatively small numbers of dereplicated, novel actinomycetes isolated from marine sediments. The success of the strategy is exemplified by the discovery of a range of novel bioactive compounds, notably atrop-abyssomicin C and proximicins A, B and C from Verrucosispora strains isolated from sediment samples taken from the Sea of Japan and the Raune Fjord, respectively, and the dermacozines derived from Dermacoccus strains isolated from the Challenger Deep of the Mariana Trench in the Pacific Ocean. The importance of current advances in prokaryotic systematics in work of this nature is stressed and a plea made that resources be sought to train, support and employ the next generation of actinobacterial systematists.     

Saccharopolyspora: an underexplored source for bioactive natural products

Actinomycetes are a rich source for secondary metabolites with a diverse array of biological activities. Among the various genera of actinomycetes, the genus Saccharopolyspora has long been recognized as a potential source for antibiotics and other therapeutic leads that belong to diverse classes of natural products. Members of the genus Saccharopolyspora have been widely reported from several natural sources including both terrestrial and marine environments. A plethora of this genus has been chemically investigated for the production of novel natural products with interesting pharmacological effects. Therefore, Saccharopolyspora is considered one of the pharmaceutical important genera that could provide further chemical diversity with potential lead compounds. In this review, the literature from 1976 until December 2018 was covered, providing a comprehensive survey of all natural products derived from this genus and their semi-synthetic derivatives along with their biological activities, whenever applicable. Moreover, the biological diversity of Saccharopolyspora species and their habitats were also discussed.     

How to discover new antibiotics: harvesting the parvome

There is a dire need for new antibiotics; commercial discovery programs have essentially dried up and there is talk of 'a return to the pre-antibiotic era'. Natural products are an inexhaustible source of bioactive compounds (antibiotics among them), and recent technical advances such as DNA sequencing and bioinformatics offer new approaches to small molecule discovery. Given that nucleotide sequence studies of actinomycetes genomes reveal the presence of 20 or more pathways for the synthesis of bioactive compounds, 'mining' these sequences offers the potential of expanding the repertoire of antibiotics and other drugs. Combined with advanced chemical separation and characterization techniques, the construction of large chemically diverse libraries of bioactive compounds for therapeutic applications is a realistic near-term goal.     

Systematic and biotechnological aspects of halophilic and halotolerant actinomycetes

More than 70 species of halotolerant and halophilic actinomycetes belonging to at least 24 genera have been validly described. Halophilic actinomycetes are a less explored source of actinomycetes for discovery of novel bioactive secondary metabolites. Degradation of aliphatic and aromatic organic compounds, detoxification of pollutants, production of new enzymes and other metabolites such as antibiotics, compatible solutes and polymers are other potential industrial applications of halophilic and halotolerant actinomycetes. Especially new bioactive secondary metabolites that are derived from only a small fraction of the investigated halophilic actinomycetes, mainly from marine habitats, have revealed the huge capacity of this physiological group in production of new bioactive chemical entities. Combined high metabolic capacities of actinomycetes and unique features related to extremophilic nature of the halophilic actinomycetes have conferred on them an influential role for future biotechnological applications.     

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.     

Triggers and cues that activate antibiotic production by actinomycetes

Actinomycetes are a rich source of natural products, and these mycelial bacteria produce the majority of the known antibiotics. The increasing difficulty to find new drugs via high-throughput screening has led to a decline in antibiotic research, while infectious diseases associated with multidrug resistance are spreading rapidly. Here we review new approaches and ideas that are currently being developed to increase our chances of finding novel antimicrobials, with focus on genetic, chemical, and ecological methods to elicit the expression of biosynthetic gene clusters. The genome sequencing revolution identified numerous gene clusters for natural products in actinomycetes, associated with a potentially huge reservoir of unknown molecules, and prioritizing them is a major challenge for in silico screening-based approaches. Some antibiotics are likely only expressed under very specific conditions, such as interaction with other microbes, which explains the renewed interest in soil and marine ecology. The identification of new gene clusters, as well as chemical elicitors and culturing conditions that activate their expression, should allow scientists to reinforce their efforts to find the necessary novel antimicrobial drugs.     

Current approaches to exploit actinomycetes as a source of novel natural products

For decades, microbial natural products have been one of the major sources of novel drugs for pharmaceutical companies, and today all evidence suggests that novel molecules with potential therapeutic applications are still waiting to be discovered from these natural sources, especially from actinomycetes. Any appropriate exploitation of the chemical diversity of these microbial sources relies on proper understanding of their biological diversity and other related key factors that maximize the possibility of successful identification of novel molecules. Without doubt, the discovery of platensimycin has shown that microbial natural products can continue to deliver novel scaffolds if appropriate tools are put in place to reveal them in a cost-effective manner. Whereas today innovative technologies involving exploitation of uncultivated environmental diversity, together with chemical biology and in silico approaches, are seeing rapid development in natural products research, maximization of the chances of exploiting chemical diversity from microbial collections is still essential for novel drug discovery. This work provides an overview of the integrated approaches developed at the former Basic Research Center of Merck Sharp and Dohme in Spain to exploit the diversity and biosynthetic potential of actinomycetes, and includes some examples of those that were successfully applied to the discovery of novel antibiotics.     

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.