海洋来源节菱孢菌ZSDS1-F3中PKS-NRPS类天然产物挖掘

【目的】以基因组信息为指导,定向激活海洋来源真菌Arthrinium arundinisZSDS1-F3中沉默的聚酮合成酶-非核糖体肽合成酶(PKS-NRPS)类生物合成基因簇,鉴定次级代谢产物结构。【方法】通过启动子工程和异源表达的策略激活实验室培养条件下沉默或低表达的生物合成基因簇,实现目标化合物的分离,通过HR-ESI-MS和NMR数据分析鉴定产物结构,结合基因重组和生物信息学分析结果推导化合物的生物合成途径。【结果】依据基因组生物信息学分析,从海洋来源真菌A. arundinis ZSDS1-F3中选取一个编码PKS-NRPS类次级代谢产物的生物合成基因簇开展研究,在宿主Aspergillus nidulansA1145中实现了基因簇的异源表达,从中分离到2个新化合物,并推导了其生物合成途径。【结论】基因组信息指导下的天然产物挖掘,可以目标明确地分离产物,加快真菌中新颖天然产物的发现步伐。     

卡伍尔氏链霉菌NA4的Ⅱ型聚酮基因簇的靶向激活及其产物鉴定

【目的】以基因组信息为导向,定向激活海洋来源卡伍尔氏链霉菌（Streptomyces cavourensis） NA4中沉默的Ⅱ型聚酮类次级代谢产物生物合成基因簇,鉴定新产生的次级代谢产物的结构和抑菌活性。【方法】通过添加启动子和敲除负调控基因的方法激活实验室培养条件下沉默或低表达的生物合成基因簇,并完成目标化合物的分离与纯化,通过电喷雾质谱（electrospray ionization-mass spectrometry,ESI-MS）和核磁共振（nuclear magnetic resonance,NMR）数据分析鉴定目标化合物结构,对目标化合物进行抑菌活性鉴定,基于生物信息学信息推导化合物的生物合成途径。【结果】根据基因组生物信息学分析,从海洋来源链霉菌Streptomyces cavourensis NA4中选取一个编码PKSⅡ型次级代谢产物的生物合成基因簇开展研究,成功激活目标基因簇,从中分离到1个PKSⅡ型化合物,推导了其生物合成途径并进行了抑菌活性鉴定。【结论】基因组导向下的天然产物挖掘,可以目标明确地分离产物,充分挖掘链霉菌编码次级代谢产物的潜力。     

链霉菌隐性次级代谢产物生物合成基因簇的激活

链霉菌基因组中存在许多隐性次级代谢产物生物合成基因簇(cryptic secondary metabolite biosynthetic gene cluster,CSMG),在实验室条件下,它们多数处于沉默状态或者很低的表达水平。但在一定条件下这些CSMG可以被激活合成次级代谢产物,这为寻找新的活性天然产物提供新来源。本文总结了目前激活链霉菌CSMG所使用的方法,包括改变发酵条件、核糖体工程、共培养、异源表达、CSR(cluster-situated regulator)基因的遗传改造等。     

后基因组时代微生物天然产物高效发掘体系设计与展望

微生物天然产物具有丰富的化学结构多样性和诱人的生物活性,持续启迪着创新医药和农药的发现。近年来,随着高通量测序技术的快速发展,巨大的微生物基因组数据揭示了多样生物合成和新颖天然产物的潜能远高于以前的认知。然而,如何高效地激活隐性的生物合成基因簇 (BGCs) 并识别相应的化合物,以及避免已知代谢产物的重复发现等挑战依然严峻。本文描述了面对这些问题时基因组学、生物信息学、机器学习、代谢组学、基因编辑和合成生物学等新技术在发现药用先导化合物过程中提供的机遇;总结并论述了在潜力菌株优选、BGCs的生物信息学预测、沉默 BGCs的高效激活以及目标产物的识别和跟踪方面的新见解;提出了基于潜力菌株选择和多组学挖掘技术从微生物天然产物中高效发现先导结构的系统线路 (SPLSD),并讨论了未来天然产物药用先导发现的机遇和挑战。     

基于微生物共培养的隐性基因簇激活策略

微生物次级代谢产物是药物先导化合物的重要源泉之一。随着测序技术的迅猛发展,越来越多的微生物基因组得以测序完成。伴随着测序技术的进步,生物信息学也得到了快速发展。基因组序列分析发现,链霉菌和丝状真菌等微生物中存在大量的已知的或未知的次级代谢物生物合成基因簇(secondary metabolite-biosynthetic gene clusters,SM-BGCs)。然而,在实验室培养条件下大部分基因簇无法表达或表达量很低,导致难以发现这些基因簇所对应的代谢产物,人们将这类基因簇称为“隐性基因簇”或“沉默基因簇”。通过调节基因簇中特异调控基因或基因簇外全局性调控基因的表达,对代谢途径的定向改造,以及将基因簇导入异源宿主等策略,能够激活部分隐性基因簇的表达。通过激活隐性基因簇的表达,能够发现通过常规实验室培养无法获得的具有独特生物活性的新结构代谢产物,成为创新药物的重要来源之一。然而,这些基因簇激活策略都严重依赖于对特定菌株或宿主的遗传操作。近年来,通过模拟自然混合培养中微生物间相互作用,开发了通过混合特定微生物菌株在厌氧或好氧条件下激活隐性基因簇的方法,称之为共培养激活策略。这种策略不...     

基因组学技术解码天然产物合成

天然产物一直以来都是新药发现的重要来源。自20世纪末以来,随着组学技术的不断发展,许多生物的基因组被破译并解析,发现基因组中潜藏着众多未知的天然产物生物合成基因簇,而这些基因簇在实验室生长条件下无法表达或低表达。因此,需要综合运用多种学科深入挖掘生物中潜藏的具有新型结构和生物活性的天然产物,使其广泛应用于人类的生产生活。文中将从天然产物合成基因簇的挖掘、“沉默”天然产物合成途径的激活和生物底盘构建3个方面简述基因组学技术在天然产物挖掘中的研究进展。     

链霉菌Streptomyces antibioticus NRRL 8167中Naphthgeranine A生物合成基因簇的分析鉴定

【背景】微生物来源的天然产物是小分子药物或药物先导物的重要来源。对链霉菌Streptomyces antibioticus NRRL 8167的基因组分析显示,其包含多个次级代谢产物的生物合成基因簇,具有产生多种新化合物的潜力。【目的】对链霉菌S. antibioticus NRRL 8167中次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并对相应产物的生物合成基因簇和生物合成途径进行解析。【方法】利用HPLC图谱结合特征性紫外吸收和LC-MS方法,排除S. antibioticus NRRL 8167产生的已知化合物,确定具有特殊紫外吸收的化合物作为挖掘对象,然后利用正、反相硅胶柱色谱、高效液相色谱等技术对次级代谢产物进行分离纯化,分离化合物。利用质谱及核磁共振光谱技术对化合物结构进行解析和鉴定;提取链霉菌S. antibioticus NRRL 8167基因组DNA,利用PacBio测序平台进行基因组测序;利用生物信息学对基因组进行注释,并对合成该化合物的基因簇进行定位分析,推导其生物合成途径。【结果】确定这个化合物是NaphthgeranineA,属于聚酮类化合物。全基因组序列分析发现S.antibioticusNRRL8167基因组含有28个次级代谢产物生物合成基因簇,其中基因簇20可能负责Naphthgeranine A的生物合成,并对其生物合成途径进行了推导。【结论】基于紫外吸收光谱和质谱特征,从S. antibioticus NRRL 8167菌株的发酵提取物中分离鉴定了一个聚酮类化合物Naphthgeranine A。该菌株的全基因组测序为其生物合成基因簇的鉴定提供了前提,对Naphthgeranine A生物合成基因簇和生物合成途径的推测为进一步研究这个化合物的生物合成机制奠定了基础。     

基因簇大片段克隆技术研究进展及挑战

天然产物结构复杂、活性多样,是新药开发的重要来源,对天然产物生物合成途径的研究,有利于探索酶催化的合成机制,促进复杂天然产物的应用。天然产物的生物合成由其对应的基因簇调控,其中大量天然产物生物合成基因簇(biosynthetic gene clusters,BGCs)在野生型菌株中无法表达或表达量低。对这些基因簇的研究,需要进行克隆表达,而如何克隆大片段基因簇并使其表达,从而发现新型天然产物是一个具有挑战性的问题。其中构建基因组文库、转化关联重组(transformation-associated recombination,TAR)、Red/ET重组等是克隆大片段基因簇的重要技术。本文从克隆技术的策略和应用两个方面,总结了这3种克隆技术目前的研究进展,讨论了目前大片段基因簇克隆技术面临的挑战,为研究大片段基因簇提供方法学借鉴。     

海洋共附生微生物天然产物生物合成基因研究进展

对海洋无脊椎动物天然产物的研究表明,很多种活性物质的真正生产者是与其共生或附生的未培养微生物.克隆这些未培养微生物中特定活性物质的生物合成基因,不仅为活性物质的来源提供遗传学证据,也使通过异源表达相关生物合成基因来大量获取目标化合物成为可能.本文综述了来源于海绵、海鞘、苔藓虫、深海管状蠕虫和深海沉积物中共附生微生物天然产物生物合成基因簇的研究进展.     

白僵菌天然产物及其药理活性和生物合成研究进展

白僵菌是重要的昆虫病原真菌,能产生多肽类、聚酮类、生物碱类、苯丙素类、萜类、核苷类等多种结构类型的天然产物,其中很多天然产物显示出优良的抗肿瘤、抗菌、抗病毒和杀虫等活性,具有极大的应用开发潜力。随着白僵菌基因组测序的完成,白僵菌素、白僵菌交酯、球孢交酯、卵孢素及纤细素等活性分子的生物合成基因簇及其生物合成机制已得到阐明,这些研究将大大促进白僵菌来源的新结构活性天然产物的基因组挖掘和发现以及已知重要活性分子的开发应用。本文对已知白僵菌产生的天然产物、药理活性及重要活性分子的生物合成途径进行了概括总结,为系统开发白僵菌天然产物资源提供参考。     

Current strategies to induce secondary metabolites from microbial biosynthetic cryptic gene clusters

The genome of actinomycetes and several other microorganisms are endowed with many cryptic gene clusters that can code for previously undetected, a plethora of complex secondary metabolites. Under standard laboratory controlled conditions, the genes regulating these biosynthetic clusters are expressed at very low levels or remain phenotypically cryptic (silent). Over the past several decades, multi-drug-resistant bacteria have been observed with increased frequency, posing a significant threat to human health worldwide. The present alarming situation urgently calls for concerted global efforts for the discovery of new antimicrobials. The present situation, if not controlled, will take us again to the pre-antibiotic era. Today, in the post-genomic era, various new strategies such as the activation of cryptic gene clusters in microorganisms rejuvenate a new conviction in the field of natural product research that may lead to the identification of yet-unidentified novel secondary metabolites of therapeutic and other use. Decryptification of this versatile endogenous genetic reservoir may provide in the near future the more concrete rationale for antibiotic discovery. The present review is an attempt to provide a comprehensive detail, outlining current strategies that have been shown successful to activate cryptic biosynthetic gene clusters in microorganisms.     

Renewed interests in the discovery of bioactive actinomycete metabolites driven by emerging technologies

Actinomycetes are prolific sources of bioactive molecules. Traditional workflows including bacterial isolation, fermentation, metabolite identification and structure elucidation have resulted in high rates of natural product rediscovery in recent years. Recent advancements in multi-omics techniques have uncovered cryptic gene clusters within the genomes of actinomycetes, potentially introducing vast resources for the investigation of bioactive molecules. While developments in culture techniques have allowed for the fermentation of difficult-to-culture actinomycetes, high-throughput metabolite screening has offered plenary tools to accelerate hits discovery. A variety of new bioactive molecules have been isolated from actinomycetes of unique environmental origins, such as endophytic and symbiotic actinomycetes. Synthetic biology and genome mining have also emerged as new frontiers for the discovery of bioactive molecules. This review covers the highlights of recent developments in actinomycete-derived natural product drug discovery.     

Microbial genomics for the improvement of natural product discovery

The quest for the discovery of novel natural products has entered a new chapter with the enormous wealth of genetic data that is now available. This information has been exploited by using whole-genome sequence mining to uncover cryptic pathways, or biosynthetic pathways for previously undetected metabolites. Alternatively, using known paradigms for secondary metabolite biosynthesis, genetic information has been 'fished out' of DNA libraries resulting in the discovery of new natural products and isolation of gene clusters for known metabolites. Novel natural products have been discovered by expressing genetic data from uncultured organisms or difficult-to-manipulate strains in heterologous hosts. Furthermore, improvements in heterologous expression have not only helped to identify gene clusters but have also made it easier to manipulate these genes in order to generate new compounds. Finally, and perhaps the most crucial aspect of the efficient and prosperous use of the abundance of genetic information, novel enzyme chemistry continues to be discovered, which has aided our understanding of how natural products are biosynthesized de novo, and enabled us to rework the current paradigms for natural product biosynthesis.     

New strategies and approaches for engineering biosynthetic gene clusters of microbial natural products

With the rapidly growing number of sequenced microbial (meta)genomes, enormous cryptic natural product (NP) biosynthetic gene clusters (BGCs) have been identified, which are regarded as a rich reservoir for novel drug discovery. A series of powerful tools for engineering BGCs has accelerated the discovery and development of pharmaceutically active NPs. Here, we describe recent advances in the strategies for BGCs manipulation, which are driven by emerging technologies, including efficient DNA recombination systems, versatile CRISPR/Cas9 genome editing tools and diverse DNA assembly methods. We further discuss how these approaches could be used for genome mining studies and industrial strain improvement.     

Culture-independent discovery of natural products from soil metagenomes

Bacterial natural products have proven to be invaluable starting points in the development of many currently used therapeutic agents. Unfortunately, traditional culture-based methods for natural product discovery have been deemphasized by pharmaceutical companies due in large part to high rediscovery rates. Culture-independent, or “metagenomic,” methods, which rely on the heterologous expression of DNA extracted directly from environmental samples (eDNA), have the potential to provide access to metabolites encoded by a large fraction of the earth’s microbial biosynthetic diversity. As soil is both ubiquitous and rich in bacterial diversity, it is an appealing starting point for culture-independent natural product discovery efforts. This review provides an overview of the history of soil metagenome-driven natural product discovery studies and elaborates on the recent development of new tools for sequence-based, high-throughput profiling of environmental samples used in discovering novel natural product biosynthetic gene clusters. We conclude with several examples of these new tools being employed to facilitate the recovery of novel secondary metabolite encoding gene clusters from soil metagenomes and the subsequent heterologous expression of these clusters to produce bioactive small molecules.     

Drug discovery prospect from untapped species: indications from approved natural product drugs

Due to extensive bioprospecting efforts of the past and technology factors, there have been questions about drug discovery prospect from untapped species. We analyzed recent trends of approved drugs derived from previously untapped species, which show no sign of untapped drug-productive species being near extinction and suggest high probability of deriving new drugs from new species in existing drug-productive species families and clusters. Case histories of recently approved drugs reveal useful strategies for deriving new drugs from the scaffolds and pharmacophores of the natural product leads of these untapped species. New technologies such as cryptic gene-cluster exploration may generate novel natural products with highly anticipated potential impact on drug discovery.     

Lessons learned from the transformation of natural product discovery to a genome-driven endeavor

Natural product discovery is currently undergoing a transformation from a phenotype-driven field to a genotype-driven one. The increasing availability of genome sequences, coupled with improved techniques for identifying biosynthetic gene clusters, has revealed that secondary metabolomes are strikingly vaster than previously thought. New approaches to correlate biosynthetic gene clusters with the compounds they produce have facilitated the production and isolation of a rapidly growing collection of what we refer to as “reverse-discovered” natural products, in analogy to reverse genetics. In this review, we present an extensive list of reverse-discovered natural products and discuss seven important lessons for natural product discovery by genome-guided methods: structure prediction, accurate annotation, continued study of model organisms, avoiding genome-size bias, genetic manipulation, heterologous expression, and potential engineering of natural product analogs.     

丝状真菌新型全局性调控因子LaeA

全局调控在丝状真菌次级代谢调控及其生长发育过程中有着重要的作用。LaeA是2004年首次在构巢曲霉中被发现的第一个丝状真菌全局性调控因子,继而在烟曲霉、黄曲霉、产黄青霉、橘青霉中相继被报道。LaeA能够全局性调控抗生素和真菌毒素等次级代谢产物的合成,影响真菌形态分化,另外还通过影响沉默基因的表达从而调控未知代谢产物的产生,因而能为真菌中天然产物的开发提供新的重要途径。本文就其在丝状真菌中的发现、功能、作用机制及其应用等方面进行综述。     

Non-ribosomal peptide synthetases: Identifying the cryptic gene clusters and decoding the natural product

Non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) present in bacteria and fungi are the major multi-modular enzyme complexes which synthesize secondary metabolites like the pharmacologically important antibiotics and siderophores. Each of the multiple modules of an NRPS activates a different amino or aryl acid, followed by their condensation to synthesize a linear or cyclic natural product. The studies on NRPS domains, the knowledge of their gene cluster architecture and tailoring enzymes have helped in the in silico genetic screening of the ever-expanding sequenced microbial genomic data for the identification of novel NRPS/PKS clusters and thus deciphering novel non-ribosomal peptides (NRPs). Adenylation domain is an integral part of the NRPSs and is the substrate selecting unit for the final assembled NRP. In some cases, it also requires a small protein, the MbtH homolog, for its optimum activity. The presence of putative adenylation domain and MbtH homologs in a sequenced genome can help identify the novel secondary metabolite producers. The role of the adenylation domain in the NRPS gene clusters and its characterization as a tool for the discovery of novel cryptic NRPS gene clusters are discussed.