Deoxysugars in bioactive natural products: development of novel derivatives by altering the sugar pattern

Bioactive natural products are frequently glycosylated with saccharide chains of variable length. These sugars are important for the biological activity of the compounds and they contribute to the interaction with the biological target. The increasing knowledge of sugar biosynthesis pathways and the isolation of a large number of sugar gene clusters from antibiotic-producing actinomycetes are providing tools for combinatorial biosynthesis approaches that can generate potentially improved derivatives with altered sugars in their architecture. Novel derivatives of known bioactive natural products can be produced either in the producer organisms or in heterologous hosts by using different combinatorial biosynthesis strategies. In this article, recent advances in the field are discussed, illustrating the alternative approaches of gene inactivation, gene expression, combining gene inactivation and gene expression, co-expression of genes from different pathways or the use of sugar cassette plasmids to endow a host with the capability of synthesizing new sugars.     

非核糖体肽类化合物的组合生物合成策略研究进展

很多微生物能够利用非核糖体肽合酶合成结构复杂、种类繁多、生物活性多样的小分子肽类化合物.组合生物合成是对控制抗生素生物合成的基因簇进行阻断、置换、重组或异源表达等遗传操作,从而达到利用生物技术和环境友好的手段构建化合物衍生物库的目的.组合生物合成在增加天然活性化合物的数量,改良天然化合物的生物学活性,提高天然化合物的产量,开发创新药物和酶制剂等领域都具有重要应用价值.近年来,非核糖体肽的组合生物合成研究取得了重要进展.本文就非核糖体肽合酶的组合生物合成研究策略,从模块定点突变、替换、插入、删除、模块“洗牌”与异源表达等角度进行了综述.     

Combinatorial biosynthesis, metabolic engineering and mutasynthesis for the generation of new aminocoumarin antibiotics

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are produced by different Streptomyces strains. They are potent inhibitors of bacterial gyrase and topoisomerase IV, and novobiocin has been licensed as antibiotic for clinical use (Albamycin). They also have potential applications in oncology. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of nearly all genes contained therein has been elucidated. Rapid and versatile methods have been developed for the heterologous expression of these biosynthetic gene clusters, and in Streptomyces coelicolor M512 as heterologous host these antibiotics were produced in yields comparable to those in the natural producer strains. lambda RED-mediated homologous recombination was used for genetic modification of the gene clusters in Escherichia coli. The phage PhiC31 attachment site and integrase functions were introduced into the cosmid backbones and employed for stable integration of the clusters into the genome of the heterologous hosts. Modification of the clusters by single or multiple gene replacements or gene deletions resulted in the formation of numerous new aminocoumarin derivatives, providing an efficient tool for the rational generation of antibiotics with modified structure. Additionally, many new antibiotics were generated by mutasynthesis experiments, i.e. the targeted deletion of genes required for the biosynthesis of a certain structural moiety of the antibiotic, and the replacement of this moiety by structural analogs which were added to the culture broth. The diversity of new structures obtained by this approach could be expanded by further genetic modifications of the gene deletion mutants, especially by expression of heterologous biosynthetic enzymes with appropriate substrate specificity.     

The biosynthetic gene clusters of aminocoumarin antibiotics

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A (1), which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.     

Plasticity of the streptomyces genome-evolution and engineering of new antibiotics

Streptomyces is a genus of soil dwelling bacteria with the ability to produce natural products that have found widespread use in medicine. Annotation of Streptomyces genome sequences has revealed far more biosynthetic gene clusters than previously imagined, offering exciting possibilities for future combinatorial biosynthesis. Experiments to manipulate modular biosynthetic clusters to create novel chemistries often result in no detectable product or product yield is extremely low. Understanding the coupling between components in these hybrid enzymes will be crucial for efficient synthesis of new compounds. We are using new algebraic approaches to predict protein properties, and homologous recombination to exploit natural evolutionary constraints to generate novel functional enzymes. The methods and techniques developed could easily be adapted to study modular, multi-interacting complex systems where appreciable biochemical and comparative sequence data are available, for example, clinically significant non-ribosomally synthesised peptides and polyketides.     

微生物次级代谢产物生物合成基因簇与药物创新

微生物产生众多结构和生物活性多样的次级代谢产物，其生物合成基因簇的克隆是药物创新和产量提高的必要前提。迄今为止已有超过150种生物合成基因簇通过各种方式被克隆，并被用于组合生物合成、体外糖类随机化、代谢工程的定向改造。我们研究室已经克隆并测定了氨基糖苷类井冈霉索／有效霉索、多烯类抗生素FR-008／克念菌索、聚醚类南昌霉索、聚酮类梅岭霉索、杂合聚酮一多肽类略唑霉索等生物合成基因簇。深入的基因功能分析揭示了他们独特的生物合成途径和调节机理，为正在进行的组合生物合成结构改造和代谢工程产量提高奠定了基础。     

海洋微生物次级代谢产物生物合成的研究进展

海洋微生物次级代谢产物往往具有新颖的化学结构,蕴含着独特的生物合成途径、酶学机理和不同于陆生放线菌次级代谢产物的生物合成机制。自从2000年第一例海洋微生物天然产物enterocin的生物合成基因簇被阐明以来,迄今已克隆和鉴定了27种海洋微生物次级代谢产物的完整生物合成基因簇。这些次级代谢产物的生物合成主要源于四种途径,包括聚酮合酶途径,非核糖体肽合成酶途径,聚酮-非核糖体肽合成酶杂合途径,以及其他途径。本文综述了近年来一些重要海洋微生物活性次级代谢产物的生物合成途径,以及组合生物合成技术在海洋微生物次级代谢产物结构多样化方面的应用。     

Using natural products for drug discovery: the impact of the genomics era

Introduction: Evolutionarily selected over billions of years for their interactions with biomolecules, natural products have been and continue to be a major source of pharmaceuticals. In the 1990s, pharmaceutical companies scaled down their natural product discovery programs in favor of synthetic chemical libraries due to major challenges such as high rediscovery rates, challenging isolation, and low production titers. Propelled by advances in DNA sequencing and synthetic biology technologies, insights into microbial secondary metabolism provided have inspired a number of strategies to address these challenges.

Areas covered: This review highlights the importance of genomics and metagenomics in natural product discovery, and provides an overview of the technical and conceptual advances that offer unprecedented access to molecules encoded by biosynthetic gene clusters.

Expert opinion: Genomics and metagenomics revealed nature’s remarkable biosynthetic potential and her vast chemical inventory that we can now prioritize and systematically mine for novel chemical scaffolds with desirable bioactivities. Coupled with synthetic biology and genome engineering technologies, significant progress has been made in identifying and predicting the chemical output of biosynthetic gene clusters, as well as in optimizing cluster expression in native and heterologous host systems for the production of pharmaceutically relevant metabolites and their derivatives.     

Enediyne natural products: biosynthesis and prospect towards engineering novel antitumor agents

This review gives a brief account on the current status of enediyne biosynthesis and the prospective of applying combinatorial biosynthesis methods to the enediyne system for novel analog production. Methods for cloning enediyne biosynthetic gene clusters are first reviewed. A unified paradigm for enediyne biosynthesis, characterized with (a) the enediyne PKS, (b) the enediyne PKS accessory enzymes, and (c) tailoring enzymes, is then presented. Strategies and tools for novel enediyne analog production by combinatorial biosynthesis are finally discussed. The results set the stage to decipher the molecular mechanism for enediyne biosynthesis and lay the foundation to engineer novel enediynes by combinatorial biosynthesis for future endeavors.     

Metagenomic sequencing-driven multidisciplinary approaches to shed light on the untapped microbial natural products

The advantage of metagenomics over the culture-based natural product (NP) discovery pipeline is the ability to access the biosynthetic potential of uncultivable microbes. Advances in DNA sequencing are revolutionizing conventional metagenomics approaches for microbial NP discovery. The genomes of (in)cultivable bugs can be resolved straightforwardly from environmental samples, enabling in situ prediction of biosynthetic gene clusters (BGCs). The predicted chemical diversities could be realized not only by heterologous expression of gene clusters originating from DNA synthesis or direct cloning, but also potentially by bioinformatic-directed organic synthesis or chemoenzymatic total synthesis. In this review, we suggest that metagenomic sequencing in tandem with multidisciplinary approaches will form a versatile platform to shed light on a plethora of microbial ‘dark matter’.     

Bacterial hosts for natural product production

Four bacterial hosts are reviewed in the context of either native or heterologous natural product production. E. coli, B. subtilis, pseudomonads, and Streptomyces bacterial systems are presented with each having either a long-standing or more recent application to the production of therapeutic natural compounds. The four natural product classes focused upon include the polyketides, nonribosomal peptides, terpenoids, and flavonoids. From the perspective of both innate and heterologous production potential, each bacterial host is evaluated according to biological properties that would either hinder or facilitate natural product biosynthesis.     

Biosynthesis of hybrid peptide-polyketide natural products

The structural and catalytic similarities between non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) support the idea of combining individual NRPS and PKS modules for combinatorial biosynthesis. Recent advances in cloning and characterization of biosynthetic gene clusters for naturally occurring hybrid polyketide-peptide metabolites have provided direct evidence for the existence of hybrid NRPS-PKS systems, thus setting the stage to investigate the molecular basis for intermodular communication between NRPS and PKS modules. Reviewed in this article are biosynthetic data pertinent to hybrid peptide-polyketide biosynthesis published up to late 2000. Hybrid peptide-polyketide natural products can be divided into two classes: (i) those whose biosyntheses do not involve functional interaction between NRPS and PKS modules; and (ii) those whose biosyntheses are catalyzed by hybrid NRPS-PKS systems involving direct interactions between NRPS and PKS modules. It is the latter systems that are most likely amenable to combinatorial biosynthesis. The same catalytic sites appear to be conserved in both hybrid NRPS-PKS and normal NRPS or PKS systems, with the exception of the ketoacyl synthase domains in hybrid NRPS-PKS systems which are unique. Specific linkers may play a critical role in communication, facilitating the transfer of the growing intermediates between the interacting NRPS and/or PKS modules. In addition, phosphopantetheinyl transferases with broad carrier protein specificity are essential for the production of functional hybrid NRPS-PKS megasynthetases. These findings should now be taken into consideration in engineered biosynthesis of hybrid peptide-polyketide natural products for drug discovery and development.     

链霉菌沉默生物合成基因簇调控的研究进展

本文以天蓝色链霉菌(Streptomyces     

链霉菌沉默生物合成基因簇调控的研究进展

本文以天蓝色链霉菌(Streptomyces coelicolor)中黄色色素coelimycin生物合成调控及开发策略的研究进展为代表,介绍了链霉菌中沉默生物合成基因簇调控激活的新进展,为链霉菌天然产物基因簇的挖掘和新次级代谢产物的发现提供研究思路。     

Advances in polyketide synthase structure and function

Recent progress in the understanding of polyketide synthase (PKS) continues to fuel the growth of combinatorial biosynthesis for natural product structural diversity. The structural analysis of many components of PKS, in particular for the modular type I 6-deoxyerythronilide B synthase (DEBS) involved in erythromycin biosynthesis, has provided structural imperatives for the observed biochemistry of DEBS and has enabled the generation of a working structural model of the entire DEBS system. New functions for PKS domains continue to be defined, such as the general control nonderepressible 5 (GCN5) N-acyltransferase strategy for polyketide chain initiation and the true identity of the elusive precursor for the methoxymalonylate extender unit. Novel molecular architectures have been continuously uncovered, including the 'AT-less' PKS and enediyne PKS, thereby expanding the known bacterial PKS paradigms beyond the prototypical type I, II and III PKSs. Finally, the genetic characterization of PKS in vivo and biochemical studies of PKS in vitro have also been greatly facilitated by the application of emerging technologies, such as RNA-mediated gene silencing, reconstitution of an entire polyketide biosynthetic pathway in a model heterologous host and Fourier-transform mass spectroscopy. The application of these technologies is discussed.     

异源表达策略在抗生素研究中的应用进展

异源表达在抗生素研究中有广泛的应用,如鉴定基因功能、克隆抗生素合成基因、调节基因表达、提高抗生素产量、组合生物合成和发现活性代谢产物等各个方面。本文简单综述异源表达策略在抗生素研究中的应用进展。     

New aminocoumarin antibiotics from genetically engineered Streptomyces strains

The aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A(1) are produced by different Streptomyces strains and are potent inhibitors of DNA gyrase. The biosynthetic gene clusters of all three antibiotics have been cloned and sequenced, and the function of most genes contained therein has been elucidated. In the last years, a number of "unnatural" aminocoumarins could be generated using the genetic information for the biosynthesis of these antibiotics. The investigated enzymes of aminocoumarin biosynthesis have less-than-perfect substrate specificity, facilitating the production of new antibiotics by various methods. Several new aminocoumarins could be produced by targeted genetic manipulation in the natural producers, but also in heterologous host Streptomyces coelicolor after expression of the respective gene cluster. Mutasynthesis experiments, i. e. generation of a cloQ-defective mutant of the clorobiocin producer and feeding of 13 different structural analogs of 3-dimethylallyl-4-hydroxybenzoic acid to this mutant, allowed the isolation of 32 new aminocoumarins. These compounds contained, instead of the genuine 3-dimethylallyl-4-hydroxybenzoyl moiety, the externally added analogs as the acyl components in their structures. Production of new aminocoumarins was also achieved by chemoenzymatic synthesis in vitro. Several biosynthetic enzymes have been heterologously expressed, purified und used for chemoenzymatic synthesis. The structures of the new aminocoumarins were elucidated by NMR and mass spectroscopy. Their inhibitory activity on gyrase in vitro as well as their antibacterial activity was determined. These results give further insight into the structure-activity relationships of aminocoumarins.     

Mining the microbial metabolome: a new frontier for natural product lead discovery

Traditionally, natural products have been important sources of new leads for the pharmaceutical industry, but with discovery rates of novel structural classes in decline, the need to bioprospect alternate sources of chemical diversity is evident. Microbial genome sequencing projects have revealed the presence of 'silent' biosynthetic gene clusters where there is no current detectable product. Likewise, culture-independent techniques have provided access to the collective genomes of environmental microflora. Both sources of molecular diversity could encode potentially valuable metabolites. The ability to measure the entire complement of metabolites within microorganisms that are used as surrogate hosts to express such gene clusters will be crucial to the exploitation of these yet untapped reservoirs of metabolic diversity for future natural product drug discovery.