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.     

Combinatorial biosynthesis for new drug discovery

Combinatorial biosynthesis involves interchanging secondary metabolism genes between antibiotic-producing microorganisms to create unnatural gene combinations or hybrid genes if only part of a gene is exchanged. Novel metabolites can be made by both approaches, due to the effect of a new enzyme on a metabolic pathway or to the formation of proteins with new enzymatic properties. The method has been particularly successful with polyketide synthase (PKS) genes: derivatives of medically important macrolide antibiotics and unusual polycyclic aromatic compounds have been produced by novel combinations of the type I and type II PKS genes, respectively. Recent extensions of the approach to include deoxysugar biosynthesis genes have expanded the possibilities for making new microbial metabolites and discovering valuable drugs through the genetic engineering of bacteria.     

On the evolution of functional secondary metabolites (natural products)

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.     

Total biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α: heterologous expression of four biosynthetic genes in Aspergillus oryzae

Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions. The resulting quadruple transformant carrying the genes of geranylgeranyl diphosphate synthase PbGGS, terpene synthase PbACS, and two monooxygenases (PbP450-1 and PbP450-2) produced aphidicolin. The double and triple transformants also respectively produced aphidicolan-16β-ol and 3-deoxyaphidicolin. Alternative host Saccharomyces cerevisiae carrying the genes, PbGGS and PbACS, produced key intermediate aphidicolan-16β-ol. This is the first example of a total biosynthesis of terpenoids using fungal hosts.     

基因组挖掘技术在海洋放线菌天然产物研究开发中的应用及展望

利用微生物的基因组信息预测其合成特定天然产物的潜能, 进而进行新化合物分离纯化和结构鉴定的基因组挖掘技术, 已经成为国内外研究的热点, 并在多种细菌和真菌的天然产物发现中得到成功应用。本文综述了基因组挖掘技术的最新进展, 包括生物信息分析和结构预测、基因组指导的天然产物的发现、沉默基因的激活和异源表达技术等, 以及我国学者开发的转录组挖掘技术, 并重点综述了影像质谱技术在基因组挖掘中的应用。目前对海洋放线菌进行基因组挖掘的研究还比较少, 而基因组挖掘技术的发展, 将极大地促进对海洋放线菌天然产物的发现和鉴定。未来除了充分挖掘可培养微生物的基因组, 对未培养微生物宏基因组的挖掘将进一步深入。此外, 除了开发利用基因组中合成天然产物的结构基因和调节基因, 还应该充分开发利用其他不同的遗传元件, 包括不同转录活性和响应不同环境条件和信号的启动子, 以及具有调节作用的RNA等。     

Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes: isolation, characterization and generation of novel glycosylated derivatives

Many bioactive natural products synthesized by actinomycetes are glycosylated compounds in which the appended sugars contribute to specific interactions with their biological target. Most of these sugars are 6-deoxyhexoses, of which more than 70 different forms have been identified, and an increasing number of gene clusters involved in 6-deoxyhexoses biosynthesis are being characterized from antibiotic-producing actinomycetes. Novel glycosylated compounds have been generated by modifying natural deoxysugar biosynthesis pathways in the producer organisms, and/or the simultaneous expression in these strains of selected deoxysugar biosynthesis genes from other strains. Non-producing strains endowed with the capacity to synthesize novel deoxysugars through the expression of engineered deoxysugar biosynthesis clusters can also be used as alternative hosts. Transfer of these deoxysugars to a multiplicity of aglycones relies upon the existence of glycosyltransferases with an inherent degree of 'relaxed substrate specificity'. In this review, we analyze how the knowledge coming out from isolation and characterization of deoxysugar biosynthesis pathways from actinomycetes is being used to produce novel glycosylated derivatives of natural products.     

The Logic,Experimental Steps,and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling >50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.     

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.     

<Emphasis Type="Italic">In silico</Emphasis> analysis and experimental improvement of taxadiene heterologous biosynthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The biosynthesis of terpenoids in heterologous hosts has become increasingly popular. Isopentenyl diphosphate (IPP) is the central precursor of all isoprenoids, and the synthesis can proceed via two separate pathways in different organisms: The 1-deoxylulose 5-phosphate (DXP) pathway and the mevalonate (MVA) pathway. In this study, an in silico comparison was made between the maximum theoretical IPP yields and the thermodynamic properties of the DXP and MVA pathways using different hosts and carbon sources. We found that Escherichia coli and its DXP pathway have the most potential for IPP production. Consequently, codon usage redesign, and combinations of chromosomal engineering and various strains were considered for optimizing taxadiene biosynthesis through the endogenic DXP pathway. A high production strain yielding 876 ± 60 mg/L taxadiene, with an overall volumetric productivity of 8.9 mg/(L × h), was successfully obtained by combining the chromosomal engineered upstream DXP pathway and the downstream taxadiene biosynthesis pathway. This is the highest yield thus far reported for taxadiene production in a heterologous host. These results indicate that genetic manipulation of the DXP pathway has great potential to be used for production of terpenoids, and that chromosomal engineering is a powerful tool for heterologous biosynthesis of natural products.     

Molecular approaches for improvement of medicinal and aromatic plants

Medicinal and aromatic plants (MAPs) are important sources for plant secondary metabolites, which are important for human healthcare. Improvement of the yield and quality of these natural plant products through conventional breeding is still a challenge. However, recent advances in plant genomics research has generated knowledge leading to a better understanding of the complex genetics and biochemistry involved in biosynthesis of these plant secondary metabolites. This genomics research also concerned identification and isolation of genes involved in different steps of a number of metabolic pathways. Progress has also been made in the development of functional genomics resources (EST databases and micro-arrays) in several medicinal plant species, which offer new opportunities for improvement of genotypes using perfect markers or genetic transformation. This review article presents an overview of the recent developments and future possibilities in genetics and genomics of MAP species including use of transgenic approach for their improvement.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

聚酮类抗生素组合生物合成

组合生物合成是公认的产生大量"非天然"的天然产物的一种有效方法,也是近年来药物创新与应用的研究热点和重要手段之一。目前,组合生物合成在聚酮类抗生素等生物活性物质的开发应用研究中已经取得了显著的成果。结合文献中的例子,回顾了运用组合生物合成在天然产物的基础上产生更多结构及功能多样性的聚酮类抗生素的方法和思路,并对某些方法所存在的问题与不足进行了讨论。     

Engineering the glycosylation of natural products in actinomycetes

Bioactive natural products are frequently glycosylated with saccharide chains of different length, in which the sugars contribute to specific interactions with the biological target. Combinatorial biosynthesis approaches are being used in antibiotic-producing actinomycetes to generate derivatives with novel sugars in their architecture. Recent advances in this area indicate that glycosyltransferases involved in the biosynthesis of natural products have substrate flexibility regarding the sugar donor but also, less frequently, with respect to the aglycon acceptor. Therefore, the possibility exists of altering the glycosylation pattern of natural products, thus enabling an increase in the structural diversity of natural products.     

Biosynthesis of Polyketides in Heterologous Hosts

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.     

Biosynthesis of polyketides in heterologous hosts.

Polyketide natural products show great promise as medicinal agents. Typically the products of microbial secondary biosynthesis, polyketides are synthesized by an evolutionarily related but architecturally diverse family of multifunctional enzymes called polyketide synthases. A principal limitation for fundamental biochemical studies of these modular megasynthases, as well as for their applications in biotechnology, is the challenge associated with manipulating the natural microorganism that produces a polyketide of interest. To ameliorate this limitation, over the past decade several genetically amenable microbes have been developed as heterologous hosts for polyketide biosynthesis. Here we review the state of the art as well as the difficulties associated with heterologous polyketide production. In particular, we focus on two model hosts, Streptomyces coelicolor and Escherichia coli. Future directions for this relatively new but growing technological opportunity are also discussed.     

It works: combinatorial biosynthesis for generating novel glycosylated compounds

Combinatorial biosynthesis is a valuable method to generate novel glycosylated natural products. By coexpression of deoxysugar gene cassettes and genes from the staurosporine biosynthetic gene cluster it has now been applied to the generation of novel staurosporine derivatives. The work of Salas and co-workers is highlighted in this article.     

Biosynthesis of deoxyaminosugars in antibiotic-producing bacteria

Deoxyaminosugars comprise an important class of deoxysugars synthesized by a variety of different microorganisms; they can be structural components of lipopolysaccharides, extracellular polysaccharides, and secondary metabolites such as antibiotics. Genes involved in the biosynthesis of the deoxyaminosugars are often clustered and are located in the vicinity of other genes required for the synthesis of the final compound. Most of the gene clusters for aminosugar biosynthesis have common features, as they contain genes encoding dehydratases, isomerases, aminotransferases, methyltransferases, and glycosyltransferases. In the present mini-review, the proposed biosynthetic pathways for deoxyaminosugar components of both macrolide and non-macrolide antibiotics are highlighted. The possibilities for genetic manipulations of the deoxyaminosugar biosynthetic pathways aimed at production of novel secondary metabolites are discussed.     

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

Over the past decade, the evolving commercial importance of so-called plant secondary metabolites has resulted in a great interest in secondary metabolism and, particularly, in the possibilities to enhance the yield of fine metabolites by means of genetic engineering. Plant alkaloids, which constitute one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the tropane alkaloids biosynthesis pathways have been cloned, making the metabolic engineering of these alkaloids possible. The content of the target chemical scopolamine could be significantly increased by various approaches, such as introducing genes encoding the key biosynthetic enzymes or genes encoding regulatory proteins to overcome the specific rate-limiting steps. In addition, antisense genes have been used to block competitive pathways. These investigations have opened up new, promising perspectives for increased production in plants or plant cell culture. Recent achievements have been made in the metabolic engineering of plant tropane alkaloids and some new powerful strategies are reviewed in the present paper.     

The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures

Fragments spanning 20 kb of Streptomyces nogalater genomic DNA were characterized to elucidate the molecular genetic basis of the biosynthetic pathway of the anthracycline antibiotic nogalamycin. Structural analysis of the products obtained by expression of the fragments in S. galilaeus and S. peucetius mutants producing aclacinomycin and daunomycin metabolites, respectively, revealed hybrid compounds in which either the aglycone or the sugar moiety was modified. Subsequent sequence analysis revealed twenty ORFs involved in nogalamycin biosynthesis, of which eleven could be assigned to the deoxysugar pathway, four to aglycone biosynthesis, while the remaining five express products with unknown function. On the basis of sequence similarity and experimental data, the functions of the products of the newly discovered genes were determined. The results suggest that the entire biosynthetic gene cluster for nogalamycin is now known. Furthermore, the compounds obtained by heterologous expression of the genes show that it is possible to use the genes in combinatorial biosynthesis to create novel chemical structures for drug screening purposes.     

Strategies for mining fungal natural products

Fungi are well known for their ability to produce a multitude of natural products. On the one hand their potential to provide beneficial antibiotics and immunosuppressants has been maximized by the pharmaceutical industry to service the market with cost-efficient drugs. On the other hand identification of trace amounts of known mycotoxins in food and feed samples is of major importance to ensure consumer health and safety. Although several fungal natural products, their biosynthesis and regulation are known today, recent genome sequences of hundreds of fungal species illustrate that the secondary metabolite potential of fungi has been substantially underestimated. Since expression of genes and subsequent production of the encoded metabolites are frequently cryptic or silent under standard laboratory conditions, strategies for activating these hidden new compounds are essential. This review will cover the latest advances in fungal genome mining undertaken to unlock novel products.