Biosynthesis of pyrrolopyrimidines

Pyrrolopyrimidine containing compounds, also known as 7-deazapurines, are a collection of purine-based metabolites that have been isolated from a variety of biological sources and have diverse functions which range from secondary metabolism to RNA modification. To date, nearly 35 compounds with the common 7-deazapurine core structure have been described. This article will illustrate the structural diversity of these compounds and review the current state of knowledge on the biosynthetic pathways that give rise to them.     

Polyene antibiotic biosynthesis gene clusters

Over the past 15 years the biosynthetic gene clusters for numerous bioactive polyketides have been intensively studied and recently this work has been extended to the antifungal polyene macrolides. These compounds consist of large macrolactone rings that have a characteristic series of conjugated double bonds, as well as an exocyclic carboxyl group and an unusual mycosamine sugar. The biosynthetic gene clusters for nystatin, pimaricin, amphotericin and candicidin have been investigated in detail. These clusters contain the largest modular polyketide synthase genes reported to date. This body of work also provides insights into the enzymes catalysing the unusual post-polyketide modifications, and the genes regulating antibiotic biosynthesis. The sequences also provide clues about the evolutionary origins of polyene biosynthetic genes. Successful genetic manipulation of the producing organisms leading to production of polyene analogues indicates good prospects for generating improved antifungal compounds via genetic engineering.     

An Introduction to the Biosynthesis of Chemicals Used in Plant-Microbe Communication

Abstract Plants accumulate a diverse array of natural products, which can serve either to defend the plant against various microbes in its environment or to attract various microbes, both beneficial and pathogenic. Plants must also attract pollinators, repel or poison herbivores, compete with other plant species, and protect themselves from environmental dangers such as high light intensities. Some compounds have been implicated in playing a role in multiple interactions. Although the structures vary immensely in size and complexity, most are derived from a limited number of core biosynthetic pathways. This review briefly summarizes the biosynthetic origins of phenylpropanoid (including simple phenolics, flavonoids, anthocyanins and isoflavonoids), polyacetate, terpenoid, and alkaloid classes of metabolites. Compounds reported to be important in plant-microbe, plant-animal, and plant-plant interactions will be given as examples of each of these classes. Other aspects of biosynthesis also will be discussed, including the timing or location of biosynthesis, the potential for genetic manipulation of these pathways, and various questions regarding the biosynthesis of these compounds.     

Synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of O-antigen repeating units

O-antigens of Gram negative bacteria are polysaccharides covalently attached to lipopolysaccharides (LPS) that have roles as virulence factors. Due to the lack of defined substrates for in vitro assays only a few of the enzymes involved in the biosynthesis of O-antigens have been studied. Many O-antigens have GlcNAc at the reducing end of the oligosaccharide chain linked to pyrophosphate-lipid. We therefore designed and synthesized a series of GlcNAc-pyrophosphate-lipid analogs of the natural GlcNAc-pyrophosphate-undecaprenol acceptor substrate for studies of the acceptor specificities of O-antigen biosynthetic enzymes. We synthesized analogs with modifications of the pyrophosphate bond as well as the lipid chain. These compounds will be useful for the specificity studies of many bacterial glycosyltransferases. Knowledge of the substrate specificities is the basis for the development of specific glycosyltransferase inhibitors that could block O-antigen biosynthesis.     

The aureolic acid family of antitumor compounds: structure, mode of action, biosynthesis, and novel derivatives

Members of the aureolic acid family are tricyclic polyketides with antitumor activity which are produced by different streptomycete species. These members are glycosylated compounds with two oligosaccharide chains of variable sugar length. They interact with the DNA minor groove in high-GC-content regions in a nonintercalative way and with a requirement for magnesium ions. Mithramycin and chromomycins are the most representative members of the family, mithramycin being used as a chemotherapeutic agent for the treatment of several cancer diseases. For chromomycin and durhamycin A, antiviral activity has also been reported. The biosynthesis gene clusters for mithramycin and chromomycin A₃ have been studied in detail by gene sequencing, insertional inactivation, and gene expression. Most of the biosynthetic intermediates in these pathways have been isolated and characterized. Some of these compounds showed an increase in antitumor activity in comparison with the parent compounds. A common step in the biosynthesis of all members of the family is the formation of the tetracyclic intermediate premithramycinone. Further biosynthetic steps (glycosylation, methylations, acylations) proceed through tetracyclic intermediates which are finally converted into tricyclic compounds by the action of a monooxygenase, a key event for the biological activity. Heterologous expression of biosynthetic genes from other aromatic polyketide pathways in the mithramycin producer (or some mutants) led to the isolation of novel hybrid compounds.Felipe Lombó and Nuria Menéndez have equally contribute to this work.     

Synthetic biological approaches to natural product biosynthesis

Small molecules produced in Nature possess exquisite chemical diversity and continue to be an inspiration for the development of new therapeutic agents. In their host organisms, natural products are assembled and modified using dedicated biosynthetic pathways. By rationally reprogramming and manipulating these pathways, unnatural metabolites containing enhanced structural features that were otherwise inaccessible can be obtained. Additionally, new chemical entities can be synthesized by developing the enzymes that carry out these complicated chemical reactions into biocatalysts. In this review, we will discuss a variety of combinatorial biosynthetic strategies, their technical challenges, and highlight some recent (since 2007) examples of rationally designed metabolites, as well as platforms that have been established for the production and modification of clinically important pharmaceutical compounds.     

海洋链霉菌Streptomyces sp. B9173合成的2-吲哚酮生物碱

【背景】海洋来源的天然产物近年来已成为小分子药物的重要来源。对海洋链霉菌Streptomyces sp. B9173的基因组分析显示,该菌包含多种天然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】挖掘B9173菌株中未知的次级代谢产物,以期发现结构新颖或生物活性独特的化合物。【方法】利用HPLC/LC-MS结合的方法,排除了该菌株产生的已知化合物,确定3个未知化合物作为挖掘对象,然后利用正、反相硅胶柱色谱、葡聚糖凝胶柱色谱和高效液相色谱等技术对次级代谢产物进行分离纯化,最后得到化合物单体。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。【结果】确定3个化合物分别是色胺酮、甲基异靛蓝和N,N-二甲基异靛蓝,三者都属于2-吲哚酮生物碱。其中色胺酮具有非常广的生物活性,包括抗菌、抗肿瘤、抗炎症等,是药物开发的良好前体,这是首次在细菌中被分离得到。甲基异靛蓝是我国临床治疗慢性粒细胞白血病的药物,这是首次在微生物发酵液中被分离得到。目前这3个化合物均主要依赖化学合成。本研究结合B9173菌株的代谢背景,推测了3个化合物的生物合成途径。【结论】基于紫外吸收光谱和质谱特征,从B9173菌株的发酵液中分离鉴定了3个2-吲哚酮生物碱,丰富了微生物活性天然产物的种类,对3个化合物生物合成途径的推测也为进一步研究色胺酮和甲基异靛蓝的生物合成机制奠定基础,后续可利用合成生物学技术重构这类化合物的生物合成途径,提供更便捷、低成本的生物合成方法。     

Bioactive natural products from marine cyanobacteria for drug discovery

The prokaryotic marine cyanobacteria continue to be an important source of structurally bioactive secondary metabolites. A majority of these molecules are nitrogen-containing compounds biosynthesized by large multimodular nonribosomal polypeptide (NRP) or mixed polyketide-NRP enzymatic systems. A total of 128 marine cyanobacterial alkaloids, published in the literature between January 2001 and December 2006, are presented in this review with emphasis on their biosynthesis and biological activities. In addition, a number of highly cytotoxic compounds such as hectochlorin, lyngbyabellins, apratoxins, and aurilides have been identified as potential lead compounds for the development of anticancer agents. A brief coverage on the distribution of natural product biosynthetic genes as well as the mechanisms of tailoring enzymes involved in the biosynthesis of cyanobacterial compounds will also be given.     

南昌链霉菌代谢产物与代谢工程研究现状的回顾与展望

南昌链霉菌是从江西农业大学校园油茶根际土壤中分离筛选到的一株链霉菌新种,它至少可以产生两种具有重要应用和基础研究价值的抗生素——南昌霉素和梅岭霉素。在国家自然科学基金、国家科技攻关计划、上海市科委的资助下,对这一链霉菌新种进行了多年全面系统的研究,本文对此进行了全面的回顾,并对后续研究进行展望。     

Secondary transport as an efficient membrane transport mechanism for plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, and phenolic compounds. Secondary metabolites have various functions including protection against pathogens and UV light in plants, and have been used as natural medicines for humans utilizing their diverse biological activities. Many of these natural compounds are accumulated in a particular compartment such as vacuoles, and some are even translocated from source cells to sink organs via long distance transport. Both primary and secondary transporters are involved in such compartmentation and translocation, and many transporter genes, especially genes belonging to the multidrug and toxin extrusion type transporter family, which consists of 56 members in Arabidopsis, have been identified as responsible for the membrane transport of secondary metabolites. Better understandings of these transporters as well as the biosynthetic genes of secondary metabolites will be important for metabolic engineering aiming to increase the production of commercially valuable secondary metabolites in plant cells.     

Studies on the biosynthesis of avermectins

To elucidate the pathway of avermectin biosynthesis, the biosynthetic relationships of avermectins A1a, A2a, B1a, B2a, and their respective monosaccharides and aglycones were studied. 14C-labeled avermectin compounds prepared from [1-14C]acetate were fed to Streptomyces avermitilis strain MA5502 and their metabolites were determined. Two furan ring-free aglycones, 6,8a-seco-6,8a-deoxy-5-keto avermectin B1a and B2a, have been isolated from the fermentation broth of a blocked mutant of S. avermitilis. Addition of the compounds and a semisynthetic compound, 5-keto avermectin B2a aglycone, to the fermentation medium of a second blocked mutant established that the two compounds are intermediates in the avermectin biosynthetic pathway immediately preceding avermectin aglycones.     

The potential role of immobilization in plant cell biotechnology

It has long been hoped that the unique biosynthetic capacity of plants could be exploited in vitro using culture systems analogous to microbial fermentations. However, the characteristics of both the growth and metabolism of plant cells in vitro differ considerably from those of microbial cells and plant cell suspension culture systems have met with limited success. Immobilizing the cells creates a new set of options for the plant biotechnologist to explore. Improvements in some process criteria are apparent although evaluating the potential of immobilized plant cells for producing commercial compounds will only be possible when the biological problems have been overcome.     

Functional evolution of biosynthetic enzymes that produce plant volatiles*

Plants synthesize volatile compounds to attract pollinators. The volatiles emitted by flowers are often complex mixtures of organic compounds; pollinators are capable of distinctly recognizing different volatile compounds. Plants also produce volatile compounds to protect themselves against herbivores and pathogens. Some of the volatile compounds produced in floral and vegetative tissues are toxic to insects and microbes. To adapt changes in the environment, plants have evolved the ability to synthesize a unique set of volatiles. Intensive studies have identified and characterized the enzymes responsible for the formation of plant volatiles. In particular, many biosynthetic genes have been isolated and their enzymatic functions have been proposed. This review describes how plants have evolved the biosynthetic pathways leading to the formation of green leaf volatiles and phenylpropene volatiles.     

How to prove the existence of metabolons?

Sequential enzymes in biosynthetic pathways are organized in metabolons. It is challenging to provide experimental evidence for the existence of metabolons as biosynthetic pathways are composed of highly dynamic protein–protein interactions. Many different methods are being applied, each with strengths and weaknesses. We will present and evaluate several techniques that have been applied in providing evidence for the orchestration of the biosynthetic pathways of cyanogenic glucosides and glucosinolates in metabolons. These evolutionarily related pathways have ER-localized cytochromes P450 that are proposed to function as anchoring site for assembly of the enzymes into metabolons. Additionally, we have included commonly used techniques, even though they have not been used (yet) on these two pathways. In the review, special attention will be given to less-exploited fluorescence-based methods such as FCS and FLIM. Ultimately, understanding the orchestration of biosynthetic pathways may contribute to successful engineering in heterologous hosts.     

Studies on the biosynthesis of bialaphos (SF-1293) Part 3. Production of phosphinic acid derivatives,MP-103, MP-104 and MP-105, by a blocked mutant of Streptomyceshygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos

During biosynthetic studies on bialaphos to reveal the formation mechanisms of carbon-phosphorous bonds in detail, three new metabolites containing a HPC bond structure were isolated from the fermentation broth of a mutant of $\text{Streptomyces}\text{hygroscopicus}$ SF-1293. Based on the spectroscopic analysis, the structures of these compounds have been determined as shown in Fig. 1. Transformation experiments of these metabolites to bialaphos suggested that the reduction of the phosphorous atom in phosphate will take place at an early biosynthetic stage.     

Exploring the biosynthesis of natural products and their inherent suitability for the rational design of desirable compounds through genetic engineering

Much effort has been invested in studying how natural products are biosynthesized, and great advances have been made in understanding how these compounds acquire their structural complexity and biological activities. In recent years, significant progress has been made due to the devoted efforts of scientists in this field and to technological advancements. Numerous details, applications, and innovative findings have been elucidated by scientists using biochemical, genetic, and molecular biological approaches. Here I present a comprehensive overview of highly valued biosynthetic proteins, polyketide synthase and nonribosomal peptide synthetase. I begin with "Diels-Alderase" a captivating enzyme that has the ability to catalyze a Diels-Alder reaction valued by chemists for its usefulness in chemical synthesis. A handful of these enzymes have been characterized and chemically authenticated. The most well understood enzyme of this category is macrophomate synthase. Secondly, I focus on the polyketide and nonribosomal peptide biosynthetic pathways and the enzyme assembly for producing its metabolite. Many important natural products are produced by this biosynthetic pathway as secondary metabolites, such as erythromycin, rifamycin, and FK520, as antibiotics and an immunosuppressive, respectively. I conclude with a discussion of nonribosomal peptides and their mechanistic pathways. Special attention will be devoted to de novo production of echinomycin in a heterologous manner, the earliest example of totally engineered biosynthesis of the biologically active form of a nonribosomal peptide host in Escherichia coli.     

De novo biosynthetic pathways: rational design of microbial chemical factories

Increasing interest in the production of organic compounds from non-petroleum-derived feedstocks, especially biomass, is a significant driver for the construction of new recombinant microorganisms for this purpose. As a discipline, Metabolic Engineering has provided a framework for the development of such systems. Efforts have traditionally been focused, first, on the optimization of natural producers, later progressing towards re-construction of natural pathways in heterologous hosts. To maximize the potential of microbes for biosynthetic purposes, new tools and methodologies within Metabolic Engineering are needed for the proposition and construction of de novo designed pathways. This review will focus on recent advances towards the design and assembly of biosynthetic pathways, and provide a Synthetic Biology perspective for the construction of microbial chemical factories.     

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.     

二硫吡咯酮类抗生素的生物合成与代谢工程应用北大核心CSCD

二硫吡咯酮类抗生素是一类具有独特的吡咯酮二硫杂环戊二烯(4H-[1,2]二硫[4,3-b]吡咯-5-酮)骨架的化合物的总称。基于N-7位酰基侧链的不同以及N-4位是否含有甲基,可分为N-methyl-Nacylpyrrothine、N-acylpyrrothine和thiomarinols等类别。迄今为止,已有27种该类化合物被报道,重要代表包括全霉素(holomycin)、硫藤黄菌素(thiolutin)、金霉素(aureothricin)以及最近发现的thiomarinols。就生物活性而言,二硫吡咯酮类抗生素具有广谱的抗细菌活性,对多种微生物,包括革兰氏阴性菌、革兰氏阳性菌以及寄生虫都有较好的杀灭活性。甚至一些二硫吡咯酮衍生物表现出较强的抗肿瘤活性。近几年来,多个二硫吡咯酮类抗生素的生物合成基因簇相继被报道,其生物合成机理也逐步被阐明。本文将针对目前国内外二硫吡咯酮类抗生素的生物合成研究进展,以及在组合生物合成与代谢工程领域所取得的成果进行综述,旨在为通过合成生物学的方法创造结构新颖、高效低毒的"非天然"二硫吡咯酮类化合物提供理论借鉴。     

Carotenoid metabolism: biosynthesis, regulation, and beyond

Carotenoids are Indispensable to plants and play a critical role in human nutrition and health. Significant progress has been made in our understanding of carotenoid metabolism in plants. The biosynthetic pathway has been extensively studied.Nearly all the genes encoding the biosynthetic enzymes have been isolated and characterized from various organisms. In recent years, there is an increasing body of work on the signaling pathways and plastid development, which might provide global control of carotenoid biosynthesis and accumulation. Herein, we will highlight recent progress on the biosynthesis,regulation, and metabolic engineering of carotenoids in plants, as well as the future research towards elucidating the regulatory mechanisms and metabolic network that control carotenoid metabolism.