Cloning of polyketide synthase genes from amphidinolide-producing dinoflagellate Amphidinium sp

Cloning of polyketide synthase (PKS) gene for amphidinolide biosynthesis was attempted from a dinoflagellate Amphidinium sp. (strain Y-42). Fourteen beta-ketoacyl synthase gene fragments were obtained by Polymerase Chain Reaction (PCR) amplification from degenerated primer sets designed on the basis of the conserved amino acid sequences of beta-ketoacyl synthase domains in known type I PKSs. The PCR analysis using primer sets designed from these fourteen beta-ketoacyl synthase gene fragments revealed that these DNA sequences exist only in the dinoflagellates producing amphidinolides. The DNA sequence of the positive clone, which was isolated from genomic DNA library of Amphidinium sp. (strain Y-42) by PCR detection using the specific primer set, was analyzed by shotgun sequencing. The deduced gene products in the positive clone showed similarity with beta-ketoacyl synthase (KS), acyl transferase (AT), dehydratase (DH), ketoreductase (KR), and acyl carrier protein (ACP) in known type I PKSs and thioesterase (TE).     

聚酮合成酶应用研究进展

聚酮合成酶在生物活性产物的合成方面有广泛应用,本综述简单介绍了三类聚酮合成酶,并阐述了几例聚酮合成酶应用的原理及合成过程,分别为八酮烯二炔核心结构生物合成应用、增加聚酮合成酶多样性应用和构建"非自然"的天然产物。     

维吉霉素类抗生素新化合物X-435-C的产生菌X-435的初步分类鉴定

菌株Ｘ－４３５是从我国土壤中分离得到的一株维吉霉素（ｖｉｒｇｉｎｉａｍｙｃｉｎ）类抗生素产生菌,它与已经 维吉霉素类抗生素的产生菌相比较,在气丝的颜色、孢子莲的形状和孢子的表面形态等方面比较接地菌株Ｓ．ｇｒａｍｉｎａｆａｃｉｅｎｓ（ＩＦＯ１３４５５）、Ｓ．ｐｒｉｓｔｉｎａｅｓｐｉｒａｌｉｓ（ＩＦＯ１３０７４）。因此,进一步对这三株菌进行ｒＤＮＡ同源性分析,结果表明,菌株Ｘ－４３５与ＩＦＯ１３０７４     

Partial activation of a silent angucycline-type gene cluster from a rubromycin beta producing Streptomyces sp. PGA64

In the course of DNA-fingerprinting our strain collection for antibiotic biosynthesis genes, two different type II polyketide synthase (PKS) gene clusters were observed from Streptomyces sp. PGA64. Phylogenetic analysis placed these together with known rubromycin and angucycline biosynthetic gene clusters. The host strain itself has a very clean production profile of secondary metabolites, which composes mainly of rubromycin beta under typical fermentation conditions. Sequencing of a 16.5 kb fragment from the putative angucycline cluster revealed eight genes that were homologous to typical type II PKS genes responsible for synthesizing aromatic polyketides. These genes were especially similar to genes from known angucycline biosynthetic gene clusters and also synteny to these clusters was observed. In addition, three genes were recognized that are needed for priming the minimal PKS complex before polyketide synthesis can initiate, but which are not normally found to cluster with antibiotic biosynthesis genes. A putative repressor gene that was dissimilar to repressor genes found from well-characterized antibiotic biosynthesis gene clusters was also discovered. Gene disruption of the repressor resulted in partial activation of the cluster and production of two angucycline metabolites, UWM6 and rabelomycin. The results confirm that the DNA-fingerprinting method we have developed can be used to correctly detect compounds that are not visible in chemical screens.     

组合生物合成研究进展

组合生物合成是通过对微生物代谢途径中一些酶的编码基因进行操作,从而获得许多新的“非天然”天然产物。本文重点介绍聚酮合酶（PKSs）和非核糖体多肽合酶（NRPSs）的分类、作用机制及其杂合系统在组合生物合成应用研究中相关报道。另外,对于PKSs和NRPSs在大肠埃希荫中的异源表达也作了简单介绍。     

农抗5102产生菌工程菌株WH─1的稳定性

无论是在连续传代的过程中，还是在不同温度条件下保存的过程中，工程菌株ＷＨ－１均不稳定，其不稳定性主要表现在培养特征的变化和产生抗生素性能的衰退两个方面，且都是一个由量变逐渐转化为质变的过程。采用连续多次单孢子分离筛选的方法可以获得比较稳定的高产菌株。本实验主要通过５次单孢子分离筛选，得到了ＷＨ－１－１、ＷＨ－１－２、ＷＨ－１－３、ＷＨ－１－４和ＷＨ－１－５共５个较稳定的高产菌株     

Phylogenetic Study of Polyketide Synthases and Nonribosomal Peptide Synthetases Involved in the Biosynthesis of Mycotoxins

Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) are large multimodular enzymes involved in biosynthesis of polyketide and peptide toxins produced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fused to a single NRPS module, are also responsible of the synthesis of peptide-polyketide metabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed to complex evolutionary mechanisms, which have determined the great number and diversity of metabolites. In this study, we considered the most important polyketide and peptide mycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSs involved in their biosynthesis was assessed using two domains for each enzyme: β-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) and condensation (C) for NRPSs. The analysis of both KS and AT domains confirmed the differentiation of the three classes of highly, partially and non-reducing PKSs. Hybrid PKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetic trees of all the domains analyzed. For most mycotoxins, the corresponding biosynthetic enzymes from distinct fungal species grouped together, except for PKS and NRPS involved in ochratoxin A biosynthesis, for which an unlike process of evolution could be hypothesized in different species.     

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。     

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.     

Motional effects on NMR structural data. Comparison of spinach and Escherichia coli acyl carrier proteins

Proteins in solution need not exist in a single rigid structure but can exist in a dynamic equilibrium among structural forms. The problems that this poses for structure determination using nuclear Overhauser effect data from two-dimensional NMR experiments are discussed and illustrated with data on functionally equivalent proteins from two different species. One of these proteins, acyl carrier protein from Escherichia coli, shows a single set of resonances, easily interpreted on the basis of a single rigid structure. However, the related protein, acyl carrier protein from spinach, shows two sets of resonances, suggesting that two conformers in dynamic equilibrium would be a better structural model.     

Analysis of Enoyl‐Acyl Carrier Protein Reductase Structure and Interactions Yields an Efficient Virtual Screening Approach and Suggests a Potential Allosteric Site

Enoyl‐acyl carrier protein reductases have an important role in fatty acid biosynthesis and are considered essential for bacterial and protozoal survival. Here, we perform a computational assessment of enoyl‐acyl carrier protein reductase structures, providing insights for inhibitor design that we incorporate into a virtual screening approach. Firstly, we analyse 80 crystal structures of 16 different enoyl‐acyl carrier protein reductases for their active site characteristics and druggability, finding these sites contain a readily druggable pocket, of varying size and shape. Interestingly, a high affinity, potentially allosteric site was identified for pfFabI. Analysis of the ligand–protein interactions of four enoyl‐acyl carrier protein reductases from different micro‐organisms (InhA, pfFabI, saFabI and ecFabI), involving 59 available crystal structures, found three commonly shared interactions; constraining these interactions in docking improved enrichment of enoyl‐acyl carrier protein reductase virtual screens, by up to 60% in the top 3% of the ranked library. This docking protocol also improved pose prediction, decreasing the root‐mean‐square deviation to crystallographic pose by up to 75% on average. The binding site analysis and knowledge‐based docking protocol presented here can potentially assist in the structure‐based design of new enoyl‐acyl carrier protein reductase inhibitors.     

A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases

Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyte-producing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.     

7-Hydroxyguanine, a novel antimetabolite from a strain of Streptomyces purpurascens. I. Taxonomy of producing organism, fermentation, isolation and biological activity

Strain A-347, an actinomycete isolated from a soil sample, was found to produce a new antimetabolite, 7-hydroxyguanine. The aerial mycelium formed spiral spore chains with spiny spore surface. The chemical composition of strain A-347 indicated that it was an actinomycete of cell wall Type I. From its morphological, cultural, physiological characteristics and direct comparison with the type culture, this strain was classified as Streptomyces purpurascens. 7-Hydroxyguanine was purified from the broth filtrate by ion exchange chromatography and crystallized from hot 2 M NH4OH. 7-Hydroxyguanine inhibited the growth of experimental tumors such as L1210 leukemia.     

Cloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin

The gene cluster encoding the deoxyoleandolide polyketide synthase (OlePKS) was isolated from the oleandomycin producing strain Streptomnyces antibioticus. Sequencing of the first two genes encoding OlePKS, together with the previously identified third gene revealed an overall genetic and protein architecture similar to that of the erythromycin gene cluster encoding the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. When the entire OlePKS (10,487 amino acids) was expressed in the heterologous host Streptomyces lividans, it produced 8,8a-deoxyoleandolide, an aglycone precursor of oleandomycin. The role of the P-450 monooxygenase, OleP, in oleandomycin biosynthesis was also examined in vivo by co-expression with DEBS in S. lividans. The production of 8,8a-dihydroxy-6-deoxyerythronolide B and other derivatives indicates that OleP is involved in the epoxidation pathway of oleandomycin biosynthesis. Since there are currently no genetic systems available for manipulation of the natural oleandomycin producing strain, the heterologous expression system reported here provides a useful tool for studying this important macrolide antibiotic.     

Combinatorial biosynthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs

Combinatorial biosynthesis utilizes the genes of biosynthetic pathways that produce microbial products to create novel chemical structures. The engineering of mondular polyketide synthase (PKS) genes has been the major focus of this effort and has led to the production of analogs of macrolide antibiotics like the erythromycins and their derived ketolides, and of the immunosuppressive macrolide FK-520 (Fujisawa Pharmaceutical Co Ltd). Approaches to making analogs of the promising antitumor compounds known as epothilones are also being explored. Lead compounds for further study have resulted and routes to analogs of other pharmacologically important compounds have been established. To facilitate this work, many new tools for manipulating and studying the multifunctional PKSs have been developed including the development of Escherichia coli as a PKS expression last. These developments have resulted in faster ways of engineering PKS to produce new compounds for the development of chemotherapeutic agents from natural products.     

Structure alteration of polyketides by recombinant DNA technology in producer organisms--prospects for the generation of novel pharmaceutical drugs

Actinomycetes are gram-positive bacteria and commercially important microorganisms. They are producers of approximately two thirds of all bioactive compounds known and they produce a great variety of compounds which have clinical application on the basis of their activity against different kinds of organisms and cells as antibacterial (macrolides, avermectins), antitumor (anthracyclines, angucyclines, aureolic acid group) and also compounds showing immunosuppresant activity (rapamycin, FK506). Most of these clinically useful pharmaceuticals produced by actinomycetes belong to the polyketide family. Polyketides comprise a wide family of chemically diverse compounds, many of which have shown bioactivity. The development of recombinant DNA technology has opened a new and exciting field of research for the generation of new bioactive compounds through genetic manipulation of the biosynthetic pathways. Researchers in this area are trying to take advantage of the enormous capability of actinomycetes to produce pharmaceutically useful compounds in order to manipulate the different biosynthetic pathways and subsequently generate novel drugs. Combinatorial biosynthesis is now emerging as a powerful tool to generate novel families of compounds by interchanging secondary metabolism genes between bioactive producing actinomycetes. Novel compounds will be the consequence of the concerted action of enzymes from different, but related, biosynthetic pathways. Insertional inactivation of selected genes and tailoring modification may also produce novel compounds that can be useful pharmaceuticals or lead compounds for further chemical modification. This minireview will present the state of the art in this field showing the different polyketides biosynthetic pathways so far characterized and how the identified genes are being used to generate structural biodiversity. Emphasis will be made on the polyketide family including type I and type II polyketides.     

The complete biosynthetic gene cluster of the 28-membered polyketide macrolactones, halstoctacosanolides, from Streptomyces halstedii HC34

Halstoctacoanolides A and B are 28-membered polyketide macrolactones and were isolated from Streptomyces halstedii HC34. The biosynthetic gene cluster (hls cluster) of halstoctacosanolides was completely identified from the genome library of Streptomyces halstedii HC34. DNA sequence analysis of ca. 100 kb region revealed that there were seven type I polyketide synthases (PKSs) and two cytochrome P450 monooxygenases in this cluster. Involvement of the gene cluster in the halstoctacosanolide biosynthesis was demonstrated by the gene disruption of P450 monooxygenase genes. The mutants produced a new deoxygenated halstoctacosanolide derivative, halstoctacosanolide C, which confirmed that the hls gene cluster was essential for the biosynthesis of halstoctacosanolides.     

褐产色小单孢菌——一株新的西索米星产生菌

从云南省大理县分得的一株褐产色小单孢菌,孢子层呈橄榄绿色,在酪氨酸琼脂上能产生褐色素,孢子表面有棘刺状突起,分类学鉴别它为小单孢菌属的一株新种。其主要发酵产物经提取、精制和微生物、理化特性、耐药谱及波谱学研究证明为西索米星。     

Promiscuous beta-strand interactions and the conformational diseases

Conformational change plays an important role in the life of all proteins, starting from when they fold, through their function and often their fate. For an increasing number of proteins inappropriate conformational change leads to a chain of events, which culminate in the deposition of proteinacious aggregates and disease. In this review we consider the current literature on a number of proteins which form part of the Conformational Disease family. We describe here two types of aggregate that can be formed, Type I aggregates are typified by the Serpin superfamily and consist of non-fibrillar polymeric species. Type II aggregates are of the classical fibrillar form formed by a diverse range of proteins. Through biochemical and biophysical analysis of the aggregation reaction of members of these two classes we show that they form these aggregates through highly similar pathways. Essentially, the whole process can be summed up in two key stages. Firstly, the existence of conditions which increase the conformational flexibility of the protein, enabling it to adopt a partially folded state. Secondly, the propensity of this intermediate conformer to form intermolecular linkages leads to multimeric forms, a step often mediated via hydrophobic or beta -strand interactions. Our understanding of these structural changes has facilitated the rationale design of specific aggregation inhibitors. We will discuss the successes and pitfalls of such approaches to demonstrate how similar approaches may be applied to any misfolding protein.