Cloning and heterologous expression of the epothilone gene cluster

The polyketide epothilone is a potential anticancer agent that stabilizes microtubules in a similar manner to Taxol. The gene cluster responsible for epothilone biosynthesis in the myxobacterium Sorangium cellulosum was cloned and completely sequenced. It encodes six multifunctional proteins composed of a loading module, one nonribosomal peptide synthetase module, eight polyketide synthase modules, and a P450 epoxidase that converts desoxyepothilone into epothilone. Concomitant expression of these genes in the actinomycete Streptomyces coelicolor produced epothilones A and B. Streptomyces coelicolor is more amenable to strain improvement and grows about 10-fold as rapidly as the natural producer, so this heterologous expression system portends a plentiful supply of this important agent.     

The crystal structure of a mammalian fatty acid synthase

Mammalian fatty acid synthase is a large multienzyme that catalyzes all steps of fatty acid synthesis. We have determined its crystal structure at 3.2 angstrom resolution covering five catalytic domains, whereas the flexibly tethered terminal acyl carrier protein and thioesterase domains remain unresolved. The structure reveals a complex architecture of alternating linkers and enzymatic domains. Substrate shuttling is facilitated by flexible tethering of the acyl carrier protein domain and by the limited contact between the condensing and modifying portions of the multienzyme, which are mainly connected by linkers rather than direct interaction. The structure identifies two additional nonenzymatic domains: (i) a pseudo-ketoreductase and (ii) a peripheral pseudo-methyltransferase that is probably a remnant of an ancestral methyltransferase domain maintained in some related polyketide synthases. The structural comparison of mammalian fatty acid synthase with modular polyketide synthases shows how their segmental construction allows the variation of domain composition to achieve diverse product synthesis.     

聚酮类化合物及其应用

由微生物和植物产生的聚酮类化合物(PK)的数量庞大,是一大类结构多样化和生物活性多样性的天然产物,已经成为新药的重要来源。综述了3种类型聚酮类化合物生物合成基因簇的特点,即以模块形式存在的I型聚酮合酶、包含一套可重复使用结构域的Ⅱ型聚酮合酶以及不需要ACP参与,以植物中的查耳酮合酶为代表的Ⅲ型聚酮合酶。介绍了近年来组合生物合成技术的基本原理、聚酮合酶的基本作用机制以及合成途径的研究进展。     

富集培养促进宏基因组途径获得聚酮酶基因的研究

进行了富集培养促进宏基因组途径获得聚酮酶基因的研究。结果表明,通过富集培养土壤样品有效提高了红树林土壤高分子量DNA的提取效率,且经分散差速离心(DDC)法预处理并由RH-土壤浸提液富集培养后,可同时检测到放线菌和聚酮化合物合成酶的特征序列,该富集培养样品有助于构建红树林土壤宏基因组BAC文库和聚酮合酶基因的筛选。     

Cis-AT和Trans-AT聚酮合酶及其特殊功能域研究进展

聚酮合酶(Polyketide synthase,PKS)是产生聚酮化合物的模块化复合酶,聚酮合酶能产生数量众多并具有生物活性的聚酮类天然产物。近年来一类与cis-AT聚酮合酶存在较大差异的trans-AT聚酮合酶被广泛研究。文章介绍cis-AT聚酮合酶与trans-AT聚酮合酶区别及trans-AT聚酮合酶基因簇中特殊功能域研究进展,展望利用功能域改造基因簇研究方向,以期为天然产物改造提供新靶点。     

GNAT-like strategy for polyketide chain initiation

An unexpected biochemical strategy for chain initiation is described for the loading module of the polyketide synthase of curacin A, an anticancer lead derived from the marine cyanobacterium Lyngbya majuscula. A central GCN5-related N-acetyltransferase (GNAT) domain bears bifunctional decarboxylase/S-acetyltransferase activity, both unprecedented for the GNAT superfamily. A CurA loading tridomain, consisting of an adaptor domain, the GNAT domain, and an acyl carrier protein, was assessed biochemically, revealing that a domain showing homology to GNAT (GNAT(L)) catalyzes (i) decarboxylation of malonyl-coenzyme A (malonyl-CoA) to acetyl-CoA and (ii) direct S-acetyl transfer from acetyl-CoA to load an adjacent acyl carrier protein domain (ACP(L)). Moreover, the N-terminal adapter domain was shown to facilitate acetyl-group transfer. Crystal structures of GNAT(L) were solved at 1.95 angstroms (ligand-free form) and 2.75 angstroms (acyl-CoA complex), showing distinct substrate tunnels for acyl-CoA and holo-ACP(L) binding. Modeling and site-directed mutagenesis experiments demonstrated that histidine-389 and threonine-355, at the convergence of the CoA and ACP tunnels, participate in malonyl-CoA decarboxylation but not in acetyl-group transfer. Decarboxylation precedes acetyl-group transfer, leading to acetyl-ACP(L) as the key curacin A starter unit.     

Biosynthesis of the enediyne antitumor antibiotic C-1027

C-1027 is a potent antitumor agent with a previously undescribed molecular architecture and mode of action. Cloning and characterization of the 85-kilobase C-1027 biosynthesis gene cluster from Streptomyces globisporus revealed (i) an iterative type I polyketide synthase that is distinct from any bacterial polyketide synthases known to date, (ii) a general polyketide pathway for the biosynthesis of both the 9- and 10-membered enediyne antibiotics, and (iii) a convergent biosynthetic strategy for the C-1027 chromophore from four building blocks. Manipulation of genes governing C-1027 biosynthesis allowed us to produce an enediyne compound in a predicted manner.     

何首乌查尔酮合成酶基因的克隆及序列分析

[目的]克隆何首乌查尔酮合成酶基因并作序列分析。[方法]以cDNA序列的保守区域设计引物,以何首乌 （ Polygonum multiflorum Thunb.）为材料,根据其他植物查尔酮合成酶（Chaleone synthase,CHS）基因eDNA序列的保守区域设计引物,利用RT-PCR和3＇-RACE进行克隆。[结果]从何首乌叶cDNA中克隆出了长度为1258bp的基因片段。序列分析表明,该片段具有典型的CHS基因家族的结构域,为何首乌的CHS基因片段,命名为PmCHS。将得到的序列提交GenBank,序列号为FJ601685。对获得的PmCHS的氨基酸序列进行比较分析,发现PmCHS含有Phe215,推测其能够催化聚酮的合成反应。何首乌CHS与其他植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的虎杖和掌叶大黄的同源性较近。[结论]何首乌查尔酮合成酶基因的成功克隆为蓼科植物中蒽醌的基因工程等研究打下了良好的基础。     

C-O bond formation by polyketide synthases

Polyketide synthases (PKSs) assemble the polyketide carbon backbone by sequential decarboxylative condensation of acyl coenzyme A (CoA) precursors, and the C-C bond-forming step in this process is catalyzed by the beta-ketoacyl synthase (KS) domain or subunit. Genetic and biochemical characterization of the nonactin biosynthesis gene cluster from Streptomyces griseus revealed two KSs, NonJ and NonK, that are highly homologous to known KSs but catalyze sequential condensation of the acyl CoA substrates by forming C-O rather than C-C bonds. This chemistry can be used in PKS engineering to increase the scope and diversity of polyketide biosynthesis.     

基于GIS和模型集成的国土生态安全评价系统

针对当前国内国土生态安全评价系统空缺问题,本文集成14 个定量评价模型,构建了国土生态安全评 价模型库,并利用ArcEngine 二次开发技术将模型库与GIS 技术有机结合,研发了一个集数据管理、国土生态安全评 价、图形输出于一体的通用性强的国土生态安全评价系统。并以湖南省作为系统应用示范区,示范应用表明,该系统 既能实现属性数据和空间数据一体化管理,又能保证评价结果的精度,还能规范绘制和输出评价专题图,能满足国 土生态安全评价工作的需求,能为国土规划、管理部门评价国土生态安全提供技术支持。     

The calicheamicin gene cluster and its iterative type I enediyne PKS

The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.     

西藏米拉山草甸土壤PKS和NRPS基因多样性

为了解西藏米拉山草甸土壤中的聚酮合成酶(PKS)基因和非核糖体多肽合成酶(NRPS)基因多样性,采用直接提取土壤总DNA,PCR扩增Ⅰ型PKS基因KS域和NRPS基因A域,并在NCBI进行Blast比对,分析其系统进化发育.得到27个PKS基因KS片段,经NCBI比对,主要来自蓝细菌(Cyanobaeteria)、α-和γ-变形菌(Proteobaeteria)和不能培养的微生物,得到NPRSA片段32个,主要属于变形菌门、蓝细菌门等.     

Molecular architecture of the rotary motor in ATP synthase

Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion. Its membrane-embedded F0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F1 domain. An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits. Each c subunit forms an alpha-helical hairpin. The interhelical loops of six to seven of the c subunits are in close contact with the gamma and delta subunits of the central stalk. The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.     

利用同框敲除技术研究Ⅲ型聚酮合酶基因orf18的代谢产物

为了阐明Streptomyces sahachiroi ATCC 33158中Ⅲ型聚酮合酶基因orf18的功能,利用同源双交换的方法对该基因进行同框敲除,采用异位表达的方法进行了相应的回补实验。通过高效液相色谱及液相色谱-质谱联用分析发现基因orf18的敲除会明显降低发酵产物中苯甲酸盐的含量,回补该基因能够在一定程度上恢复苯甲酸盐的产量,表明orf18在原宿主中极有可能参与苯甲酸盐的生物合成。     

Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli

The macrocyclic core of the antibiotic erythromycin, 6-deoxyerythronolide B (6dEB), is a complex natural product synthesized by the soil bacterium Saccharopolyspora erythraea through the action of a multifunctional polyketide synthase (PKS). The engineering potential of modular PKSs is hampered by the limited capabilities for molecular biological manipulation of organisms (principally actinomycetes) in which complex polyketides have thus far been produced. To address this problem, a derivative of Escherichia coli has been genetically engineered. The resulting cellular catalyst converts exogenous propionate into 6dEB with a specific productivity that compares well with a high-producing mutant of S. erythraea that has been incrementally enhanced over decades for the industrial production of erythromycin.     

真菌rDNA的特点及在外生菌根菌鉴定中的应用

核糖体DNA(rDNA)是真菌基因组中的一类中度或高度重复序列,每一重复单位包括高度保守、中度保守和不保守三类区段,在真菌的系统发育分析和分类鉴定中,rDNA是很重要的靶序列。本文主要评述了真菌rDNA的结构特点及应用,特别是内转录间隔区(ITS)在外生菌根菌鉴定上的应用,并对一些名词概念进行了讨论。     

Structure of fungal fatty acid synthase and implications for iterative substrate shuttling

We report crystal structures of the 2.6-megadalton alpha6beta6 heterododecameric fatty acid synthase from Thermomyces lanuginosus at 3.1 angstrom resolution. The alpha and beta polypeptide chains form the six catalytic domains required for fatty acid synthesis and numerous expansion segments responsible for extensive intersubunit connections. Detailed views of all active sites provide insights into substrate specificities and catalytic mechanisms and reveal their unique characteristics, which are due to the integration into the multienzyme. The mode of acyl carrier protein attachment in the reaction chamber, together with the spatial distribution of active sites, suggests that iterative substrate shuttling is achieved by a relatively restricted circular motion of the carrier domain in the multifunctional enzyme.     

分布式数据检索系统的设计与实现

提出了一套对非结构化数据进行分析和检索系统的设计方案.采用分布式技术在windows平台上建立了一套以任务分发服务器为中心,运算终端为任务处理单元,带有反馈和容错机制的分布式数据检索系统.在给出系统模型和设计思想基础上,对各个功能部件进行了模块化设计,并对各模块故障进行了测试.结果表明,系统能够在部分模块出错的情况下稳定运行,能够满足在实际工作中的需求.     

细菌Ⅲ型聚酮合酶研究进展

Ⅲ型聚酮合酶（PKS）被认为是结构最简单的聚酮合酶,这一类酶广泛存在于植物细菌和真菌中。随着基因组学的发展,细菌中Ⅲ型聚酮合酶引起越来越多的关注。Ⅲ型聚酮合酶在不同细菌中表现出不同的功能,其产物的结构和生物学活性也各不相同。对细菌Ⅲ型聚酮合酶的生物工程学改造也一直是研究的热点。综述了目前已经完成功能鉴定的细菌Ⅲ型聚酮合酶的研究进展,按照细菌Ⅲ型聚酮合酶产物的不同将其分为五类,并分别论述了各类细菌Ⅲ型聚酮合酶的结构、功能以及特点,对深入研究不同细菌Ⅲ型聚酮合酶以及进行生物工程学改造具有重要意义。