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.     

Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions

Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity.     

硫藤黄链霉菌基因组表达文库的构建及筛选

为了探讨硫藤黄链霉菌中可能存在的次级代谢产物合成及调控机制,以链霉菌整合型质粒pJTU2554为载体,构建了硫藤黄链霉菌的基因组表达文库。以模式菌株变铅青链霉菌为异源表达宿主,通过生物活性筛选获得9株具有抑菌活性的异源表达突变菌株,同时通过PCR筛选获得多个含有聚酮合酶和非核糖体多肽合成酶基因的克隆子。TLC及HPLC-MS分析发现6个携带有aureothin生物合成基因簇的cosmid,通过异源表达在变铅青链霉菌中成功合成聚酮类抗生素aureothin,证实文库异源表达及筛选的有效性。     

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。     

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.     

多烯大环类抗生素合成酶结构域的差异进化分析及应用于一株产四霉素链霉菌菌株的筛选

多烯大环类抗生素的生物合成酶属于典型的Ⅰ类聚酮合酶(PKS),这些生物大分子有着非常相似的化学结构和模块合成的形式.在本研究中,每个多烯聚酮合酶的单一结构域被作为系统进化分析的对象.我们发现,各类结构域在进化上出现了不同方向的倾向,其中,KS域在进化树中归成三大类,分别对应多烯结构的三大块区域;相对于KS域和ACP域表...     

Calicheamicin gamma 1I: an antitumor antibiotic that cleaves double-stranded DNA site specifically

Calicheamicin gamma 1I is a recently discovered diyne-ene--containing antitumor antibiotic with considerable potency against murine tumors. In vitro, this drug interacts with double-helical DNA in the minor groove and causes site-specific double-stranded cleavage. It is proposed that the observed cleavage specificity is a result of a unique fit of the drug and DNA followed by the generation of a nondiffusible 1,4-dehydrobenzene--diradical species that initiates oxidative strand scission by hydrogen abstraction on the deoxyribose ring. The ability of calicheamicin gamma 1I to cause double-strand cuts at very low concentrations may account for its potent antitumor activity.     

1个含有SDR结构域PKS/NRPS基因的克隆

聚酮和非核糖体多肽的复合化合物具有独特的生理活性,它们由聚酮合酶/非核糖体肽合成酶（PKS/NRPS）催化合成。目前球孢白僵菌Beauveria bassiana中含SDR结构域的PKS/NRPS酶的生物合成机制尚不清楚,采用基因挖掘技术从球孢白僵菌基因组中分离得到1个PKS/NRPS基因（命名Bbpks2）,利用生物信息学分析对其功能进行预测并检测该基因在以6.0 g·L-1麦芽提取物和3.0 g·L-1酵母提取物为基本氮源培养基,7种碳源添加物和以1.8 g·L-1麦芽糖和6.0 g·L-1葡萄糖为基本碳源培养基,4种氮源添加物培养基上的具体表达情况,其中每种添加物含量为4.0 g·L-1。结果显示:Bbpks2基因长度为12 051 bp,编码4 016个氨基酸;其结构域组织顺序为KS-AT-DH-MT-KR-ACP-C-A-PP-SDR,是一种含有SDR结构域的PKS/NRPS;系统进化分析发现,BbPKS2与球孢白僵菌JEF007菌株（PMB64475.1）、头状虫草Tolypocladium capitatum（PNY25600.1）等的PKS/NRPS蛋白聚在同个分支中,可能参与一种聚酮/非核糖体多肽类化合物的生物合成;比较不同氮源、碳源添加物对Bbpks2基因表达的影响,发现该基因在添加了乳糖的培养基上的表达量是其他碳源添加物的3.4倍以上,添加了牛肉浸粉的培养基上表达量是其他氮源添加物的1.3倍以上。该研究为下一步通过异源表达鉴定Bbpks2基因的具体功能,及其调控机理研究和基因资源利用奠定基础。     

Resistance to enediyne antitumor antibiotics by CalC self-sacrifice

Antibiotic self-resistance mechanisms, which include drug elimination, drug modification, target modification, and drug sequestration, contribute substantially to the growing problem of antibiotic resistance among pathogenic bacteria. Enediynes are among the most potent naturally occurring antibiotics, yet the mechanism of resistance to these toxins has remained a mystery. We characterize an enediyne self-resistance protein that reveals a self-sacrificing paradigm for resistance to highly reactive antibiotics, and thus another opportunity for nonpathogenic or pathogenic bacteria to evade extremely potent small molecules.     

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。     

白色链霉菌JA3453中垩唑霉素生物合成基因簇甲氧丙二酰ACP生物合成基因ozmF的遗传分析

恶唑霉素（oxazolomycin,OZM）是由白色链霉菌JA3453产生的杂合聚酮聚肽抗生素,其结构特征为螺旋链接的β-内酯/γ-内酰胺,垩唑环,（E,E）二烯和（Z,Z,E）三烯链。恶唑霉素具有抗肿瘤,抗病毒和一定的抗细菌的生物活性。前面工作中我们已经从白色链霉菌JA3453中定位了135kb的DNA区域,这个区域覆盖了整个ozm基因簇,对部分片段测序揭示了负责甲氧丙二酰ACP生物合成位点的6个基因。本文通过突变以及互补分析其中的甲基转移酶基因ozmF并证实其与恶唑霉素生物合成相关。这些结论有助于我们通过组合生物化学的方法产生新的OZM衍生物。     

松仁蛋白的研究进展

红松种子仁是优质的植物资源,其所含的蛋白质更是非常优质的蛋白质来源,近年来得到国内外学者的广泛关注和研究。综述了松仁蛋白的提取方法、理化性质和生理活性以及主要成分的生理功能,以期为松仁蛋白进一步的开发利用提供参考。     

Transmembrane protein structure: spin labeling of bacteriorhodopsin mutants

Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.     

细菌芳香聚酮研究进展

鉴于芳香聚酮化合物在抗菌、抗肿瘤、抗病毒等方面具有重要的临床药用价值,该文综述了细菌芳香聚酮化合物及其生物合成研究的主要进展,重点讨论了四类芳香聚酮的生物活性和化学结构以及芳香聚酮生物合成机理研究的基础理论意义。在此基础上对组合生物合成新的具有一定生物活性化合物的研究前景进行了展望。     

浅述植物萜类物质的生物合成途径

本文简述了植物萜类物质的主要生物合成途径,以期为萜类物质的生物合成提供一定的理论依据。     

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.     

PKS-NRPS数据库的研究进展

非核糖体多肽合成酶(NRPS)和聚酮合成酶(PKS)是调控次级代谢物聚酮类物质和非核糖体多肽合成的关键酶。随着生物息学的快速发展,国外的NRPS-PKS数据库和预测软件不断推出。本文对几个重要NRPS和PKS领域的数据库与预测软件的研究做一综述     

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.     

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。     

链丝菌素生物合成基因簇的克隆及其异源表达

通过基因组粘粒文库的高通量接合转移、异源表达和生物活性测定,结合DNA序列测定,从刺孢吸水链霉菌AA97026中筛选具有广谱抗菌活性的小分子化合物及其生物合成基因簇,获得1个对革兰氏阳性细菌和红酵母均有抑制活性的阳性克隆1H5,其部分DNA序列与链丝菌素生物合成基因相似。含1H5的异源链霉菌宿主的发酵液均能检测到链丝菌素的不同组份,表明1H5含有完整的链丝菌素生物合成基因簇。