Alternative modular polyketide synthase expression controls macrolactone structure

Modular polyketide synthases are giant multifunctional enzymes that catalyse the condensation of small carboxylic acids such as acetate and propionate into structurally diverse polyketides that possess a spectrum of biological activities. In a modular polyketide synthase, an enzymatic domain catalyses a specific reaction, and three to six enzymatic domains involved in a condensation-processing cycle are organized into a module. A fundamental aspect of a modular polyketide synthase is that its module arrangement linearly specifies the structure of its polyketide product. Here we report a natural example in which alternative expression of the pikromycin polyketide synthase results in the generation of two macrolactone structures. Expression of the full-length modular polyketide synthase PikAIV in Streptomyces venezuelae generates the 14-membered ring macrolactone narbonolide, whereas expression of the amino-terminal truncated form of PikAIV leads to 'skipping' of the final condensation cycle in polyketide biosynthesis to generate the 12-membered ring macrolactone 10-deoxymethynolide. Our findings provide insight into the structure and function of modular polyketide synthases, as well as a new set of tools to generate structural diversity in polyketide natural products.     

松科植物萜类合成酶及其基因家族研究进展

萜类化合物是松科(Pinaceae)植物中重要的代谢物,它们与松科植物生长发育、信息传递、气候适应和化学防御等关系密切,在植物生理和生态等方面具有重要功能。松科植物萜类化合物还广泛应用在制药、生物燃料以及合成化学等工业领域,具有重要的经济价值。松科植物通过甲羟戊酸途径和甲基赤藓糖磷酸途径合成所有萜类物质合成所必需的5碳前体,并在异戊烯基转移酶家族、萜类合成酶家族作用下合成单萜、倍半萜和二萜等不同长度碳链的萜类分子骨架,并进一步在细胞色素P450酶家族的作用下发生甲基化、羟基化、过氧化、糖基化等酶促反应形成具有结构极为丰富的萜类化合物。和其他次生代谢过程类似,多种酶及其基因在萜烯化合物形成过程中起到了至关重要的作用,同时,萜类化合物结构多样性的形成也主要依赖于萜类合成酶及其基因。植物中已经发现了大量的萜类合成酶,由于大量植物基因组、转录组等组学数据的公布,不断有新的萜类合成酶被报道。笔者介绍了植物萜类化合物前体的合成途径及其关键酶基因、植物萜类合成酶的结构和类型,着重阐述松科植物萜类合成酶结构、功能以及相应基因家族鉴定和系统分类的研究进展,并针对松科植物萜类合成酶及其基因研究领域存在的研究树种偏少、松科植物萜烯类代谢可能存在的特异代谢路径重视程度不够、适用于针叶树种相关基因的功能研究平台搭建欠缺、多基因网络调控松科植物萜烯类合成机制研究未得到系统开展、产脂和抗逆相关的松科植物关键基因未得到挖掘与利用等相关问题提出了建议,以期为松科植物萜类生物合成机制解析及松科植物遗传改良提供参考。     

何首乌中一种Ⅲ型PKS基因的克隆及生物信息学分析

Ⅲ型聚酮合酶即查耳酮合酶超家族,能催化生成一系列结构各异、具有不同生理活性、含有查耳酮合酶基本骨架的植物次生代谢产物。根据不同植物Ⅲ型PKSs基因的保守序列,设计简并引物,采用RT - PCR和RACE技术,首次从传统中药材何首乌(Polygonum multiflorum Thunb.)中克隆到一种Ⅲ型PKS基因,其cDNA全长1228bp, 编码379个氨基酸,命名为FmPKS (GenBank登录号: GQ411431)。该基因含有三个内含子, 与迄今报道的绝大部分Ⅲ型PKS含有一个内含子不同,而与最近报道的虎杖中克隆的PcPKS2基因相同。通过生物信息学方法对其编码蛋白的序列进行同源性分析,构建了系统进化树,并对其等电点、疏水性及二级结构、跨膜区域等理化性质进行了初步预测,为进一步研究其功能奠定了基础。     

汕头南澳海绵Halichondria sp.放线菌分离的研究

海绵相关微生物是药物开发的重要资源.本文利用6种培养基对汕头南澳普遍存在的海绵Halichondria sp.的共附生放线菌进行分离和培养,共得到36株放线菌.基于16S rRNA基因序列的系统发育分析表明,这些放线菌分别属于Streptomyces、Nocardiopsis和Micromonospora属.抗菌活性筛选结果显示,分离菌株分别对Staphylococcus aureus、Bacillus subtilis和Escherichia coli等的生长有抑制作用.同时分子方法筛选显示这些放线菌具有潜在产生聚酮类、角蒽环类和非核糖体多肽类的能力.本研究结果为海绵相关微生物在药物开发方面积累了基础数据.     

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.     

A new pathway for polyketide synthesis in microorganisms.

Chalcone synthases, which biosynthesize chalcones (the starting materials for many flavonoids), have been believed to be specific to plants. However, the rppA gene from the Gram-positive, soil-living filamentous bacterium Streptomyces griseus encodes a 372-amino-acid protein that shows significant similarity to chalcone synthases. Several rppA-like genes are known, but their functions and catalytic properties have not been described. Here we show that a homodimer of RppA catalyses polyketide synthesis: it selects malonyl-coenzyme-A as the starter, carries out four successive extensions and releases the resulting pentaketide to cyclize to 1,3,6,8-tetrahydroxynaphthalene (THN). Site-directed mutagenesis revealed that, as in other chalcone synthases, a cysteine residue is essential for enzyme activity. Disruption of the chromosomal rppA gene in S. griseus abolished melanin production in hyphae, resulting in 'albino' mycelium. THN was readily oxidized to form 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin), which then randomly polymerized to form various coloured compounds. THN formed by RppA appears to be an intermediate in the biosynthetic pathways for not only melanins but also various secondary metabolites containing a naphthoquinone ring. Therefore, RppA is a chalcone-synthase-related synthase that synthesizes polyketides and is found in the Streptomyces and other bacteria.     

An unusually large multifunctional polypeptide in the erythromycin-producing polyketide synthase of Saccharopolyspora erythraea

Erythromycin A, a clinically important polyketide antibiotic, is produced by the Gram-positive bacterium Saccharopolyspora erythraea. In an arrangement that seems to be generally true of antibiotic biosynthetic genes in Streptomyces and related bacteria like S. erythraea, the ery genes encoding the biosynthetic pathway to erythromycin are clustered around the gene (ermE) that confers self-resistance on S. erythraea. The aglycone core of erythromycin A is derived from one propionyl-CoA and six methylmalonyl-CoA units, which are incorporated head-to-tail into the growing polyketide chain, in a process similar to that of fatty-acid biosynthesis, to generate a macrolide intermediate, 6-deoxyerythronolide B. 6-Deoxyerythronolide B is converted into erythromycin A through the action of specific hydroxylases, glycosyltransferases and a methyltransferase. We report here the analysis of about 10 kilobases of DNA from S. erythraea, cloned by chromosome 'walking' outwards from the erythromycin-resistance determinant ermE, and previously shown to be essential for erythromycin biosynthesis. Partial sequencing of this region indicates that it encodes the synthase. Our results confirm this, and reveal a novel organization of the erythromycin-producing polyketide synthase, which provides further insight into the mechanism of chain assembly.     

Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria

The metabolic repertoire in nature is augmented by generating hybrid metabolites from a limited set of gene products. In mycobacteria, several unique complex lipids are produced by the combined action of fatty acid synthases and polyketide synthases (PKSs), although it is not clear how the covalently sequestered biosynthetic intermediates are transferred from one enzymatic complex to another. Here we show that some of the 36 annotated fadD genes, located adjacent to the PKS genes in the Mycobacterium tuberculosis genome, constitute a new class of long-chain fatty acyl-AMP ligases (FAALs). These proteins activate long-chain fatty acids as acyl-adenylates, which are then transferred to the multifunctional PKSs for further chain extension. This mode of activation and transfer of fatty acids is contrary to the previously described universal mechanism involving the formation of acyl-coenzyme A thioesters. Similar mechanisms may operate in the biosynthesis of other lipid-containing metabolites and could have implications in engineering novel hybrid products.     

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.     

Cloning and analysis of the antagonistic related genes of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> B8

To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.     

DNA cloning in Streptomyces: resistance genes from antibiotic-producing species

The biochemical and morphological differentiation of actinomycetes makes them academically and economically interesting. Their secondary metabolites provide the majority of medically and agriculturally important antibiotics (streptomycete genes may also be the primary source of clinically important antibiotic resistance); their complex morphological developmental cycle involves a series of changes from vegetative mycelial growth to spore formation. Recombinant DNA technology would add a powerful new dimension to the analysis of these various aspects of actinomycete biology and would also facilitate the development of industrial strains with increased antibiotic yield, or capable of making new antibiotics. For most of these purposes, cloning of genes within and between actinomycetes is required to study the expression of particular genes in genetic backgrounds defined by mutations of the characters under study. To achieve this, we have now developed a method for molecular cloning involving the transfer of genes between unrelated streptomycetes.     

Engineered reversal of the β-oxidation cycle for the synthesis of fuels and chemicals

Advanced (long-chain) fuels and chemicals are generated from short-chain metabolic intermediates through pathways that require carbon-chain elongation. The condensation reactions mediating this carbon-carbon bond formation can be catalysed by enzymes from the thiolase superfamily, including β-ketoacyl-acyl-carrier protein (ACP) synthases, polyketide synthases, 3-hydroxy-3-methylglutaryl-CoA synthases, and biosynthetic thiolases. Pathways involving these enzymes have been exploited for fuel and chemical production, with fatty-acid biosynthesis (β-ketoacyl-ACP synthases) attracting the most attention in recent years. Degradative thiolases, which are part of the thiolase superfamily and naturally function in the β-oxidation of fatty acids, can also operate in the synthetic direction and thus enable carbon-chain elongation. Here we demonstrate that a functional reversal of the β-oxidation cycle can be used as a metabolic platform for the synthesis of alcohols and carboxylic acids with various chain lengths and functionalities. This pathway operates with coenzyme A (CoA) thioester intermediates and directly uses acetyl-CoA for acyl-chain elongation (rather than first requiring ATP-dependent activation to malonyl-CoA), characteristics that enable product synthesis at maximum carbon and energy efficiency. The reversal of the β-oxidation cycle was engineered in Escherichia coli and used in combination with endogenous dehydrogenases and thioesterases to synthesize n-alcohols, fatty acids and 3-hydroxy-, 3-keto- and trans-Δ(2)-carboxylic acids. The superior nature of the engineered pathway was demonstrated by producing higher-chain linear n-alcohols (C?≥?4) and extracellular long-chain fatty acids (C?>?10) at higher efficiency than previously reported. The ubiquitous nature of β-oxidation, aldehyde/alcohol dehydrogenase and thioesterase enzymes has the potential to enable the efficient synthesis of these products in other industrial organisms.     

Functional inactivation of genes by dominant negative mutations

Molecular biologists are increasingly faced with the problem of assigning a function to genes that have been cloned. A new approach to this problem involves the manipulation of the cloned gene to create what are known as 'dominant negative' mutations. These encode mutant polypeptides that when overexpressed disrupt the activity of the wild-type gene. There are many precedents for this kind of behaviour in the literature--some oncogenes might be examples of naturally occurring dominant negative mutations.     

通过S-柠檬烯合酶突变体的动力学分析揭示萜烯合酶进化的机制

使用孔雀绿比色法分析了S-柠檬烯合酶(S-limonene synthase,S-LS)野生型和突变体的动力学特性.研究表明,萜烯合酶经过引入少数残基突变合成新型萜烯化合物仍然可以保持酶的催化活性.该特性可以解释植物中是如何进化出新的萜烯合酶,从而解释了自然界中萜烯化合物的多样性.本研究提出了萜烯合酶的进化模型.同时针对生物合成中萜烯化合物产量不高的难题,为定向改造萜烯合酶提供了解决方法.     

Molecular cloning of the whole biosynthetic pathway of a Streptomyces antibiotic and its expression in a heterologous host

The application of molecular cloning to antibiotic-producing microorganisms should lead to enhanced antibiotic productivity and to the biosynthesis of novel antibiotics by in vitro interspecific recombination. To allow such approaches, the genes for antibiotic synthesis must be isolated, analysed and perhaps modified. Certain Streptomyces species produce nearly two-thirds of the known natural antibiotics; the recent development of cloning systems in the genus makes it possible to isolate and analyse Streptomyces genes. However, antibiotics are metabolites which require sets of several enzymes for their synthesis and attempts to isolate the corresponding genes have so far yielded clones carrying either individual genes of the set, or only incomplete gene sets. We describe here the isolation of a large continuous segment of Streptomyces coelicolor DNA which apparently carries the complete genetic information required for synthesis of an antibiotic, actinorhodin , from simple primary metabolites. Not only can the cloned DNA 'complement' all available classes of actinorhodin non-producing mutants of S. coelicolor but, on introduction into a different host, Streptomyces parvulus , it directs the synthesis of the antibiotic. The tendency for the genes for antibiotic synthesis to be clustered together on the chromosomes of Streptomyces species and the availability of plasmid vectors which can carry stable inserts of DNA larger than 30 kilobase pairs (kb) and which can be introduced efficiently into Streptomyces protoplasts, suggest that the experiments described have general significance for this area of biotechnology.     

Human disease genes

The complete human genome sequence will facilitate the identification of all genes that contribute to disease. We propose that the functional classification of disease genes and their products will reveal general principles of human disease. We have determined functional categories for nearly 1,000 documented disease genes, and found striking correlations between the function of the gene product and features of disease, such as age of onset and mode of inheritance. As knowledge of disease genes grows, including those contributing to complex traits, more sophisticated analyses will be possible; their results will yield a deeper understanding of disease and an enhanced integration of medicine with biology.     

高效热不对称交互式PCR技术克隆地黄基因

利用高效热不对称交互式PCR(hiTAIL-PCR)技术从地黄基因组中扩增出3个DNA片段,经过电泳检测、回收、纯化和测序获得2个片段的碱基序列.其中一个由578个bp组成,包含一个453bp的开放阅读框(ORF),编码151个氨基酸,与一些已知纤维素合酶基因在DNA水平和氨基酸水平的同源性分别高达81%~91%和83%~99%,而且推测蛋白质具有纤维素合酶的结构域;另一个由385个bp组成,没有ORF,不编码蛋白质,但是,预测其包含启动子元件.这些结果表明使用hiTAIL-PCR技术可以克隆地黄基因,克隆的基因将为地黄基因分子作用机制和分子育种研究奠定基础.     

Designed divergent evolution of enzyme function

It is generally believed that proteins with promiscuous functions divergently evolved to acquire higher specificity and activity, and that this process was highly dependent on the ability of proteins to alter their functions with a small number of amino acid substitutions (plasticity). The application of this theory of divergent molecular evolution to promiscuous enzymes may allow us to design enzymes with more specificity and higher activity. Many structural and biochemical analyses have identified the active or binding site residues important for functional plasticity (plasticity residues). To understand how these residues contribute to molecular evolution, and thereby formulate a design methodology, plasticity residues were probed in the active site of the promiscuous sesquiterpene synthase gamma-humulene synthase. Identified plasticity residues were systematically recombined based on a mathematical model in order to construct novel terpene synthases, each catalysing the synthesis of one or a few very different sesquiterpenes. Here we present the construction of seven specific and active synthases that use different reaction pathways to produce the specific and very different products. Creation of these enzymes demonstrates the feasibility of exploiting the underlying evolvability of this scaffold, and provides evidence that rational approaches based on these ideas are useful for enzyme design.     

水环境中抗生素和抗性基因污染特征及控制措施

 抗生素作为一类抗菌类药物被广泛用于医疗、农业和畜牧业等领域，因其使用量大并能诱导产生耐药菌株，对人类健康和生态环境造成巨大威胁。在梳理近年来地表水环境中抗生素相关研究的基础上，阐述了水环境中抗生素和抗性基因的污染来源和污染特征，分析了环境浓度水平下抗生素污染对人群和生态环境的影响，讨论了水环境中抗生素污染的控制措施及目前研究的主要问题，并对今后的研究进行了展望。     

利用RNAi技术鉴定哈茨木霉聚酮合酶基因pkst-1的功能

哈茨木霉(Trichoderma harzianum)是优良的生物防治真菌,能产生抗生性聚酮化合物(polyketides).目前没有关于催化木霉聚酮化合物合成的聚酮合成酶(polyketide synthase,PKS)的相关编码基因的报道,这限制了对聚酮化合物代谢合成途径的研究.该研究通过融合PCR技术获得哈茨木霉的聚酮合成酶pkst-1基因的编码区序列,成功构建了RNAi介导的pkst-1基因沉默表达载体,使pkst-1基因的表达量在mRNA水平上显著降低.pkst-1基因失活使转化子发酵液产生了类似基因敲除的抑菌效果,降低了哈茨木霉抑菌能力,研究表明pkst-1基因编码催化抗生性聚酮化合物合成的关键酶.此外,RNA干扰技术在研究中的成功应用为哈茨木霉或其它丝状真菌的次级代谢合成途径相关基因的功能鉴定提供新的研究思路.