176 resultados para polyketide synthases
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Mycobacterium tuberculosis (Mtb) adaptation to hypoxia is considered crucial to its prolonged latent persistence in humans. Mtb lesions are known to contain physiologically heterogeneous microenvironments that bring about differential responses from bacteria. Here we exploit metabolic variability within biofilm cells to identify alternate respiratory polyketide quinones (PkQs) from both Mycobacterium smegmatis (Msmeg) and Mtb. PkQs are specifically expressed in biofilms and other oxygen-deficient niches to maintain cellular bioenergetics. Under such conditions, these metabolites function as mobile electron carriers in the respiratory electron transport chain. In the absence of PkQs, mycobacteria escape from the hypoxic core of biofilms and prefer oxygenrich conditions. Unlike the ubiquitous isoprenoid pathway for the biosynthesis of respiratory quinones, PkQs are produced by type III polyketide synthases using fatty acyl-CoA precursors. The biosynthetic pathway is conserved in several other bacterial genomes, and our study reveals a redox-balancing chemicocellular process in microbial physiology.
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The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.
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Coronafacic acid (CFA) is the polyketide component of the phytotoxin coronatine, a virulence factor of the plant pathogen Pseudomonas syringae. Our current knowledge of polyketide biosynthesis largely is based on the analysis of polyketide synthases (PKSs) in actinomycetes and other Gram-positive bacteria. Consequently, the cloning and characterization of the CFA biosynthetic gene cluster will contribute significantly to our knowledge of polyketide synthesis in Pseudomonas. In this report, we describe two genes in the CFA biosynthetic gene cluster that encode PKSs that are structurally and functionally similar to the multifunctional modular PKSs, which catalyze the synthesis of macrolide antibiotics. The CFA PKS genes were overproduced in Escherichia coli and shown to cross-react with antisera made to a modular PKS involved in erythromycin synthesis. A scheme for CFA biosynthesis is presented that incorporates the activities of all proteins in the CFA PKS. In this report a gene cluster encoding a pseudomonad polyketide has been completely sequenced and the deduced gene functions have been used to develop a biosynthetic scheme.
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Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^
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Ⅰ 虎杖聚酮类化合物生物合成相关基因的克隆及功能分析 虎杖 (Polygonum cuspidatum Sieb. et Zucc) 属于蓼科蓼属多年生草本植物,在中国和日本民间曾被广泛用于动脉粥样硬化、高血压、咳嗽、化脓性皮肤炎以及淋病的治疗,具有祛风利湿、散瘀定痛、止咳化痰等功效。而在现代医学上最令人瞩目和具有发展前景的是其在抗肿瘤、心血管保护、抗氧化方面的作用,相关疗效主要来自于虎杖中结构迥异、种类丰富的聚酮化合物及其衍生物资源。这些聚酮类化合物主要包括蒽醌、大黄素、大黄素-甲醚、大黄酚、芪类以及类黄酮化合物等。其中,大部分聚酮类化合物生物合成途径机制尚不明确,但可以肯定的是植物类型III聚酮合酶type III polyketide synthases (PKSs) 在这些聚酮化合物的生物合成起始反应中行使着关键的作用。因此,除了我们所熟悉的类黄酮化合物、芪类化合物之外,进一步分离和分析虎杖中其它重要聚酮类化合物生物合成所涉及的类型III聚酮合酶基因的是非常值得期待的。 目前,已经有14个植物类型III PKS基因被克隆和功能分析。植物类型III PKS的共同特征包括基因结构、序列相似性、保守的活性中心、酶学性质以及共同的催化机制等。显花植物(裸子植物和被子植物)中,植物类型III PKS的基因结构绝对保守,除了一个早期报道的金鱼草(Antirrhinum majus)查尔酮合酶chalcone synthase (CHS) 含有第二个内含子外,迄今为止所有已知的植物类型III PKS基因均含有一个内含子且该内含子位置保守。有趣的是,在本研究中,两个含有3个内含子的类型III PKS基因从虎杖中被分离,且两个基因3个内含子的位置完全保守,这是三内含子类型III PKS基因首次得到分离。除了新奇的基因结构外,体外功能分析显示上述两个基因还具有特殊的酶学性质和功能。 本论文围绕上述2个三内含子基因开展了以下工作: 虎杖中一个由三内含子基因编码的新型类型III聚酮合酶 一个类型III PKS的cDNA及其相应的基因(PcPKS2)从药用植物虎杖中被克隆。序列分析结果表明,PcPKS2的开放阅读框被3个内含子分隔,这是一个出人意料的发现,因为截至到目前为止,除了金鱼草一个CHS基因外,所有已知的类型III PKS基因均在固定位置上含有一个内含子。除了特殊的基因结构外,PcPKS2显示了一些有趣的特性:(i) CHS“守卫”苯丙氨酸——Phe215和Phe265在PcPKS2中双双缺失,它们分别被亮氨酸和半胱氨酸取代;(ii) 体外功能分析结果表明,当酶促反应体系的pH值为6.5-8.5时,大肠杆菌中过表达的重组PcPKS2高效地合成丁烯酮非环化产物——4-香豆酰甘油酸内酯(4-coumaroyltriacetic acid lactone (CTAL))为主产物,而丙烯酮非环化产物bis-noryangonin (BNY) 以及苯亚甲基丙酮为副产物;而当酶促反应体系的pH值为9.0时,PcPKS2高效地合成苯亚甲基丙酮为主产物,而CTAL、BNY为副产物。另外,除了上述3种产物外,在不同的pH条件下,还有痕量的柚皮素查尔酮能被检测到。此外,在4-香豆酰辅酶A(4-coumaroyl-CoA)的类似化合物中,除了4-香豆酰辅酶A外,只有feruloyl-CoA能够被PcPKS2接受作为起始底物。PcPKS2不接受脂肪酰辅酶A——异丁酰基辅酶A(isobutyryl-CoA)、异戊酰基辅酶A(isovaleryl-CoA)以及乙酰辅酶A(acetyl-CoA)作为起始底物。Southern blot杂交结果表明,在虎杖基因组中存在2-4个PcPKS2基因的拷贝。Northern blot杂交结果表明,在根茎和幼叶中,PcPKS2表达量很高,而在根中无表达。叶中的PcPKS2的表达受病原菌诱导,但不受伤诱导。 虎杖中一个编码双功能类型III聚酮合酶的三内含子基因的鉴定 显花植物中,所有已知的类型III PKS 基因均含有一个内含子且位置绝对保守。本研究中,综合运用PCR技术,从富含聚酮类化合物的植物虎杖中克隆得到一个类型III PKS 基因(PcPKS1)及其cDNA。序列分析结果表明,PcPKS1含有3个内含子。系统发育分析结果表明,PcPKS1与其它植物的CHSs归为一类。然而,体外功能分析结果表明,当酶促反应体系pH值为7.0时,大肠杆菌中过表达的重组PcPKS1高效地合成柚皮素查尔酮(naringenin)为单一产物;而当pH值为9.0时,苯亚甲基丙酮(p-hydroxybenzalacetone)几乎为重组PcPKS1的唯一产物。后续的研究表明,与典型的CHSs相比,PcPKS1具有另外一些不同的特点:在pH值为9.0时(PcPKS1的苯亚甲基丙酮合成活性最适pH值),在4-香豆酰辅酶A的类似化合物中,只有feruloyl-CoA能够被PcPKS1接受作为起始底物。与CHSs展现出的对脂肪酰辅酶A宽泛的底物特异性不同,在不同的pH条件下,PcPKS1不接受异丁酰基辅酶A(isobutyryl-CoA)、异戊酰基辅酶A(isovaleryl-CoA)以及乙酰辅酶A(acetyl-CoA)作为起始底物。以上数据指出重组PcPKS1是一个具有查尔酮合酶(CHS)和苯亚甲基丙酮合酶(BAS)活性的双功能酶。Southern blot杂交结果表明,在虎杖基因组中存在2-4个PcPKS1基因的拷贝。Northern blot杂交结果表明,PcPKS1可能在防御病原菌和草食动物方面起着重要作用。PcPKS1和PcPKS2共同从虎杖中被分离的事实极有可能暗示了苯丁烷类化合物(phenylbutanoid)及其衍生物存在于虎杖中。 Ⅱ 高山红景天酪醇生物合成代谢途径机制研究 高山红景天(Rhodiola sachalinensis A. Bor)是景天科(Crassulaceae)红景天属多年生草本植物,作为一种适应原性中草药在中国的应用史已经超过800年。最近红景天提取物作为一种重要的商业药用制剂资源,其应用遍及欧洲、亚洲和美国,其主要治疗范围包括抗变应性和消炎,提高心理机敏性等。目前已经非常明确,红景天甙(salidroside)和甙元酪醇(tyrosol)是红景天属植物的主要功效成分,主要分布于这类植物的根中并且具有抗缺氧、抗疲劳、延缓衰老、预防紫外线辐射伤害等功效。红景天甙为酪醇8-O-β-D葡萄糖甙,是酪醇在葡萄糖基转移酶UDP-glucosyltransferase (UGT) 的催化下糖基化后形成的,可以认为是酪醇在植物体内的贮存形式。酪醇作为一种重要的活性分子,同样存在于橄榄树和葡萄酒中。 虽然已经非常明确酪醇来自于莽草酸代谢途径,然而其具体的生物合成途径及其调控仍不明确。总结以往的报道,在酪醇的生物合成上主要存在两种观点:一是酪醇可能来自于苯丙烷代谢途径产生的4-香豆酸(4-coumaric acid)前体;二是来自于酪氨酸的酪胺(tyramine)可能是酪醇生物合成的直接前体。我们的工作兴趣主要围绕着鉴别高山红景天中的酪醇生物合成途径展开: 高山红景天内源苯丙氨酸解氨酶PALrs1的过表达对红景天甙积累的影响 红景天甙是来自于药用植物高山红景天的一种适应原性新型药物,其生物合成途径可能起始于苯丙氨酸或酪氨酸。由于高山红景天野生植物资源的匮乏和相对含量很低,阐明红景天甙的生物合成途径对于增加红景天甙的供给至关重要。在我们以前的工作中,运用cDNA末端快速扩增技术(RACE),一个编码苯丙氨酸解氨酶phenylalanine ammonia-lyase (PAL)的cDNA从高山红景天中被克隆,命名为PALrs1。在本研究中,PALrs1置于35S启动子+Ω增强子序列的控制下通过农杆菌(Agrobacterium tumefaciens)介导法转化回高山红景天。PCR 和 PCR–Southern blot分析结果表明,PALrs1已经整合到了转基因植物的基因组上。Northern blot杂交结果表明,PALrs1已经获得在转录水平上的高水平表达。与预期的结果相同,高效液相色谱High-performance liquid chromatography (HPLC)测定结果显示PALrs1的过表达引起4-香豆酸含量增长3.3倍。然而,与之相反的是,酪醇和红景天甙含量与对照相比反而分别下降4.7和7.7倍。此外,我们发现PALrs1的过表达造成酪氨酸含量下降2.6倍。这些数据暗示着PALrs1的过表达和4-香豆酸的积累并不能促进酪醇的生物合成。酪醇,作为一种苯乙烷类衍生物并非来自苯丙氨酸,而酪氨酸含量的下降则极有可能是酪醇生物合成和红景天甙积累大规模下降的直接原因。
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Marine sponges have been an abundant source of new metabolites in recent years. The symbiotic association between the bacteria and the sponge has enabled scientists to access the bacterial diversity present within the bacterial/sponge ecosystem. This study has focussed on accessing the bacterial diversity in two Irish coastal marine sponges, namely Amphilectus fucorum and Eurypon major. A novel species from the genus Aquimarina has been isolated from the sponge Amphilectus fucorum. The study has also resulted in the identification of an α–Proteobacteria, Pseudovibrio sp. as a potential producer of antibiotics. Thus a targeted based approach to specifically cultivate Pseudovibrio sp. may prove useful for the development of new metabolites from this particular genus. Bacterial isolates from the marine sponge Haliclona simulans were screened for anti–fungal activity and one isolate namely Streptomyces sp. SM8 displayed activity against all five fungal strains tested. The strain was also tested for anti–bacterial activity and it showed activity against both against B. subtilis and P. aeruginosa. Hence a combinatorial approach involving both biochemical and genomic approaches were employed in an attempt to identify the bioactive compounds with these activities which were being produced by this strain. Culture broths from Streptomyces sp. SM8 were extracted and purified by various techniques such as reverse–phase HPLC, MPLC and ash chromatography. Anti–bacterial activity was observed in a fraction which contained a hydroxylated saturated fatty acid and also another compound with a m/z 227 but further structural elucidation of these compounds proved unsuccessful. The anti–fungal fractions from SM8 were shown to contain antimycin–like compounds, with some of these compounds having different retention times from that of an antimycin standard. A high–throughput assay was developed to screen for novel calcineurin inhibitors using yeast as a model system and three putative bacterial extracts were found to be positive using this screen. One of these extracts from SM8 was subsequently analysed using NMR and the calcineurin inhibition activity was con rmed to belong to a butenolide type compound. A H. simulans metagenomic library was also screened using the novel calcineurin inhibitor high–throughput assay system and eight clones displaying putative calcineurin inhibitory activity were detected. The clone which displayed the best inhibitory activity was subsequently sequenced and following the use of other genetic based approaches it became clear that the inhibition was being caused by a hypothetical protein with similarity to a hypothetical Na+/Ca2+ exchanger protein. The Streptomyces sp. SM8 genome was sequenced from a fragment library using Roche 454 pyrosequencing technology to identify potential secondary metabolism clusters. The draft genome was annotated by IMG/ER using the Prodigal pipeline. The Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMPN00000000. The genome contains genes which appear to encode for several polyketide synthases (PKS), non–ribosomal peptide synthetases (NRPS), terpene and siderophore biosynthesis and ribosomal peptides. Transcriptional analyses led to the identification of three hybrid clusters of which one is predicted to be involved in the synthesis of antimycin, while the functions of the others are as yet unknown. Two NRPS clusters were also identified, of which one may be involved in gramicidin biosynthesis and the function of the other is unknown. A Streptomyces sp. SM8 NRPS antC gene knockout was constructed and extracts from the strain were shown to possess a mild anti–fungal activity when compared to the SM8 wild–type. Subsequent LCMS analysis of antC mutant extracts confirmed the absence of the antimycin in the extract proving that the observed anti–fungal activity may involve metabolite(s) other than antimycin. Anti–bacterial activity in the antC gene knockout strain against P. aeruginosa was reduced when compared to the SM8 wild–type indicating that antimycin may be contributing to the observed anti–bacterial activity in addition to the metabolite(s) already identified during the chemical analyses. This is the first report of antimycins exhibiting anti–bacterial activity against P. aeruginosa. One of the hybrid clusters potentially involved in secondary metabolism in SM8 that displayed high and consistent levels of gene–expression in RNA studies was analysed in an attempt to identify the metabolite being produced by the pathway. A number of unusual features were observed following bioinformatics analysis of the gene sequence of the cluster, including a formylation domain within the NRPS cluster which may add a formyl group to the growing chain. Another unusual feature is the lack of AT domains on two of the PKS modules. Other unusual features observed in this cluster is the lack of a KR domain in module 3 of the cluster and an aminotransferase domain in module 4 for which no clear role has been hypothesised.
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The direct addition of enolizable aldehydes and a-halo thioesters to produce beta-hydroxy thioesters enabled by reductive soft enolization is reported. The transformation is operationally simple and efficient and has the unusual feature of giving high syn-selectivity, which is the opposite of that produced for (thio)esters under conventional conditions. Moreover, excellent diastereoselectivity results when a chiral nonracemic alpha-hydroxy aldehyde derivative is used.
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Pós-graduação em Microbiologia Agropecuária - FCAV
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Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with gamma-aminobutanoyl-CoA but with 4-guanidinylbutanoyl-CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final-stage de-amidination of the corresponding guanidino-substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective-group chemistry.
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The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675–679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.
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Aspergillus nidulans contains two functionally distinct fatty acid synthases (FASs): one required for primary fatty acid metabolism (FAS) and the other required for secondary metabolism (sFAS). FAS mutants require long-chain fatty acids for growth, whereas sFAS mutants grow normally but cannot synthesize sterigmatocystin (ST), a carcinogenic secondary metabolite structurally and biosynthetically related to aflatoxin. sFAS mutants regain the ability to synthesize ST when provided with hexanoic acid, supporting the model that the ST polyketide synthase uses this short-chain fatty acid as a starter unit. The characterization of both the polyketide synthase and FAS may provide novel means for modifying secondary metabolites.
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Plants produce a diversity of secondary metabolites, i.e., low-molecular-weight compounds that have primarily ecological functions in plants. The flavonoid pathway is one of the most studied biosynthetic pathways in plants. In order to understand biosynthetic pathways fully, it is necessary to isolate and purify the enzymes of the pathways to study individual steps and to study the regulatory genes of the pathways. Chalcone synthases are key enzymes in the formation of several groups of flavonoids, including anthocyanins. In this study, a new chalcone synthase enzyme (GCHS4), which may be one of the main contributors to flower colour, was characterised from the ornamental plant Gerbera hybrida. In addition, four chalcone synthase-like genes and enzymes (GCHS17, GCHS17b, GCHS26 and GCHS26b) were studied. Spatial expression of the polyketide synthase gene family in gerbera was also analysed with quantitative RT-PCR from 12 tissues, including several developmental stages and flower types. A previously identified MYB transcription factor from gerbera, GMYB10, which regulates the anthocyanin pathway, was transferred to gerbera and the phenotypes were analysed. Total anthocyanin content and anthocyanidin profiles of control and transgenic samples were compared spectrophotometrically and with HPLC. The overexpression of GMYB10 alone was able to change anthocyanin pigmentation: cyanidin pigmentation was induced and pelargonidin pigmentation was increased. The gerbera 9K cDNA microarray was used to compare the gene expression profiles of transgenic tissues against the corresponding control tissues to reveal putative target genes for GMYB10. GMYB10 overexpression affected the expression of both early and late biosynthetic genes in anthocyanin-accumulating transgenic tissues, including the newly isolated gene GCHS4. Two new MYB domain factors, named as GMYB11 and GMYB12, were also upregulated. Gene transfer is not only a powerful tool for basic research, but also for plant breeding. However, crop improvement by genetic modification (GM) remains controversial, at least in Europe. Many of the concerns relating to both human health and to ecological impacts relate to changes in the secondary metabolites of GM crops. In the second part of this study, qualitative and quantitative differences in cytotoxicity and metabolic fingerprints between 225 genetically modified Gerbera hybrida lines and 42 non-GM Gerbera varieties were compared. There was no evidence for any major qualitative and quantitative changes between the GM lines and non-GM varieties. The developed cell viability assays offer also a model scheme for cell-based cytotoxicity screening of a large variety of GM plants in standardized conditions.
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The enantioselective synthesis of the polyketide unit present in depsipeptides aetheramide A and B, which possess potent HIV-inhibitory activity, is accomplished from a chiral furyl carbinol.