Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula

Curacin A (1) is a potent cancer cell toxin obtained from strains of the tropical marine cyanobacterium Lyngbya majuscula found in Cura?ao. Its structure is unique in that it contains the sequential positioning of a thiazoline and cyclopropyl ring, and it exerts its potent cell toxicity through interaction with the colchicine drug binding site on microtubules. A series of stable isotope-labeled precursors were fed to cultures of curacin A-producing strains and, following NMR analysis, allowed determination of the metabolic origin of all atoms in the natural product (one cysteine, 10 acetate units, two S-adenosyl methionine-derived methyl groups) as well as several unique mechanistic insights. Moreover, these incorporation experiments facilitated an effective gene cloning strategy that allowed identification and sequencing of the approximately 64 kb putative curacin A gene cluster. The metabolic system is comprised of a nonribosomal peptide synthetase (NRPS) and multiple polyketide synthases (PKSs) and shows a very high level of collinearity between genes in the cluster and the predicted biochemical steps required for curacin biosynthesis. Unique features of the cluster include (1) all but one of the PKSs are monomodular multifunctional proteins, (2) a unique gene cassette that contains an HMG-CoA synthase likely responsible for formation of the cyclopropyl ring, and (3) a terminating motif that is predicted to function in both product release and terminal dehydrative decarboxylation.     

Structure and absolute stereochemistry of hectochlorin,a potent stimulator of actin assembly

Hectochlorin (1) was isolated from marine isolates of Lyngbya majuscula collected from Hector Bay, Jamaica, and Boca del Drago Beach, Bocas del Toro, Panama. The planar structure was deduced by one- and two-dimensional NMR spectroscopy. X-ray crystallography was used to determine the absolute stereochemistry of hectochlorin as 2S,3S,14S,22S. Hectochlorin is equipotent to jasplakinolide (5) in its ability to promote actin polymerization, but unlike jasplakinolide, is unable to displace a fluorescent phalloidin analogue from polymerized actin. In addition, hectochlorin shows both a unique profile of cytotoxicity by the COMPARE algorithm and potent inhibitory activity toward the fungus Candida albicans. Structurally, hectochlorin resembles dolabellin and the recently reported lyngbyabellin class of compounds.     

Biochemical characterization of the SgcA1 alpha-D-glucopyranosyl-1-phosphate thymidylyltransferase from the enediyne antitumor antibiotic C-1027 biosynthetic pathway and overexpression of sgcA1 in Streptomyces globisporus to improve C-1027 production

Sequence analysis of the biosynthetic gene cluster for the enediyne antitumor antibiotic C-1027 from Streptomyces globisporus has previously suggested that the sgcA1 gene encodes a alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase (Glc-1-P-TT) catalyzing the first step in the biosynthesis of the 4-deoxy-4-(dimethylamino)-5,5-dimethyl-d-ribopyranose moiety by activating alpha-d-glucopyranosyl-1-phosphate (Glc-1-P) into deoxythymidine diphosphate-alpha-d-glucose (dTDP-Glc). Here we report the overexpression of sgcA1 in E. coli, purification of the overproduced SgcA1 to homogenetity, biochemical and kinetic characterization of the purified SgcA1 as a Glc-1-P-TT, and yield improvement for C-1027 production by overexpression of sgcA1 and its flanking gene in S. globisporus. These findings provide biochemical evidence supporting the genetics-based hypothesis for C-1027 biosynthesis, set the stage for further investigation of the deoxysugar biosynthetic pathway, and demonstrate the utility of sugar biosynthesis genes in natural product yield improvement via combinatorial biosynthesis methods. In contrast to the homotetrameric quaternary structure known for Glc-1-P-TT enzymes from primary metabolic pathways, Glc-1-P-TT enzymes such as SgcA1 from secondary metabolic pathways are monomeric in solution. Sequence differences between the two subclasses of Glc-1-P-TT enzymes were noted. The monomeric structural feature of the latter enzymes could be exploited in engineering Glc-1-P-TT enzymes with broad substrate specificity for structural diversity via the glycorandomization strategy.     

Identification of the putative bryostatin polyketide synthase gene cluster from "Candidatus Endobugula sertula", the uncultivated microbial symbiont of the marine bryozoan Bugula neritina

The bryostatins are protein kinase C modulators with unique structural features and potential anticancer and neurological activities. These complex polyketides were isolated from the marine bryozoan Bugula neritina, but recent studies indicate that they are produced by the uncultured symbiotic bacterium "Candidatus Endobugula sertula" ("E. sertula"). Here we present the putative biosynthetic genes: five modular polyketide synthase (PKS) genes, a discrete acyltransferase, a beta-ketosynthase, a hydroxy-methyl-glutaryl CoA synthase (HMG-CS), and a methyltransferase. The cluster was sequenced in two closely related "E. sertula" strains from different host species. In one strain the gene cluster is contiguous, while in the other strain it is split into two loci, with one locus containing the PKS genes and the other containing the accessory genes. Here, we propose a hypothesis for the biosynthesis of the bryostatins. Thirteen PKS modules form the core macrolactone ring, and the pendent methyl ester groups are added by the HMG-CS gene cassette. The resulting hypothetical compound bryostatin 0 is the common basis for the 20 known bryostatins. As "E. sertula" is to date uncultured, heterologous expression of this biosynthetic gene cluster has the potential of producing the bioactive bryostatins in large enough quantities for development into a pharmaceutical.     

Cloning and characterization of the bleomycin biosynthetic gene cluster from Streptomyces verticillus ATCC15003

Bleomycin (BLM) biosynthesis has been studied as a model for hybrid peptide-polyketide natural product biosynthesis. Cloning, sequencing, and biochemical characterization of the blm biosynthetic gene cluster from Streptomyces verticillus ATCC15003 revealed that (1) the BLM hybrid peptide-polyketide aglycon is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules; (2) BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems; (3) the catalytic sites appear to be conserved in both hybrid NRPS/PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (4) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate the transfer of the growing intermediates between the interacting NRPS and/or PKS modules; (5) post-translational modification of the BLM megasynthetase has been accomplished by a single PPTase with broad carrier protein specificity; and (6) BlmIV/BlmIII-templated assembly of the BLM bithiazole moiety requires intriguing protein juxtaposition and modular recognition. These results lay the foundation to investigate the molecular basis for intermodular communication between NRPS and PKS in hybrid peptide-polyketide natural product biosynthesis and set the stage for engineering novel BLM analogues by genetic manipulation of genes governing BLM biosynthesis.     

Isolation, structure elucidation, and biosynthesis of an unusual hydroxamic acid ester-containing siderophore from Actinosynnema mirum

In this study we report the isolation, structure elucidation, and biosynthesis of mirubactin (1), a siderophore containing an unprecedented chemical functionality in natural products, namely, an O-acyl hydroxamic acid ester. Mirubactin represents the first siderophore isolated from the genus Actinosynnema and the first natural product produced by Actinosynnema mirum whose biosynthetic gene cluster could be identified. Structure elucidation was accomplished through a combination of spectroscopic (NMR, IR, and UV/vis) and mass spectrometric methods and revealed the presence of an unusual ester bond between the δ-N-hydroxyl group of δ-N-formyl-δ-N-hydroxyornithine and a 2,3-dihydroxybenzoate moiety. Bioinformatic analysis of the A. mirum genome and subsequent biochemical characterization of the putative biosynthetic machinery identified the gene cluster responsible for mirubactin assembly. The proposed biosynthesis of mirubactin comprises the iterative use of a stand-alone carrier-protein-bound substrate, as well as an ester-bond-forming step catalyzed by a C-terminal condensation domain, thus revealing an interesting system for further biochemical studies to gain a deeper understanding of nonribosomal peptide synthetase-catalyzed siderophore biosynthesis.     

基于转录组测序的山茱萸次生代谢生物合成相关基因的挖掘

为探讨山茱萸中环烯醚萜等次生代谢产物合成的遗传基础,采用新一代高通量测序技术对其果实进行转录组测序,共获得得96 032条unigenes,平均长度590.53 bp;其中共有35 478条unigene能被NR,Swissprot,COG,GO,KOG,Pfam和KEGG等7个公共数据库注释。通过对注释所得的unigene进行KEGG代谢通路的分析发现,共有84个unigene与环烯醚萜类成分的生物合成有关;487条unigene参与山茱萸其他次生代谢相关物质代谢调控。研究发现,共有153条unigene参与山茱萸次生代谢产物的氧化/羟基化;72条unigene参与次生代谢产物的糖基化。该研究首次对山茱萸转录组进行了分析,并获得了山茱萸环烯醚萜类等次生代谢生物合成相关的候选基因,为山茱萸的分子生物学研究提供了丰富的数据资源,也为后续探讨候选基因的功能奠定了基础。     

Polyketide chain skipping mechanism in the biosynthesis of the hybrid nonribosomal peptide-polyketide antitumor antibiotic leinamycin in Streptomyces atroolivaceus S-140

A fundamental feature of modular polyketide synthases (PKSs) is the highly predictable relationship between the domain order and the chemical functional groups of resultant polyketide products. Sequence analysis and biochemical characterization of the leinamycin (LNM) biosynthetic gene cluster from Streptomyces atroolivaceus S-140 has revealed a gene, lnmJ, that encodes five PKS modules but with six acyl carrier protein (ACP) domains. The LnmJ PKS module 6 contains two ACP domains, ACP(6-1) and ACP(6-2), separated by a C-methyltransferase domain. Site-directed mutagenesis experiments were carried out with each of these ACPs to test alternative mechanisms proposed for their role in polyketide chain elongation. The in vivo results revealed a new type of polyketide chain "skipping" mechanism, in which either ACP is sufficient for LNM biosynthesis. Biochemical characterization in vitro showed that both ACPs can be loaded with a malonate extender unit by the LnmG acyl transferase; however, ACP(6-2) appears to be preferred because the loading efficiency is about 5-fold that of ACP(6-1). The results are consistent with ACP(6-2) being used for the initial chain elongation step wth ACP(6-1) being involved in the ensuing C-methylation process. These findings provide new insights into the polyketide chain skipping mechanism for modular PKSs.     

细胞色素P450在人参皂苷生物合成途径中的研究进展

细胞色素P450(CYP450)是一类超基因家族编码的单加氧酶,参与萜类、生物碱和甾醇类等多种次生代谢产物的合成与代谢。CYP450对人参皂苷三萜碳环骨架进行羟基化和氧化等一系列复杂修饰作用,是人参皂苷生物合成途径中的关键酶。近年来利用新一代测序技术及生物信息学分析等方法,从CYP450家族中筛选出参与人参皂苷生物合成的相关CYP450s,并对候选基因(CYP716A47)进行了生物功能验证,进一步阐明了人参皂苷合成途径。本文对人参皂苷生物合成途径做简要介绍,并对近年来CYP450在人参皂苷生物合成途径中的研究进行综述,为阐明人参皂苷合成途径及通过基因工程手段合成人参皂苷提供理论依据。     

Isolation, structure determination, and biological activity of Lyngbyabellin A from the marine cyanobacterium lyngbya majuscula

Lyngbyabellin A (1), a significantly cytotoxic compound with unusual structural features, was isolated from a Guamanian strain of the marine cyanobacterium Lyngbya majuscula. This novel peptolide is structurally related to dolabellin (2) in that both depsipeptides bear a dichlorinated beta-hydroxy acid and two functionalized thiazole carboxylic acid units. Its gross structure has been elucidated by spectral analysis, including 2D NMR techniques. The absolute stereochemistry of 1 was determined by chiral HPLC analysis of hydrolysis products and by characterization of the degradation products methyl 7,7-dichloro-3-hydroxy-2,2-dimethyloctanoate (3) and the corresponding acid 4. The total structure was further supported by molecular modeling studies. The isolation of 1 from L. majuscula once more supports the proposal that many compounds originally isolated from the sea hare Dolabella auricularia are of cyanobacterial origin. Lyngbyabellin A (1) was shown to be a potent disrupter of the cellular microfilament network.     

系统生物学方法在药用植物次生代谢产物研究中的应用

次生代谢产物是植物在其生长发育和对环境的适应过程中形成的,通常是药用植物中的主要活性成分,药材品质的物质基础。但次生代谢产物的生源途径复杂,其产生和积累受到自身遗传和环境中各种生物和非生物因素的调控,影响了药用植物作为药材的品质控制及其活性成分的开发利用。系统生物学思维与方法是系统全面探索生物的有力工具,随着现代分子生物学技术及生物信息学的发展,系统整合基因组、转录组、蛋白组和代谢组等组学技术,将为药用植物次生代谢产物研究带来新的机遇。这种整体、系统的研究方法在药用植物次生代谢产物形成的生物合成途径、信号转导、生态环境及其代谢工程等研究中的应用,构建次生代谢物生物合成基因表达调控系统模型,对于系统阐释药用植物有效成分成因和道地药材形成机制、代谢工程产生药用植物活性成分、和药用植物资源合理开发利用等具有重要意义。     

The use of recombinant DNA techniques to study tylosin biosynthesis and resistance in Streptomyces fradiae

A substantial amount of information on the biosynthesis of tylosin has been obtained over the past ten years. Physiological studies and experiments with tylosin-blocked (tyl) mutants have suggested the probable pathway by which tylactone is converted to tylosin. The development of recombinant DNA methodology for streptomycetes in general, and for Streptomyces fradiae in particular, has allowed us to apply gene cloning techniques in further studies of tylosin biosynthesis in S. fradiae. The macrocin O-methyltransferase (MOMT), which catalyzes the last step in tylosin biosynthesis, was purified, and the sequence of the 35 amino acids at its amino-terminus was determined. A synthetic 44 base oligonucleotide probe was constructed on the basis of the amino acid sequence. The probe was used to identify sequences containing the MOMT structural gene in bacteriophage and cosmid libraries of S. fradiae DNA. Complementation of tyl mutants with the cloned DNA sequences identified nine tyl biosynthetic genes (tylC, D, E, F, H, J, K, L, and M) in a 42 kb stretch of DNA. Genes complementing four mutant classes, tylA, B, G, and I were not found. A tylosin-resistance gene, tlrB, was located just left of the tyl gene cluster. Tylosin-sensitive mutants of S. fradiae, which were isolated from regenerated protoplasts and which have pleiotropic deficiencies in tylosin biosynthesis, contained deletions which included at least some of the identified tyl loci and one or both of two tylosin-resistance genes, tlrB and tlrC. Possible schemes for the functional organization of the tyl region of the S. fradiae genome are discussed.     

Diverse secondary metabolites from a Puerto Rican collection of Lyngbyamajuscula

Extensive fractionation of the crude organic extract from a Puerto Rican collection of Lyngbya majuscula led to the discovery of three new secondary metabolites: a quinoline alkaloid (1), malyngamide T (2), and a tryptophan derivative (3). In addition, several previously reported compounds, including the potent neurotoxins antillatoxin, antillatoxin B, and kalkitoxin, were identified. The structures of 1, 2, and 3 were deduced by NMR and mass spectral data interpretation and suggest the existence of a convergent biosynthetic pathway for these new and unusual metabolites.     

多花黄精根茎的转录组分析与甾体皂苷生物合成相关基因发掘

为了丰富多花黄精Polygonatum cyrtonema幼苗期根茎发育的转录组数据,发掘参与甾体皂苷生物合成的功能基因,为多花黄精活性成分的合成代谢途径与调控机制研究提供遗传资源,该研究基于Illumina深度测序平台,对多花黄精幼苗期根茎进行转录组测序及生物信息学分析,包括序列数据的组装拼接、unigene序列的功能注释、分类和代谢途径分析,并对次生代谢途径中甾体皂苷的生物合成通路进行深入解析。结果显示,多花黄精根茎的转录组数据经拼接共产生了126 546条unigene序列,其中47 226条被注释。共有16 499条unigene被定位到了KEGG数据库中的132个代谢通路,其中2 768条被鉴定出参与了22个次生代谢物生物合成通路。鉴定出的113条unigene序列,分别编码27个与甾体皂苷生物合成相关的代谢酶,与45个拟南芥酶基因同源。该研究从多花黄精转录组数据库中发掘到一系列与活性成分甾体皂苷生物合成相关的酶基因,对这些基因的深入研究,有助于从分子水平上解析多花黄精中甾体皂苷的生物合成途径。该研究也为多花黄精的转录组学研究提供了丰富的参考数据,对于多花黄精的功能基因组学研究具有重要意义。     

Thailandepsins: bacterial products with potent histone deacetylase inhibitory activities and broad-spectrum antiproliferative activities

Histone deacetylase (HDAC) inhibitors have emerged as a new class of anticancer drugs, with one synthetic compound, SAHA (vorinostat, Zolinza; 1), and one natural product, FK228 (depsipeptide, romidepsin, Istodax; 2), approved by FDA for clinical use. Our studies of FK228 biosynthesis in Chromobacterium violaceum no. 968 led to the identification of a cryptic biosynthetic gene cluster in the genome of Burkholderia thailandensis E264. Genome mining and genetic manipulation of this gene cluster further led to the discovery of two new products, thailandepsin A (6) and thailandepsin B (7). HDAC inhibition assays showed that thailandepsins have selective inhibition profiles different from that of FK228, with comparable inhibitory activities to those of FK228 toward human HDAC1, HDAC2, HDAC3, HDAC6, HDAC7, and HDAC9 but weaker inhibitory activities than FK228 toward HDAC4 and HDAC8, the latter of which could be beneficial. NCI-60 anticancer screening assays showed that thailandepsins possess broad-spectrum antiproliferative activities with GI50 for over 90% of the tested cell lines at low nanomolar concentrations and potent cytotoxic activities toward certain types of cell lines, particularly for those derived from colon, melanoma, ovarian, and renal cancers. Thailandepsins thus represent new naturally produced HDAC inhibitors that are promising for anticancer drug development.     

丹参酮合成相关的SmCYP81C16基因克隆和功能研究

目的:丹参(Salvia miltiorrhiza)根中所含的丹参酮为二萜醌类化合物,而鉴定参与丹参酮合成的CYP450基因成为解析丹参酮生物合成途径分子机制的关键步骤。方法:本实验从丹参基因组和转录组数据中筛选到SmCYP81C16基因,采用RT-PCR方法克隆获得基因cDNA全长序列,利用生物信息学分析方法分析其所编码蛋白质的理化性质,预测该蛋白质二级结构、保守结构域等,建立系统发育进化树;利用实时荧光定量PCR方法检测了该基因的组织/器官表达特异性;通过遗传转化方法获得了该基因的过表达转基因毛状根株系,通过化学检测及代谢组学分析丹参酮类化合物在对照株系和过表达株系中的含量变化。利用实时荧光定量PCR方法检测了毛状根中丹参酮合成途径关键酶基因中的相对表达量。结果:SmCYP81C16基因全长1497 bp,编码498个氨基酸残基,该蛋白质相对分子质量为55.8 kDa;SmCYP81C16在丹参茎和根的周皮部高表达;通过化学检测及代谢组学分析发现,与对照株系比较,在SmCYP81C16过表达阳性株系中,丹参酮类化合物的含量升高;过表达毛状根中丹参酮合成途径关键酶基因的表达量显著上升。结论:本研究结果表明SmCYP81C16具有正向调控丹参酮生物合成的功能。本研究为利用生物技术方法提高丹参酮类化合物含量奠定基础。     

Plant-like biosynthetic pathways in bacteria: from benzoic acid to chalcone

Although phenylpropanoids and flavonoids are common plant natural products, these major classes of biologically active secondary metabolites are largely absent from bacteria. The ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are key biosynthetic catalysts in phenylpropanoid and flavonoid assembly, respectively. Until recently, few bacterial counterparts were known, thus reflecting the dearth of these plant natural products in bacteria. This review highlights our progress on the biochemical and genetic characterization of recently identified streptomycete biosynthetic pathways to benzoic acid and type III polyketide synthase (PKS)-derived products. The sediment-derived bacterium "Streptomyces maritimus" produces benzoyl-CoA in a plant-like manner from phenylalanine involving a PAL-mediated reaction through cinnamic acid during the biosynthesis of the polyketide antibiotic enterocin. All but one of the genes encoding benzoyl-CoA biosynthesis in "S. maritimus" have been cloned, sequenced, and inactivated, providing a model for benzoate biosynthesis not only in this bacterium, but in plants where benzoic acid is an important constituent of many products. The recent discovery that bacteria harbor homodimeric PKSs belonging to the plant CHS superfamily of condensing enzymes has further linked the biosynthetic capabilities of plants and bacteria. A bioinformatics approach led to the prediction that the model actinomycete Streptomyces coelicolor A3(2) contains up to three type III PKSs. Biochemical analysis of one of the recombinant type III PKSs from S. coelicolor demonstrated activity as a 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). A homology model of THNS based upon the known three-dimensional structure of CHS was constructed to explore the structural and mechanistic details of this new subclass of bacterial PKSs.     

三萜皂苷生物合成途径及关键酶的研究进展

三萜皂苷是植物界中具有多种生物活性的一类重要的植物次生代谢产物,是许多药用植物或中药材的重要活性成分之一,阐明三萜皂苷的生物合成途径具有重要的理论意义与广阔的应用前景。近年来,三萜皂苷整个生物合成途径的阐明,尤其是三萜碳环骨架合成及环上复杂官能化等相关酶基因的确定与克隆,仍是众多学者关注与研究的热点。本文对三萜皂苷的生物合成途经和有关“鲨烯及三萜碳环骨架合成和碳环上的复杂官能化”的关键酶进行了综述,为深入阐明三萜皂苷生物合成途径及人工生物合成提供理论基础。     

参与植物三萜生物合成的细胞色素P450酶研究进展

三萜是许多药用植物的活性成分,目前已对其生物合成途径进行了大量研究并取得了重大进展。细胞色素P450(CYP450)主要参与三萜的后修饰过程,对三萜的多样性起着关键作用。目前,已从多种植物中克隆得到与三萜生物合成相关的CYP450基因。对参与三萜生物合成的CYP450的功能研究进行综述,为三萜生物合成途径的解析及CYP450的功能研究提供借鉴。