Phylogenetic analysis of polyketide synthase genes fromAspergillus ochraceus

A number of polyketide synthase gene sequences fromAspergillus ochraceus were isolated by both SSH-PCR and degenerate PCR. The deduced amino acid sequences of the corresponding clonedpks DNA fragments were then aligned with the amino acid sequences of other polyketide synthase enzymes. One of thesepks genes is essential for ochratoxin A biosynthesis (OTA-PKS). The OTA-PKS was most similar to methylsalicylic acid synthase (MSAS) type PKS proteins based on the alignment of the ketosynthase domains while if the acyl transferase domains were aligned it appeared to be more similar to PKS enzymes fromCochliobolus heterostrophus. The three PKS proteins identified by degenerate PCR were all from different PKS types, one was a MSAS type enzyme, the second was similar to the PKS proteins involved in lovastatin biosynthesis while the third was not similar to any of the other phylogenetic groupings. Data is presented which suggests that the use of phylogenetic analysis to predict the function of PKS proteins/genes is likely to be significantly enhanced by analyzing more than one domain of the protein. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Irish Government under the National Development Plan 2000–2006     

Phylogenetic analysis of type I polyketide synthase and nonribosomal peptide synthetase genes in Antarctic sediment

The modular polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) have been found to be involved in natural product synthesis in many microorganisms. Study on their diversities in natural environment may provide important ecological insights, in addition to opportunities for antibacterial drugs development. In this study, the PKS and NRPS gene diversities in two coast sediments near China Zhongshan Station were studied. The phylogenetic analysis of amino acid (AA) sequences indicated that the identified ketosynthase (KS) domains were clustered with those from diverse bacterial groups, including Proteobacteria, Firmicutes, Planctomycetes, Cyanobacteria, Actinobacteria, and some uncultured symbiotic bacteria. One new branch belonging to hybrid PKS/NRPS enzyme complexes and five independent clades were found on the phylogenetic tree. The obtained adenylation (A) domains were mainly clustered within the Cyanobacteria and Proteobacteria group. Most of the identified KS and A domains showed below 80 and 60% identities at the AA level to their closest matches in GenBank, respectively. The diversities of both KS and A domains in natural environmental sample were different from those in sewage-contaminated sample. These results revealed the great diversity and novelty of both PKS and NRPS genes in Antarctic sediment.     

Characterization of a novel asparaginase from soil metagenomic libraries generated from forest soil

Objectives

To screen soil metagenomic libraries for novel enzymes with enhanced activities.

Results

To screen soil metagenomic libraries for novel enzymes with enhanced activities. A novel l-asparaginase was identified from forest soil metagenome and its characteristics were studied. The purified protein had a specific activity of 696 IU mg^?1 and optimum activity at pH 7 and 35 °C. Enhanced enzyme activities were observed in the presence of Mg²⁺, Ca²⁺ and K⁺. The K_m value, 2 mM, and enzyme specificity constant 7.7 mM^?1s^?1 indicated that the recombinant enzyme has good substrate affinity to l-asparagine compared with commercially-available Escherichia coli asparaginase. The IC₅₀ value of 0.78 µg ml^?1 (0.47 IU ml^?1) was observed with HL60 cell line and 0.39 µg ml^?1(0.23 IU ml^?1) with MOLT-3 and MOLT-4 cell lines, which is better than that of commercially-available drugs.

Conclusion

The soil metagenome derived l-asparaginase with enhanced activities could be a potential candidate to develop as a drug in Acute Lymphoblastic Leukemia (ALL) therapy.

     

Phylogenetic analysis of homologous fatty acid synthase and polyketide synthase involved in aflatoxin biosynthesis

The first two steps of aflatoxin biosynthesis are catalyzed by the HexA/B and by the Pks protein. The phylogenetic analysis clearly distinguished fungal HexA/B from FAS subunits and from other homologous proteins. The phylogenetic trees of the HexA and HexB set of proteins share the same clustering. Proteins involved in the synthesis of fatty acids or in the aflatoxin or sterigmatocystin biosynthesis cluster separately. The Pks phylogenetic tree also differentiates the aflatoxin-related polypeptide sequences from those of other kinds of secondary metabolism. The function of some of the A. flavus Pks homologues may be deduced from the phylogenetic analysis. The conserved sequence motifs of protein domains shared by HexA/B and Pks - namely, β-polyketide synthase (KS), acetyl transferase (AT) and acyl carrier protein (ACP) - have been identified, and the HexA/B and Pks involved in aflatoxin biosynthesis have been distinguished from those involved in primary metabolism or other kinds of secondary metabolism.     

SEARCHPKS: A program for detection and analysis of polyketide synthase domains

SEARCHPKS is a software for detection and analysis of polyketide synthase (PKS) domains in a polypeptide sequence. Modular polyketide synthases are unusually large multi-enzymatic multi-domain megasynthases, which are involved in the biosynthesis of pharmaceutically important natural products using an assembly-line mechanism. This program facilitates easy identification of various PKS domains and modules from a given polypeptide sequence. In addition, it also predicts the specificity of the potential acyltransferase domains for various starter and extender precursor units. SEARCHPKS is a user-friendly tool for correlating polyketide chemical structures with the organization of domains and modules in the corresponding modular polyketide synthases. This program also allows the user to extensively analyze and assess the sequence homology of various polyketide synthase domains, thus providing guidelines for carrying out domain and module swapping experiments. SEARCHPKS can also aid in identification of polyketide products made by PKS clusters found in newly sequenced genomes. The computational approach used in SEARCHPKS is based on a comprehensive analysis of various characterized clusters of modular polyketide synthases compiled in PKSDB, a database of modular polyketide synthases. SEARCHPKS can be accessed at http://www.nii.res.in/searchpks.html.     

四种红树植物根际土壤微生物Ⅰ型和Ⅱ型PKS基因的检测与多样性分析

[目的]探究红树林土壤中聚酮合酶(Polyketide synthase,PKS)基因的多样性和新颖性.[方法]用Ⅰ型和Ⅱ型PKS基因酮基合成酶(Ketosynthase,KS)域的简并引物对海南清澜港红树林海莲、黄槿、银叶、老鼠簕4种红树根际土壤样品中DNA进行PCR扩增,之后利用PCR-限制性酶切片段多样性(PCR-RFLP)和测序分析法对Ⅰ型和Ⅱ型PKS基因的多样性进行探讨.[结果]对得到的72条Ⅰ型PKS基因的酮基合成酶(Ketosynthase,KS)域DNA序列进行PCR-RFLP分析,共得到51个可操作分类单元(Operational taxonomic unit,OTUs),其中37个OTUs为单克隆产生,没有明显的优势OTU.选取了26个代表不同OTU的克隆进行测序分析,这些序列与GenBank中已知序列的最大相似率均未超过85％. KS域氨基酸序列的系统发育分析显示,所得KS域来源广泛,包括蓝细菌门(Cyanobacteria)、变形杆菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和一些未可培养细菌;对55条PKSⅡ基因KS域DNA序列的PCR-RFLP分析后共得到25个OTUs,有两个明显的优势OTUs,代表的克隆子数所占比例超过10％.[结论]PCR-RFLP分析表明红树林根际土壤中存在着丰富多样的Ⅰ型和Ⅱ型PKS基因,且前者多样性更高;低的序列相似度表明所获得的PKSⅠ基因KS域序列独特;系统发育分析表明得到的PKSⅠ基因来源广泛.     

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.     

Purifying selection is a prevailing motif in the evolution of ketoacyl synthase domains of polyketide synthases from lichenized fungi

We analysed ketoacyl synthase domains of type I polyketide synthase (PKS) gene fragments of 163 lichenized and 51 non-lichenized fungi in a Bayesian phylogenetic framework. Lichenized taxa from several unrelated taxonomic groups, some of which produce identical secondary metabolites, were included. We found 12 clades of non-reducing PKS genes, which represent monophyletic PKS paralogues. PAML and SELECTON analyses indicated that purifying selection is the prevailing selective force in the evolution of the keto synthase domain of these paralogues. We detected no unambiguous correlation between PKS clades and the distribution of lichen substances. Together with the strong evidence for purifying selection, the wide distribution of certain paralogues in ascomycetes suggested early gene duplication events in the evolutionary history of this gene family in the Ascomycota.     

Phylogenetic and metagenomic analysis of Verrucomicrobia in former agricultural grassland soil

The bacterial phylum Verrucomicrobia has a widespread distribution, and is known to be one of the most common and diverse phyla in soil habitats. However, members of this phylum have typically been recalcitrant to cultivation methods, hampering the study of this presumably important bacterial group. In this study, we examine the phylogenetic diversity of the Verrucomicrobia in a former agricultural field and gain access to genomic information via a metagenomic approach. We examined Verrucomicrobia -like 16S rRNA gene sequences recovered from general bacterial and phylum-specific libraries, revealing a dominance of subdivisions 1 and 2. A PCR-based screening method was developed to identify inserts containing verrucomicrobial 16S rRNA genes within a large-insert metagenomic library, and on screening of 28 800 clones, four fosmids were identified as containing verrucomicrobial genomic DNA. Full-length sequencing of fosmid inserts and gene annotation identified a total of 98 ORFs, representing a range of functions. No conservation of gene order was observed adjacent to the ribosomal operons. Fosmid inserts were further analyzed for tetranucleotide frequencies to identify remnants of past horizontal gene transfer events. The metagenomic approach utilized proved to be suitable for the recovery of verrucomicrobial genomic DNA, thereby providing a window into the genomes of members of this important, yet poorly characterized, bacterial phylum.     

Polyketide synthase pathways identified from a metagenomic library are derived from soil Acidobacteria

Polyketides are structurally diverse secondary metabolites, many of which have antibiotic or anticancer activity. Type I modular polyketide synthase (PKS) genes are typically large and encode repeating enzymatic domains that elongate and modify the nascent polyketide chain. A fosmid metagenomic library constructed from an agricultural soil was arrayed and the macroarray was screened for the presence of conserved ketosynthase [β-ketoacyl synthase (KS)] domains, enzymatic domains present in PKSs. Thirty-four clones containing KS domains were identified by Southern hybridization. Many of the KS domains contained within metagenomic clones shared significant similarity to PKS or nonribosomal peptide synthesis genes from members of the Cyanobacteria or the Proteobacteria phyla. However, analysis of complete clone insert sequences indicated that the blast analysis for KS domains did not reflect the true phylogenetic origin of many of these metagenomic clones that had a %G+C content and significant sequence similarity to genes from members of the phylum Acidobacteria. This conclusion of an Acidobacteria origin for several clones was further supported by evidence that cultured soil Acidobacteria from different subdivisions have genetic loci closely related to PKS domains contained within metagenomic clones, suggesting that Acidobacteria may be a source of novel polyketides. This study also demonstrates the utility of combining data from culture-dependent and -independent investigations in expanding our collective knowledge of microbial genomic diversity.     

Biochemical characterization of the minimal polyketide synthase domains in the lovastatin nonaketide synthase LovB

The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.     

Construction and validation of two metagenomic DNA libraries from Cerrado soil with high clay content

A challenge of metagenomic studies is in the extraction and purification of DNA from environmental samples. The soils of the Cerrado region of Brazil present several technical difficulties to DNA extraction: high clay content (>55% w/w), low pH (4.7) and high iron levels (146 ppm). Here we describe for the first time the efficient recovery and purification of microbial DNA associated with these unusual soil characteristics and the construction and validation of two metagenomic libraries: a 150,000 clones library with insert size of approximately 8 kb and a 65,000 clones library with insert size of approximately 35 kb. The construction of these metagenomic libraries will allow the biotechnological exploitation of the microbial community present in the soil from this endangered biome.     

Identification of unique type II polyketide synthase genes in soil

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS(alpha) (ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS(alpha) genes as determined by TRFLP analysis of KS(alpha) PCR products. KS(alpha) PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS(alpha) genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS(alpha) TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS(alpha) involved in ardacin (glycopeptide antibiotic) production by Kibdelosporangium aridum. The remaining sequences showed greatest similarity to the KS(alpha) genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.     

Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase

Chain initiation on many modular polyketide synthases is mediated by acyl transfer from the CoA ester of a dicarboxylic acid, followed by decarboxylation in situ by KSQ, a ketosynthase-like decarboxylase domain. Consistent with this, the acyltransferase (AT) domains of all KSQ-containing loading modules are shown here to contain a key arginine residue at their active site. Site-specific replacement of this arginine residue in the oleandomycin (ole) loading AT domain effectively abolished AT activity, consistent with its importance for catalysis. Substitution of the ole PKS loading module, or of the tylosin PKS loading module, for the erythromycin (ery) loading module gave polyketide products almost wholly either acetate derived or propionate derived, respectively, instead of the mixture found normally. An authentic extension module AT domain, rap AT2 from the rapamycin PKS, functioned appropriately when engineered in the place of the ole loading AT domain, and gave rise to substantial amounts of C13-methylerythromycins, as predicted. The role of direct acylation of the ketosynthase domain of ex-tension module 1 in chain initiation was confirmed by demonstrating that a mutant of the triketide synthase DEBS1-TE, in which the 4'-phosphopante-theine attachment site for starter acyl groups was specifically removed, produced triketide lactone pro-ducts in detectable amounts.     

Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries

A high-throughput method (≥ 10⁶ of clones can be analysed on a single agar plate) for the selection of ester-hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′-O-tri-acetyluridine and 2′,3′,5′-O-tri-hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.     

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.     

Kinetic analysis of the actinorhodin aromatic polyketide synthase.

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [(14)C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.     

云南土壤宏基因组文库中替加环素耐药基因的筛选与分析

[背景]随着抗生素的广泛应用以及滥用,出现了多重耐药的"超级细菌",并且在临床上已出现对治疗"超级细菌"感染的最后一线抗生素——替加环素耐药的细菌.[目的]对土壤环境中存在的替加环素耐药基因进行筛选调查,探讨替加环素耐药机制,为临床抗生素治疗提供借鉴.[方法]利用功能宏基因组学技术对土壤宏基因组文库进行替加环素耐药阳性...     

Cloning of modular type I polyketide synthase genes from salinomycin producing strain of Streptomyces albus

Cloning of polyether polyketide synthase (PKS) genes for salinomycin biosynthesis was attempted from Streptomyces albus. Seven beta-ketoacyl synthase (KS) core regions were obtained by PCR amplification using primers designed based on the conserved KS domains of type I PKSs. Using the KS fragment as a probe, screening of an S. albus genomic DNA library was carried out by colony hybridization. From the positive cosmid clone isolated, a 4.5-kbBamHI fragment was subcloned and sequenced. It showed high homology with bacterial type I PKSs and was deduced to code for KS, malonyl transferase, and ketoreductase motifs. By gene disruption with this 4.5-kb BamHI fragment, the cloned gene was shown to be a part of the salinomycin biosynthetic gene cluster of S. albus.     

Identification of domains within megalomicin and erythromycin polyketide synthase modules responsible for differences in polyketide production levels in Escherichia coli

The megalomicin and erythromycin polyketide synthases (PKSs) produce the same aglycon product, 6-deoxyerythronolide B (6-dEB). Both PKSs were examined in an Escherichia coli strain metabolically engineered to support complex polyketide biosynthesis. Production of 6-dEB in shake flask fermentations was undetectable by mass spectrometry in the strain expressing the megalomicin (Meg) PKS genes, whereas 31 mg/L 6-dEB was produced by the strain with the erythromycin (DEBS) PKS. The genes for each of the three subunits comprising the PKSs were expressed in different combinations from three compatible expression vectors (e.g., DEBS1, DEBS2, and MegA3) to identify two Meg PKS subunits, MegA1 and MegA3, which conferred lower 6-dEB titers than their DEBS counterparts. Comparison of protein expression levels and 6-dEB titers by engineered hybrid DEBS/Meg PKS genes further defined regions within modules 2 and 6 of MegA1 and MegA3, respectively, which limit protein expression and 6-dEB production in E. coli. Meg module 2 + TE (M2 + TE) and a hybrid DEBS M2/Meg M2 + TE protein were engineered and purified for in vitro comparisons with DEBS M2 + TE. The specific activity of the hybrid M2 + TE was approximately 16-fold lower than DEBS M2 + TE and only twice as high as the Meg M2 + TE enzyme in diketide elongation assays. Since the hybrid M2 worked comparably to DEBS M2 in vivo, this suggests that boosting subunit concentration could serve as a useful approach to overcome enzyme deficiencies in heterologous polyketide production.