ThioFinder: A Web-Based Tool for the Identification of Thiopeptide Gene Clusters in DNA Sequences

Thiopeptides are a growing class of sulfur-rich, highly modified heterocyclic peptides that are mainly active against Gram-positive bacteria including various drug-resistant pathogens. Recent studies also reveal that many thiopeptides inhibit the proliferation of human cancer cells, further expanding their application potentials for clinical use. Thiopeptide biosynthesis shares a common paradigm, featuring a ribosomally synthesized precursor peptide and conserved posttranslational modifications, to afford a characteristic core system, but differs in tailoring to furnish individual members. Identification of new thiopeptide gene clusters, by taking advantage of increasing information of DNA sequences from bacteria, may facilitate new thiopeptide discovery and enrichment of the unique biosynthetic elements to produce novel drug leads by applying the principle of combinatorial biosynthesis. In this study, we have developed a web-based tool ThioFinder to rapidly identify thiopeptide biosynthetic gene cluster from DNA sequence using a profile Hidden Markov Model approach. Fifty-four new putative thiopeptide biosynthetic gene clusters were found in the sequenced bacterial genomes of previously unknown producing microorganisms. ThioFinder is fully supported by an open-access database ThioBase, which contains the sufficient information of the 99 known thiopeptides regarding the chemical structure, biological activity, producing organism, and biosynthetic gene (cluster) along with the associated genome if available. The ThioFinder website offers researchers a unique resource and great flexibility for sequence analysis of thiopeptide biosynthetic gene clusters. ThioFinder is freely available at http://db-mml.sjtu.edu.cn/ThioFinder/.     

Heterologous production of thiostrepton A and biosynthetic engineering of thiostrepton analogs

Thiostrepton A 1, produced by Streptomyces laurentii ATCC 31255 (S. laurentii), is one of the more well-recognized thiopeptide metabolites. Thiostrepton A 1 and other thiopeptides are of great interest due to their potent activities against emerging antibiotic-resistant Gram-positive pathogens. Although numerous lines of evidence have established that the thiopeptides arise from the post-translational modification of ribosomally-synthesized peptides, few details have been revealed concerning this elaborate process. Alteration to the primary amino acid sequence of the precursor peptide provides an avenue to probe the substrate specificity of the thiostrepton post-translational machinery. Due to the difficulties in the genetic manipulation of S. laurentii, the heterologous production of thiostrepton A 1 from an alternate streptomycete host was sought to facilitate the biosynthetic investigations of the peptide metabolite. The production of thiostrepton A 1 from the non-cognate hosts did not lend itself to be as robust as S. laurentii-based production, therefore an alternate strategy was pursued for the production of thiostrepton variants. The introduction of a fosmid used in the heterologous production of thiostrepton A 1, harboring the entire thiostrepton biosynthetic gene cluster, into the tsrA deletion mutant permitted restoration of thiostrepton A 1 production near to that of the wild-type level. The fosmid was then engineered to enable the replacement of wild-type tsrA. Introduction of expression fosmids encoding alternate TsrA sequences into the S. laurentii tsrA deletion mutant led to the production of thiostrepton variants retaining antibacterial activity, demonstrating the utility of this expression platform toward thiopeptide engineering.     

Thiazole/oxazole-modified microcins: complex natural products from ribosomal templates

With billions of years of evolution under its belt, Nature has been expanding and optimizing its biosynthetic capabilities. Chemically complex secondary metabolites continue to challenge and inspire today's most talented synthetic chemists. A brief glance at these natural products, especially the substantial structural variation within a class of compounds, clearly demonstrates that Nature has long played the role of medicinal chemist. The recent explosion in genome sequencing has expanded our appreciation of natural product space and the vastness of uncharted territory that remains. One small corner of natural product chemical space is occupied by the recently dubbed thiazole/oxazole-modified microcins (TOMMs), which are ribosomally produced peptides with posttranslationally installed heterocycles derived from cysteine, serine and threonine residues. As with other classes of natural products, the genetic capacity to synthesize TOMMs has been widely disseminated among bacteria. Over the evolutionary timescale, Nature has tested countless random mutations and selected for gain of function in TOMM biosynthetic gene clusters, yielding several privileged molecular scaffolds. Today, this burgeoning class of natural products encompasses a structurally and functionally diverse set of molecules (i.e. microcin B17, cyanobactins, and thiopeptides). TOMMs presumably provide their producers with an ecological advantage. This advantage can include chemical weapons wielded in the battle for nutrients, disease-promoting virulence factors, or compounds presumably beneficial for symbiosis. Despite this plethora of functions, many TOMMs await experimental interrogation. This review will focus on the biosynthesis and natural combinatorial diversity of the TOMM family.     

Elucidating and engineering thiopeptide biosynthesis

Initially discovered in the mid-twentieth century, thiopeptides constitute a diverse family of bacterially produced natural products exhibiting a remarkable array of biological properties. Only in the last several years have the details of thiopeptide biosynthesis been uncovered by a combination of genomic, genetic, and biochemical approaches. Thiopeptides are now known to be ribosomally synthesized and subsequently densely modified to carry azol(in)es, dehydro amino acids, and various other pathway-specific decorations. The defining feature of thiopeptides is a central six-membered nitrogenous ring that constrains peptide macrocycles of varying sequences and sizes. Recent landmark studies have defined the precisely orchestrated posttranslational modification cascade culminating in thiopeptide product formation. Because diverse thiopeptides are processed by a relatively small number of well-conserved enzymes, it has been suggested that artificial diversification of the precursor peptide could allow a vast new chemical space to be explored for clinically important activities. The success of this strategy depends on the plasticity of thiopeptide processing machinery, an open question that warrants further investigation. There is an urgent need therefore to leverage established thiopeptide research platforms to investigate substrate-enzyme specificity and devise intelligent diversification strategies for library generation. Meanwhile, the distinct genomic signatures of conserved thiopeptide-associated genes will enable the continued mining of nature for novel compounds and processing enzymes.     

Identification and Analysis of the Biosynthetic Gene Cluster Encoding the Thiopeptide Antibiotic Cyclothiazomycin in Streptomyces hygroscopicus 10-22

Thiopeptide antibiotics are an important class of natural products resulting from posttranslational modifications of ribosomally synthesized peptides. Cyclothiazomycin is a typical thiopeptide antibiotic that has a unique bridged macrocyclic structure derived from an 18-amino-acid structural peptide. Here we reported cloning, sequencing, and heterologous expression of the cyclothiazomycin biosynthetic gene cluster from Streptomyces hygroscopicus 10-22. Remarkably, successful heterologous expression of a 22.7-kb gene cluster in Streptomyces lividans 1326 suggested that there is a minimum set of 15 open reading frames that includes all of the functional genes required for cyclothiazomycin production. Six genes of these genes, cltBCDEFG flanking the structural gene cltA, were predicted to encode the enzymes required for the main framework of cyclothiazomycin, and two enzymes encoded by a putative operon, cltMN, were hypothesized to participate in the tailoring step to generate the tertiary thioether, leading to the final cyclization of the bridged macrocyclic structure. This rigorous bioinformatics analysis based on heterologous expression of cyclothiazomycin resulted in an ideal biosynthetic model for us to understand the biosynthesis of thiopeptides.The thiopeptides are a family of highly modified, sulfur-containing macrocyclic peptides, such as thiostrepton, thiocillins, and micrococcinic acid (3, 11). Their structures have several common features: a tri- or tetrasubstituted nitrogen heterocycle central domain, a macrocyclic framework, and heavily modified amino acid residues, including thiazoles, oxazoles, and dehydroamino acids (Fig. ​(Fig.1)1) (3). Members of this family exhibit various biological properties, such as inhibition of ribosomal protein synthesis (24), rennin inhibitory activity (2), and induction of TipA (20). Moreover, many thiopeptide antibiotics show bioactivity against some bacterial strains resistant to most conventional treatments, including methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant enterococci (VRE) (3, 17).Open in a separate windowFIG. 1.Structures of thiostrepton, thiocillin, microncoccinate, and cyclothiazomycin (3).Early in vitro investigations of these special heterocycles of thiopeptides were performed by organic chemists and included stereoselective synthesis of a γ-lactam acidic hydrolysate of cyclothiazomycin (4) and total synthesis of thiostrepton (21). Extensive research on microccins (5, 19) and lantibiotics (6, 29) described biosynthesis of the thiazole- and dehydroamino acid-containing polypeptides that were derived from ribosomally synthesized prepeptides. Similarly, Lee et al. described a widely conserved gene cluster for toxin biosynthesis and suggested a ribosome biosynthetic pathway for the modified polypeptide containing thiazoles and oxazoles (16).Recently, Wieland Brown et al. (28) and Kelly et al. (14) identified the gene clusters encoding thiocillin and thiostrepton, respectively, with a probe that targeted hypothetic prepeptide genes, whereas Liao and coworkers (17) took advantage of the conservation of one putative cyclodehydratase. Although most of the biochemical reactions involved in the biosynthetic pathway remain obscure, it is clear that thiopeptides are synthesized ribosomally and then there is a series of posttranslational modifications.Cyclothiazomycin, which was isolated as a novel selective inhibitor of human plasma rennin, is a unique bridged macrocyclic thiopeptide (2) whose stereo structure was recently revealed by degradation experiments and spectroscopic analysis (10). It contains a dehydroserine, two dehydrothreonine residues, three thiazolines, three thiazoles, and a trisubstituted pyridine. Compared with common thiopeptides, it lacks the characteristic 2- and 3-azole substituent on the central pyridine domain; instead, it has an alanine-derived heterocyclic residue with the (R) configuration, a quaternary sulfide, and two macrocyclic peptide loops (Fig. ​(Fig.1).1). Moreover, a pair of convertible isomers of cyclothiazomycin B, the cyclothiazomycin analogues produced by Streptomyces sp. strain A307 (10), were identified as Z and E configurations caused by the tautomerization of the dehydrated threonine. These structural traits may indicate that some new genetic elements are likely involved in posttranslational modification.Here we reported cloning and sequencing of the cyclothiazomycin biosynthetic gene cluster of Streptomyces hygroscopicus 10-22 (23). In addition, an analysis of heterologous expression in Streptomyces lividans 1326 and a deletion analysis were also performed, which indicated that a gene cluster at least 22.7 kb long is required for biosynthesis. A bioinformatics-based approach to analysis of this gene cluster postulated that there is a posttranslational modification pathway in which eight proteins are involved in the biosynthetic machinery.     

Characterization of a Novel Plasmid-Borne Thiopeptide Gene Cluster in Staphylococcus epidermidis Strain 115

Thiopeptides are small (12- to 17-amino-acid), heavily modified peptides of bacterial origin. This antibiotic family, with more than 100 known members, is characterized by the presence of sulfur-containing heterocyclic rings and dehydrated residues within a macrocyclic peptide structure. Thiopeptides, including micrococcin P1, have garnered significant attention in recent years for their potent antimicrobial activity against bacteria, fungi, and even protozoa. Micrococcin P1 is known to target the ribosome; however, like those of other thiopeptides, its biosynthesis and mechanisms of self-immunity are poorly characterized. We have discovered an isolate of Staphylococcus epidermidis harboring the genes for thiopeptide production and self-protection on a 24-kb plasmid. Here we report the characterization of this plasmid, identify the antimicrobial peptide that it encodes, and provide evidence of a target replacement-mediated mechanism of self-immunity.     

Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.     

Chloroplast biogenesis 80. Proposal of a unified multibranched chlorophyll a/b biosynthetic pathway

A unified multibranched chlorophyll (Chl) biosynthetic pathway is proposed. The proposed pathway takes into account the following considerations: (a) that the earliest putative precursor of monovinyl Chl b that has been detected in higher plants is monovinyl protochlorophyllide b, (b) that in most cases, Chl b biosynthesis has its roots in the Chl a biosynthetic pathway, (c) that the Chl a biosynthetic pathway exhibits extensive biosynthetic heterogeneity, (d) that Chl biosynthesis may proceed differently at different stages of greening and in different greening groups of plants. Integration of the Chl a and b biosynthetic pathways into a unified multibranched pathway offers the functional flexibility to account for the structural and biosynthetic complexity of photosynthetic membranes. In this context, it is proposed that the unified, multibranched Chl a/b biosynthetic pathway represents the template of a Chl-protein biosynthesis center where photosystem (PS) 1, PS2, and light-harvesting Chl-protein complexes are assembled into functional photosynthetic units. The individual biosynthetic routes or groups of two to three adjacent biosynthetic routes may constitute Chl-protein biosynthesis subcenters, where specific Chl-protein complexes are assembled. This revised version was published online in September 2006 with corrections to the Cover Date.     

Altered mitochondrial function and cholesterol synthesis influences protein synthesis in extended HepG2 spheroid cultures

Cultures of hepatocytes and HepG2 cells provide useful in vitro models of liver specific function. In this study, we investigated metabolic and biosynthetic function in 3-D HepG2 spheroid cultures, in particular to characterise changes on prolonged culture. We show that HepG2 cells cultured in spheroids demonstrate a reduction in mitochondrial membrane potential and respiration following 10 days of culture. This coincides with a modest reduction in glycolysis but an increase in glucose uptake where increased glycogen synthesis occurs at the expense of the intracellular ATP pool. Lowered biosynthesis coincides with and is linked to mitochondrial functional decline since low glucose-adapted spheroids, which exhibit extended mitochondrial function, have stable biosynthetic activity during extended culture although biosynthetic function is lower. This indicates that glucose is required for biosynthetic output but sustained mitochondrial function is required for the maintenance of biosynthetic function. Furthermore, we show that cholesterol synthesis is markedly increased in spheroids cf. monolayer culture and that inhibition of cholesterol synthesis by lovastatin extends mitochondrial and biosynthetic function. Therefore, increased cholesterol synthesis and/or its derivatives contributes to mitochondrial functional decline in extended HepG2 spheroid cultures.     

Intramolecular H-bonds and thioamide rotational isomerism in thiopeptides

Mono- and dithionated N-acyl amino acid and dipeptide N'-methylamides were synthesized using Lawesson's reagent and S-thioacetyl thioglycolic acid. The conformation of the thionated models was characterized by IR, 13C, and 1H NMR spectroscopy, including NOE experiments. The formation of -C = S...H-N-C = X (X = O or S) intramolecular H-bonds of the type 2----2, 1----3 and 1----4 was evidenced by the characteristic shifts of the IR stretching frequencies of the NH group. Act-Pro-NHCH3(4) and Act-Prot-NHCH3(5) were found to be present as mixtures of rotational isomers about the CS-N bond. 13C chemical shifts of the gamma- and beta-carbons of the proline ring elucidated the conformation (Z or E) of the tertiary thioamide group. Our results suggest that the conformation of thiopeptides is determined by two factors: 1) the H-bond donating and accepting ability of the thioamide group and 2) the repulsion between the thiocarbonyl sulfur atom and the side chain groups of the neighbouring amino acid residues.     

植物花青素苷生物合成及调控的研究进展

花青素苷( anthocyanin)是植物新陈代谢过程中产生的类黄酮物质,决定被子植物花、果实、种皮、茎、叶和根等的颜色,具有重要的营养价值和药理作用.近年来关于花青素生物合成途径的研究已取得突破,综述了植物花青素苷基因研究现状和发展趋势,包括植物花青素生物合成途径、参与生物合成途径中相关的结构基因和调控基因及功能研究以及影响花青素苷生物合成的环境因素等的研究进展.     

The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.     

植物花青素合成途径中的调控基因研究进展

花青素广泛分布于高等植物中,是一种水溶性的植物色素,与农作物的多种品质性状密切相关。虽长期受到关注,但其生物合成途径则是近年来随着拟南芥等植物突变体研究的深入才取得突破的。对于花、果实和种子中的花青素研究始终是热点,近来国内外有很多关于花青素合成与基因调控发明研究的报道。随着研究的深入不仅可以为医疗保健等提供科学依据,而且有助于其在农业生产中应用。本文综述了植物花青素基因的研究现状和发展趋势,包括植物花青素生物合成途径,生物合成途径中相关转录因子的调控,以及已经分离和克隆的调控基因在功能方面的研究进展。     

Effect of the relA gene on derepression of amino acid biosynthetic enzymes in growing Escherichia coli depends on the pathway being derepressed.

Derepression of an enzyme in the arginine biosynthetic pathway, but not of an enzyme in the tryptophan biosynthetic pathway, is inhibited during the stringent response produced by a partial deprivation of valyl transfer ribonucleic acid in a rel+ strain. In contrast, derepression of the tryptophan biosynthetic enzyme, but not of the arginine biosynthetic enzyme, was inhibited during the relaxed response produced in an isogenic relA strain by the partial deprivation of valyl transfer ribonucleic acid.     

Improving key enzyme activity in phenylpropanoid pathway with a designed biosensor

Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.     

Chemical variation and the species concept in lichenized ascomycetes

Differences in secondary metabolites produced by lichens are not always genetically based, and even if genetically based may represent only a one gene difference. Taxonomic decision involving secondary metabolism should be based on the degree of difference demonstrated between biosynthetic pathways, not on the individual products. No taxonomic status should be accorded to entities which differ only in products from a single biosynthetic pathway, but varietal status should be given to those which have different biosynthetic pathways. Species status is justified if chemistry is correlated with morphological or proven physiological difference, or if more than one major biosynthetic system is involved. While ecological and biogeographic differences point to the likelihood of differences being found, if no differences can be demonstrated which in themselves justify taxonomic separation, then features ought not be allowed to influence the taxonomic decision.