Daslav Hranueli

• Prof. Dr..
• University of Zagreb

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis...

Most of the biosynthetic gene clusters (BGC) encoding the biosynthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, robust technologies are required to secure the production of natural products for both drug discovery and any subsequent commercial fermentation processes. Indust...

Three metagenomic libraries were constructed using surface sediment samples from the northern Adriatic Sea. Two of the samples were taken from a highly polluted and an unpolluted site respectively. The third sample from a polluted site had been enriched using crude oil. The results of the metagenome analyses were incorporated in the REDPET relation...

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to ge...

Gene duplication followed by adaptive selection is a well-accepted process leading to toxin diversification in venoms. However, emergent genomic, transcriptomic and proteomic evidence now challenges this role to be at best equivocal to other processess . Cnidaria are arguably the most ancient phylum of the extant metazoa that are venomous and such...

An important mechanism for the evolution of toxins in venomous animals is believed to be the acquisition of genes encoding proteins that switch from physiological to toxic roles following gene duplication. The 'reverse recruitment' hypothesis pertains that these genes can also revert back to physiological functions, although such events are thought...

Shipboard experiments were performed over a two day period to examine the proteomic response of the symbiotic coral Acropora microphthalma exposed to acute conditions of high temperature / low light or high light / low temperature stress. During these treatments, corals had noticeably bleached. The photosynthetic performance of residual algal endos...

The genome sequence of Streptomyces rimosus R6-500, an industrially improved strain which produces high titers of the important antibiotic oxytetracycline, is reported, as well as the genome sequences of two derivatives arising due to the genetic instability of the strain.

Streptomyces olindensis DAUFPE 5622, which was isolated from a Brazilian soil sample, produces the antitumor anthracycline cosmomycin D. The genome sequence is 9.4 Mb in length, with a G+C content of 71%. Thirty-four putative secondary metabolite biosynthetic gene clusters were identified, including the cosmomycin D cluster.

Successful genome mining is dependent on accurate prediction of protein function from sequence. This often involves dividing protein families into functional subtypes (e.g., with different substrates). In many cases, there are only a small number of known functional subtypes, but in the case of the adenylation domains of nonribosomal peptide synthe...

Actinomycetes are a very important source of natural products for the pharmaceutical industry and other applications. Most of the strains belong to Streptomyces or related genera, partly because they are particularly amenable to growth in the laboratory and industrial fermenters. It is unlikely that chemical synthesis can fulfil the needs of the ph...

Streptomyces rapamycinicus strain NRRL 5491 produces the important drug rapamycin. It has a large genome of 12.7 Mb, of which over 3 Mb consists of 48 secondary metabolite biosynthesis clusters.

Background Contemporary coral reef research has firmly established that a genomic approach is urgently needed to better understand the effects of anthropogenic environmental stress and global climate change on coral holobiont interactions. Here we present KEGG orthology-based annotation of the complete genome sequence of the scleractinian coral Acr...

Modular biosynthetic clusters are responsible for the synthesis of many important pharmaceutical products. They include polyketide synthases (PKS clusters), non-ribosomal synthetases (NRPS clusters), and mixed clusters (containing both PKS and NRPS modules). The ClustScan database (CSDB) contains highly annotated descriptions of 170 clusters. The d...

The recent achievement of synthesising a functioning bacterial chromosome marks a coming of age for engineering living organisms. In the future this should allow the construction of novel organisms to help solve the problems facing the human race, including health care, food, energy and environmental protection. In this minireview, the current stat...

The high G+C-content and large genome size make sequencing and assembly of Streptomyces genomes more difficult than for those of other bacteria. Many pharmaceutically important natural products are synthesized by modular polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS). Analysing such gene clusters is difficult if the genome...

Soil bacteria live in a very competitive environment and produce many secondary metabolites; there appears to be strong selective pressure for evolution of new compounds. Secondary metabolites are the most important source of chemical structures for the pharmaceutical industry and an understanding of the evolutionary process should help in finding...

Modular polyketide synthases (PKSs) from Streptomyces and related genera of bacteria produce many important pharmaceuticals. A program called CompGen was developed to carry out in silico homologous recombination between gene clusters encoding PKSs and determine whether recombinants have cluster architectures compatible with the production of polyke...

Attempts at generating novel chemistries by genetically manipulating polyketide synthases (PKSs) usually result in no detectable or poor product yield. Understanding processes that drive the evolution of PKSs might provide a solution to this problem. The synonymous-to-non-synonymous nucleotide substitution ratios across alignments of well-character...

The gel shows PCR amplification of genetically pure DNA templates using primers designed on the sequences of the putative MAA pathway and indicates that these sequences are encoded within the coral and not the algal symbiont. (0.18 MB PPT)

Comparison of active site residues from 2-epi-5-epi-valiolone synthase and 3-dehydroquinate synthase enzymes from non-MAA producing prokaryotes and from cyanobacteria known or are likely to produce MAAs. (Alignment is available on request). (0.10 MB DOC)

Profile of gene expression in A. microphthalma exposed to high solar irradiance. (0.04 MB XLS)

Known cyanobacterial MAA-producers contain a fused aroB (3-dehydroquinate synthase) and O-methyltransferase gene, which could theoretically combine the shikimic acid pathway production of 3-dehydroquinate with the first O-methylation step of MAA biosynthesis. The data was generated by BLAST searching using as a seed the protein sequence of the fuse...

Chemical structures of MAAs identified in the coral Acropora microphthalma. (0.14 MB TIF)

Amino acid alignments for hypothetical enzymes predicted for MAA biosynthesis. (0.03 MB DOC)

UV-absorption spectra of methanolic extracts of the coral Acropora microphthalma before and after high UV/light exposure. (0.01 MB PDF)

Laboratory codes and corresponding GenBank Accession numbers for the 141 cDNA clones. (0.03 MB DOC)

A discussion supporting vesicular transport and exocytosis as a cellular mechanism of coral bleaching. (0.08 MB DOC)

The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppress...

Distribution of genes encoding enzymes of the shikimic acid pathway in Archaea.

Distribution of the gene encoding KDPGal aldolase in non-host-associated (free-living) Bacteria.

Comparison of the distribution of the shikimic acid pathway in 33 Archaea using HMM and BioCyc database searching.

Distribution of genes encoding enzymes of the shikimic acid pathway in non-host associated (free-living) Bacteria.

Distribution of the gene encoding KDPGal aldolase in host- associated Bacteria.

Distribution of the gene encoding KDPGal aldolase in Archaea.

Genetic architecture of the shikimic acid pathway across a range of taxonomically different prokaryote genomes shows that the genes encoding the pathway are not clustered.

Distribution of genes encoding enzymes of the shikimic acid pathway in host-associated Bacteria.

Comparison of the distribution of the shikimic acid pathway in 85 non-host associated bacteria using HMM and BioCyc database searching.

Comparison of the distribution of the shikimic acid pathway in 204 host-associated bacteria using HMM and BioCyc database searching.

A central tenet in biochemistry for over 50 years has held that microorganisms, plants and, more recently, certain apicomplexan parasites synthesize essential aromatic compounds via elaboration of a complete shikimic acid pathway, whereas metazoans lacking this pathway require a dietary source of these compounds. The large number of sequenced bacte...

An in silico model for homoeologous recombination between gene clusters encoding modular polyketide synthases (PKS) or non-ribosomal peptide synthetases (NRPS) was developed. This model was used to analyze recombination between 12 PKS clusters from Streptomyces species and related genera to predict if new clusters might give rise to new products. I...

Rapid mining of large genomic and metagenomic data sets for modular polyketide synthases, non-ribosomal peptide synthetases and hybrid polyketide synthase/non-riboso-mal peptide synthetase biosynthetic gene clusters has been achieved using the generic computer program packages ClustScan and CompGen. These program packages perform the annotation wit...

Detailed output of the evolutionary split and clustering programs. For each of the protein families (nucleotidyl cyclases, protein kinases, dehydrogenases, acyl transferases, ketoreductases and small heat shock proteins) there are two tables. The evolutionary split table has the following columns: residue position in the alignment (only residues th...

Alignments. The alignments used to test the clustering method: nucleotidyl cyclases, protein kinases, dehydrogenases, acyl transferases, ketoreductases and small heat shock proteins.

The number of protein family members defined by DNA sequencing is usually much larger than those characterised experimentally. This paper describes a method to divide protein families into subtypes purely on sequence criteria. Comparison with experimental data allows an independent test of the quality of the clustering. An evolutionary split statis...

Aklanonic acid is synthesized by a type II polyketide synthase (PKS) composed of eight protein subunits. The network of protein interactions within this complex was investigated using a yeast two-hybrid system, by coaffinity chromatography and by two different computer-aided protein docking simulations. Results suggest that the ketosynthase (KS) al...

The program package ‘ClustScan’ (Cluster Scanner) is designed for rapid, semi-automatic, annotation of DNA sequences encoding modular biosynthetic enzymes including polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and hybrid (PKS/NRPS) enzymes. The program displays the predicted chemical structures of products as well as allowin...

Polyketides and non-ribosomal peptides represent a large class of structurally diverse natural products much studied over recent years because the enzymes that synthesise them, the modular polyketide synthases (PKSs) and the non-ribosomal peptide synthetases (NRPSs), share striking architectural similarities that can be exploited to generate 'un-na...

The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditi...

Motivation: The genome of the social amoeba Dictyostelium discoideum contains an unusually large number of polyketide synthase (PKS) genes. An analysis of the genes is a first step towards understanding the biological roles of their products and exploiting novel products. Results: A total of 45 Type I iterative PKS genes were found, 5 of which a...

When and where? Modular polyketide syntheses catalyse the Claisen condensation of simple organic acids with inversion of configuration to give a 2R methyl centre. However, according to Celmer's rules, the ultimate stereochemical configuration of many polyketides requires epimerisation to the 25 isomer. It has been generally believed that the epimer...

Polyketides and non-ribosomal peptides represent a large class of structurally diverse natural products much studied over recent years because the enzymes that synthesise them, the modular polyketide synthases (PKSs) and the non-ribosomal peptide synthetases (NRPSs), share striking architectural similarities that can be exploited to generate "un-na...

Mathematical models were applied to define the behaviour kinetics distinction among microbial strains. In the first series of experiments the growth kinetics of microbial colonies of several S. rimosus mutant strains cultivated on agar plates were compared. Then, the interest was focused on the chosen two strains, in order to express mathematically...

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and...

Post-translationally modified ribosomal peptides are unusual natural products and many have potent biological activity. The biosynthetic processes involved in their formation have been delineated for some, but the patellamides represent a unique group of these metabolites with a combination of a macrocycle, small heterocycles and d-stereocentres. T...

Natural products from symbiotic or commensal associations between marine invertebrate and microbial organisms show exceptional promise as pharmaceuticals in many therapeutic areas. An economic and sustainable global market supply due to difficulty of synthesis is cited as the main obstacle for exploitation of these otherwise exciting marine bioacti...

Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic...

Streptomyces is a genus of soil dwelling bacteria with the ability to produce natural products that have found widespread use in medicine. Annotation of Streptomyces genome sequences has revealed far more biosynthetic gene clusters than previously imagined, offering exciting possibilities for future combinatorial biosynthesis. Experiments to manipu...

Streptomycetes are important antibiotic producing bacteria that often exhibit genetic instability. One or both ends of the linear Streptomyces chromosome are lost spontane-ously, resulting in viable mutant strains sometimes lacking hundreds of genes. We exam-ined some strains of Streptomyces rimosus and Streptomyces lividans, which had been classi-...

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Introduction Mutation and Selection Bacteria Plasmids Yeast and Fungi Animal and Plant Cells Viruses Consequences for Good Manufacturing Practice Bibliography

To recognise microbial strains capable to produce substances with antimicrobial activity the culture conditions enabling, both, the microorganism growth and the expression of antibiotic production, should be applied. Since the optimal culture conditions vary depending on properties of particular strains one can recommend the application of various...

Streptomyces species and related genera synthesize a large number of secondary meta-bolites, many of which are biologically active. Amongst them, polyketides is the largest class. Polyketides are a structurally diverse family of natural products with a broad range of biological activities. The formation of polyketides is very similar to the biosynt...

Oxytetracycline is a polyketide antibiotic made by Streptomyces rimosus. From DNA sequencing, the gene product of otcD1 is deduced to function as a bifunctional cyclase/aromatase involved in ring closure of the polyketide backbone. Although otcD1 is contiguous with the ketoreductase gene, they are located an unusually large distance from the genes...

The whole oxytetracycline (OTC) gene cluster was cloned from the strain S. rimosus R6. Its restriction map is indistinguishable from that of the strain S. rimosus M15883, which means that results from the two strains can probably be combined. Constructions to induce gene disruptions of otcD1 and otcC were undertaken within the chromosome of S. rimo...

Background and purpose: Streptomyces are the genetically best characterised bacteria which posses a linear chromosome. The chromosomes are 8 Mb in size. Linear plasmids are also common and can integrate into the linear chromosome or gain chromosomal genes to form plasmid primes. DNA sequences near the chromosome ends can undergo deletions and ampli...

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had...

A restriction map of the 8 Mb linear chromosome of Streptomyces rimosus R6-501 was constructed for the enzymes Asel (13 fragments) and Dral (7 fragments). Linking clones for all 12 Asel sites and 5 of the 6 Dral sites were isolated. The chromosome has terminal inverted repeats of 550 kb, which are the longest yet reported for a Streptomyces species...

The linear plasmid pPZG101 of Streptomyces rimosus R6 was restriction mapped with the enzymes AseI, BfrI, DraI and XbaI. It is 387 kb in size and the ends are inverted repeats of at least 95 kb in length. Twenty spontaneous morphological variants and seventeen auxotrophic mutants were screened for changes in the plasmid. Two strains were found that...

The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on 'cured' S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA...

During a strain selection program to improve oxytetracycline production in Streptomyces rimosus R6, isolates that showed extreme morphological instability appeared. Propagation via spores gave much higher instability than did propagation via mycelial fragments. Five phenotypic traits were affected: sporulation, pigmentation, colony morphology, oxyt...

An experimental system was designed to permit the detection of recombination events occurring via unequal crossing over between sister bacterial chromosomes in Bacillus subtilis. It exploits the fact that during spore development, genetic and metabolic cooperation occurs between two different cell types, only one of which survives. During the early...

Genetic instability is very common in Streptomyces species, and was one of the first reported properties (Beijerinck, 1913). Usually genetic instability has been detected as influencing easily scored phenotypes such as pigment production (Gregory and Huang, 1964), sporulation, auxotrophy (Redshaw et al., 1979) and antibiotic resistance (Freeman et...

One of the basic techniques for DNA cloning in Streptomyces is the preparation of protoplasts and the efficient regeneration of normal mycelia. In order to develop an oxytetracycline producing S. rimosus strain as a host for molecular cloning, the efficiencies of protoplast preparation and cell wall regeneration were compared with those obtained us...

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Ther...

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. Aft...

The infection of Streptomyces rimosus by the virulent actinophage RP1 was partially characterized. RP1 infection of the host cells results in a dramatic decrease in viable cell count, followed by reduced antibiotic production. Phage-resistant mutants were isolated after mutagenic treatment and RP1 selective pressure. Characterization of the isolate...

While searching for true lysogens among oxytetracycline-producing Streptomyces rimosus strains, free phage particles were detected and isolated from a liquid culture of S. rimosus ATCC 10970 (R7). The actinophage, designated RP2, appears to be a typical temperate DNA phage producing turbid plaques on the sensitive strain S. rimosus R6. Electron mic...

As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23...

The application of functional genomics can and will be found in three main areas. These are the fields of: (a) human health, (b) agriculture and cattle breeding, and (c) industrial microbiology. Genomics and proteomics in human health The sequencing of the human genome was important for understanding the molecular bases of disease and for identifyi...

In his book 'The Biotech Century' published in 1999 (1), Jeremy Rifkin has claimed that never before in the history of humanity, have human beings been faced with such gigantic new technological and economic challenges as those that lie ahead. He believes that by the year 2025, our children and we might be living in a world entirely different from...