Gloria Blanco

Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin

BACKGROUND Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tü2353. Structurally is a tetracyclic aromatic polyketide derived from the condensation of 10 acetate units. Its chromophoric aglycon is glycosylated with a permethylated L-rhamnose moiety at the C-8 hydroxy group. Only limited information is available about the genes involved in the biosynthesis of elloramycin. From a library of chromosomal DNA from S. olivaceus, a cosmid (16F4) was isolated that contains part of the elloramycin gene cluster and when expressed in Streptomyces lividans resulted in the production of a non-glycosylated intermediate in elloramycin biosynthesis, 8-demethyl-tetracenomycin C (8-DMTC). RESULTS The expression of cosmid 16F4 in several producers of glycosylated antibiotics has been shown to produce tetracenomycin derivatives containing different 6-deoxysugars. Different experimental approaches showed that the glycosyltransferase gene involved in these glycosylation events was located in 16F4. Using degenerated oligoprimers derived from conserved amino acid sequences in glycosyltransferases, the gene encoding this sugar flexible glycosyltransferase (elmGT) has been identified. After expression of elmGT in Streptomyces albus under the control of the erythromycin resistance promoter, ermEp, it was shown that elmG can transfer different monosaccharides (both L- and D-sugars) and a disaccharide to 8-DMTC. Formation of a diolivosyl derivative in the mithramycin producer Streptomyces argillaceus was found to require the cooperative action of two mithramycin glycosyltransferases (MtmGI and MtmGII) responsible for the formation of the diolivosyl disaccharide, which is then transferred by ElmGT to 8-DMTC. CONCLUSIONS The ElmGT glycosyltransferase from S. olivaceus Tü2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several L- and D-sugars and also a diolivosyl disaccharide into the aglycon 8-DMTC. ElmGT is an example of sugar flexible glycosyltransferase and can represent an important tool for combinatorial biosynthesis.

The Biosynthetic Gene Cluster for the β-Lactam Carbapenem Thienamycin in Streptomyces cattleya

beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

Oxidative cleavage of premithramycin B is one of the last steps in the biosynthesis of the antitumor drug mithramycin

BACKGROUND Mithramycin is a member of the clinically important aureolic acid group of antitumor drugs that interact with GC-rich regions of DNA nonintercalatively. These drugs contain a chromophore aglycon that is derived from condensation of ten acetate units (catalyzed by a type II polyketide synthase). The aglycones are glycosylated at two positions with different chain length deoxyoligosaccharides, which are essential for the antitumor activity. During the early stages of mithramycin biosynthesis, tetracyclic intermediates of the tetracycline-type occur, which must be converted at later stages into the tricyclic glycosylated molecule, presumably through oxidative breakage of the fourth ring. RESULTS Two intermediates in the mithramycin biosynthetic pathway, 4-demethyl-premithramycinone and premithramycin B, were identified in a mutant lacking the mithramycin glycosyltransferase and methyltransferase genes and in the same mutant complemented with the deleted genes, respectively. Premithramycin B contains five deoxysugars moieties (like mithramycin), but contains a tetracyclic aglycon moiety instead of a tricyclic aglycon. We hypothesized that transcription of mtmOIV (encoding an oxygenase) was impaired in this strain, preventing oxidative breakage of the fourth ring of premithramycin B. Inactivating mtmOIV generated a mithramycin nonproducing mutant that accumulated premithramycin B instead of mithramycin. In vitro assays demonstrated that MtmOIV converted premithramycin B into a tricyclic compound. CONCLUSIONS In the late stages of mithramycin biosynthesis by Strepyomyces argillaceus, a fully glycosylated tetracyclic tetracycline-like intermediate (premithramycin B) is converted into a tricyclic compound by the oxygenase MtmOIV. This oxygenase inserts an oxygen (Baeyer-Villiger oxidation) and opens the resulting lactone. The following decarboxylation and ketoreduction steps lead to mithramycin. Opening of the fourth ring represents one of the last steps in mithramycin biosynthesis.

Characterization of two glycosyltransferases involved in early glycosylation steps during biosynthesis of the antitumor polyketide mithramycin by Streptomyces argillaceus

Abstract A 2580-bp region of the chromosome of Streptomyces argillaceus, the producer of the antitumor polyketide mithramycin, was sequenced. Analysis of the nucleotide sequence revealed the presence of two genes (mtmGIII and mtmGIV ) encoding proteins that showed a high degree of similarity to glycosyltransferases involved in the biosynthesis of various antibiotics and antitumor drugs. Independent insertional inactivation of both genes produced mutants that did not synthesize mithramycin but accumulated several mithramycin intermediates. Both mutants accumulated premithramycinone, a non-glycosylated intermediate in mithramycin biosynthesis. The mutant affected in the mtmGIII gene also accumulated premithramycin A1, which contains premithramycinone as the aglycon unit and a D-olivose attached at C-12a-O. These experiments demonstrate that the glycosyltransferases MtmGIV and MtmGIII catalyze the first two glycosylation steps in mithramycin biosynthesis. A model is proposed for the glycosylation steps in mithramycin biosynthesis.

Identification of transcriptional activators for thienamycin and cephamycin C biosynthetic genes within the thienamycin gene cluster from Streptomyces cattleya

Two regulatory genes, thnI and thnU, were identified in the thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR‐type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT‐PCR). Deletion of thnI abolished thienamycin production showing its involvement in thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for thienamycin biosynthesis that regulates the expression of nine genes involved in thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.

Deciphering the biosynthetic origin of the aglycone of the aureolic acid group of anti-tumor agents

BACKGROUND Mithramycin, chromomycin, and olivomycin belong to the aureolic acid family of clinically important anti-tumor agents. These natural products share a common aromatic aglycone. Although isotope labeling studies have firmly established the polyketide origin of this aglycone, they do not distinguish between alternative biosynthetic models in which the aglycone is derived from one, two or three distinct polyketide moieties. We set out to determine the biosynthetic origin of this moiety using a recombinant approach in which the ketosynthase and chain-length factor proteins from the antibiotic-producer strain, which determine the chain length of a polyketide, are produced in a heterologous bacterial host. RESULTS The ketosynthase and chain-length factor genes from the polyketide synthase gene cluster from the mithramycin producer, Streptomyces argillaceus ATCC 12956, and the acyl carrier protein and ketoreductase genes from the actinorhodin polyketide synthase were expressed in Streptomyces coelicolor CH999. The recombinant strain produced a 20-carbon polyketide, comprising the complete backbone of the aglycone of mithramycin. CONCLUSIONS The aglycone moieties of mithramycin, chromomycin, and olivomycin are derived from a single polyketide backbone. The nascent polyketide backbone must undergo a series of regiospecific cyclizations to form a tetracenomycin-like tetracyclic intermediate. The final steps in the aglycone biosynthetic pathway presumably involve decarboxylation and oxidative cleavage between C-18 and C-19, followed by additional oxidation, reduction, and methylation reactions.

Hybridization and DNA sequence analyses suggest an early evolutionary divergence of related biosynthetic gene sets encoding polyketide antibiotics and spore pigments in Streptomyces spp.

The whiE gene cluster of Streptomyces coelicolor, which is related to gene sets encoding the biosynthesis of polycyclic aromatic polyketide antibiotics, determines a spore pigment. Southern blotting using probes from three different parts of the whiE cluster revealed related gene sets in about half of a collection of diverse Streptomyces strains. A 5.2-kb segment of one such cluster, sch, previously shown to determine spore pigmentation in Streptomyces halstedii, was sequenced. Seven open reading frames (ORFs), two of them incomplete, were found. Six of the ORFs resemble the known part of the whiE cluster closely. The derived gene products include a ketosynthase (= condensing enzyme) pair, acyl carrier protein and cyclase, as well as two of unidentified function. The seventh ORF diverges from the main cluster and encodes a protein that resembles a dichlorophenol hydroxylase. Comparison with sequences of related gene sets for the biosynthesis of antibiotics suggests that gene clusters destined to specify pigment production diverged from those destined to specify antibiotics early in the evolution of the Streptomyces genus.

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse‐labelled with [35 S]‐methionine, separated by two‐dimensional poly‐acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ‐galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10‐L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.

Allozyme variation in natural populations of Atlantic salmon in Asturias (northern Spain)

Abstract This study describes the genetic variation (based on analysis of 24 protein-coding genes) within and between two natural salmon populations of Asturian rivers. Information on the genetic structure of populations at the southern distribution limit of Salmo salar in Europe is provided. In the natural populations, five of 24 protein-coding loci were polymorphic ( Aat-2 , Idh-3 , Mdh-3,4 , Sdh-1 and Sdh-2 ). Non-significant allele frequency differences were found between populations. At the Mdh-3,4 locus the frequency of the 87 allele is much higher than the values given for other European populations. The mean heterozygosity in these samples (based on 24 loci investigated) was 2.18%. Nevertheless, the amount of variability in Asturian natural populations is somewhat lower than the average estimated in the Atlantic salmon population.

Superior developmental stability of heterozygotes at enzyme loci in Salmo salar L.

Abstract The degree of heterozygosity of five enzymatic loci is related to the degree of fluctuating asymmetry of three bilateral traits in two farm stocks and in two natural populations of Salmo salar . In all cases we verified the existence of an inverse correlation of the degree of heterozygosity with the number of asymmetrical traits per individual as well as with the total magnitude of asymmetry that the individuals present. We also detected the association at the populational levels: a higher average populational heterozygosity implies a lower total magnitude of asymmetry. The phenomenon is general and the possibility that the correlation is due to the direct effect of a particular locus is excluded. The use of fluctuating asymmetry is proposed as an indicator of the loss of heterozygosity in salmon populations.