Carlos Olano | University of Oviedo / Universidad de Oviedo (original) (raw)

Papers by Carlos Olano

The Journal of Antibiotics, 2011

Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide co... more Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide compounds, in particular, those produced by actinomycetes. Hutch principally centered his efforts to study the biosynthesis of bioactive compounds, antibiotic and antitumor drugs, and to develop new derivatives with improved therapeutic properties. After dedicating 40 years to the study of polyketides, Hutch leaves us, as legacy, the knowledge that he and his collaborators have accumulated and shared with the scientific community. The best tribute we can offer to him is keeping on the study of polyketides and other bioactive compounds, in an effort to generate more safer and useful drugs. In this review, the work on the polyketides, borrelidin, steffimycin and streptolydigin, performed at the laboratory of Professors Salas and Mé ndez at University of Oviedo (Spain) during the last 10 years, is summarized.

J Antibiot, 2011

Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide co... more Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide compounds, in particular, those produced by actinomycetes. Hutch principally centered his efforts to study the biosynthesis of bioactive compounds, antibiotic and antitumor drugs, and to develop new derivatives with improved therapeutic properties. After dedicating 40 years to the study of polyketides, Hutch leaves us, as legacy, the knowledge that he and his collaborators have accumulated and shared with the scientific community. The best tribute we can offer to him is keeping on the study of polyketides and other bioactive compounds, in an effort to generate more safer and useful drugs. In this review, the work on the polyketides, borrelidin, steffimycin and streptolydigin, performed at the laboratory of Professors Salas and Mé ndez at University of Oviedo (Spain) during the last 10 years, is summarized.

ChemBioChem, 2015

Streptomyces sp. Tü 6176, known as producer of cytotoxic benzoxazoles AJI9561, nataxazole and 5-h... more Streptomyces sp. Tü 6176, known as producer of cytotoxic benzoxazoles AJI9561, nataxazole and 5-hydroxy-nataxazole, has been found to produce also a fourth benzoxazole, UK-1. All of them derive from 3-hydroxy-anthranilate synthesized by nataxazole biosynthesis machinery. However, biosynthesis of AJI9561, nataxazole and 5-hydroxy-nataxazole requires 6-methylsalicylic acid also provided by nataxazole biosynthesis pathway, while biosynthesis of UK-1 utilizes salicylic acid produced by a salicylate synthase from coelibactin biosynthesis pathway. This clearly suggests cross-talk between nataxazole and coelibactin pathways.Furthermore, Streptomyces sp. Tü 6176 also produces siderophore enterobactin in an iron-free medium. Enterobactin production can be induced in an iron-independent manner by inactivating natAN that encodes an anthranilate synthase involved in nataxazole production. All these results indicate a close relationship between nataxazole, enterobactin and coelibactin pathways.

ChemBioChem, 2015

Bioinformatic analysis of the genome of Streptomyces sp. Tü 6176, nataxazole producer, predicts t... more Bioinformatic analysis of the genome of Streptomyces sp. Tü 6176, nataxazole producer, predicts the presence of 38 secondary metabolite biosynthesis gene clusters, including those involved in the biosynthesis nataxazole, antibiotic hygromycin B and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster has been identified and characterized showing the absence of an O-methyltransferase coding gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism since AJI9561 shows antibiotic activity while nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster resulted in the production of AJI9561. Benzoxazole, 5-hydroxynataxazole, also produced by Streptomyces sp. Tü 6176, derives from nataxazole by the activity of a yet unidentified oxygenase, which might imply a cross-talk between the nataxazole biosynthesis pathway and a still unknown pathway.

Antimicrobial Compounds, 2013

ABSTRACT Most bioactive natural products currently known are synthesized by members of the Actino... more ABSTRACT Most bioactive natural products currently known are synthesized by members of the Actinomycetales order. The development of genetic engineering provides novel genetic tools for the modification of known antibiotics and other bioactive compounds to generate derivatives with improved therapeutic properties. This new technology, named combinatorial biosynthesis, is able of introducing structural modifications in bioactive compounds not easily accessible by chemical means. Furthermore, progress in genome sequencing in this group of microorganisms shows that actinomycetes have a greater potential of synthesizing bioactive compounds than was anticipated. Each genome sequenced shows the presence of 18–37 gene clusters potentially directing the biosynthesis of bioactive compounds that have not been previously identified. Novel strategies are being developed to activate these cryptic or silent gene clusters in these microorganisms, allowing the identification of novel potentially bioactive compounds. This chapter will revise the state of the art in this field of research.

Microbiology, 2014

Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ... more Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ClmR1, belonging to the TetR-family, and the LuxR-family transcriptional regulator ClmR2. Inactivation of clmR1 led to a moderate increase of collismycin A yields along with an early onset of its production, suggesting an inhibitory role for the product of this gene. Inactivation of clmR2 abolished collismycin A biosynthesis, whereas overexpression of ClmR2 led to a fourfold increase in production yields, indicating that ClmR2 is an activator of collismycin A biosynthesis. Expression analyses of the collismycin gene cluster in the wild-type strain and in ΔclmR1 and ΔclmR2 mutants confirmed the role proposed for both regulatory genes, revealing that ClmR2 positively controls the expression of most of the genes in the cluster and ClmR1 negatively regulates both its own expression and that of clmR2. Additionally, production assays and further transcription analyses confirmed the existence of a higher regulatory level modulating collismycin A biosynthesis in response to iron concentrations in the culture medium. Thus, high iron levels inhibit collismycin A biosynthesis through the repression of clmR2 transcription. These results have allowed us to propose a regulatory model that integrates the effect of iron as the main environmental stimulus controlling collismycin A biosynthesis.

Microbiology, 2012

The streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three p... more The streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three putative regulatory genes, slgR1, slgR2 and slgY, encoding proteins belonging to TetR and LuxR transcriptional regulator families and ATP/GTP-binding proteins of DNA and RNA helicase superfamily I, respectively. Inactivation of slgR1 or slgR2 resulted in the abolition of streptolydigin production, suggesting that these proteins are pathway-specific positive regulators. In the case of the slgR1 mutant, low amounts of streptolydigin C were produced instead of streptolydigin. RT-PCR transcription analysis of streptolydigin biosynthesis genes revealed a hierarchical regulation process. SlgY was found to control the expression of the regulator slgR2. SlgR2 regulates the expression of structural genes involved in the formation of the streptolydigin bicyclic ketal moiety, incorporation and processing of 3-methylaspartate, and the regulator slgR1. On the other hand, SlgR1 controls the expression of slgE1-E2, involved in the conversion of glutamate to 3-methylaspartate, and putative glycoside hydrolases slgC1 and slgC2. Ectopic expression of slgR1, slgR2 and slgY regulatory genes in S. lydicus led to considerable increases in streptolydigin yields, 18-, 11-and 8.5-fold, respectively. Ectopic expression of slgY in an slgR1 mutant led to a 14-fold increase of streptolydigin C yields, while no effect was observed to result from expression of slgR2.

Microbial Biotechnology, 2011

Natural products are traditionally the main source of drug leads. In particular, many antitumour ... more Natural products are traditionally the main source of drug leads. In particular, many antitumour compounds are either natural products or derived from them. However, the search for novel antitumour drugs active against untreatable tumours, with fewer side-effects or with enhanced therapeutic efficiency, is a priority goal in cancer chemotherapy. Microorganisms, particularly actinomycetes, are prolific producers of bioactive compounds, including antitumour drugs, produced as secondary metabolites. Structural genes involved in the biosynthesis of such compounds are normally clustered together with resistance and regulatory genes, which facilitates the isolation of the gene cluster. The characterization of these clusters has represented, during the last 25 years, a great source of genes for the generation of novel derivatives by using combinatorial biosynthesis approaches: gene inactivation, gene expression, heterologous expression of the clusters or mutasynthesis. In addition, these techniques have been also applied to improve the production yields of natural and novel antitumour compounds. In this review we focus on some representative antitumour compounds produced by actinomycetes covering the genetic approaches used to isolate and validate their biosynthesis gene clusters, which finally led to generating novel derivatives and to improving the production yields.

Microbial Biotechnology, 2012

Two genes of the streptolydigin gene cluster in Streptomyces lydicus cluster encode putative fami... more Two genes of the streptolydigin gene cluster in Streptomyces lydicus cluster encode putative family 16 glycoside hydrolases. Both genes are expressed when streptolydigin is produced. Inactivation of these genes affects streptolydigin production when the microorganism is grown in minimal medium containing either glycerol or D-glucans as carbon source. Streptolydigin yields in S. lydicus were increased by overexpression of either slgC1 or slgC2.

Microbial Biotechnology, 2014

Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of second... more Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the highperformance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.

Journal of Bacteriology, 2011

The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the ... more The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the biosynthesis of the tetramic acid streptolydigin in Streptomyces lydicus. Inactivation of slgZ led to a novel streptolydigin derivative. Overexpression of slgZ, slgM, or both in S. lydicus led to a considerable increase in streptolydigin production.

The Journal of Antibiotics, 2012

The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene... more The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene, dnrN, that activates the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius. Gene disruption and replacement of dnrO produced the dnrO ::aphII mutant strain and resulted in the complete loss of daunorubicin biosynthesis. Suppression of the dnrO ::aphII mutation by the introduction of dnrN or dnrI on a plasmid suggested that DnrO is required for the transcription of dnrN, whose product is known to be required for dnrI expression. These conclusions were supported by the effects of the dnrO mutation on expression of dnrO, dnrN and dnrI, as viewed by melC fusions to each of these regulatory genes. DnrO was overexpressed in Escherichia coli and the cell-free extract was used to conduct mobility shift DNA-binding assays. The results showed that DnrO binds specifically to the overlapping dnrN/dnrO p1 promoter region. Thus, DnrO may regulate the expression of both the dnrN and dnrO genes.

Molecular Microbiology, 1993

Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring ... more Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.

Antimicrobial Agents and Chemotherapy, 2000

A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. S... more A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2,6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl-and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.

Journal of Bacteriology, 2009

Cheminform, 2009

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Fems Microbiology Letters, 1995

Abstract A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a larg... more Abstract A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis. The oleP gene was expressed in Escherichia coli as a fusion protein to a maltose-binding protein. Using polyclonal antibodies against this fusion protein it was observed that the synthesis of the cytochrome P450 was in parallel to that of oleandomycin. The cytochrome P450 encoded by the oleP gene could be responsible for the epoxidation of carbon 8 of the oleandomycin lactone ring.

Natural Product Reports, 2010

The Journal of Antibiotics, 2011

Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide co... more Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide compounds, in particular, those produced by actinomycetes. Hutch principally centered his efforts to study the biosynthesis of bioactive compounds, antibiotic and antitumor drugs, and to develop new derivatives with improved therapeutic properties. After dedicating 40 years to the study of polyketides, Hutch leaves us, as legacy, the knowledge that he and his collaborators have accumulated and shared with the scientific community. The best tribute we can offer to him is keeping on the study of polyketides and other bioactive compounds, in an effort to generate more safer and useful drugs. In this review, the work on the polyketides, borrelidin, steffimycin and streptolydigin, performed at the laboratory of Professors Salas and Mé ndez at University of Oviedo (Spain) during the last 10 years, is summarized.

J Antibiot, 2011

Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide co... more Professor Charles Richard Hutchinson (Hutch) dedicated his research to the study of polyketide compounds, in particular, those produced by actinomycetes. Hutch principally centered his efforts to study the biosynthesis of bioactive compounds, antibiotic and antitumor drugs, and to develop new derivatives with improved therapeutic properties. After dedicating 40 years to the study of polyketides, Hutch leaves us, as legacy, the knowledge that he and his collaborators have accumulated and shared with the scientific community. The best tribute we can offer to him is keeping on the study of polyketides and other bioactive compounds, in an effort to generate more safer and useful drugs. In this review, the work on the polyketides, borrelidin, steffimycin and streptolydigin, performed at the laboratory of Professors Salas and Mé ndez at University of Oviedo (Spain) during the last 10 years, is summarized.

ChemBioChem, 2015

Streptomyces sp. Tü 6176, known as producer of cytotoxic benzoxazoles AJI9561, nataxazole and 5-h... more Streptomyces sp. Tü 6176, known as producer of cytotoxic benzoxazoles AJI9561, nataxazole and 5-hydroxy-nataxazole, has been found to produce also a fourth benzoxazole, UK-1. All of them derive from 3-hydroxy-anthranilate synthesized by nataxazole biosynthesis machinery. However, biosynthesis of AJI9561, nataxazole and 5-hydroxy-nataxazole requires 6-methylsalicylic acid also provided by nataxazole biosynthesis pathway, while biosynthesis of UK-1 utilizes salicylic acid produced by a salicylate synthase from coelibactin biosynthesis pathway. This clearly suggests cross-talk between nataxazole and coelibactin pathways.Furthermore, Streptomyces sp. Tü 6176 also produces siderophore enterobactin in an iron-free medium. Enterobactin production can be induced in an iron-independent manner by inactivating natAN that encodes an anthranilate synthase involved in nataxazole production. All these results indicate a close relationship between nataxazole, enterobactin and coelibactin pathways.

ChemBioChem, 2015

Bioinformatic analysis of the genome of Streptomyces sp. Tü 6176, nataxazole producer, predicts t... more Bioinformatic analysis of the genome of Streptomyces sp. Tü 6176, nataxazole producer, predicts the presence of 38 secondary metabolite biosynthesis gene clusters, including those involved in the biosynthesis nataxazole, antibiotic hygromycin B and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster has been identified and characterized showing the absence of an O-methyltransferase coding gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism since AJI9561 shows antibiotic activity while nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster resulted in the production of AJI9561. Benzoxazole, 5-hydroxynataxazole, also produced by Streptomyces sp. Tü 6176, derives from nataxazole by the activity of a yet unidentified oxygenase, which might imply a cross-talk between the nataxazole biosynthesis pathway and a still unknown pathway.

Antimicrobial Compounds, 2013

ABSTRACT Most bioactive natural products currently known are synthesized by members of the Actino... more ABSTRACT Most bioactive natural products currently known are synthesized by members of the Actinomycetales order. The development of genetic engineering provides novel genetic tools for the modification of known antibiotics and other bioactive compounds to generate derivatives with improved therapeutic properties. This new technology, named combinatorial biosynthesis, is able of introducing structural modifications in bioactive compounds not easily accessible by chemical means. Furthermore, progress in genome sequencing in this group of microorganisms shows that actinomycetes have a greater potential of synthesizing bioactive compounds than was anticipated. Each genome sequenced shows the presence of 18–37 gene clusters potentially directing the biosynthesis of bioactive compounds that have not been previously identified. Novel strategies are being developed to activate these cryptic or silent gene clusters in these microorganisms, allowing the identification of novel potentially bioactive compounds. This chapter will revise the state of the art in this field of research.

Microbiology, 2014

Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ... more Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ClmR1, belonging to the TetR-family, and the LuxR-family transcriptional regulator ClmR2. Inactivation of clmR1 led to a moderate increase of collismycin A yields along with an early onset of its production, suggesting an inhibitory role for the product of this gene. Inactivation of clmR2 abolished collismycin A biosynthesis, whereas overexpression of ClmR2 led to a fourfold increase in production yields, indicating that ClmR2 is an activator of collismycin A biosynthesis. Expression analyses of the collismycin gene cluster in the wild-type strain and in ΔclmR1 and ΔclmR2 mutants confirmed the role proposed for both regulatory genes, revealing that ClmR2 positively controls the expression of most of the genes in the cluster and ClmR1 negatively regulates both its own expression and that of clmR2. Additionally, production assays and further transcription analyses confirmed the existence of a higher regulatory level modulating collismycin A biosynthesis in response to iron concentrations in the culture medium. Thus, high iron levels inhibit collismycin A biosynthesis through the repression of clmR2 transcription. These results have allowed us to propose a regulatory model that integrates the effect of iron as the main environmental stimulus controlling collismycin A biosynthesis.

Microbiology, 2012

The streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three p... more The streptolydigin biosynthetic gene cluster from Streptomyces lydicus NRRL 2433 contains three putative regulatory genes, slgR1, slgR2 and slgY, encoding proteins belonging to TetR and LuxR transcriptional regulator families and ATP/GTP-binding proteins of DNA and RNA helicase superfamily I, respectively. Inactivation of slgR1 or slgR2 resulted in the abolition of streptolydigin production, suggesting that these proteins are pathway-specific positive regulators. In the case of the slgR1 mutant, low amounts of streptolydigin C were produced instead of streptolydigin. RT-PCR transcription analysis of streptolydigin biosynthesis genes revealed a hierarchical regulation process. SlgY was found to control the expression of the regulator slgR2. SlgR2 regulates the expression of structural genes involved in the formation of the streptolydigin bicyclic ketal moiety, incorporation and processing of 3-methylaspartate, and the regulator slgR1. On the other hand, SlgR1 controls the expression of slgE1-E2, involved in the conversion of glutamate to 3-methylaspartate, and putative glycoside hydrolases slgC1 and slgC2. Ectopic expression of slgR1, slgR2 and slgY regulatory genes in S. lydicus led to considerable increases in streptolydigin yields, 18-, 11-and 8.5-fold, respectively. Ectopic expression of slgY in an slgR1 mutant led to a 14-fold increase of streptolydigin C yields, while no effect was observed to result from expression of slgR2.

Microbial Biotechnology, 2011

Natural products are traditionally the main source of drug leads. In particular, many antitumour ... more Natural products are traditionally the main source of drug leads. In particular, many antitumour compounds are either natural products or derived from them. However, the search for novel antitumour drugs active against untreatable tumours, with fewer side-effects or with enhanced therapeutic efficiency, is a priority goal in cancer chemotherapy. Microorganisms, particularly actinomycetes, are prolific producers of bioactive compounds, including antitumour drugs, produced as secondary metabolites. Structural genes involved in the biosynthesis of such compounds are normally clustered together with resistance and regulatory genes, which facilitates the isolation of the gene cluster. The characterization of these clusters has represented, during the last 25 years, a great source of genes for the generation of novel derivatives by using combinatorial biosynthesis approaches: gene inactivation, gene expression, heterologous expression of the clusters or mutasynthesis. In addition, these techniques have been also applied to improve the production yields of natural and novel antitumour compounds. In this review we focus on some representative antitumour compounds produced by actinomycetes covering the genetic approaches used to isolate and validate their biosynthesis gene clusters, which finally led to generating novel derivatives and to improving the production yields.

Microbial Biotechnology, 2012

Two genes of the streptolydigin gene cluster in Streptomyces lydicus cluster encode putative fami... more Two genes of the streptolydigin gene cluster in Streptomyces lydicus cluster encode putative family 16 glycoside hydrolases. Both genes are expressed when streptolydigin is produced. Inactivation of these genes affects streptolydigin production when the microorganism is grown in minimal medium containing either glycerol or D-glucans as carbon source. Streptolydigin yields in S. lydicus were increased by overexpression of either slgC1 or slgC2.

Microbial Biotechnology, 2014

Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of second... more Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the highperformance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.

Journal of Bacteriology, 2011

The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the ... more The asparaginyl-tRNA synthetase-like SlgZ and methyltransferase SlgM enzymes are involved in the biosynthesis of the tetramic acid streptolydigin in Streptomyces lydicus. Inactivation of slgZ led to a novel streptolydigin derivative. Overexpression of slgZ, slgM, or both in S. lydicus led to a considerable increase in streptolydigin production.

The Journal of Antibiotics, 2012

The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene... more The dnrO gene is located adjacent to and divergently transcribed from the response regulator gene, dnrN, that activates the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius. Gene disruption and replacement of dnrO produced the dnrO ::aphII mutant strain and resulted in the complete loss of daunorubicin biosynthesis. Suppression of the dnrO ::aphII mutation by the introduction of dnrN or dnrI on a plasmid suggested that DnrO is required for the transcription of dnrN, whose product is known to be required for dnrI expression. These conclusions were supported by the effects of the dnrO mutation on expression of dnrO, dnrN and dnrI, as viewed by melC fusions to each of these regulatory genes. DnrO was overexpressed in Escherichia coli and the cell-free extract was used to conduct mobility shift DNA-binding assays. The results showed that DnrO binds specifically to the overlapping dnrN/dnrO p1 promoter region. Thus, DnrO may regulate the expression of both the dnrN and dnrO genes.

Molecular Microbiology, 1993

Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring ... more Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several trans-membrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9kb Pst1-Sph1 fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.

Antimicrobial Agents and Chemotherapy, 2000

A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. S... more A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2,6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl-and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.

Journal of Bacteriology, 2009

Cheminform, 2009

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Fems Microbiology Letters, 1995

Abstract A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a larg... more Abstract A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a large open reading frame encoding a type I polyketide synthase subunit and an oleandomycin resistance gene (oleB). Sequencing of a 1.4-kb DNA fragment adjacent to oleB revealed the existence of an open reading frame (oleP) encoding a protein similar to several cytochrome P450 monooxygenases from different sources, including the products of the eryF and eryK genes from Saccharopolyspora erythraea that participate in erythromycin biosynthesis. The oleP gene was expressed in Escherichia coli as a fusion protein to a maltose-binding protein. Using polyclonal antibodies against this fusion protein it was observed that the synthesis of the cytochrome P450 was in parallel to that of oleandomycin. The cytochrome P450 encoded by the oleP gene could be responsible for the epoxidation of carbon 8 of the oleandomycin lactone ring.

Natural Product Reports, 2010