Carmen Méndez - Academia.edu (original) (raw)

Papers by Carmen Méndez

Microbial Cell Factories, 2020

Background Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptom... more Background Mithramycin is an anti-tumor compound of the aureolic acid family produced by Streptomyces argillaceus. Its biosynthesis gene cluster has been cloned and characterized, and several new analogs with improved pharmacological properties have been generated through combinatorial biosynthesis. To further study these compounds as potential new anticancer drugs requires their production yields to be improved significantly. The biosynthesis of mithramycin proceeds through the formation of the key intermediate 4-demethyl-premithramycinone. Extensive studies have characterized the biosynthesis pathway from this intermediate to mithramycin. However, the biosynthesis pathway for 4-demethyl-premithramycinone remains unclear. Results Expression of cosmid cosAR7, containing a set of mithramycin biosynthesis genes, in Streptomyces albus resulted in the production of 4-demethyl-premithramycinone, delimiting genes required for its biosynthesis. Inactivation of mtmL, encoding an ATP-depende...

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Marine drugs, Jan 31, 2018

Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurall... more Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurally, JAs are formed by a modified β-methylphenylalanine residue, whose biosynthesis involves a methyltransferase that in has been identified as MppJ. Up to date, three JA members (A⁻C) and a few other natural products containing β-methylphenylalanine have been discovered from soil-derived microorganisms. Herein, we report the identification of a gene () coding for a putative methyltransferase highly identical to MppJ in the chromosome of the marine actinobacteria GUA-06-05-006A. In its 5' region, clusters with two polyketide synthases (PKS) (, ), a nonribosomal peptide synthetase (NRPS) () and a thioesterase gene (), possibly conforming a single transcriptional unit. Insertion of a strong constitutive promoter upstream of P1 led to the detection of JA A, along with at least two novel JA family members (D and E). Independent inactivation of , and abolished production of JA A, JA D and J...

Microbial Cell Factories, 2016

Background: Antitumor compounds PM100117 and PM100118 are glycosylated polyketides derived from t... more Background: Antitumor compounds PM100117 and PM100118 are glycosylated polyketides derived from the marine actinobacteria Streptomyces caniferus GUA-06-05-006A. The organization and characterization of the PM100117/18 biosynthesis gene cluster has been recently reported. Results: Based on the preceding information and new genetic engineering data, we have outlined the pathway by which PM100117/18 are glycosylated. Furthermore, these genetic engineering experiments have allowed the generation of novel PM100117/18 analogues. Deletion of putative glycosyltranferase genes and additional genes presumably involved in late biosynthesis steps of the three 2,6-dideoxysugars appended to the PM100117/18 polyketide skeleton, resulted in the generation of a series of intermediates and novel derivatives. Conclusions: Isolation and identification of the novel compounds constitutes an important contribution to our knowledge on PM100117/18 glycosylation, and set the basis for further characterization of specific enzymatic reactions, additional genetic engineering and combinatorial biosynthesis approaches.

ChemBioChem, 2015

Streptomyces sp. Tü 6176 produces cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the... more Streptomyces sp. Tü 6176 produces cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of thirty eight putative secondary metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, antibiotic hygromycin B and ChemBioChem 10.1002/cbic.201500153 ionophores enterobactin and coelibactin. 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, expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the heterologous production of AJI9561. Nataxazole biosynthesis requires shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to 4-fold by deregulating the gene cluster expression. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5hydroxynataxazole 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.

Natural Products: Discourse, Diversity, and Design, 2014

Molecular Microbiology, 2005

The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It conta... more The indolocarbazole staurosporine is a potent inhibitor of a variety of protein kinases. It contains a sugar moiety attached through C-N linkages to both indole nitrogen atoms of the indolocarbazole core. Staurosporine biosynthesis was reconstituted in vivo in a heterologous host Streptomyces albus by using two different plasmids: the 'aglycone vector' expressing a set of genes involved in indolocarbazole biosynthesis together with staG (encoding a glycosyltransferase) and/or staN (coding for a P450 oxygenase), and the 'sugar vector' expressing a set of genes responsible for the biosynthesis of the sugar moiety. Attachment of the sugar to the two indole nitrogens of the indolocarbazole core was dependent on the combined action of StaG and StaN. When StaN was absent, the sugar was attached only to one of the nitrogen atoms, through an N-glycosidic linkage, as in the indolocarbazole rebeccamycin. The StaG glycosyltransferase showed flexibility with respect to the sugar donor. When the 'sugar vector' was substituted by constructs directing the biosynthesis of L-rhamnose, L-digitoxose, L-olivose and D-olivose, respectively, StaG and StaN were able to transfer and attach all of these sugars to the indolocarbazole aglycone.

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. Streptomyces species are Gram-positive bacteria widely distributed in terrestrial and aquatic ecosystems (McCarthy and Williams, 1992; Stach and Bull, 2005; Gao and Gupta, 2012). They exhibit diverse physiological and metabolic properties, such as the production of a wide variety of secondary metabolites (Berdy, 2005). In addition, they are important for soil biodegradation and humus formation by decomposing and recycling complex mixtures of polymers in dead plant, animal and fungal materials using extracellular enzymes (McCarthy and Williams, 1992; Chater et al., 2010). Among these polymers are polysaccharides such as cellulose, chitin, b-glucans, starch, glycogen, inulin, pullulan and xylan, which can be degraded by hydrolytic enzymes (Guillén et al., 2010). Streptolydigin (Fig. 1A) is a potent inhibitor of bacterial RNA polymerase and eukaryotic terminal deoxynucleotidyl transferase produced by Streptomyces lydicus NRRL 2344

Microbial Biotechnology, 2010

Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in... more Mithramycin and chromomycin A3 are two structurally related antitumour compounds, which differ in the glycosylation profiles and functional group substitutions of the sugars. Chromomycin contains two acetyl groups, which are incorporated during the biosynthesis by the acetyltransferase CmmA in Streptomyces griseus ssp. griseus. A bioconversion strategy using an engineered S. griseus strain generated seven novel acetylated mithramycins. The newly formed compounds were purified and characterized by MS and NMR. These new compounds differ from their parental compounds in the presence of one, two or three acetyl groups, attached at 3E, 4E and/or 4D positions. All new mithramycin analogues showed antitumour activity at micromolar of lower concentrations. Some of the compounds showed improved activities against glioblastoma or pancreas tumour cells. The CmmA acetyltransferase was located in the cell membrane and was shown to accept several acyl-CoA substrates. All these results highlight the potential of CmmA as a tool to create structural diversity in these antitumour compounds.

Metabolic Engineering, 2008

Production of secondary metabolites is a process influenced by several physico-chemical factors i... more Production of secondary metabolites is a process influenced by several physico-chemical factors including nutrient supply, oxygenation, temperature and pH. These factors have been traditionally controlled and optimized in industrial fermentations in order to enhance metabolite production. In addition, traditional mutagenesis programs have been used by the pharmaceutical industry for strain and production yield improvement. In the last years, the development of recombinant DNA technology has provided new tools for approaching yields improvement by means of genetic manipulation of biosynthetic pathways. These efforts are usually focused in redirecting precursor metabolic fluxes, deregulation of biosynthetic pathways and overexpression of specific enzymes involved in metabolic bottlenecks. In addition, efforts have been made for the heterologous expression of biosynthetic gene clusters in other organisms, looking not only for an increase of production levels but also to speed the process by using rapidly growing and easy to manipulate organisms compared to the producing organism. In this review, we will focus on these genetic approaches as applied to bioactive secondary metabolites produced by actinomycetes.

Journal of Natural Products, 2008

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-a... more Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activatedL-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a "flooding" of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDPactivated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1-4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, L-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-β-D-digitoxosyl-MTM) and 3 (deoliosyl-3C-β-Dmycarosyl-MTM) show improved activity (64.8 ± 2% and 50.3 ± 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 ± 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 ± 2% and 12.6 ± 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 ± 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.

The Journal of Antibiotics, 2012

Streptolydigin is a tetramic acid antibiotic produced by Streptomyces lydicus NRRL 2433 and invol... more Streptolydigin is a tetramic acid antibiotic produced by Streptomyces lydicus NRRL 2433 and involving a hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) system in its biosynthesis. The streptolydigin amino-acid precursor, 3-methylaspartate, has been proposed to be condensed to the polyketide portion of the molecule by a NRPS composed by three enzymes (SlgN1, SlgN2 and SlgL). On the other hand, biosynthesis of the polyketide moiety involves the participation of cytochrome P450 SlgO2 for the correct cyclization of the characteristic bicyclic ketal. Independent disruption of slgN1, slgN2, slgL or slgO2 resulted in S. lydicus mutants unable to produce the antibiotic thus confirming the involvement of these genes in the biosynthesis of the antibiotic. These mutants did not accumulate any streptolydigin biosynthesis intermediate or shunt product derived from early polyketides released from the PKS. However, they produced three novel compounds identified as 4-(2-carboxy-propylamino)-3-chloro-benzoic acid, 4-(2-carboxy-propylamino)-3-hydroxy-benzoic acid and 4-(2-carboxypropylamino)-benzoic acid, which were designated as christolane A, christolane B and christolane C, respectively. These compounds have been shown to exert some antibiotic activity.

Gene, 1996

Mithramycin (Mtm) is an aromatic polyketide which shows antibacterial and antitumor activity. Fro... more Mithramycin (Mtm) is an aromatic polyketide which shows antibacterial and antitumor activity. From a chromosomal cosmid library of Streptomyces argillaceus, a Mtm producer, a clone (cosAR7) was isolated by homology to the actI/III region of S. coelicolor and the strDEM genes of S. griseus. From this clone, a 5.3-kb DNA region was sequenced and found to encode six open reading frames (designated as mtmQXPKST1), five of them transcribed in the same direction. The deduced products of five of these genes resembled components of type-II polyketide synthases. The mtm genes would code for an aromatase (mtmQ), a polypeptide of unknown function (mtmX), a beta-ketoacylsynthase (mtmP) and a related 'chain length factor' (mtmK), an acyl carrier protein (mtmS) and a beta-ketoreductase (mtmT1). The involvement of this gene cluster in Mtm biosynthesis was demonstrated by the Mtm non-producing phenotype of mutants generated in two independent insertional inactivation experiments.

FEMS Microbiology Letters, 1995

A cosmid clone from an oleandomycin producer, Streptomyces antibioticus, contains a large open re... more 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 01eB 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 Succharopolysporu erythrueu that participate in erythromycin biosynthesis. The oZeP gene was expressed in Exherichiu cob 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.

FEMS Microbiology Letters, 2000

Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two gene... more Mithramycin is an antitumor aromatic polyketide synthesized by Streptomyces argillaceus. Two genes (mtrX and mtrY) of the mithramycin gene cluster were inactivated by gene replacement. Inactivation of mtrX, that encodes an ABC excission nuclease system for DNA repair, produced a mutant that was affected in the normal rate of growth. Expression of mtrX in Streptomyces albus in a multicopy plasmid vector conferred a low increase in resistance to mithramycin. Inactivation of mtrY, that encodes a protein of unknown function, produced a 50% decrease in mithramycin biosynthesis. When mtrY was expressed in the wild-type S. argillaceus in a multicopy plasmid, this caused about 47% increase in the levels of mithramycin production. It is proposed that mtrX and mtrY could code for a secondary defense mechanism and a mithramycin regulatory element, respectively.

FEMS Microbiology Letters, 1996

The OleC ABC transporter of Streptomyces antibioticus is constituted by an ATP-binding protein (O... more The OleC ABC transporter of Streptomyces antibioticus is constituted by an ATP-binding protein (OleC) and a hydrophobic protein (OleC5). Here we present experimental evidence demonstrating that the 01eC5 protein is an integral membrane protein and we propose a topological model for its integration into the membrane. This model is based on the generation of hybrid proteins between different regions of OleC5 and a Escherichia coli B-lactamase (BlaM) and the determination of the minimal inhibitory concentrations to ampicillin in these constructions. Fusions were generated both by cloning specific fragments of oZeC5 and by creating ExoIII nested deletions of the gene. In the topological model proposed there will be six a-helix transmembrane regions, two cytoplasmic and four periplasmic loops and a hydrophobic linker domain.

Chemistry & Biology, 1999

Background: Mithramycin is a member of the clinically important aureolic acid group of antitumor ... more 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.

Chemistry & Biology, 2009

The biosynthetic gene cluster for the dienoyltetramic acid streptolydigin was identified and char... more The biosynthetic gene cluster for the dienoyltetramic acid streptolydigin was identified and characterized from the producer organism Streptomyces lydicus NRRL2433. Sequence analysis of an 80.8 kb DNA region revealed the presence of 38 ORFs, 29 of which are probably involved in streptolydigin biosynthesis and would code for all activities required for its biosynthesis. Six insertional inactivation mutants were generated in the sequenced region to prove its involvement in streptolydigin biosynthesis, to define the boundaries of the cluster, to functionally characterize some genes, and to generate novel derivatives. A model for streptolydigin biosynthesis is proposed that includes a probable domain skipping in the streptolydigin PKS and the participation of a freestanding adenylation domain protein. Some bioactive derivatives of streptolydigin with altered glycosylation pattern have been produced by combinatorial biosynthesis showing a certain degree of flexibility of the L-rhodinosyl transferase SlgG for the recognition of 2,3,6-trideoxyhexoses and 2,6-dideoxyhexoses, both in D-and L-configuration.

Chemistry & Biology, 2013

Collismycin A is a member of the 2,2 0-bipyridyl family of natural products that shows cytotoxic ... more Collismycin A is a member of the 2,2 0-bipyridyl family of natural products that shows cytotoxic activity. Structurally, it belongs to the hybrid polyketidesnonribosomal peptides. After the isolation and characterization of the collismycin A gene cluster, we have used the combination of two different approaches (insertional inactivation and biocatalysis) to increase structural diversity in this natural product class. Twelve collismycin analogs were generated with modifications in the second pyridine ring of collismycin A, thus potentially maintaining biologic activity. None of these analogs showed better cytotoxic activity than the parental collismycin. However, some analogs showed neuroprotective activity and one of them (collismycin H) showed better values for neuroprotection against oxidative stress in a zebrafish model than those of collismycin A. Interestingly, this analog also showed very poor cytotoxic activity, a feature very desirable for a neuroprotectant compound.

Chemistry & Biology, 2001

Background: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which... more Background: Elloramycin is an anthracycline-like antitumor drug related to tetracenomycin C which is produced by Streptomyces olivaceus Tu « 2353. 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 Land 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 Tu « 2353 can transfer different sugars into the aglycon 8-DMTC. In addition to its natural sugar substrate L-rhamnose, ElmGT can transfer several Land 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.