RESUMEN
BACKGROUND: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis. RESULTS: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose. CONCLUSIONS: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.
Asunto(s)
Antihelmínticos/metabolismo , Desoxiazúcares/biosíntesis , Desoxiazúcares/metabolismo , Hexosas/metabolismo , Ivermectina/metabolismo , Streptomyces/química , Técnicas Químicas Combinatorias , Desoxiazúcares/genética , Expresión Génica , Ivermectina/análogos & derivados , Familia de Multigenes/genética , Plásmidos , Ingeniería de Proteínas , Racemasas y Epimerasas/metabolismo , Estereoisomerismo , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
A fermentation method that bypasses the low-yielding semisynthesis of epirubicin (4'-epidoxorubicin) and 4'-epidaunorubicin, important cancer chemotherapy drugs, has been developed for Streptomyces peucetius. This bacterium normally produces the anthracycline antibiotics, doxorubicin and daunorubicin; the 4'-epimeric anthracyclines are formed by introducing the heterologous Streptomyces avermitilis avrE or Saccharopolyspora eryBIV genes into an S. peucetius dnmV mutant blocked in the biosynthesis of daunosamine, the deoxysugar component of these antibiotics. Product yields were enhanced considerably by replacing the chromosomal copy of dnmV with avrE and by introducing further mutations that can increase daunorubicin and doxorubicin yields in the wild-type strain. This method demonstrates that valuable hybrid antibiotics can be made by combinatorial biosynthesis with bacterial deoxysugar biosynthesis genes.
Asunto(s)
Antibióticos Antineoplásicos/biosíntesis , Epirrubicina/biosíntesis , Profármacos/metabolismo , Streptomyces/metabolismo , Cartilla de ADN , Fermentación , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Ingeniería Genética , Genotipo , Hexosaminas/biosíntesis , Mutación/genética , Plásmidos , Streptomyces/genéticaRESUMEN
Streptomyces avermitilis produces a series of eight potent anthelmintic compounds called avermectins (AVM). AVM are pentacyclic, macrocyclic lactone compounds containing an oleandrose disaccharide. Labeling studies have shown that AVM is a polyketide derived from the condensation of 12 acyl units (five propionates and seven acetates) to an isobutyl or 2-methylbutyryl starter unit. The genes required for AVM biosynthesis have been cloned, and deletion mapping has located the AVM gene cluster to a 95-kb region. Partial DNA sequencing of this region indicates two 30-kb segments encode large, multifunctional peptides of the AVM polyketide synthase (PKS). The PKS proteins contain at least 49 domains with homology to the domains in fatty acid synthase and erythromycin PKS. These domains are arranged as 12 modular repeats that each encode a PKS unit with various subsets of the FAS-like functions. The predicted functions required to form the side groups on the AVM macrocyclic ring were compared to the functions found in the 12 PKS units. This comparison suggests that each PKS unit is specific for condensation and reduction of one acyl unit. If the various domains can be manipulated without disrupting the PKS, it may be possible to synthesize a variety of AVM derivatives.
Asunto(s)
Ivermectina/análogos & derivados , Macrólidos , Complejos Multienzimáticos/genética , Antihelmínticos/química , Antihelmínticos/metabolismo , Antibacterianos/metabolismo , Antiprotozoarios/química , Antiprotozoarios/metabolismo , Diseño de Fármacos , Eritromicina/metabolismo , Genes Bacterianos , Ingeniería Genética , Ivermectina/química , Ivermectina/metabolismo , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
A human glucagon receptor has been cloned from human liver tissue. The 1578-bp cDNA clone encodes a protein of 477 amino acids with 82% identity to the rat glucagon receptor. The predicted secondary structure and homology to known proteins places this receptor within the superfamily of seven transmembrane domain G protein coupled receptors. Transfection of the human glucagon receptor into COS-7 cells confers upon them high affinity binding for [125I] glucagon. In membranes prepared from COS-7 cells transfected with the human glucagon receptor, the binding of [125I] glucagon is inhibited with the rank order of potency glucagon > oxyntomodulin > glucagon-like peptide 1 (7-36) amide >> glucagon-like peptide 2 = gastric inhibitory peptide = secretin.
Asunto(s)
Hígado/metabolismo , Receptores de Glucagón/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular/métodos , Cartilla de ADN , ADN Complementario/metabolismo , Expresión Génica , Glucagón/metabolismo , Humanos , Riñón , Cinética , Datos de Secuencia Molecular , Poli A/aislamiento & purificación , Poli A/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ARN/aislamiento & purificación , ARN/metabolismo , ARN Mensajero , Ratas , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The sites for 24 Tn5seq1 insertions into G+C-rich DNA of Streptomyces avermitilis were determined. Although Tn5-based transposons usually generate a 9-bp duplication at their insertion site, one Tn5seq1 insertion generated a 10-bp duplication and another insertion an 8-bp duplication.
Asunto(s)
Elementos Transponibles de ADN , Mutagénesis Insercional , Plásmidos , Streptomyces/genética , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Streptomyces/metabolismoRESUMEN
New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922. The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors. The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.
Asunto(s)
Escherichia coli/genética , Eliminación de Gen , Vectores Genéticos , Plásmidos , Streptomyces/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Datos de Secuencia MolecularRESUMEN
The avermectin (Av) polyketide synthase (PKS) and erythromycin (Er) PKS are encoded by modular repeats of DNA, but the genetic organization of the modules encoding Av PKS is more complex than Er PKS. Sequencing of several related DNA fragments from Streptomyces avermitilis that are part of the Av biosynthetic gene cluster, revealed that they encode parts of large multifunctional PKS proteins. The Av PKS proteins show strong similarity to each other, as well as similarity to Er PKS proteins [Donadio et al., Science 252 (1991) 675-679] and fatty acid synthases. Partial DNA sequencing of the 65-kb region containing all the related sequence elements in the avr genes provides evidence for twelve modular repeats encoding FAS-like domains. The genes encoding the Av PKS are organized as two sets of six modular repeats which are convergently transcribed.
Asunto(s)
Genes Bacterianos , Ivermectina/análogos & derivados , Complejos Multienzimáticos/genética , Familia de Multigenes , Streptomyces/genética , Eritromicina/biosíntesis , Ivermectina/química , Sustancias Macromoleculares , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Complejos Multienzimáticos/fisiología , Streptomyces/enzimología , Streptomyces/fisiologíaRESUMEN
We describe the isolation and characterization of mutations in ompF that alter the pore properties of the OmpF porin. The selection makes use of the fact that maltodextrins larger than maltotriose are too large to diffuse through the normal OmpF pore. By demanding growth on maltodextrins (Dex+) in the absence of the LamB protein, which is normally required for the uptake of these large sugars, we are able to obtain ompF mutations. These include transversions, transitions and small deletions. We obtained almost exclusively ompF mutations in spite of the fact that analogous alterations in ompC can result in similar phenotypes. Fifteen independent point mutations identify residues R42, R82, D113 and R132 of the mature peptide as important in pore function. The alterations result in uncharged amino acids being substituted for charged amino acids. Growth tests, antibiotic sensitivities and rates of [14C]maltose uptake suggest that the alterations result in an increased pore size. Small deletions of six to 15 amino acid residues in the region between A108 and V133 of mature OmpF dramatically alter outer membrane permeability to hydrophobic antibiotics and detergents as well as conferring a Dex+ phenotype. We suggest that these mutations affect both the pore function and interactions with other outer membrane components. A model of OmpF protein structure based on general rules for folding membrane proteins and these mutations is presented.