RESUMEN
The most important tools in killing and overcoming on the microbes and pathogens that cause diseases in medicine and/or in agriculture are the antibiotics. The discovery and synthesis of the microbial natural products or antibiotics has greatly developed genetically and biotechnologically quickly in the last decades. It is necessary to access this great genetic diversity by finding ways to increase the level of expression of these biosynthetic pathways. In this study, we carried out an improvement in the antibiotic production of weak Streptomyces graminofaciens strain NBR9 that has high lipid content; using Ultra-Violet irradiation mutagenesis. This strain was isolated from the Northern Region in the kingdom of Saudi Arabia and identified biochemically and confirmed genetically by sequencing of the 16S rRNA gene as Streptomyces graminofaciens NBR9; Accession No. (MN640578). The resultant mutant strain showed increasing in their antimicrobial activities. The methods and techniques used for the antibiotic extraction, purification, characterization and identification proved that the obtained antibiotic is same with antibiotic Carbomycin.
Asunto(s)
Vías Biosintéticas/genética , Microbiología Industrial , Leucomicinas/biosíntesis , Lípidos/genética , Mutación/genética , Streptomyces/genética , Antibacterianos/biosíntesis , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Leucomicinas/aislamiento & purificación , Leucomicinas/farmacología , Mutagénesis , ARN Ribosómico 16S/genéticaRESUMEN
The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction. Whole-cell assays using Escherichia coli cells expressing RosC mutants with single and double amino acid substitutions derived from RM30 indicated that P107S/L176Q, P107S/V277A, P107S/I319N, L176Q/V277A, L176Q/I319N, and S254N/V277A significantly reduced the catalytic activity of the second reaction, which is alcohol oxidation. Of the previously mentioned mutants, double mutants containing L176Q, which was presumed to occur in the FG loop region, lost the total catalytic activity of the third reaction (aldehyde oxidation). Additionally, an engineered M. rosaria strain with rosC disruption, which introduced the gene encoding the RosC mutants P107S/L176Q and P107S/V277A preferentially produced 20-dihydrorosamicin, which is formed after the first oxidation reaction of RosC.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Leucomicinas/biosíntesis , Micromonospora/enzimología , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Escherichia coli/genética , Mutación , Oxidación-ReducciónRESUMEN
Carbomycins are 16-membered macrolide antibiotics produced by Streptomyces thermotolerans ATCC 11416T. To characterize gene cluster responsible for carbomycin biosynthesis, the draft genome sequences for strain ATCC 11416T were obtained, from which the partial carbomycin biosynthetic gene cluster was identified. This gene cluster was approximately 40 kb in length, and encoding 30 ORFs. Two putative transcriptional regulatory genes, acyB2 and cbmR, were inactivated by insertion of the apramycin resistance gene, and the resulting mutants were unable to produce carbomycin, thus confirming the involvement of two regulatory genes in carbomycin biosynthesis. Overexpression of acyB2 greatly improved the yield of carbomycin; however, overexpression of cbmR blocked carbomycin production. The qPCR analysis of the carbomycin biosynthetic genes in various mutants indicated that most genes were highly expressed in acyB2-overexpressing strains, but few expressed in cbmR-overexpressing strains. Furthermore, acyB2 co-expression with 4â³-isovaleryltransferase gene (ist), resulted in efficient biotransformation of spiramycin into bitespiramycin in S. lividans TK24, whereas ist gene regulated by acyB2 and cbmR would cause the lower efficiency of spiramycin biotransformation. These results indicated that AcyB2 was a pathway-specific positive regulator of carbomycin biosynthesis. However, CbmR played a dual role in the carbomycin biosynthesis by acting as a positive regulator, and as a repressor at cbmR high expression levels.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Genes Reguladores/genética , Leucomicinas/biosíntesis , Streptomyces/genética , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Familia de Multigenes/genética , Espiramicina/análogos & derivados , Espiramicina/metabolismo , Streptomyces/metabolismoRESUMEN
Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The D-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M. rosaria IFO13697. The resulting engineered strains, M. rosaria TPMA0054 and TPMA0069, produced mycinosyl rosamicin derivatives, IZIV and IZV, respectively. IZIV was identified as a novel mycinosyl rosamicin derivative, 23-O-mycinosyl-20-deoxo-20-dihydrorosamicin.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/genética , Leucomicinas/biosíntesis , Micromonospora/genética , Micromonospora/metabolismo , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Ingeniería Genética , Leucomicinas/química , Micromonospora/enzimología , Estructura Molecular , Mutación , Sintasas Poliquetidas/genéticaRESUMEN
The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Leucomicinas/biosíntesis , Micromonospora/enzimología , Micromonospora/genética , Proteínas Bacterianas/genética , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/enzimología , Escherichia coli/genética , Eliminación de Gen , Prueba de Complementación Genética , Familia de MultigenesRESUMEN
Spiramycin and midecamycin are 16-membered macrolide antibiotics with very similar chemical structures. Spiramycin has three components, namely spiramycin I, II and III. Spiramycin II and III are, respectively, the O-acetyl and propionyl derivatives at C3-hydroxyl group of spiramycin I. Midecamycin has four components, and the C3-hydroxyl group of midecamycin is all O-propionylated. The enzyme adding acyl group(s) at the C3-hydroxyl group during the biosynthesis of spiramycin and midecamycin is 3-O-acyltransferase. The 3-O-acyltransferases for spiramycin and midecamycin are also very similar, and presume to function when exchanged. To explore whether the 3-O-acyltransferase for midecamycin biosynthesis hold still the character of selective and efficient propionylation for spiramycin I at its C3-hydroxyl group, we inserted mdmB, the 3-O-acyltransferase gene from Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis, into a mutant strain of S. spiramyceticus F21, in which the 3-O-acyltransferase gene for spiramycin biosynthesis, sspA, was deleted; and the mdmB was integrated exactly into the chromosomal site where the sspA was deleted. We name this "hybrid" strain as SP-mdmB. HPLC analysis of the spiramycin produced by SP-mdmB showed that spiramycin I was still the major component, although the relative proportions of both spiramycin II and III increased significantly. We thus conclude that MdmB from Streptomyces mycarofaciens ATCC 21454 for midecamyicn biosynthesis do not hold the character of selective and efficient propionylation for spiramycin I within S. spiramyceticus F21, and this character is possibly limited in Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis.
Asunto(s)
Aciltransferasas/metabolismo , Leucomicinas/biosíntesis , Espiramicina/biosíntesis , Streptomyces/enzimología , Acilación , Aciltransferasas/genética , Medios de Cultivo , Genes Bacterianos , Ingeniería Genética/métodos , Streptomyces/genética , Especificidad por SustratoRESUMEN
Forty two strains of enterococci were isolated from feces of healthy adolescents. Strains were selected according to their antagonistic effects associated with bacteriocinogenic and microcinogenic activity. Resistance of enterococci to antibiotics, various concentrations of hydrochloric acid and bile, their level of production of organic acids and adhesiveness were determined. Characteristics related to pathogenicity were investigared in 5 microcinogenic strains of E. faecium with broad spectrum of antagonistic activity. Non-pathogenic microcin-producing strains of E. faecium resistant to physiological concentrations of hydrochloric acid and bile with broad spectrum of antagonistic activity against obligatory pathogenic and opportunistic microorganisms can be considered as possessing probiotic activity.
Asunto(s)
Enterococcus faecium/fisiología , Adolescente , Antibacterianos/farmacología , Antibiosis , Adhesión Bacteriana , Bacteriocinas/biosíntesis , Bilis , Niño , Farmacorresistencia Bacteriana Múltiple , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/aislamiento & purificación , Heces/microbiología , Femenino , Humanos , Ácido Clorhídrico/farmacología , Leucomicinas/biosíntesis , Masculino , Probióticos/aislamiento & purificación , Probióticos/farmacologíaRESUMEN
Two subclusters from Streptomyces mycarofaciens, a midecamycin producer, were cloned and partially sequenced. One region was located at the 5' end of the mid polyketide synthase (PKS) genes and contained the genes midA, midB and midC. The other region was at the 3' end of the PKS genes and contained midK, midI and midH. Analysis of the nucleotide sequence revealed that these genes encode dTDP-glucose synthase (midA), dTDP-glucose dehydratase (midB), aminotransferase (midC), methyltransferase (midK), glycosyltransferase (midI) and an assistant gene (midH). All of these genes are involved in the biosynthesis of dTDP-D-mycaminose, the first deoxysugar of midecamycin, and in transferring the mycaminose to the midecamycin aglycone in S. mycarofaciens. Similar to gene pairs desVIII/desVII in S. venezuelae and tylMIII/tylMII in S. fradiae, the product of midH probably functions as an auxiliary protein required by the MidI protein for efficient glycosyltransfer in midecamycin biosynthesis.
Asunto(s)
Genes Bacterianos , Glucosamina/análogos & derivados , Leucomicinas/biosíntesis , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/biosíntesis , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Glucosamina/biosíntesis , Macrólidos/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Nucleótidos de Timina/metabolismoRESUMEN
Development of host microorganisms for heterologous expression of polyketide synthases (PKS) that possess the intrinsic capacity to overproduce polyketides with a broad spectrum of precursors supports the current demand for new tools to create novel chemical structures by combinatorial engineering of modular and other classes of PKS. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors--malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA--used by modular PKS, and is a host that is amenable to genetic manipulation. We have expanded the utility of an overproducing S. fradiae strain for engineered biosynthesis of polyketides by engineering a biosynthetic pathway for methoxymalonyl-ACP, a fourth precursor used by many 16-membered macrolide PKS. This was achieved by introducing a set of five genes, fkbG-K from Streptomyces hygroscopicus, putatively encoding the methoxymalonyl-ACP biosynthetic pathway, into the S. fradiae chromosome. Heterologous expression of the midecamycin PKS genes in this strain resulted in 1 g/l production of a midecamycin analog. These results confirm the ability to engineer unusual precursor pathways to support high levels of polyketide production, and validate the use of S. fradiae for overproduction of 16-membered macrolides derived from heterologous PKS that require a broad range of precursors.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Macrólidos/metabolismo , Malonil Coenzima A/análogos & derivados , Streptomyces/metabolismo , Fermentación , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Ingeniería Genética , Leucomicinas/biosíntesis , Malonil Coenzima A/biosíntesis , Malonil Coenzima A/metabolismo , Streptomyces/genéticaRESUMEN
Two primers were designed and synthesized according to the polyketide ketoreductase from midecamycin producing strain gene(MKR) sequence data which has been reported, MKR gene was amplified using PCR technique. The amplified MKR gene was subcloned into NdeI and BamHI sites of the T7RNA polymerase-dependent pT7-7 expression vector, and introduced into E. coli K38/pGP1-2. Proteins were isolated from transformant. The result of exclusive labeling by L-35S-methionine of plasmid-encoded proteins SDS-PAGE analysis showed that the MKR was produced in E. coli. The expressed MKR has bioactivity.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Proteínas Bacterianas , Escherichia coli/genética , Leucomicinas/biosíntesis , Oxidorreductasas de Alcohol/biosíntesis , Clonación Molecular , Escherichia coli/metabolismo , Amplificación de Genes , Expresión Génica , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesisRESUMEN
We explored transposition in Streptomyces fradiae (Sf) as a means to insert a second copy of the tylF gene to improve tylosin (Ty) production. Transposons Tn5096 and Tn5099 transposed relatively randomly in Sf, and many of the insertions caused no deleterious effects on Ty production yields. Tn5098, a derivative of Tn5096 containing tylF and tylJ genes, recombined into the chromosome into the tyl gene cluster and transposition was not observed. However, following the tagging of a neutral site (NS) by Tn5099 transposition, tylF was effectively inserted into the NS by homologous recombination (transposon exchange). Recombinants obtained by transposon exchange produced higher yields of Ty.
Asunto(s)
Elementos Transponibles de ADN/genética , Mutagénesis Insercional/genética , Streptomyces/metabolismo , Tilosina/biosíntesis , Antibacterianos/biosíntesis , Southern Blotting , Sondas de ADN , Dosificación de Gen , Leucomicinas/biosíntesis , Plásmidos/genética , Recombinación Genética/genética , Streptomyces/genéticaRESUMEN
The promoter region of the midecamycin 4"-hydroxyl propionyltransferase gene (mpt) gene was reconstructed by PCR, and ligated with a fragment from the midecamycin producing strain (Streptomyces mycarofaciens var. 68) which contained fairly strong promoter activity (PLF). Recombinant plasmids pCHFPE2 (promoter region of mpt was reconstructed by combining the PLF in the upstream of its own promoter) and pCHFPE3 (the promoter of mpt was replaced by PLF) were obtained. The extent of expression of mpt was measured according to the amount of propionylspiramycin bioconverted from exogenous spiramycin by transformants of S. lividans TK24 containing pCHFPE2 and pCHFPE3. The results showed that the PLF could increase the expression of mpt in S. lividans TK24 up to 89.02% and 58.53%, respectively, and also enhance the expression in the industrial spiramycin producing strain S. spiramyceticus to a great extent.
Asunto(s)
Aciltransferasas/genética , Leucomicinas/biosíntesis , Regiones Promotoras Genéticas , Streptomyces/genética , Antibacterianos/biosíntesis , Cartilla de ADN , Expresión Génica , Reacción en Cadena de la Polimerasa , Streptomyces/metabolismoRESUMEN
A 3.2-kb DNA fragment of the carbomycin biosynthetic region including the 3-O-acyltransferase gene (acyA) from Streptomyces thermotolerans was sequenced, and four ORFs were found in the fragment. The second ORF, designated ORF-A, was transcribed in the opposite direction to the other three ORFs. The first ORF was identified as carA, a gene for carbomycin resistance. The amino acid sequence of ORF-A was homologous to proteins of the cytochrome P-450 family. Streptomyces lividans transformed with pCB20, in which ORF-A was subcloned, epoxidized carbomycin B at its C-12, 13 positions, thus producing carbomycin A. The third ORF, the amino acid sequence of which showed a homology to macrolide antibiotics O-acyltransferases was identified as acyA. The last ORF (ORF-B), which starts just 3 bp downstream from the TGA termination codon of acyA, was thought to be a carbomycin 4-O-methyltransferase gene, because the amino acid sequence deduced from ORF-B showed high homology to a putative midecamycin 4-O-methyltransferase encoded on mdmC.
Asunto(s)
Acetiltransferasas/genética , Proteínas Bacterianas , Leucomicinas/biosíntesis , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Carbohidratos , Clonación Molecular , ADN Bacteriano , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Streptomyces/enzimologíaRESUMEN
A DNA fragment containing promoter activity has been cloned from midecamycin producing strain (S. mycarofaciens 1748)., using promoter-probe plasmid vector pIJ486. The molecular size of this fragment was 2.3kb as shown by restriction analysis. A HindIII-HindIII 2.08KB DNA fragment obtained from the original fragment has been analysed by subcloning it into polylinker of vector pIJ486/7 which have opposite direction. The result showed that HindIII-HindIII 2.08kb DNA fragment has promoter activity in both direction. Transformants of plasmid containing this fragment in vector pIJ487 in S. lividans TK24 were resistant to Km in the level of 20mg/ml, but in vector pIJ 486 were resistant to the level of 3mg/ml. It indicated that a rather strong promoter activity region was in the HindIII/XbaI-HindIII direction. BamHI fragments (A-0.79kg, B-0.67kb, C-0.62kb) in 2.08kb DNA fragment have been studied in regards of their promoter activity. The result suggested that A-0.79kb region has the same promoter activity as in HindIII-HindIII 2.08kb DNA fragment.
Asunto(s)
ADN Bacteriano/genética , Regiones Promotoras Genéticas , Streptomyces/genética , Clonación Molecular , Leucomicinas/biosíntesis , Fragmentos de Péptidos/genética , Plásmidos , Streptomyces/clasificación , Streptomyces/metabolismoRESUMEN
This paper presents the results about the expression of the polyketide ketoreductase gene from midecamycin-producing strain (S. mycarofaciens 1748), gene localization and nucleotide sequences analysis. A BamHI-BamHI 4.0 kg fragment isolated from a genomic library of midecamycin-producing strains containing the actIII homologous DNA was inserted into E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCB4 was obtained and introduced into a 2-hydroxyaklavinone producer S. galilaeus ATCC 31671 that was a polyketide ketoreductase gene deficient mutant. The transformant produced aklavinone according to TLC and HPLC analyses. The BamHI-BamHI 4.0-kb fragment was inserted into pWHM3 in the reverted orientation with pCB4 and a recombinant plasmid pCBR4 was obtained. Introduction pCBR4 into S. galilaeus ATCC 31671 also resulted in the production of aklavinone. Thus we demonstrated that this gene encoded a polyketide ketoreductase which results in deoxygenation of 2-hydroxyaklavinone in C-2 position and that this gene has its own promoter. Restriction map analysis of pCB4 indicated that there were no sites for EcoRI and HindIII, but there were sites for BssHII, SalI, SphI and XhoI on the cloned gene fragment. The polyketide ketoreductase gene was located on a BssHII-BamHI 1.3-kb fragment from Southern hybridization result, using actIII gene as a probe, and that was confirmed by gene expression in S. galilaeus ATCC 31671. The nucleotide sequence analysis showed that the BssHII-BamHI 1.3-kb fragment contained an open reading frame (ORF). The protein coding region was assumed to start with an ATG start codon, end at an TAG stop codon. There was a 5nt (GGAGG) SD sequence at the upstream of start codon. A presumptive promoter consisted of 7 nt AACCGGA at the -10 region and 5 nt TTCGA at the -35 region. The deduced amino acid sequences of MPKR gene consisted of 261 aa residues. Its amino acid were compared with actIII gene coding protein sequences. The similarity was 77.4% and the identity was 66.7%.
Asunto(s)
Cetona Oxidorreductasas/genética , Streptomyces/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Vectores Genéticos , Cetona Oxidorreductasas/química , Leucomicinas/biosíntesis , Datos de Secuencia Molecular , Plásmidos , Análisis de Secuencia de ADN , Streptomyces/genéticaRESUMEN
It has been demonstrated that in the cellular proteins of Streptomyces hygroscopicus JA 6599 and Streptomyces noursei JA 3890 b, the producers of the antibiotics turimycin and nourseothricin, respectively, phosphorylated proteins are present. Numbers and concentrations of phosphorylated proteins decreased during the idiophase as characterized by phosphate limitation, antibiotic biosynthesis and phosphatase formation. Phosphoamino acids of serine, threonine and tyrosine were found in the hydrolysates of proteins. Protein tyrosyl kinase was demonstrated in the cellular extracts. The results supports the hypothesis that protein phosphorylation possesses a function in the regulation of growth and secondary product formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Leucomicinas/biosíntesis , Organofosfatos/metabolismo , Streptomyces/metabolismo , Estreptotricinas/biosíntesis , Electroforesis en Gel Bidimensional , Fosforilación , Streptomyces/crecimiento & desarrolloRESUMEN
The Streptomyces mycarofaciens mdmB gene encodes a 3-O-acyltransferase that catalyzes the addition of acetyl and propionyl groups to position 3 of the lactone ring in 16-member macrolide antibiotics like midecamycin and spiramycin. A putative O-methyltransferase gene (mdmC) is immediately downstream of mdmB, and both of these genes are closely linked to the mdmA midecamycin resistance gene.
Asunto(s)
Acetiltransferasas/genética , Genes Bacterianos , Leucomicinas/biosíntesis , Streptomyces/genética , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
A stable mutant No. 68 was obtained by treatment of S. mycarofaciens 1748 spores at high temperature. The electromicroscopic examination has shown that the mutant No. 68 and parent strain 1748 both have the spore chains of the spiratype. The spores of both strain are cylindrical in shape. The only difference is that the spores of the mutant No. 68 are of smooth surface, but the 1748 are of thorny. The physiological characteristics of both strains are also very similar with slight differences in utilization of few carbon sources and in cultural characters in few medium. Feeding experiment has shown that the mutant No. 68 was blocked in the formation of the macrolide lactone in the midecamycin biosynthetic pathway. This suggested that the mutant No. 68 might be a polyketide synthase genes deficient mutant. The ability of the mutant No. 68 to convert spiramycin into 4"-propionylspiramycin indicated that the mutant No. 68 contained the midecamycin 4"-propionyltransferase and could be used for microbial bioconversion of spiramycin into 4"-propionylspiramycin.
Asunto(s)
Leucomicinas/biosíntesis , Streptomyces/aislamiento & purificación , Mutación , Espiramicina/metabolismo , Streptomyces/genética , Streptomyces/fisiologíaRESUMEN
A 2.4 kb fragment containing the midecamycin polyketide synthase genes (mps) was subcloned from the preliminary clone pCN8B12 out of the genomic library of midecamycin-producing strain S. mycarofaciens 1748, by using the homologous DNA of the actinorhodin polyketide synthase gene (act I) as hybridization probe. This DNA fragment was subcloned onto Streptomyces/E. coli shuttle vector pMHM3. A recombinant plasmid pCG2 was obtained. The transformation of the polyketide synthase deficient mutant of actinorhodin-producing strain, S. colicolor TK17, with pCG2 DNA resulted in the production of an antibacterial compound which was similar neither to actinorhodin nor to midecamycin. The transformation of spiramycin-producing strain S. ambofaciens with pCG2 DNA increased spiramycin production in the fermentation broth. The transformation of the regulatory mutant of daunorubicin-producing strain with pCG2 DNA resulted in the production of epsilon-rhodomycinone verified by TLC and HPLC analyses. The pCG2 DNA also could be functionally expressed in tetracenomycin C-producing strain S. glaucescens. However, it could not be expressed in the blocked mutants of erythromycin-producing strain Saccharopolyspora erythrarea WMH 15,261. These suggest that the pCG2 DNA may complement polyketide synthase gene deficiency or have some regulatory function in certain polyketide antibiotic-producing strains.
Asunto(s)
Genes Bacterianos/genética , Leucomicinas/biosíntesis , Complejos Multienzimáticos/genética , Streptomyces/genética , Clonación Molecular , ADN Bacteriano/análisis , Vectores Genéticos , Complejos Multienzimáticos/biosíntesis , Plásmidos/genética , Proteínas Recombinantes/biosíntesisRESUMEN
Antibiotics 6108 A1, B, C and D, a new series of analogues of rosaramicin, were found together with rosaramicin, juvenimicin A4 and M-4365 A1 from the cultured broth of strain BA06108 which was assigned to be a new species of Micromonospora. 6108 A1 and C showed inhibitory activity against Gram-positive and some Gram-negative bacteria as potent as rosaramicin and exhibited low acute toxicity in mice. However, 6108 B showed less potent antimicrobial activity and 6108 D showed higher toxicity than those two antibiotics.