WO2000008168A1 - Improvements to strains of streptomyces through the use of biosynthetic genes of tylosine - Google Patents

Improvements to strains of streptomyces through the use of biosynthetic genes of tylosine Download PDF

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WO2000008168A1
WO2000008168A1 PCT/ES1999/000248 ES9900248W WO0008168A1 WO 2000008168 A1 WO2000008168 A1 WO 2000008168A1 ES 9900248 W ES9900248 W ES 9900248W WO 0008168 A1 WO0008168 A1 WO 0008168A1
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seq
gene sequence
encoded product
tylosin
fradiae
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French (fr)
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Roberto Fouces Martinez
Encarnación MELLADO DURAN
Bruno Diez Garcia
Manuel Esteban Morales
Ermanno Bernasconi
José Luis BARREDO FUENTE
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Antibioticos, S.A.U.
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Priority to AU52911/99A priority Critical patent/AU5291199A/en
Publication of WO2000008168A1 publication Critical patent/WO2000008168A1/en

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Definitions

  • the present invention relates to a method based on the use of genes of the tylosin biosynthetic cluster for increasing the production of tylosin in S. fradiae or for the production of new hybrid metabolites in related microorganisms ⁇ Streptomyces spp. ). It describes: (I) a method to isolate a DNA fragment of S. fradiae containing 11 genes, 10 of them belonging to the cluster of biosynthetic genes of tylosin and (II) the expression of said genes in the microorganism producing tylosin S. fradiae, as well as in other related microorganisms ⁇ Streptomyces spp. ) achieving an increase in tylosin production in the first case and the production of new hybrid antibiotics in the second.
  • Tylosin is a macrolide antibiotic composed of a 16-member macrocyclic lactone or ring to which 3 modified sugars bind: micarous, micaminous and microsane.
  • microorganisms capable of producing tylosin are the aforementioned S. fradiae (Seno, ET et ai., 1977. Antimicrob. Agents. Chemother. 21, 758), S. rimosus (Pape, H. and Brillinger, RH 1973. Arch. Microbiol. 88, 25-35) and S. hygroscopicus (Jensen, A. L. et al., 1964. In: Antimicrobial agents and chemotherapy-1963, JC Sylvester ed., American Society for Microbiology, Washington D. C). For the industrial production of tylosin S is used. fradiae.
  • Tylosin biosynthesis begins with the formation of the macrocyclic ring, by a process similar to that of fatty acid biosynthesis, but which differs from the latter in the great variety of isolated precursors that can be incorporated and in the partial or total absence of reactions that reduce the ⁇ -ketone group formed after each chain extension.
  • the macrocyclic ring is composed of 2 acetates, 5 propionates and a butyrate.
  • the enzymatic activity responsible for synthesizing it, called polyketide synthase is encoded by the tylG gene.
  • Said reactions are as follows: (I) addition of micaminose in the 5-OH position of the macrolide ring to form O-micamino-siltilactone; (II) oxidation of the methyl group in C-20 to form 20-dihydro-23-deoxy-O-mycaminosyllactone; (III) oxidation of the hydroxymethyl in C-20 to formyl group to produce 23-deoxy-O-micaminosiltilactone; (IV) oxidation of methyl group in C-23 to hydroxymethyl to form a 0-mycosylamino-methylonolide; (V) addition of 6-deoxyalose to the C-23 OH group of the macrolide ring to form demethyllactone; (VI) addition of micarose to 4'OH of the micaminose to form demethyl-macrocin; (VII) methylation of 2 '' OH of 6-deoxyalose to form macrocin and (VIII) methylation of 3 ''OH of 6-de
  • the technique called combinatorial biosynthesis, has allowed the biosynthesis, among other compounds, of erythromycin analogues and peptide antibiotics, of spiramycin and tylosin derivatives, as well as of numerous aromatic and reduced metabolites by manipulating genes that code for polyketide synthases (Madduri, K. et al. 1998. Nature Biotechnology 16: 69-74).
  • This new approach has been used for the production of isovaleryl spiramycin by Streptomyces ambofaciens strains in which the StEptomyces thermotolerans carE gene has been expressed, which codes for the deoxy hexose O-acyltransferase enzymatic activity.
  • This enzyme causes the conversion of the 4 '' -hydroxyl group of the spicamycin micarous residue into its isovaleryl ester (Epp, JK et al. 1989. Gene 85: 293-301).
  • the production of the antitumor compound 4 '-epidoxorubicin has been described in a strain of Streptomyces peucetius in which the dnmV gene has been inactivated and the avrE and / or eryBIV genes of Streptomyces avermi tilis and Saccharopolyspora erythraea respectively (Madduri) have been introduced , K. et al. 1998. Nature Biotechnology 16: 69-74).
  • tylosin biosynthetic genes described in the present invention into microorganisms producing macrolide or related antibiotics can be used for the production of new hybrid antibiotics.
  • the genes involved in the biosynthesis of the sugars included in the tylosin molecule could be introduced into microorganisms producing macrolide antibiotics to achieve biosynthesize hybrid antibiotics.
  • genes responsible for the myosin biosynthesis expressed in an erythromycin-producing microorganism will enable the production of erythromycin-derived mycinosil.
  • fradiae ATCC 19609 both as a source of deoxyribonucleic acid (hereinafter referred to as DNA) and ribonucleic acid (hereinafter referred to as RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • fradiae ATCC 19609 a library was constructed in the lambda-GEM12 (Promega) phage vector according to standard procedures (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press).
  • Plasmid pALFIA Figure 2 was constructed by subcloning an 11.5 kb Sacl fragment into pBluescript I KS (+). Likewise, a PvuII fragment was purified, which included both the recombinant phage insert and a part belonging to the lambda-GEM12 vector arm.
  • Plasmid pALFIA was propagated in E. coli strain DH5 ⁇ in order to obtain, by means of the alkaline lysis procedure (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press), sufficient quantity of DNA for successive processes.
  • the location of the tylF gene was determined by hybridization by the Southern technique of the above-mentioned synthetic oligonucleotide (5 ' GCTCGATGTAGAGATCG 3') with digestions of the plasmid pALFIA with a series of endo- restriction nucleases.
  • the tylF gene was identified in a 5.7 kb BamHI fragment and in another 4.1 kb BglII-.Ba.7iHI.
  • a preliminary restriction map of the area was prepared ( Figure 2), which was used as the basis for the sequencing process.
  • the determination of the nucleotide sequence (example 1) of a fragment of 12,905 base pairs (hereinafter referred to as bp) showed the existence of 11 open reading frames (hereinafter referred to as ORF), of which 10 belong to the cluster tylosin biosynthetic.
  • ORF 11 open reading frames
  • Plasmids, pALF287 or pALFIB, which contain a series of tylosin biosynthetic genes, were introduced into the S. fradiae ATCC 19609 collection strain and into the tylosin S superproductive strains. fradiae 302 and S. fradiae 455, achieving a decrease in the accumulated macrocin levels and an increase in the level of tylosin production with respect to parental strains.
  • Plasmid pALF250 was also constructed ( Figure 5), which is formed by pULVK99 plus a DNA fragment of approximately 1.4 kb containing the trlB gene, a determinant of tylosin resistance of S. fradiae.
  • pALF250 was introduced by transformation into S host cells. lividans ATCC 1326 and S. parvulus DSM 40048, using the tylosin resistance marker as a selective method to maintain plasmid pALF250 autonomously in said host. The transformants of S.
  • Carrier lividans of said plasmid were selected using tylosin concentrations of 250 to 500 ⁇ g / ml, although some of them were capable of growing in concentrations of 3.5 mg / ml, the minimum inhibitory concentration of tylosin being for S. untransformed lividans of 200 ⁇ g / ml.
  • the transformants of S. Parvulus were able to grow in concentrations of 200 ⁇ g / ml of tylosin, the minimum inhibition concentration of tylosin being for S. untransformed parvulus of 100 ⁇ g / ml.
  • This new resistance marker is very useful, since the number of selectable genetic markers in Streptomyces spp. It is not very broad and the use of the tlrB gene expands that restricted field.
  • Plasmids pALF17 and pALF18 were subdivided into smaller fragments using the 2 Ba ⁇ nHI restriction sites and the unique Sacl site. The resulting fragments were subcloned into the pBluescript I KS (+) vector giving rise to the following plasmids: pALF71, pALF72, pALF73, pALF74, pALF76 and pALF77 ( Figure 2).
  • plasmid pALF32 was double digested with the enzymes BamHI and BglII, the fragments obtained (4.1 kb and 165 bp) were subcloned into pBluescript II KS (+).
  • the plasmids obtained were called pALF2 and pALFIO (4.1 kb fragment) and pALF21 (165 bp fragment) ( Figure 2).
  • pALFl ⁇ and pALF267 were obtained. The first covers the BamHI restriction site that separates the 4.1 kb BglI l-BamUl fragment and the 1 kb BamHI fragment and the second does the same on the BamHI site that separates the latter from the 2.1 kb BamHI fragment (figure 2).
  • ORFs correspond to the following genes: 0RF1 with the ddcA gene, ORF2 with the tlrB gene, ORF3 with the tylN gene, ORF4 with the tylE gene, ORF5 with the tylD gene, ORF6 with the tylH2 gene, ORF7 with the tylHl gene, ORF8 with the tylF gene, ORF9 with the tylO gene, ORF10 with the tylP gene and ORF11 with the tylQ gene.
  • the preparation of S protoplasts Fradiae was performed by inoculating 50 ml of TSB medium supplemented with 0.4% glycine and 5 mM MgCl 2 , in a 500 ml flask, with a spore suspension of the strain to be transformed. The microorganism was grown at 30 ° C and 250 rpm for 48 hours. After that time, the mycelium was collected by centrifugation at 3,000 rpm for 7 minutes at temperature Ambience in two Sorvall SS34 tubes. The sediment was washed with 25 ml of 10% sucrose, centrifuging under the above conditions.
  • the sediment from 25 ml of culture was resuspended in 5 ml of buffer P containing 1 mg / ml lysozyme and 0.4 mm diameter glass beads were added.
  • the rest of the culture can be frozen at -20 ° C, resuspended in 25 ml of 10% sucrose, to be used in subsequent transformations.
  • the mycelium resuspended in buffer P was incubated at 19 ° C by periodically shaking the tube to homogenize the mycelium with the glass balls. The incubation was prolonged until it was observed under the microscope that a drop of mycelium suspension was completely smooth by adding a drop of a 10% SDS solution.
  • the transformation process began by defrosting one of the aliquots of the protoplast suspension in a 37 ° C bath. Next, 1 ⁇ g of the DNA we wanted to introduce was added and mixed by gentle agitation of the tube, immediately adding 500 ⁇ l of 50% PEG dissolved in P buffer. Next, the contents of the tube were mixed by rapid inversion and the transformation mixture was incubated for 3-5 minutes at room temperature. Subsequently, 5 ml of soft R5 (R5 medium containing 0.6-1% agar) were plated, with the transformed protoplasts (protoplasts, DNA and PEG), in 1-2 Petri dishes of R5 medium.
  • the transforming strains obtained by the procedure described in Example 2 were fermented according to previously described protocols (Baltz RH and Seno ET, 1981, Antimicrobial Agents and Chemotherapy 20: 214-225; Cox KL et al. 1994. European Patent Specification 0238323B1), adding thiostreptone (antibiotic to which the plasmid confers resistance) at a concentration of 5 ⁇ g / ml.
  • the transformants corresponding to plasmids pALFIB (figures 2 and 3) and pALF287 (figures 2 and 4) showed significant increases (10-50% depending on the host strain) in tylosin production when compared with the parental strain. Also, because both plasmids include the tylF gene, the transformants showed a drastic decrease (25-50 times) in the accumulation of the macrocin precursor in fermentation broths.
  • FIGURE 1 S genome region. fradiae that includes the grouping of biosynthetic genes of tylosin.
  • FIGURE 2 Restriction map of the S. fradiae genome region that includes the clustering of tylosin biosynthetic genes.
  • the different constructed plasmids are shown at the bottom.
  • the insert corresponding to plasmids pALFIA and pALF2A consist of fragments of 11.5 kb Sacl and 16 kb PvuII respectively, while the insert of plasmid pALFIB corresponds to a fragment of 12.9 kb BglII-BamHI.
  • the clones that appear with arrowheads are those used in the sequencing process.
  • FIGURE 3 Restriction map of the 20.8 kb plasmid pALFIB, which includes the grouping of biosynthetic tylosin genes described in the present invention.
  • FIGURE 4 Restriction map of the 13.5 kb plasmid pALF287, which includes part of the tylosin biosynthetic gene cluster described in the present invention.
  • FIGURE 5 Restriction map of the 9.2 kb plasmid pALF250, which includes the tyrosine resistance gene t rB described in the present invention.

Abstract

The invention relates to a process based on the use of genes of the biosynthetic cluster of tylosine to increase the production of tylosine in S. fradiae. The invention discloses (I) a method for isolating a DNA fragment of S.fradiae which contains 11 genes, 10 of which belong to the cluster of biosynthetic genes of tylosine and (II) the expression of said genes in the microorganism producing the tylosine S. fradiae, as well as in other related microorganisms (Streptomyces spp.) resulting in an increase of the production of tylosine in the first case and in the production of new hybrid antibiotics in the second case.

Description

MEJORA DE CEPAS DE STREPTOMYCES MEDIANTE LA UTILIZACIÓN DE GENES BIOSINTETICOS DE TILOSINAIMPROVEMENT OF STREPTOMYCES STRAPS BY USING TILOSINE BIOSYNTHETIC GENES
CAMPO DE LA INVENCIÓNFIELD OF THE INVENTION
La presente invención se refiere a un procedimiento basado en la utilización de genes del cluster biosintético de tilosina para el incremento de la producción de tilosina en S . fradiae o para la producción de nuevos metabolitos híbridos en microorganismos relacionados { Streptomyces spp . ) . En él se describe: (I) un método para aislar un fragmento de ADN de S. fradiae que contiene 11 genes, 10 de ellos pertenecientes al cluster de genes biosintéticos de tilosina y (II) la expresión de dichos genes en el microorganismo productor de tilosina S . fradiae, asi como en otros microorganismos relacionados { Streptomyces spp . ) consiguiendo un incremento en la producción de tilosina en el primer caso y la producción de nuevos antibióticos híbridos en el segundo.The present invention relates to a method based on the use of genes of the tylosin biosynthetic cluster for increasing the production of tylosin in S. fradiae or for the production of new hybrid metabolites in related microorganisms {Streptomyces spp. ). It describes: (I) a method to isolate a DNA fragment of S. fradiae containing 11 genes, 10 of them belonging to the cluster of biosynthetic genes of tylosin and (II) the expression of said genes in the microorganism producing tylosin S. fradiae, as well as in other related microorganisms {Streptomyces spp. ) achieving an increase in tylosin production in the first case and the production of new hybrid antibiotics in the second.
ESTADO DE LA TÉCNICASTATE OF THE TECHNIQUE
La tilosina es un antibiótico macrólido compuesto por una lactona o anillo macrociclico de 16 miembros al cual se unen 3 azúcares modificados: micarosa, micaminosa y micino- sa. Entre los microorganismos capaces de producir tilosina se hallan el ya mencionado S . fradiae (Seno, E.T. et ai., 1977. Antimicrob. Agents. Chemother. 21, 758), S . rimosus (Pape, H. and Brillinger, R. H. 1973. Arch. Microbiol. 88, 25-35) y S . hygroscopicus (Jensen, A. L . et al . , 1964. In: Antimicrobial agents and chemotherapy-1963, J. C. Sylvester ed., American Society for Microbiology, Washington D. C). Para la producción industrial de tilosina se emplea S . fradiae .Tylosin is a macrolide antibiotic composed of a 16-member macrocyclic lactone or ring to which 3 modified sugars bind: micarous, micaminous and microsane. Among the microorganisms capable of producing tylosin are the aforementioned S. fradiae (Seno, ET et ai., 1977. Antimicrob. Agents. Chemother. 21, 758), S. rimosus (Pape, H. and Brillinger, RH 1973. Arch. Microbiol. 88, 25-35) and S. hygroscopicus (Jensen, A. L. et al., 1964. In: Antimicrobial agents and chemotherapy-1963, JC Sylvester ed., American Society for Microbiology, Washington D. C). For the industrial production of tylosin S is used. fradiae.
El estudio de la ruta de biosintesis de tilosina ha determinado la existencia de al menos 13 loci implicados (Fishman, S. E. et al. 1987. Proc. Nati. Acad. Sci. USA 84, 8248-8252); Merson-Davies, L. A. and Cundliffe, E., 1994. Mol. Microbiol. 13(2), 349-355; Gandecha, A. R. and Cundliffe, E. 1997. Gene 184, 197-203), demostrándose además la localización de todos ellos en un agrupamiento o cluster genético que ocupa alrededor de 85 kilobases del genoma de S. fradiae .The study of the tylosin biosynthesis pathway has determined the existence of at least 13 involved loci (Fishman, S. E. et al. 1987. Proc. Nati. Acad. Sci. USA 84, 8248-8252); Merson-Davies, L.A. and Cundliffe, E., 1994. Mol. Microbiol 13 (2), 349-355; Gandecha, A. R. and Cundliffe, E. 1997. Gene 184, 197-203), also demonstrating the location of all of them in a genetic cluster or cluster that occupies about 85 kilobases of the S. fradiae genome.
La biosintesis de tilosina comienza por la formación del anillo macrociclico, mediante un proceso similar al de la biosintesis de ácidos grasos, pero que difiere de este último en la gran variedad de precursores aislados que pueden incorporarse y en la parcial o total ausencia de reacciones que reducen el grupo β-cetónico formado después de cada extensión de la cadena. El anillo macrociclico está compuesto por 2 acetatos, 5 propionatos y un butirato. La actividad enzimática encargada de sintetizarlo, denominada poliquétido sintasa, se halla codificada por el gen tylG. La secuencia de reacciones subsiguientes que conducen al producto final, asi como algunas de las enzimas que participan en dichos procesos han sido caracterizadas mediante idiotrofias, utilizando imitantes bloqueados en uno o más pasos de la ruta biosintética (Cox, K. L. et al . 1986. Journal of Natural Products 49(6), 971-980). Dichas reacciones son las siguientes: (I) adición de micaminosa en la posición 5-OH del anillo macrólido para formar O-micamino- siltilactona; (II) oxidación del grupo metilo en C-20 para formar 20-dihidro-23-deoxi-O-micaminosiltilactona; (III) oxidación del hidroximetil en C-20 a grupo formil para producir 23-deoxi-O-micaminosiltilactona; (IV) oxidación del grupo metilo en C-23 a hidroximetil para formar un 0- micaminosiltilonolido; (V) adición de 6-desoxialosa al grupo OH en C-23 del anillo macrólido para formar demetil- lactona; (VI) adición de micarosa al 4 ' OH de la micaminosa para formar demetil-macrocina; (VII) metilación del 2' 'OH de la 6-desoxialosa para formar macrocina y (VIII) metilación del 3' ''OH de la 6-desoxialosa para dar lugar a tilosina . Por otra parte, se ha observado que al fermentar industrialmente cepas superproductoras de tilosina desarrolladas por métodos convencionales de mutagénesis, junto con tilosina se acumulan una serie de compuestos precursores. Particularmente importantes son las cantidades detectadas de macrocina, compuesto precursor inmediato de tilosina. La actividad enzimática que interviene en este último paso biosintético se denomina macrocina-O-metiltransferasaTylosin biosynthesis begins with the formation of the macrocyclic ring, by a process similar to that of fatty acid biosynthesis, but which differs from the latter in the great variety of isolated precursors that can be incorporated and in the partial or total absence of reactions that reduce the β-ketone group formed after each chain extension. The macrocyclic ring is composed of 2 acetates, 5 propionates and a butyrate. The enzymatic activity responsible for synthesizing it, called polyketide synthase, is encoded by the tylG gene. The sequence of subsequent reactions leading to the final product, as well as some of the enzymes involved in these processes, have been characterized by idiotrophies, using imitators blocked in one or more steps of the biosynthetic pathway (Cox, KL et al. 1986. Journal of Natural Products 49 (6), 971-980). Said reactions are as follows: (I) addition of micaminose in the 5-OH position of the macrolide ring to form O-micamino-siltilactone; (II) oxidation of the methyl group in C-20 to form 20-dihydro-23-deoxy-O-mycaminosyllactone; (III) oxidation of the hydroxymethyl in C-20 to formyl group to produce 23-deoxy-O-micaminosiltilactone; (IV) oxidation of methyl group in C-23 to hydroxymethyl to form a 0-mycosylamino-methylonolide; (V) addition of 6-deoxyalose to the C-23 OH group of the macrolide ring to form demethyllactone; (VI) addition of micarose to 4'OH of the micaminose to form demethyl-macrocin; (VII) methylation of 2 '' OH of 6-deoxyalose to form macrocin and (VIII) methylation of 3 '''OH of 6-deoxyalose to give rise to tylosin. On the other hand, it has been observed that when industrially fermenting tylosin-producing strains developed by conventional methods of mutagenesis, a series of precursor compounds accumulate together with tylosin. Particularly important are the detected amounts of macrocin, an immediate precursor compound of tylosin. The enzymatic activity involved in this last biosynthetic step is called macrocina-O-methyltransferase
(MOMT) y está codificada por el gen tylF. La introducción de copias adicionales del gen tylF en S . fradiae origina una reducción de los niveles de macrocina en los caldos de fermentación (Seno, E. T. and Baltz, R. H. 1982. Antimicrob. Agents Chemother. 21, 758-763). De igual modo, la introducción de copias adicionales de genes implicados en la biosintesis de tilosina podria contribuir a un incremento en los niveles de producción del antibiótico. Durante los últimos 50 años, el descubrimiento de nuevos antibióticos para el tratamiento de enfermedades en humanos, animales o plantas se ha conseguido fundamentalmente mediante el aislamiento de nuevos microorganismos productores. Sin embargo, desde finales de la década de los 70, el ritmo de descubrimiento de nuevos compuestos ha decaído notablemente. Este hecho, junto con el rápido incremento en la aparición de microorganismos patógenos resistentes a algunos de los antibióticos tradicionalmente em- pleados, ha obligado a diseñar vias alternativas para el desarrollo de nuevos antibióticos. Una de dichas vias consiste en la biosintesis de nuevos antibióticos mediante la manipulación, por técnicas de ADN recombinante, de genes del metabolismo secundario implicados en la biosintesis de antibióticos. Este nuevo campo de experimentación está permitiendo la obtención de nuevos compuestos que mejoran las propiedades de los antibióticos tradicionalmente conocidos, aumentando su espectro de acción y mejorando tanto la efectividad como la tolerancia de los mismos.(MOMT) and is encoded by the tylF gene. The introduction of additional copies of the tylF gene in S. fradiae causes a reduction in macrocin levels in fermentation broths (Seno, ET and Baltz, RH 1982. Antimicrob. Agents Chemother. 21, 758-763). Similarly, the introduction of additional copies of genes involved in tylosin biosynthesis could contribute to an increase in antibiotic production levels. During the last 50 years, the discovery of new antibiotics for the treatment of diseases in humans, animals or plants has been achieved primarily by the isolation of new producing microorganisms. However, since the late 1970s, the pace of discovery of new compounds has declined markedly. This fact, together with the rapid increase in the appearance of pathogenic microorganisms resistant to some of the traditionally used antibiotics pleados, has forced to design alternative ways for the development of new antibiotics. One such route consists in the biosynthesis of new antibiotics by manipulating, by recombinant DNA techniques, secondary metabolism genes involved in antibiotic biosynthesis. This new field of experimentation is allowing the obtaining of new compounds that improve the properties of traditionally known antibiotics, increasing their spectrum of action and improving both their effectiveness and tolerance.
La técnica, denominada biosintesis combinatoria, ha permitido la biosintesis, entre otros compuestos, de análogos de eritromicina y antibióticos peptidicos, de deri- vados de espiramicina y tilosina, asi como de numerosos metabolitos aromáticos y reducidos mediante la manipulación de genes que codifican para poliquétido sintasas (Madduri, K. et al. 1998. Nature Biotechnology 16: 69-74). Este nuevo enfoque ha sido utilizado para la producción de isovaleril espiramicina por cepas de Streptomyces ambofaciens en las que se ha expresado el gen carE de Streptomyces thermotole- rans, el cual codifica para la actividad enzimática desoxi- hexosa O-aciltransferasa. Esta enzima provoca la conversión del grupo 4 ' ' -hidroxilo del residuo micarosa de la espira- micina en su isovaleril éster (Epp, J. K. et al. 1989. Gene 85: 293-301). Asimismo, la producción del compuesto antitumoral 4 '-epidoxorubicina ha sido descrita en una cepa de Streptomyces peucetius en la que se ha inactivado el gen dnmV y se han introducido los genes avrE y/o eryBIV de Streptomyces avermi tilis y Saccharopolyspora erythraea respectivamente (Madduri, K. et al. 1998. Nature Biotechnology 16: 69-74). La introducción de los genes biosintéticos de tilosina descritos en la presente invención en microorganismos productores de antibióticos macrólidos o relacionados, puede ser usada para la producción de nuevos antibióticos híbridos. De este modo, los genes implicados en la biosintesis de los azúcares incluidos en la molécula de tilosina (micarosa, micaminosa y micinosa) podrían ser introducidos en microorganismos productores de antibióticos macrólidos para conseguir biosintetizar antibióticos híbridos. Más concretamente, genes responsables de la biosintesis de micinosa expresados en un microorganismo productor de eri- tromicina posibilitarán la producción de micinosil derivados de eritromicina.The technique, called combinatorial biosynthesis, has allowed the biosynthesis, among other compounds, of erythromycin analogues and peptide antibiotics, of spiramycin and tylosin derivatives, as well as of numerous aromatic and reduced metabolites by manipulating genes that code for polyketide synthases (Madduri, K. et al. 1998. Nature Biotechnology 16: 69-74). This new approach has been used for the production of isovaleryl spiramycin by Streptomyces ambofaciens strains in which the StEptomyces thermotolerans carE gene has been expressed, which codes for the deoxy hexose O-acyltransferase enzymatic activity. This enzyme causes the conversion of the 4 '' -hydroxyl group of the spicamycin micarous residue into its isovaleryl ester (Epp, JK et al. 1989. Gene 85: 293-301). Likewise, the production of the antitumor compound 4 '-epidoxorubicin has been described in a strain of Streptomyces peucetius in which the dnmV gene has been inactivated and the avrE and / or eryBIV genes of Streptomyces avermi tilis and Saccharopolyspora erythraea respectively (Madduri) have been introduced , K. et al. 1998. Nature Biotechnology 16: 69-74). The introduction of the tylosin biosynthetic genes described in the present invention into microorganisms producing macrolide or related antibiotics can be used for the production of new hybrid antibiotics. In this way, the genes involved in the biosynthesis of the sugars included in the tylosin molecule (micarous, micaminous and myxinose) could be introduced into microorganisms producing macrolide antibiotics to achieve biosynthesize hybrid antibiotics. More specifically, genes responsible for the myosin biosynthesis expressed in an erythromycin-producing microorganism will enable the production of erythromycin-derived mycinosil.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNDETAILED DESCRIPTION OF THE INVENTION
En la presente invención se utiliza el actinomiceto S . fradiae ATCC 19609 tanto como fuente de ácido desoxirribo- nucleico (en lo sucesivo referido como ADN) como de ácido ribonucleico (en lo sucesivo referido como ARN) . Con la finalidad de clonar y caracterizar el extremo izquierdo del agrupamiento de genes biosintéticos de tilosina de S . fradiae ATCC 19609, se construyó una genoteca en el vector fágico lambda-GEM12 (Promega) de acuerdo con procedimientos standard (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press) . Para ello se purificaron a través de un gradiente de sacarosa fragmentos de ADN de 17-20 kb procedentes de diges- tiones parciales Sa u3AI . Dichos fragmentos se ligaron a los brazos del fago lambda-GEM12 obtenidos mediante digestión BamHI y purificados a través de un gradiente de sacarosa. Las reacciones de ligación se encapsidaron con el kit Giga- pack II Gold (Stratagene) y seguidamente se infectaron en E. coli NM539 obteniendo alrededor de 35.000 ufps. El rastreo de la genoteca se realizó con un oligonucleótido sintético (5'GCTCGATGTAGAGATCG 3') diseñado en función de la secuencia nucleotidica del gen tylF previamente descrita (Fishman, S. E. et al. 1987. Proc. Nati. Acad. Sci. USA 84, 8248-8252; Cox et al. 1987. European Patent Specification 0238323B1). De esta forma se consiguieron purificar una serie de clones portadores de un inserto que hibridaba específicamente con el oligonucleótido anteriormente mencionado. El plásmido pALFIA (figura 2) se construyó mediante subclonación de un fragmento Sacl de 11,5 kb en pBluescript I KS (+ ) . Asimismo, se purificó un fragmento PvuII, el cual incluía tanto inserto del fago recombinante como una parte perteneciente al brazo del vector lambda- GEM12. Una vez eliminada la parte correspondiente a lamda- GEM12, se subclonó el fragmento resultante (aproximadamente 16 kb) en pBluescript II SK ( +) dando lugar al plásmido pALF2A (figura 2) . La cepa E . coli DH5α portadora del plásmido pALF2A ha sido depositada con fecha 19.06.1998 en la Colección Española de Cultivos Tipo (CECT) , Universidad de Valencia, Campus de Burjasot, 46100 BURJASOT (Valencia) con el número de acceso CECT 5060.Actinomycete S is used in the present invention. fradiae ATCC 19609 both as a source of deoxyribonucleic acid (hereinafter referred to as DNA) and ribonucleic acid (hereinafter referred to as RNA). In order to clone and characterize the left end of the cluster of biosynthetic genes of S tylosin. fradiae ATCC 19609, a library was constructed in the lambda-GEM12 (Promega) phage vector according to standard procedures (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press). To do this, 17-20 kb DNA fragments from partial digestions Sa u3AI were purified through a sucrose gradient. These fragments were ligated to the lambda-GEM12 phage arms obtained by BamHI digestion and purified through a sucrose gradient. The ligation reactions were encapsidated with the Giga- kit Pack II Gold (Stratagene) and then became infected in E. coli NM539, obtaining around 35,000 pfu. The screening of the library was performed with a synthetic oligonucleotide (5'GCTCGATGTAGAGATCG 3 ') designed based on the nucleotide sequence of the previously described tylF gene (Fishman, SE et al. 1987. Proc. Nati. Acad. Sci. USA 84, 8248-8252; Cox et al. 1987. European Patent Specification 0238323B1). In this way, a series of clones carrying an insert that specifically hybridized with the aforementioned oligonucleotide were purified. Plasmid pALFIA (Figure 2) was constructed by subcloning an 11.5 kb Sacl fragment into pBluescript I KS (+). Likewise, a PvuII fragment was purified, which included both the recombinant phage insert and a part belonging to the lambda-GEM12 vector arm. Once the part corresponding to lamda-GEM12 was removed, the resulting fragment (approximately 16 kb) was subcloned into pBluescript II SK (+) giving rise to plasmid pALF2A (Figure 2). The strain E. coli DH5α carrying plasmid pALF2A has been deposited on 19.06.1998 in the Spanish Type Culture Collection (CECT), University of Valencia, Burjasot Campus, 46100 BURJASOT (Valencia) with the access number CECT 5060.
El plásmido pALFIA se propagó en la cepa E. coli DH5α con objeto de obtener, mediante el procedimiento de lisis alcalina (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press) , suficiente cantidad de ADN para los procesos sucesivos. La localización del gen tylF se determinó mediante hibridación por la técnica de Southern del oligonucleótido sintético anteriormente mencionado (5 ' GCTCGATGTAGAGATCG 3') con digestiones del plásmido pALFIA con una serie de endo- nucleasas de restricción. El gen tylF se identificó en un fragmento BamHI de 5,7 kb y en otro BglII-.Ba.7iHI de 4,1 kb. Asimismo, se elaboró un mapa de restricción preliminar de la zona (figura 2), el cual se utilizó como base para el proceso de secuenciación. La determinación de la secuencia de nucleótidos (ejemplo 1) de un fragmento de 12.905 pares de bases (en adelante referido como pb) mostró la existencia de 11 marcos abiertos de lectura (en adelante referidos como ORF) , de los cuales 10 pertenecen al cluster biosintético de tilosina. A continuación se abordó la tarea de construir plásmidos recombinantes que incluyen uno o varios de los genes comprendidos en esa zona, utilizando el plásmido bifuncional E . coli - Streptomyces spp . denominado pULVK99 (Chary VK, et al . 1997. Appl Environ Microbiol 63: 2977-2982) . Mediante digestión del DNA correspondiente a los fagos recombinantes previamente purificados con las endonucleasas de restricción Sacl, BglII, BamHI, PvuII o Xhol se obtuvieron fragmentos de ADN que fueron subclonados en el vector pULVK99. De esta forma se obtuvo el plásmido pALFIB, el cual incluye la región secuenciada completa (figuras 2 y 3) . Tomando como base el mapa de restricción previamente elaborado, el fragmento BamHI de 5,7 kb fue sub- clonado en el vector pULVK99 dando lugar al plásmido pALF287 (figuras 2 y 4) . Los plásmidos, pALF287 ó pALFIB, los cuales contienen una serie de genes biosintéticos de tilosina, se introdujeron en la cepa de colección S. fradiae ATCC 19609 y en las cepas superproductoras de tilosina S . fradiae 302 y S . fradiae 455, consiguiéndose una disminución en los niveles acumulados de macrocina y un incremento en el nivel de producción de tilosina respecto de las cepas parentales.Plasmid pALFIA was propagated in E. coli strain DH5α in order to obtain, by means of the alkaline lysis procedure (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press), sufficient quantity of DNA for successive processes. The location of the tylF gene was determined by hybridization by the Southern technique of the above-mentioned synthetic oligonucleotide (5 ' GCTCGATGTAGAGATCG 3') with digestions of the plasmid pALFIA with a series of endo- restriction nucleases. The tylF gene was identified in a 5.7 kb BamHI fragment and in another 4.1 kb BglII-.Ba.7iHI. Likewise, a preliminary restriction map of the area was prepared (Figure 2), which was used as the basis for the sequencing process. The determination of the nucleotide sequence (example 1) of a fragment of 12,905 base pairs (hereinafter referred to as bp) showed the existence of 11 open reading frames (hereinafter referred to as ORF), of which 10 belong to the cluster tylosin biosynthetic. Next, the task of constructing recombinant plasmids that include one or more of the genes included in that area, using bifunctional plasmid E, was discussed. coli - Streptomyces spp. denominated pULVK99 (Chary VK, et al. 1997. Appl Environ Microbiol 63: 2977-2982). By digesting the DNA corresponding to the recombinant phages previously purified with the restriction endonucleases Sacl, BglII, BamHI, PvuII or Xhol, DNA fragments were obtained that were subcloned into the vector pULVK99. In this way the plasmid pALFIB was obtained, which includes the entire sequenced region (Figures 2 and 3). Based on the previously developed restriction map, the 5.7 kb BamHI fragment was subcloned into the vector pULVK99 giving rise to plasmid pALF287 (Figures 2 and 4). Plasmids, pALF287 or pALFIB, which contain a series of tylosin biosynthetic genes, were introduced into the S. fradiae ATCC 19609 collection strain and into the tylosin S superproductive strains. fradiae 302 and S. fradiae 455, achieving a decrease in the accumulated macrocin levels and an increase in the level of tylosin production with respect to parental strains.
Asimismo se construyó el plásmido pALF250 (figura 5) , el cual esta formado por pULVK99 más un fragmento de ADN de aproximadamente 1,4 kb que contiene el gen trlB , determinante de resistencia a tilosina de S . fradiae . pALF250 se introdujo mediante transformación en células hospedantes de S . lividans ATCC 1326 y S. parvulus DSM 40048, utilizando el marcador de resistencia a tilosina como método selectivo para mantener el plásmido pALF250 autónomamente en dicho hospedador. Los transformantes de S . lividans portadores de dicho plásmido se seleccionaron empleando concentraciones de tilosina de 250 a 500 μg/ml, aunque algunos de ellos eran capaces de crecer en concentraciones de 3,5 mg/ml, siendo la concentración mínima inhibitoria de tilosina para S . lividans sin transformar de 200 μg/ml. Por su parte, los transformantes de S . parvulus eran capaces de crecer en concentraciones de 200 μg/ml de tilosina, siendo la concen- tración mínima inhibitoria de tilosina para S . parvulus sin transformar de 100 μg/ml. Este nuevo marcador de resistencia es de gran utilidad, ya que el número de marcadores genéticos seleccionables en Streptomyces spp . no es muy amplio y el empleo del gen tlrB expande ese restringido campo.Plasmid pALF250 was also constructed (Figure 5), which is formed by pULVK99 plus a DNA fragment of approximately 1.4 kb containing the trlB gene, a determinant of tylosin resistance of S. fradiae. pALF250 was introduced by transformation into S host cells. lividans ATCC 1326 and S. parvulus DSM 40048, using the tylosin resistance marker as a selective method to maintain plasmid pALF250 autonomously in said host. The transformants of S. Carrier lividans of said plasmid were selected using tylosin concentrations of 250 to 500 µg / ml, although some of them were capable of growing in concentrations of 3.5 mg / ml, the minimum inhibitory concentration of tylosin being for S. untransformed lividans of 200 μg / ml. On the other hand, the transformants of S. Parvulus were able to grow in concentrations of 200 μg / ml of tylosin, the minimum inhibition concentration of tylosin being for S. untransformed parvulus of 100 μg / ml. This new resistance marker is very useful, since the number of selectable genetic markers in Streptomyces spp. It is not very broad and the use of the tlrB gene expands that restricted field.
La presente invención se ilustra con más detalle en los ejemplos siguientes.The present invention is illustrated in more detail in the following examples.
EJEMPLO 1EXAMPLE 1
Clonación y secuenciación de un fragmento de ADN correspondiente al extremo izquierdo de cluster biosintético de tilosina de S. fradiae .Cloning and sequencing of a DNA fragment corresponding to the left end of the tyrosine biosynthetic cluster of S. fradiae.
Con la finalidad de secuenciar el extremo izquierdo del cluster de biosintesis de tilosina se subclonaron en los vectores pBluescript I KS (+) y pBC KS (+) los siguientes fragmentos de ADN (figura 2): (I) fragmento BglII de 5,5 kb que dio lugar a los plásmidos pALF17 y pALF18 (ambas orientaciones del mismo inserto); (II) fragmento BamHI de 5,7 kb que solapa con el anterior e incluye el gen tylF y que dio lugar a los plásmidos pALF32 y pALF33; (III) fragmento BamRI de 1 kb adyacente al anteriormente mencionado de 5,5 kb que dio lugar a los plásmidos pALF13 y pALF15; (IV) fragmento BamHI de 2,1 kb adyacente al anterior que dio origen a los plásmidos pALF14 y pALF20. Pósteriormente, los plásmidos pALF17 y pALF18 fueron subdivi- didos en fragmentos menores empleando los 2 sitios de restricción BaτnHI y el sitio único Sacl. Los fragmentos resultantes fueron subclonados en el vector pBluescript I KS (+) dando lugar a los plásmidos siguientes: pALF71, pALF72, pALF73, pALF74, pALF76 y pALF77 (figura 2) . Asimismo, el plásmido pALF32 fue digerido doblemente con las enzimas BamHI y BglII, los fragmentos obtenidos (de 4,1 kb y 165 pb) fueron subclonados en pBluescript II KS (+) . Los plásmidos obtenidos se denominaron pALF2 y pALFIO (fragmento de 4,1 kb) y pALF21 (fragmento de 165 pb) (figura 2) . Además, mediante el empleo de enzimas de restricción se obtuvieron dos nuevos plásmidos, pALFlδ y pALF267. El primero cubre el sitio de restricción BamHI que separa el fragmento BglI l-BamUl de 4,1 kb y el fragmento BamHI de 1 kb y el segundo hace lo propio sobre el sitio BamHI que separa este último del fragmento BamHI de 2,1 kb (figura 2) .With the purpose of sequencing the left end of the tylosin biosynthesis cluster, the following pBluescript I KS (+) and pBC KS (+) vectors were subcloned DNA fragments (Figure 2): (I) 5.5 kb BglII fragment that gave rise to plasmids pALF17 and pALF18 (both orientations of the same insert); (II) 5.7 kb BamHI fragment that overlaps with the previous one and includes the tylF gene and that gave rise to plasmids pALF32 and pALF33; (III) 1 kb BamRI fragment adjacent to the aforementioned 5.5 kb which gave rise to plasmids pALF13 and pALF15; (IV) 2.1 kb BamHI fragment adjacent to the previous one that gave rise to plasmids pALF14 and pALF20. Subsequently, plasmids pALF17 and pALF18 were subdivided into smaller fragments using the 2 BaτnHI restriction sites and the unique Sacl site. The resulting fragments were subcloned into the pBluescript I KS (+) vector giving rise to the following plasmids: pALF71, pALF72, pALF73, pALF74, pALF76 and pALF77 (Figure 2). Likewise, plasmid pALF32 was double digested with the enzymes BamHI and BglII, the fragments obtained (4.1 kb and 165 bp) were subcloned into pBluescript II KS (+). The plasmids obtained were called pALF2 and pALFIO (4.1 kb fragment) and pALF21 (165 bp fragment) (Figure 2). In addition, by using restriction enzymes, two new plasmids, pALFlδ and pALF267 were obtained. The first covers the BamHI restriction site that separates the 4.1 kb BglI l-BamUl fragment and the 1 kb BamHI fragment and the second does the same on the BamHI site that separates the latter from the 2.1 kb BamHI fragment (figure 2).
Los plásmidos mencionados anteriormente, se propagaron en E . coli DH5 y seguidamente se abordó la tarea de obte- ner clones de secuencia apropiados con el kit comercialThe plasmids mentioned above, propagated in E. coli DH5 and then the task of obtaining appropriate sequence clones with the commercial kit was discussed
"Erase a base" (Promega) , generando deleciones secuenciales de alrededor de 300 a 500 pb . Todos los plásmidos asi obtenidos fueron introducidos en E. coli WK6 con el fin de convertirlos en ADN de cadena sencilla con ayuda del fago M13K07 de acuerdo con técnicas standard (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press). Los plásmidos de cadena sencilla se emplearon como molde para las reacciones de se- cuenciación por el método de los didesoxinucleótidos (San- ger, F. et al. 1977. Proc. Nati. Acad. Sci. 74: 5463-5467) utilizando el kit comercial "Sequenase 2.0" (U.S. Bio- chemicals) y el isótopo 35S. El conjunto de todos los clones secuenciados se agrupó finalmente en una secuencia continua de ADN de 12.905 pb . (SEQ ID NO: 1 y su hebra complementaria SEG ID NO: 2) . Mediante su análisis con el programa GENEPLOT (DNASTAR), empleando un patrón de uso de codones apropiado para Strepto/nyces spp. , se detectó la presencia de 11 ORFs, los cuales se corresponden con los siguientes genes: 0RF1 con el gen ddcA, ORF2 con el gen tlrB, ORF3 con el gen tylN, ORF4 con el gen tylE, ORF5 con el gen tylD, ORF6 con el gen tylH2, ORF7 con el gen tylHl , ORF8 con el gen tylF, ORF9 con el gen tylO, ORF10 con el gen tylP y ORF11 con el gen tylQ."Erase a base" (Promega), generating sequential deletions of around 300 to 500 bp. All plasmids like that obtained were introduced in E. coli WK6 in order to convert them into single stranded DNA with the help of phage M13K07 according to standard techniques (Sambrook, J. et al. 1989. Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press ). Single chain plasmids were used as template for sequencing reactions by the dideoxynucleotide method (Sanger, F. et al. 1977. Proc. Nati. Acad. Sci. 74: 5463-5467) using the commercial kit "Sequenase 2.0" (US Biochemicals) and the 35 S isotope. The set of all sequenced clones was finally grouped into a continuous DNA sequence of 12,905 bp. (SEQ ID NO: 1 and its complementary thread SEG ID NO: 2). Through its analysis with the GENEPLOT (DNASTAR) program, using an appropriate codon usage pattern for Strepto / nyces spp. , the presence of 11 ORFs was detected, which correspond to the following genes: 0RF1 with the ddcA gene, ORF2 with the tlrB gene, ORF3 with the tylN gene, ORF4 with the tylE gene, ORF5 with the tylD gene, ORF6 with the tylH2 gene, ORF7 with the tylHl gene, ORF8 with the tylF gene, ORF9 with the tylO gene, ORF10 with the tylP gene and ORF11 with the tylQ gene.
EJEMPLO 2EXAMPLE 2
Transformación de S. fradiae .S. fradiae transformation.
La preparación de protoplastos de S . fradiae se realizó inoculando 50 mi de medio TSB suplementado con 0.4% de glicina y 5 mM de MgCl2, en un matraz de 500 mi, con una suspensión de esporas de la cepa a transformar. El microorganismo se creció a 30°C y 250 r.p.m. durante 48 horas. Transcurrido ese tiempo, el micelio se recogió por centrifugación a 3.000 r.p.m. durante 7 minutos a temperatura ambiente en dos tubos Sorvall SS34. El sedimento se lavó con 25 mi de sacarosa 10%, centrifugando en las condiciones anteriores. El sedimento procedente de 25 mi de cultivo se resuspendió en 5 mi de buffer P conteniendo 1 mg/ml de lisozima y se añadieron bolas de vidrio de 0.4 mm de diámetro. El resto del cultivo puede congelarse a -20°C, resuspendido en 25 mi de sacarosa 10%, para ser utilizado en posteriores transformaciones. El micelio resuspendido en el buffer P se incubó a 19°C agitando periódicamente el tubo para homogeneizar el micelio con las bolas de vidrio. La incubación se prolongó hasta que se observó al microscopio que una gota de suspensión del micelio se liso completamente al añadir una gota de una solución de SDS al 10%. Finalizada la formación de protoplastos, se añadieron 5 mi más de buffer P y se filtró toda la suspensión a través de un algodón estéril, recogiéndose los protoplastos en un nuevo tubo de centrifuga. El filtrado se centrifugó a 2.500 r.p.m. durante 15 minutos a temperatura ambiente para sedimentar los protoplastos y eliminar la solución de lisozima. El sedimento se resuspendió en la gota de buffer que quedó después de centrifugar. Este proceso se repitió resuspendiendo los protoplastos en 5 mi de buffer P y volviendo a centrifugar como en el punto anterior. Al eliminar el sobrenadante se resuspendieron los protoplastos en un volumen de buffer P determinado de acuerdo con el volumen del sedimento (nunca en más de 2 mi) . Posteriormente, se repartieron en alícuotas de 200 μl, las cuales se almacenaron congeladas a -80°C.The preparation of S protoplasts. Fradiae was performed by inoculating 50 ml of TSB medium supplemented with 0.4% glycine and 5 mM MgCl 2 , in a 500 ml flask, with a spore suspension of the strain to be transformed. The microorganism was grown at 30 ° C and 250 rpm for 48 hours. After that time, the mycelium was collected by centrifugation at 3,000 rpm for 7 minutes at temperature Ambience in two Sorvall SS34 tubes. The sediment was washed with 25 ml of 10% sucrose, centrifuging under the above conditions. The sediment from 25 ml of culture was resuspended in 5 ml of buffer P containing 1 mg / ml lysozyme and 0.4 mm diameter glass beads were added. The rest of the culture can be frozen at -20 ° C, resuspended in 25 ml of 10% sucrose, to be used in subsequent transformations. The mycelium resuspended in buffer P was incubated at 19 ° C by periodically shaking the tube to homogenize the mycelium with the glass balls. The incubation was prolonged until it was observed under the microscope that a drop of mycelium suspension was completely smooth by adding a drop of a 10% SDS solution. After the formation of protoplasts, 5 ml more of P buffer was added and the entire suspension was filtered through a sterile cotton, the protoplasts being collected in a new centrifuge tube. The filtrate was centrifuged at 2,500 rpm for 15 minutes at room temperature to sediment the protoplasts and remove the lysozyme solution. The sediment was resuspended in the drop of buffer that remained after centrifuging. This process was repeated by resuspending the protoplasts in 5 ml of P buffer and centrifuging again as in the previous point. Upon removal of the supernatant, the protoplasts were resuspended in a volume of buffer P determined according to the volume of the sediment (never more than 2 ml). Subsequently, they were distributed in 200 μl aliquots, which were stored frozen at -80 ° C.
El proceso de transformación comenzó por descongelar en un baño a 37°C una de las alícuotas de la suspensión de protoplastos. Seguidamente, se añadió 1 μg del ADN que pretendíamos introducir y se mezcló mediante agitación suave del tubo, añadiendo inmediatamente después 500 μl de PEG 50% disuelto en buffer P. Seguidamente, el contenido del tubo se mezcló mediante inversiones rápidas y la mezcla de transformación se incubó durante 3-5 minutos a temperatura ambiente. Posteriormente se plaquearon 5 mi de R5 soft (medio R5 conteniendo 0.6-1% de agar) , con los protoplastos transformados (protoplastos, ADN y PEG) , en 1-2 placas Petri de medio R5. Dichas placas se incubaron a 30°C durante 20-27 horas y seguidamente se añadieron a cada placa 3 mi de medio SNA (Hopwood, D.A. et al. 1985. Genetic manipulation of Streptomyces . A laboratoty Manual. The John Innes Foundation) conteniendo una concentración del antibiótico deseado del orden de 7 veces superior a la concentración final deseada en la placa (el antibiótico debe difun- dir por toda la placa, y por tanto, ha de tenerse en cuenta el volumen final que tendremos después de añadir el agar de selección) . En el caso de utilizar tiostreptona se añadió a una concentración final de 50 μg/ml, mientras que cuando se utilizó tilosina se añadió a una concentración final de 500 μg/ml para S . lividans y de 200 μg/ml para S . parvul us . Por último, se incubaron las placas a 30°C durante 5 a 8 dias tiempo suficiente para detectar la aparición de transformantes y seleccionar el fenotipo deseado.The transformation process began by defrosting one of the aliquots of the protoplast suspension in a 37 ° C bath. Next, 1 μg of the DNA we wanted to introduce was added and mixed by gentle agitation of the tube, immediately adding 500 μl of 50% PEG dissolved in P buffer. Next, the contents of the tube were mixed by rapid inversion and the transformation mixture was incubated for 3-5 minutes at room temperature. Subsequently, 5 ml of soft R5 (R5 medium containing 0.6-1% agar) were plated, with the transformed protoplasts (protoplasts, DNA and PEG), in 1-2 Petri dishes of R5 medium. Said plates were incubated at 30 ° C for 20-27 hours and then 3 ml of SNA medium (Hopwood, DA et al. 1985. Genetic manipulation of Streptomyces. A laboratoty Manual. The John Innes Foundation) containing one concentration of the desired antibiotic of the order of 7 times higher than the desired final concentration in the plaque (the antibiotic must spread throughout the plaque, and therefore, the final volume that we will have after adding the agar must be taken into account selection). In the case of using thiostreptone it was added to a final concentration of 50 μg / ml, while when tylosin was used it was added to a final concentration of 500 μg / ml for S. lividans and 200 μg / ml for S. parvul us. Finally, the plates were incubated at 30 ° C for 5 to 8 days long enough to detect the appearance of transformants and select the desired phenotype.
EJEMPLO 3EXAMPLE 3
Fermentación de S. fradiae .Fermentation of S. fradiae.
Las cepas transformantes obtenidas mediante el proce- dimiento descrito en el ejemplo 2 fueron fermentadas de acuerdo con protocolos previamente descritos (Baltz R.H. and Seno E.T., 1981, Antimicrobial Agents and Chemotherapy 20: 214-225; Cox K.L. et al. 1994. European Patent Specifi- cation 0238323B1) , adicionando tiostreptona (antibiótico al que confiere resistencia el plásmido) a una concentración de 5 μg/ml. Los transformantes correspondientes a los plás- midos pALFIB (figuras 2 y 3) y pALF287 (figuras 2 y 4) mostraron incrementos significativos (10-50% dependiendo de la cepa hospedadora) en la producción de tilosina cuando se compararon con la cepa parental. Asimismo, debido a que ambos plásmidos incluyen el gen tylF, los transformantes presentaban una drástica disminución (25-50 veces) en la acumulación del precursor macrocina en los caldos de fermentación. The transforming strains obtained by the procedure described in Example 2 were fermented according to previously described protocols (Baltz RH and Seno ET, 1981, Antimicrobial Agents and Chemotherapy 20: 214-225; Cox KL et al. 1994. European Patent Specification 0238323B1), adding thiostreptone (antibiotic to which the plasmid confers resistance) at a concentration of 5 μg / ml. The transformants corresponding to plasmids pALFIB (figures 2 and 3) and pALF287 (figures 2 and 4) showed significant increases (10-50% depending on the host strain) in tylosin production when compared with the parental strain. Also, because both plasmids include the tylF gene, the transformants showed a drastic decrease (25-50 times) in the accumulation of the macrocin precursor in fermentation broths.
DESCRIPCIÓN DE LAS FIGURASDESCRIPTION OF THE FIGURES
FIGURA 1.- Región del genoma de S . fradiae que incluye el agrupamiento de genes biosintéticos de tilosina. 1: ORF1 ( ddcA) 2: ORF2 { tlrB) 3: OFR3 ( tylN) 4: ORF4 { tylE) 5: ORF5 ( tylD) 6: ORF6 ( tylH2) 7: ORF7 { tylHl ) 8 : ORF8 ( tylF) 9: ORF9 ( tylO) 10: ORF10 (tylP) 11: ORF11 { tylQ) 12: tlrD 13: ccr 14: tylMl 15: tylM2FIGURE 1.- S genome region. fradiae that includes the grouping of biosynthetic genes of tylosin. 1: ORF1 (ddcA) 2: ORF2 {tlrB) 3: OFR3 (tylN) 4: ORF4 {tylE) 5: ORF5 (tylD) 6: ORF6 (tylH2) 7: ORF7 {tylHl) 8: ORF8 (tylF) 9: ORF9 (tylO) 10: ORF10 (tylP) 11: ORF11 {tylQ) 12: tlrD 13: ccr 14: tylMl 15: tylM2
16: ORF desconocido 17 : tylG 18: tyll 19: tylB 20: tylAl 21: tylA216: unknown ORF 17: tylG 18: tyll 19: tylB 20: tylAl 21: tylA2
22 : ORF desconocido 23: tlrC22: unknown ORF 23: tlrC
FIGURA 2.- Mapa de restricción de la región del genoma de S. fradiae que incluye el agrupamiento de genes biosintéticos de tilosina. En la parte inferior se muestran los diferentes plásmidos construidos . El inserto correspondiente a los plásmidos pALFIA y pALF2A consiste en fragmentos de 11,5 kb Sacl y 16 kb PvuII respectivamente, mientras que el inserto del plásmido pALFIB se corresponde con un fragmento de 12,9 kb BglII-BamHI . Los clones que aparecen con punta de flecha son aquellos utilizados en el proceso de secuen- ciación.FIGURE 2.- Restriction map of the S. fradiae genome region that includes the clustering of tylosin biosynthetic genes. The different constructed plasmids are shown at the bottom. The insert corresponding to plasmids pALFIA and pALF2A consist of fragments of 11.5 kb Sacl and 16 kb PvuII respectively, while the insert of plasmid pALFIB corresponds to a fragment of 12.9 kb BglII-BamHI. The clones that appear with arrowheads are those used in the sequencing process.
1: ddcA (ORF1)1: ddcA (ORF1)
2: tlrB (ORF2)2: tlrB (ORF2)
3: tylN (ORF3) 4: tylF (ORF4)3: tylN (ORF3) 4: tylF (ORF4)
5: tylD (ORF5)5: tylD (ORF5)
6: tylH2 (ORF6)6: tylH2 (ORF6)
7: tylHl (ORF7)7: tylHl (ORF7)
8: tylF (ORF8) 9: tylO (ORF9) 10: tylP (ORF10) 11: tylQ (ORF11)8: tylF (ORF8) 9: tylO (ORF9) 10: tylP (ORF10) 11: tylQ (ORF11)
FIGURA 3.- Mapa de restricción del plásmido de 20,8 kb pALFIB, el cual incluye el agrupamiento de genes biosintéticos de tilosina descrito en la presente invención.FIGURE 3.- Restriction map of the 20.8 kb plasmid pALFIB, which includes the grouping of biosynthetic tylosin genes described in the present invention.
FIGURA 4.- Mapa de restricción del plásmido de 13,5 kb pALF287, el cual incluye parte del agrupamiento de genes biosintéticos de tilosina descrito en la presente invención.FIGURE 4. Restriction map of the 13.5 kb plasmid pALF287, which includes part of the tylosin biosynthetic gene cluster described in the present invention.
FIGURA 5.- Mapa de restricción del plásmido de 9,2 kb pALF250, el cual incluye el gen de resistencia a tilosina t rB descrito en la presente invención. FIGURE 5. Restriction map of the 9.2 kb plasmid pALF250, which includes the tyrosine resistance gene t rB described in the present invention.

Claims

REIVINDICACIONES
1. Un procedimiento para incrementar los niveles de producción del antibiótico tilosina y/o antibióticos híbridos en Streptomyces spp . caracterizado por las siguientes operaciones :1. A procedure to increase the production levels of the antibiotic tylosin and/or hybrid antibiotics in Streptomyces spp. characterized by the following operations:
(a) Aislar un fragmento de ADN del genoma de S . fradiae que ' contiene las secuencias de nucleótidos descritas como(a) Isolate a DNA fragment from the genome of S . fradiae which 'contains the nucleotide sequences described as
SEQ ID NO: 1 y su hebra complementaria SEQ ID NO : 2.SEQ ID NO: 1 and its complementary strand SEQ ID NO: 2.
(b) Insertar dicho fragmento de ADN en un vector apropiado para las células hospedadoras , de modo que pueda mantenerse dentro de ellas. (c) Transformar la célula hospedante con dicho plásmido re- combinante . (d) Cultivar las células hospedantes transformadas en un medio de cultivo adecuado que permita la producción de tilosina . (b) Insert said DNA fragment into a vector appropriate for the host cells, so that it can be maintained within them. (c) Transform the host cell with said recombinant plasmid. (d) Cultivate the transformed host cells in a suitable culture medium that allows the production of tylosin.
2. Un procedimiento para la obtención de antibióticos híbridos, de acuerdo con la reivindicación 1, caracterizado por introducir genes biosintéticos de tilosina en microorganismos productores de antibióticos.2. A procedure for obtaining hybrid antibiotics, according to claim 1, characterized by introducing tylosin biosynthetic genes into antibiotic-producing microorganisms.
3. Un procedimiento de acuerdo con las reivindica- ciones 1 y 2, caracterizado porque el microorganismo hospedante productor de antibióticos es Streptoi77yces spp .3. A procedure according to claims 1 and 2, characterized in that the antibiotic-producing host microorganism is Streptoi77yces spp.
4. Un procedimiento de acuerdo con las reivindicaciones 1 a 3, caracterizado porque los microorganismos hospedantes son S . fradiae, S . rimosus o S . hygroscopicus . 4. A procedure according to claims 1 to 3, characterized in that the host microorganisms are S. fradiae, S. rimosus or S. hygroscopicus.
5. Un procedimiento para seleccionar transformantes de Streptomyces spp . caracterizado por las siguientes operaciones :5. A procedure to select transformants of Streptomyces spp. characterized by the following operations:
(a) Aislar un fragmento de ADN del genoma de S . fradiae que contiene las secuencias de nucleótidos representadas en SEQ ID NO: 1 y SEQ ID NO : 2. (b) Insertar dicho fragmento de ADN en un vector apropiado para las células hospedadoras , de modo que pueda mantenerse dentro de ellas.(a) Isolate a DNA fragment from the genome of S . fradiae containing the nucleotide sequences represented in SEQ ID NO: 1 and SEQ ID NO: 2. (b) Insert said DNA fragment into a vector appropriate for the host cells, so that it can be maintained within them.
(c) Transformar la célula hospedadora con dicho plásmido recombinante .(c) Transform the host cell with said recombinant plasmid.
(d) Cultivar las células hospedantes transformadas en un medio de cultivo adecuado que permita su crecimiento en presencia de concentraciones inhibitorias de tilosina para la cepa parental sin transformar. (d) Cultivate the transformed host cells in a suitable culture medium that allows their growth in the presence of inhibitory concentrations of tylosin for the untransformed parental strain.
6. Un procedimiento de acuerdo con la reivindicación 5, caracterizado porque las células hospedadoras son S . lividans o S . parvulus .6. A method according to claim 5, characterized in that the host cells are S. lividans or S. parvulus.
7. La secuencia de nucleótidos descrita en SEQ ID No: 1 y su hebra complementaria SEQ ID NO: 2, que incluye al menos los genes ddcA, tlrB, tylN, tylE, tylD, tylH2 , tylHl , tylF, tylO, tylP y tylQ.7. The nucleotide sequence described in SEQ ID No: 1 and its complementary strand SEQ ID NO: 2, which includes at least the genes ddcA, tlrB, tylN, tylE, tylD, tylH2, tylHl, tylF, tylO, tylP and tylQ .
8. La secuencia génica ddcA, cuyo producto codificado se muestra en SEQ ID NO: 3.8. The ddcA gene sequence, the encoded product of which is shown in SEQ ID NO: 3.
9. La secuencia géπica tlrB, cuyo producto codificado se muestra en SEQ ID NO: 4.9. The tlrB gene sequence, the encoded product of which is shown in SEQ ID NO: 4.
10. La secuencia génica tylN, cuyo producto codificado se muestra en SEQ ID ΝO: 5.10. The tylN gene sequence, the encoded product of which is shown in SEQ ID ΝO: 5.
11. La secuencia génica tylE, cuyo producto codificado se muestra en SEQ ID ΝO: 6. 11. The tylE gene sequence, the encoded product of which is shown in SEQ ID ΝO: 6.
12. La secuencia génica tylD, cuyo producto codificado se muestra en SEQ ID ΝO : 7.12. The tylD gene sequence, the encoded product of which is shown in SEQ ID ΝO: 7.
13. La secuencia génica tylH2 , cuyo producto codificado se muestra en SEQ ID ΝO: 8.13. The tylH2 gene sequence, the encoded product of which is shown in SEQ ID ΝO: 8.
14. La secuencia génica tylHl , cuyo producto codifica- do se muestra en SEQ ID ΝO: 9.14. The tylHl gene sequence, the encoded product of which is shown in SEQ ID ΝO: 9.
15. La secuencia génica tylF, cuyo producto codificado se muestra en SEQ ID ΝO : 10.15. The tylF gene sequence, the encoded product of which is shown in SEQ ID ΝO: 10.
16. La secuencia génica tylO, cuyo producto codificado se muestra en SEQ ID ΝO : 11. 16. The tylO gene sequence, the encoded product of which is shown in SEQ ID ΝO: 11.
17. La secuencia génica tylP, cuyo producto codificado se muestra en SEQ ID ΝO : 12. 17. The tylP gene sequence, the encoded product of which is shown in SEQ ID ΝO: 12.
18. La secuencia génica tylQ, cuyo producto codificado se muestra en SEQ ID NO: 13.18. The tylQ gene sequence, the encoded product of which is shown in SEQ ID NO: 13.
19. Vectores caracterizados por contener, total o parcialmente, el fragmento de ADN descrito en SEQ ID NO: 119. Vectors characterized by containing, totally or partially, the DNA fragment described in SEQ ID NO: 1
5 y su hebra complementaria SEQ ID NO: 2 correspondiente al fragmento comprendido entre las posiciones Okb y 13 kb de la figura 1 correspondiente al cluster biosintético de tilosina de S . fradiae .5 and its complementary strand SEQ ID NO: 2 corresponding to the fragment between positions Okb and 13 kb of Figure 1 corresponding to the tylosin biosynthetic cluster of S. fradiae.
20. Células hospedantes caracterizadas por ser transió formadas con los vectores indicados en la reivindicación20. Host cells characterized by being transformed with the vectors indicated in the claim
19.19.
21. Células hospedantes transformadas según la reivindicación 20, caracterizadas por consistir en una cepa de Streptomyces spp. , sus mutantes o sus derivados trans-21. Transformed host cells according to claim 20, characterized by consisting of a strain of Streptomyces spp. , their mutants or their trans-derivatives
15 formados.15 trained.
22. Células hospedantes transformadas según la reivindicación 21, caracterizadas por consistir en una cepa de S . fradiae, sus mutantes o sus derivados transformados.22. Transformed host cells according to claim 21, characterized by consisting of a strain of S. fradiae, its mutants or its transformed derivatives.
23. Células hospedantes transformadas según la reivin- 20 dicación 20, caracterizadas por consistir en una cepa pura de E. coli CECT 5060. 23. Host cells transformed according to claim 20, characterized by consisting of a pure strain of E. coli CECT 5060.
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Publication number Priority date Publication date Assignee Title
EP1457558A4 (en) * 2001-10-19 2005-10-12 Sumitomo Chemical Co Weed controller metabolism proteins, genes thereof and use of the same
US7745699B2 (en) 2001-10-19 2010-06-29 Sumitomo Chemical Company, Limited Weed controller metabolism proteins, genes thereof and use of the same
US8293979B2 (en) 2001-10-19 2012-10-23 Sumitomo Chemical Company, Limited Weed controller metabolism proteins, genes, thereof and use of the same
US10336790B2 (en) * 2014-03-10 2019-07-02 Rensselaer Polytechnic Institute Anti-microbial peptides and method for designing novel anti-microbial peptides
US11202449B2 (en) 2014-03-10 2021-12-21 Rensselaer Polytechnic Institute Anti-microbial peptides and method for designing novel anti-microbial peptides

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