Eli Lilly and Company

Eugene T Seno

Richard H Baltz

Gene M Wild

Karen L Cox

Virginia A Birmingham

Herbert A Kirst

R Nagaraja Rao

Mark A Richardson

Robert J Beckmann

Robert L Hamill

Charles L Hershberger

Brigitte E Schoner

Stuart A Kuhstoss

Scott E Fishman

Gerald L Huff

A method for increasing the antibiotic-producing ability of an antibiotic-producing microbial host cell is disclosed. The method involves transforming an antibiotic-producing microorganism with a recombinant DNA cloning vector that codes for the expression of an antibiotic biosynthetic enzyme or other gene product. The gene preferably codes for a product that is rate-limiting in the antibiotic biosynthetic pathway in Streptomyces. Plasmids that are useful for increasing tylosin production in accordance with the present method include plasmids pHJL280, pHJL284, pHJL309, pHJL311, and pHJL315. The invention further comprises microorganisms transformed with plasmids pHJL280, pHJL284, pHJL309, pHJL311, and pHJL315 and also other microorganisms and vectors used in accordance with the present method.

Method for increasing the antibiotic-producing ability of

The present invention provides a method for transforming an actinomycete with an integrating vector which has the advantages of high transformation rates into a broad host range, site-specific integration, and stable maintenance without antibiotic selection. Also provided are methods for the increased production of antibiotics and for the production of hybrid antibiotics.

Use of the site-specific integrating function of phage .phi.C31

The present invention, utilizing recombinant DNA technology, provides a novel method for obtaining 20-deoxotylosin from a tylosin producing microorganism. 20-deoxotylosin is useful as an antibiotic with a microbial inhibitory activity similar to tylosin.

Method for producing 20-deoxotylosin

Provided are gene sequences encoding tylosin biosynthetic gene products. In particular, recombinant DNA vectors comprising DNA sequences encoding the tylA, tylB, tylI and tylG activities of Streptomyces fradiae are provided. Also provided are host cells transformed with the noted vectors and a method for increasing the tylosin-producing ability of a tylosin-producing organism.

Tylosin biosynthetic genes tylA, tylB and tylI

Spiramycin antibiotic biosynthetic genes of Streptomyces ambofaciens are provided by the present invention, in addition to a variety of recombinant DNA vectors. The genes also function in other macrolide producing organisms. The genes can be used to increase or otherwise alter the macrolide antibiotic-producing ability of an organism. The present invention also provides host strains comprising mutant spiramycin biosynthetic genes which can be used to generate novel antibiotics. Also provided is a method for preparing the mutant gene comprising mutating cloned spiramycin biosynthetic DNA by transposon mutagenesis with subsequent transformation into a macrolide-antibiotic producing host and homologous recombination into its genome, to generate stable mutant cell lines.

Macrolide biosynthetic genes for use in streptomyces and other organisms

The present invention discloses a method for producing a novel antibiotic, 2''-O-demethyltylosin. The novel antibiotic, utilizing recombinant DNA technology, can be produced from a transformed mutant of a tylosin producing microorganism. By transforming for example Streptomyces fradiae GS 16 with plasmid pHJL284, the transformant can produce 2''-O-demethyltylosin. Streytomyces fradiae GS16 is a tylosin producing species that contain a mutation in the tylE gene found in the tylosin biosynthetic pathway. The tylE gene codes for demethylmacrocin 2''-O-methyltransferase enzyme (DMOMT), the enzyme that methylates the 2-hydroxyl position of the 6-deoxyallose moiety. Plasmid pHJL284 contains the cloned tylF gene which codes for the macrocin 3''-O-methyltransferase enzxyme (MOMT), the enzyme that methylates the 3''-hydroxyl position, but it does not contain the tylE gene. Transformation of S. fradiae GS16 with plasmid pHJL284 results in increased copies of the tylF gene in the absence of the tylE gene, therefore allowing an increased rate of methylation of the 3''-hydroxyl position in the absence of the methyl group at the 2''-hydroxyl position. The resulting transformant, therefore, can produce 2''-O-demethyltylosin in appreciable quantities. The present invention is not limited to S. fradiae.For example, two other tylosin producing species such as Streptomyces rimosus and Streptomyces hygroscopicus, may also be used for purposes of the invention disclosed. 2''-O-Demethyltylosin is useful as an antibiotic with a microbial inhibitory activity similar to tylosin.

Method for producing 2''-O-demethyltylosin

The tlrC gene is a novel tylosin resistance-conferring gene isolated from Streptomyces fradiae and used to construct a number of cloning vectors for use in Streptomyces. One such cloning vector, plasmid pSKC10, can be obtained in S. fradiae JS87 under the accession number NRRL 18072. S. fradiae JS87 is the preferred host when the tlrC gene is used to select tylosin-resistant Streptomyces transformants.

Tylosin resistance-conferring gene, designated tlrC, for use in

The tlrB gene is a novel tylosin resistance-conferring gene isolated from Streptomyces fradiae and used to construct a number of cloning vectors for use in Streptomyces and related organisms. One such cloning vector, plasmid pSVB9, can be obtained in S. lividans under the accession number NRRL 18073. S. lividans is the preferred host when the tlrB gene is used to select tylosin-resistant Streptomyces transformants.

Tylosin resistance-conferring gene, designated tlrB, for use in

A novel method of selecting Streptomyces recombinant DNA-containing host cells and vectors useful in the method are described. The vectors confer tylosin resistance to sensitive Streptomyces host cells and thus provide a convenient method of selecting Streptomyces transformants. The novel tylosin resistance-conferring gene described can be isolated on an .about.2.6 kb KpnI restriction fragment from plasmid pSVB2. Plasmid pSVB2 can be isolated from Streptomyces lividans TK23/pSVB2 (NRRL 15880).

Method of selecting recombinant DNA-containing streptomyces

23-Demycinosyltylosin (DMT) which has the formula: ##STR1## 20-dihydro-DMT, specified acyl ester derivatives, and their acid addition salts are useful antibacterial agents. Improved methods of making 5-O-mycaminosyltylonolide (OMT) and 20-dihydro-OMT by mild acid hydrolysis of DMT and 20-dihydro-DMT, respectively, are included.

Process for the production of mycaminosyltylonolide

2''-O-demethylmacrocin (DOMM) which has the formula: ##STR1## 20-dihydro-DOMM, 2''-O-demethyllactenocin (DOML), 20-dihydro-DOML, specified acyl ester derivatives, and their acid addition salts are useful antibacterial agents.

Demethylmacrocin compounds and derivatives thereof

A new microorganism, Streptomyces fradiae NRRL 12201, which produces mycarosyltylactone (5-O-mycarosyl-20-dihydro-20,23-dideoxytylonolide) and a process for preparing tylactone (20-dihydro-20,23-dideoxytylonolide) and mycarosyltylactone by submerged aerobic fermentation of this microorganism, or a mycarosyltylactone-producing mutant or recombinant thereof, are provided.

Process for preparing mycarosyltylactone

20-Dihydro-20-deoxy-23-demycinosyltylosin (DH-DO-DMT), 20-dihydro-20-deoxy-5-O-mycaminosyltylonolide (DH-DO-OMT), specified acyl ester derivatives, and their acid addition salts are useful intermediates and antibacterial agents. Methods of preparing DH-DO-DMT and DH-DO-OMT by fermentation of Streptomyces fradiae and the microorganism S. fradiae ATCC 31733 are included.

Growing community of inventors

Norwich, United Kingdom

Eugene T Seno