Eli Lilly and Company

Mark A Richardson

Jeffrey T Fayerman

Eugene T Seno

R Nagaraja Rao

Karen L Cox

Brigitte E Schoner

Steven Kovacevic

James R Miller

Stuart A Kuhstoss

Robert J Beckmann

Ramamoorthy Belagaje

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

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

A novel gene conferring resistance to spiramycin in Streptomyces and related organisms was cloned from a genomic library of Streptomyces ambofaciens DNA. A thirty-one Kb fragment of S. ambofaciens DNA including the spiramycin-resistance gene was isolated from this library on a cosmid designated pKC592. The novel spiramycin-resistance gene can be isolated on an .about.2.9 Kb BamHI fragment by subcloning restriction fragments obtained from the pKC592 insert DNA. This BamHI fragment contains all of the information required for the expression of the spiramycin resistant phenotype in Streptomyces. Vectors and transformants containing the novel spiramycin resistance gene are provided.

Spiramycin resistance-conferring cloning vectors

The present invention is a method for expressing functional polypeptides in Streptomyces using a recombinant DNA expression vector comprising a novel transcriptional- and translational- activating sequence. The novel activating sequence can be synthesized by conventional methods and used in Streptomyces expression vectors. One such vector, plasmid pFJ350, expresses and confers hygromycin resistance in Streptomyces host cells.

Method for using a novel transcriptional- and translational-activating

A method for expressing a functional polypeptide in Streptomyces comprises transforming a Streptomyces host cell with a recombinant DNA expression vector and then culturing the transformed cell under conditions suitable for cell growth. The recombinant DNA expression vector comprises the veg or any other homologous Bacillus promoter, a naturally occurring or modified ribosome binding site-containing DNA sequence and a gene that codes for a functional polypeptide such as human pre-proinsulin. The method is specifically exemplified by use of expression plasmids pOW529, pOW539 and transformants, Streptomyces ambofaciens/pOW529 and Streptomyces ambofaciens/pOW539. The method is broadly applicable and is particularly useful in economically important Streptomyces taxa.

Method for using an homologous bacillus promoter and associated natural

The present invention discloses selectable recombinant DNA cloning vectors for use in Streptomyces and related organisms.

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Indianapolis, IN, United States of America

Mark A Richardson