Eli Lilly and Company

R Nagaraja Rao

Stuart A Kuhstoss

Richard K Stanzak

Eugene T Seno

Karen L Cox

Mark A Richardson

Richard H Baltz

Brigitte E Schoner

Patti J Treadway

Thomas D Ingolia

Robert J Beckmann

Ramamoorthy Belagaje

Kevin R Kaster

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 transcription terminator is disclosed comprising a DNA sequence capable of functioning as a transcription terminator in a microorganism of the order Actinomycetales. These transcription terminators are functional in both gram-positive and gram-negative microorganisms, especially Streptomyces ambofaciens. Additionally, a series of recombinant DNA cloning vectors suitable for isolating the disclosed transcription terminators are set forth.

Transcription terminator and related recombinant DNA vectors and

A modified hygromycin B resistance-conferring gene either alone or in translational reading phase with a gene or portion of a gene is disclosed. The invention further comprises recombinant DNA cloning vectors and transformants of the aforementioned DNA.

Modified antibiotic resistance gene

The present invention is a method for isolating antibiotic biosynthetic genes. To practice the method, an antibiotic resistance-conferring DNA segment is labelled and used as a probe to find, via DNA hydridization, homologous DNA in a genetic library which comprises chromosomal and plasmid DNA of an antibiotic-producing organism. Individual vectors of the genetic library which hybridize to the antibiotic resistance-conferring gene, and which comprise .about.1-45 kb of contiguous DNA from the antibiotic-producing organism, which also comprise an antibiotic biosynthetic gene. The present method is exemplified by using the erythromycin resistance-conferring gene of Streptomyces erythreus to clone the erythromycin biosynthetic pathway from the same organism. The erythromycin biosynthetic pathway isolated with the present method synthesizes erythromycin when introduced into S. lividans TK23.

Method of isolating antibiotic biosynthetic genes

Novel recombinant DNA cosmid shuttle vectors and a method of using them in the construction of genomic DNA libraries are described. The vectors demonstrate the incorporation of both the size selection and in vitro packaging mechanisms of lambda into a Streptomyces-E. coli shuttle vector by the incorporation of two or more COS sequences of bacteriophage lambda.

Novel recombinant DNA cosmid shuttle vectors

A novel gene conferring resistance to spiramycin in Streptomyces griseofuscus was cloned from a genomic library of Streptomyces ambofaciens DNA. An eighteen kb fragment of S. ambofaciens DNA surrounding the spiramycin resistance gene was isolated from this library on a plasmid cosmid designated pKC514. The novel spiramycin-resistance gene can be isolated on an .about.3.4 kb Sau3AI fragment by subcloning restriction fragments obtained from the pKC514 insert DNA. This Sau3AI 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.

Novel plasmid shuttle vectors conferring spiramycin resistance in

A novel method of selecting Streptomyces recombinant DNA-containing host cells and vectors useful in exemplifying the method are described. The vectors confer apramycin resistance to sensitive Streptomyces host cells and thus provide a convenient method of selecting Streptomyces transformants. The apramycin resistance-conferring gene used in the method is an acetyltransferase aac(3)IV gene and can be isolated from E. coli K12 BE1041/pKC309 (NRRL B-15827) on an .about.1.5 kb PstI-EcoRI restriction fragment.

Method of selecting recombinant DNA-containing streptomyces

The invention relates to a method and cloning vehicle for the expression of a functional polypeptide in Streptomyces. A recombinant DNA cloning vehicle was genetically engineered to bring the expression of the neomycin phosphotransferase gene under the control of the Escherichia coli bacteriophage .lambda.p.sub.L promoter.

Method of using bacteriophage lambda p.sub.1 promoter to produce a

The present transcription terminators are DNA sequences characterized by their ability to terminate the transcription of DNA sequences. The present sequences are portable, have a variety of restriction endonuclease sites and are useful in controlling the expression in hosts of DNA sequences coding for prokaryotic, eukaryotic and viral proteins and polypeptides.

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Indianapolis, IN, United States of America

R Nagaraja Rao