Eli Lilly and Company

Thomas D Ingolia

Paul Luther Skatrud

Stephen W Queener

Kevin R Kaster

Suellen M Samson

Gary David Gustafson

Otis W Godfrey

R Nagaraja Rao

Steven Kovacevic

James R Miller

Barbara J Weigel

Cheryl A Beckage

Barbara J Wiegel

Martin Bard

Lucinda G Carr

Gretchen Kirchner

Jean L Roberts

Jerry L Chapman, Jr

The present invention provides DNA compounds that encode the expandase/hydroxylase enzyme of,. The compounds can be used to construct recombinant DNA expression vectors for a variety of host cells, including,Penicillium, and Cephalosporium.

Recombinant DNA expression vectors and DNA compounds that encode deacetoxycephalosporin C synthetase and deacetylcephalosporin C synthetase

Novel fusion reporter genes, fusion reporter proteins, and an improved reporter system for measuring the relative activity of a promoter sequence. A luxAB fusion gene of the present invention is particularly useful as a reporter gene and is derived from the fusion of a luxA gene and a luxB gene from Vibrio harveyi. The gene products of the luxA and luxB genes are the .alpha.- and .beta.-subunits, respectively, of a bacterial luciferase. A fusion protein encoded by a luxAB fusion gene is a single active protein and is particularly useful as a reporter protein having luciferase activity. An advantage of such a reporter system to assay gene expression in many cells which contain FMNH.sub.2, such as bacterial and yeast cells, is that an immediate and quantitative assessment of gene expression may be made from real-time light measurements using intact cells.

Fusion reporter gene for bacterial luciferase

The present invention provides DNA compounds that encode deacetoxycephalosporin C synthetase (DAOCS) activity. The compounds can be used to construct recombinant DNA expression vectors for a wide variety of host cells, including E. coli, Penicillium, Streptomyces, Aspergillus, and Cephalosporium.

Recombinant DNA expression vectors and DNA compounds that encode

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

DNA compounds encoding the Streptomyces lipmanii isopenicillin N synthetase (IPNS) gene are useful for constructing a variety of recombinant DNA vectors. The vectors are useful in producing IPNS in a wide variety of host cells, such as Streptomyces, Penicillium, and Cephalosporium. DNA compounds encoding the transcription and translation activating sequence and transcription termination sequence of the S. lipmanii IPNS gene are also useful in the construction of expression vectors, especially Streptomyces expression vectors. The S. lipmanii IPNS gene can be isolated from plasmid pOGO239, available from the Northern Regional Research Center under accession number NRRL B-18250.

The present invention is a novel method for maintaining and selecting recombinant DNA-containing host cells wherein the DNA encoding a selectable phenotype and the DNA encoding a useful polypeptide are the same. The aforementioned DNA is useful for expressing .delta.-aminolevulinic acid synthetase (ALAS) for the ultimate expression of .delta.-aminolevulinic acid (ALA) in yeast and related organisms. The invention further comprises plasmids pIT300, pIT301, pIT302, pIT304, pIT305, pIT306 and related Saccharomyces ALA deficient transformants. ALA is a five carbon amino acid that is useful as a light dependent herbicide.

Novel DNA for expression of delta-aminolevulinic acid synthetase and

The present invention comprises novel DNA compounds that encode isopenicillin N synthetase. The invention also comprises methods, transformants, and polypeptides related to the novel DNA compounds. The novel isopenicillin N synthetase-encoding DNA, together with its associated transcription and translation activating sequence, was isolated from Penicillium chrysogenum. The isopenicillin N synthetase-encoding DNA can be used to construct novel E. coli expression vectors that drive expression of isopenicillin N synthetase in E. coli. The intact P. chrysogenum isopenicillin N synthetase-encoding DNA and associated transcription and translation activating sequence can also be used to construct expression vectors that drive expression of the isopenicillin N synthetase in P. chrysogenum and Cephalosporium acremonium. The transcription and translation activating sequence can be fused to a hygromycin phosphotransferase-encoding DNA segment and placed onto expression vectors that function in P. chrysogenum and C. acremonium. The transcription termination and mRNA polyadenylation signals of the P. chrysogenum isopenicillin N synthetase can be used to increase ultimate expression of a product encoded on a recombinant DNA vector.

DNA compounds and recombinant DNA expression vectors that encode and drive expression in recombinant host cells of the isopenicillin N synthetase activity of Aspergillus nidulans are useful to produce isopenicillin N synthetase and to improve the yield of .beta.-lactam-containing antibiotics from antibiotic-producing organisms. The isopenicillin N synthetase gene of A. nidulans can be isolated from plasmid pOGOO4, available from the Northern Regional Research Center under the accession number NRRL B-18171.

The present invention comprises novel DNA compounds that encode isopenicillin N synthetase and also comprises related methods, transformants, and polypeptides. The novel isopenicillin N synthetase-encoding DNA, together with its associated transcriptional and translational activating sequence, was isolated from Cephalosporium acremonium and cloned into an E. coli cloning vector. The isopenicillin N synthetase-encoding DNA has been used to construct novel E. coli expression vectors that drive expression of a stable, active, and novel isopenicillin N synthetase in E. coli. The intact C. acremonium isopenicillin N synthetase-encoding DNA and associated transcriptional and translational activating sequence have also been used to construct C. acremonium expression vectors that drive expression of the isopenicillin N synthetase in C. acremonium. The C. acremonium transcriptional and translational activating sequence has further been fused to a hygromycin phosphotransferase-encoding DNA segment and placed onto C. acremonium expression vectors. Useful derivatives of the novel compounds and vectors are also described.

Recombinant DNA expression vectors and DNA compounds which encode

A method for transforming Cephalosporium and other lower eukaryotes is disclosed. The method involves inserting a recombinant DNA cloning vector comprising a Saccharomyces cerevisiae transcriptional and translational activating sequence positioned for expression of hygromycin phosphotransferase into a host cell and then growing the host cell under selective conditions. The vectors optionally further comprise Cephalosporium ribosomal DNA and also sequences that allow for replication and selection in E. coli and Streptomyces.

Vectors and conditions which allow genetic transformation of

Disclosed are a novel method for inducing the high expression of a nucleotide sequence which is under the transcriptional and translational control of the yeast YG100 gene and the novel vectors, transformants and selectable DNA for the practice thereof.

Method, vectors and transformants for high expression of heterologous

The present invention relates to a novel transcriptional and translational activating sequence. The novel activating sequence can be either chemically synthesized or isolated on a 0.17 kb PstI-SacI restriction fragment from plasmid pKC203, a plasmid of E. coli JR225 (ATCC 31912). The activating sequence directs expression of the aminoglycoside acetyltransferase aac(3)IV and hygromycin phosphotransferase aph(4) genes present on plasmid pKC203. A series of expression vectors have been constructed in which the activating sequence directs the expression of beta-galactosidase or hygromycin phosphotransferase. These vectors can be readily modified and have been designed to facilitate the subsequent cloning and expression of any gene of research or commercial interest. The expression and cloning vectors have been transformed into E. coli and other host cells in which the activating sequence functions.

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Indianapolis, IN, United States of America

Thomas D Ingolia