Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1988-12-12
2001-01-30
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S047000, C435S189000, C435S252300, C435S252330, C435S254110, C435S254300, C435S254400, C536S023200, C536S024100
Reexamination Certificate
active
06180361
ABSTRACT:
SUMMARY OF THE INVENTION
The present invention comprises a DNA sequence that encodes deacetoxycephalosporin C synthetase and deacetylcephalosporin C synthetase activities. Deacetoxycephalosporin C synthetase (DAOCS) catalyzes the reaction in which deacetoxycephalosporin C (DAOC) is formed from penicillin N and is often referred to as expandase. Deacetylcephalosporin C synthetase (DACS) catalyzes the formation of deacetylcephalosporin C (DAC) from DAOC, a hydroxylation reaction. These reactions are critical steps in the biosynthesis of important antibiotics, such as cephalosporins from
Cephalosporium acremonium
and 7&agr;-methoxycephalosporins from
Streptomyces clavuligerus.
The DNA compounds of the present invention encode the DAOCS and DACS in a single open reading frame. Transcription of this open reading frame, followed by translation of the resulting mRNA, yields a single polypeptide chain that possesses both DAOCS and DACS activities. Because of this dual nature of the compounds of the present invention, the terms “DAOCS/DACS,” “DACS/DAOCS,” “expandase/hydroxylase,” and “EXP/HYD” are used to denote that a given gene, coding sequence, and enzyme possesses both DACS and DAOCS attributes. The linkage of these activities is not universally observed; for example, cephamycin-producing species of Streptomyces utilize two different polypeptides to express the DAOCS and DACS activities. Although concomitant expression of the DAOCS and DACS activities is convenient, and usually most preferred, it is not a critical aspect of the invention. Genetic engineering techniques, as exemplified herein, provide a ready means to separate the genetic information encoding DAOCS from DACS and so separately express either the DAOCS or DACS activity. Thus, the dual nature of the illustrative compounds, vectors, and enzymatic activities of the invention is not a limitation, for the invention also comprises single nature molecules: those that demonstrate only DACS or DAOCS attributes.
The DNA compound that encodes the DACS/DAOCS activities was isolated from
Cephalosporium acremonium
genomic DNA and used to construct recombinant DNA expression vectors. Three types of these expression vectors are especially useful: the first drives high-level expression of the DAOCS/DACS activities in
E. coli;
the second in
C. acremonium;
and the third in
Penicillium chrysogenum.
The
E. coli
-produced DAOCS/DACS activities catalyze the formation of DAOC from penicillin N and DAC from DAOC. Crude cell extracts of these
E. coli
transformants of the invention exhibited DAOCS/DACS activities without any prior activation treatment. These
E. coli
expression vectors and transformants provide an efficient means for obtaining large amounts of active DAOCS/DACS enzyme. The DAOCS/DACS enzyme is useful not only for the production of DAOC and DAC but also for the expansion of penicillins other than penicillin N and hydroxylation of cephalosporins other than DAOC to form novel antibiotics.
The Cephalosporium vectors of the present invention are useful for purposes of constructing strains for use by the pharmaceutical industry. Cephalosporium is an economically important organism used to produce penicillin and cephalosporin antibiotics. Transformation of Cephalosporium with the expression vectors of this invention results in higher intracellular levels of DAOCS and DACS in the transformants. These transformants can be used to increase the efficiency of, and yield of antibiotic in, industrial fermentation.
The Penicillium vectors of the present invention are most useful to introduce cephalosporin synthesizing activities into high-level penicillin producing Penicillium strains. The DAOCS activity is useful for conversion of the various penicillins to cephalosporins, either alone or in conjunction with other activities, such as DACS or epimerase. For example, concomitant expression of isopenicillin N epimerase activity (from, e.g.,
Cephalosporium acremonium
) and DAOCS activity in Penicillium leads to production of DAOC, a heretofore unknown metabolite in Penicillium.
The present invention also provides an improved method for introducing recombinant DNA molecules to Penicillium species. This novel transformation system utilizes the amdS gene of
Aspergillus nidulans
. Using acetamide (CH
3
CONH
2
) as the sole carbon or nitrogen source on transformation plates, one can select Penicillium host cells transformed with a vector containing the amdS gene from their untransformed counter-parts, which are unable to grow on the plates because wild-type Penicillium express no acetamidase activity.
The DNA compounds encoding DAOCS/DACS are readily modified to construct expression vectors that increase the efficiency and yield of fermentations involving other organisms, such as Paecilomyces and Streptomyces, especially
S. clavuligerus.
Although the DACS/DAOCS-encoding DNA of the present invention was isolated from
Cephalosporium acremonium,
this DNA can be used to construct vectors that drive expression of DACS/DAOCS in a wide variety of host cells, as illustrated by the
E. coli
expression vectors described above. The construction protocols utilized for the
E. coli,
Cephalosporium, and Penicillium vectors of the invention can be followed to construct analogous vectors for other organisms, merely by substituting, if necessary, the appropriate regulatory elements. Most organisms that produce penicillins and cephalosporins utilize the common precursor penicillin N, a substrate for DAOCS. The DACS/DAOCS-encoding DNA compounds of the present invention can be used to construct expression vectors useful for improving the efficiency and yield of fermentations involving a wide variety of penicillin and cephalosporin antibiotic-producing organisms.
The DNA compounds of the present invention are derived from genomic DNA of
Cephalosporium acremonium
and are significantly homologous in nucleotide sequence to the DNA compounds encoding DAOCS and/or DACS activity in other cephalosporin-producing organisms, such as
Streptomyces clavuligerus.
Because of this homology, the DACS/DAOCS-encoding DNA compounds of the present invention can be labelled and used to screen genomic libraries of organisms that produce cephalosporin C or similar compounds for the presence of either DACS or DAOCS-encoding DNA. Many organisms comprise DACS/DAOCS activity-encoding DNA that can be identified and isolated using the DNA compounds of the present invention, and the present invention comprises these equivalent, homologous DNA compounds.
The DACS/DAOCS-encoding DNA compounds of the present invention were isolated in conjunction with the regulatory sequences that control transcription and, ultimately, expression of the
Cephalosporium acremonium
DAOCS/DACS activities. The present invention comprises these novel regulatory sequences, which can be used, as disclosed herein, to drive transcription, translation, and expression of any gene product in Cephalosporium. These regulatory sequences are especially preferred for use in
C. acremonium.
The present invention also comprises the regulatory signals of the DACS/DAOCS gene located at the 3′ end of the coding strand of the coding region of the
Cephalosporium acremonium
DACS/DAOCS gene. These 3′ regulatory sequences encode the transcription termination and mRNA polyadenylation and processing signals of the DACS/DAOCS gene. Placing these signals at the 3′ end of the coding strand of the coding region of the gene to be expressed enhances expression of the desired gene product in Cephalosporium.
The following section provides a more detailed description of the present invention. For purposes of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following items are defined below.
amdS—an acetamidase gene; also used in the Figures to denote the
Asperigillus nidulans
acetamidase gene.
AmR—the apramycin resistance-conferring gene; also used to denote the apramycin-resistant phenotype.
Antibiotic—a substance produced by a micro-organism that, either
Ingolia Thomas D.
Queener Stephen W.
Samson Suellen M.
Skatrud Paul L.
Achutamurthy Ponnathapu
Eli Lilly and Company
Hamilton Amy E.
Maciak Ronald S.
Moore William
LandOfFree
Recombinant DNA expression vectors and DNA compounds that... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Recombinant DNA expression vectors and DNA compounds that..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Recombinant DNA expression vectors and DNA compounds that... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2519156