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US5801032A
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Vectors and process for producing high purity 6,12-dideoxyerythromycin A by fermentation
Assignee: Abbott Laboratories
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Filing: 1996-08-01
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Granted: 1998-09-01
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Expires: 2016-08-01
Abstract
Abstract A process for producing high purity 6,12-dideoxyerythromycin A using recombinant DNA technology is disclosed. The erythromycin producing strain, Saccharopolyspora erythraea, lacking the erythromycin C-12 and C-6 hydroxylases produces a mixture of 6,12-dideoxyerythromycin A and the precursor molecule, 6-deoxyerythromycin D. To achieve conversion of the precursor to the final product, a second copy of eryG is inserted into a non-essential region of the Sac. erythraea chromosome resulting in high purity 6,12-dideoxyerythromycin A.
Inventors
- 👤 Diane L. Stassi
- 👤 Gregory T. Maine
- 👤 David A. Post
- 👤 Mark T. Satter
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-09 00:33
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/0004; C12N9/0012; C12N15/00; C12N15/09; C12N15/63; C12N15/74; C12N15/76; C12N15/87; C12N15/90; C12N9/10; C12N9/1003; C12N9/1007; C12P; C12P19/00; C12P19/44; C12P19/60; C12P19/62
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
68 words
A recombinant DNA vector for integrating a gene of interest into the chromosome of an erythromycin producing host cell, said vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome wherein said DNA sequence contains SEQ ID NO:1, a second DNA sequence which contains the origin of replication from plasmid pCD1, and a third DNA sequence encoding a selectable maker gene.
Claim 2
Dependent Claim
68 words
A recombinant DNA vector for integrating a gene of interest into the chromosome of an erythromycin producing host cell, said vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome wherein said DNA sequence contains SEQ ID NO:1, a second DNA sequence which contains the origin of replication from plasmid pCD1, and a third DNA sequence encoding a selectable maker gene.
Claim 3
Dependent Claim
28 words
The DNA vector of claim 1 further comprising a DNA sequence which encodes the ermE* promoter wherein said ermE* promoter is operably linked to said gene of interest.
Claim 4
Dependent Claim
15 words
The DNA vector of claim 2 wherein said gene of interest is the eryG gene.
Claim 5
Dependent Claim
12 words
The DNA vector of claim 1 further comprising a multiple cloning site.
Claim 6
Dependent Claim
12 words
The DNA vector of claim 1 wherein said vector is plasmid pKAS37.
Claim 7
Dependent Claim
11 words
The DNA vector of claim 5 further comprising the eryG gene.
Claim 8
Dependent Claim
12 words
The DNA vector of claim 1 wherein said vector is plasmid pDPE35.
Claim 9
Independent Claim
127 words
A method for increasing 3"-O-methyltransferase activity in host cells which produce at least one substrate for said 3"-methyltransferase activity, said method comprising: a. introducing an integrative recombinant vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome for integrating said vector into the chromosome of said host cells wherein said DNA sequence contains SEQ ID NO: 1, a second DNA sequence which contains the origin of replication from plasmid pCD1, a third DNA sequence encoding a selectable maker gene, and a fourth DNA sequence which is the eryG gene, said eryG gene encoding said 3"-O-methyltransferase activity; and b. selecting for stable integrants of said host cells, said integrants having said eryG gene stable integrated into said chromosome of said integrants.
Claim 10
Independent Claim
127 words
A method for increasing 3"-O-methyltransferase activity in host cells which produce at least one substrate for said 3"-methyltransferase activity, said method comprising: a. introducing an integrative recombinant vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome for integrating said vector into the chromosome of said host cells wherein said DNA sequence contains SEQ ID NO: 1, a second DNA sequence which contains the origin of replication from plasmid pCD1, a third DNA sequence encoding a selectable maker gene, and a fourth DNA sequence which is the eryG gene, said eryG gene encoding said 3"-O-methyltransferase activity; and b. selecting for stable integrants of said host cells, said integrants having said eryG gene stable integrated into said chromosome of said integrants.
Claim 11
Dependent Claim
16 words
The method of claim 8 wherein said integrative recombinant vector is the plasmid of claim 7.
Claim 12
Dependent Claim
17 words
The method of claim 8 wherein said integrative recombinant DNA vector is the plasmid of claim 8.
Claim 13
Independent Claim
126 words
A method for making a modified deoxyerythromycin producing host strain, wherein said modification is an increase in 3"-O-methyltransferase activity, said method comprising: a. introducing an integrative recombinant vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome for integrating said vector into the chromosome of said host cells, wherein said DNA sequence contains SEQ ID NO:1, a second DNA sequence which contains the origin of replication from plasmid pCD1, a third DNA sequence encoding a selectable maker gene, and a fourth DNA sequence which is the eryG gene, said eryG gene encoding said 3"-O-methyltransferase activity; and b. selecting for stable integrants of said host cells, said integrants having said eryG gene stable integrated into said chromosome of said integrants.
Claim 14
Independent Claim
126 words
A method for making a modified deoxyerythromycin producing host strain, wherein said modification is an increase in 3"-O-methyltransferase activity, said method comprising: a. introducing an integrative recombinant vector comprising a first DNA sequence having an 11 kb HindIII fragment of the Saccharopolyspora erythraea chromosome for integrating said vector into the chromosome of said host cells, wherein said DNA sequence contains SEQ ID NO:1, a second DNA sequence which contains the origin of replication from plasmid pCD1, a third DNA sequence encoding a selectable maker gene, and a fourth DNA sequence which is the eryG gene, said eryG gene encoding said 3"-O-methyltransferase activity; and b. selecting for stable integrants of said host cells, said integrants having said eryG gene stable integrated into said chromosome of said integrants.
Claim 15
Dependent Claim
16 words
The method of claim 8 wherein said integrative recombinant vector is the plasmid of claim 7.
Claim 16
Dependent Claim
17 words
The method of claim 11 wherein said integrative recombinant DNA vector is the plasmid of claim 8.
Claim 17
Dependent Claim
29 words
A host strain which produces an erythromycin derivative, said host strain being modified to overproduce 3"-O -methyltransferase activity wherein said modification is achieved by the method of claim 8.
References Cited (Backward Citations)
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