Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2002-03-06
2004-08-17
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S320100, C435S069100, C435S325000, C435S252300, C435S194000, C435S189000, C435S227000, C435S252330, C536S023200, C530S350000
Reexamination Certificate
active
06777209
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the production of pyrimidines, purines and derivatives thereof e.g. deoxyribonucleosides, using genetically modified cells comprising novel DNA constructs.
BACKGROUND OF THE INVENTION
Thymidine is useful as a pharmaceutical intermediate, particularly for the chemical synthesis of azidothymidine (“AZT,” sold under the trademark ZIDOVUDINE). Although ZIDOVUDINE-type AZT was one of the first therapies developed for HIV/AIDS, it continues to have important and expanded use (Langreth, R.,
The Wall Street Journal
, Nov. 21, 1995, pp B12). ZIDOVUDINE-type AZT is valuable particularly when used in combination therapies such as a combination with lamivudine (also known as 3TC), sold under the trademark EPIVIR. This lamvudine and 3TC combination is sold under the trademark COMBIVIR. Although the HIV virus can mutate to form resistance to either AZT or 3TC, COMBIVIR-type nucleotide-analog combination is particularly effective because the reverse transcriptase apparently cannot be resistant to both nucleoside analogues at the same time (Larder, B. A. et al.,
Science
269: 696-699, 1995). ZIDOVUDINE-type AZT is also useful in conjunction with HIV protease inhibitor type drugs (Waldholz, M.,
The Wall Street Journal
, Jan. 30, 1996, pp B1), and in the treatment of HIV infected pregnant women in order to reduce the frequency of infection of the fetus at birth. In 1997 about 600,000 children died of AIDS contracted from their mothers at birth. ZIDOVUDINE-type AZT taken for several months prior to birth can reduce the transmission of the virus to infants by two-thirds. Thymidine produced by chemical synthesis used in the manufacture of AZT is a very significant cost.
In U.S. Pat. No. 5,213,972 (McCandliss & Anderson, hereinafter “the '972 patent”), the entire contents of which are incorporated herein by reference and to which the reader is specifically referred, a process for the production of pyrimidine deoxyribonucleoside (PdN) is disclosed (see in particular examples 7 to 14 of the '972 patent). A replicatable microorganism comprising and expressing a DNA sequence encoding a pyrimidine deoxyribonucleotide phosphohydrolase that converts a PdN monophosphate to a pyrimidine deoxyribonucleoside is taught. More particularly, McCandliss & Anderson, supra, describe a fermentation method that can be used to produce thymidine that involves the expression of deoxythymidylate phosphohydrolase (dTMPase) from the Bacillus bacteriophage PBS1. This type of enzyme has been found in nature expressed by bacteriophages that do not contain thymidine in their DNA, but instead incorporates compounds like deoxyuridine or hydroxymethyl-deoxyuridine.
In the thymidine fermentation described in the '972 patent, the enzymes that degrade thymidine (thymidine phosphorylase and uridine phosphorylase) have been removed by mutation so that thymidine accumulates. Thus, the use of the dTMPase enzyme helps create the pathway to allow thymidine synthesis. An expression of dTMPase alone, however, may not assure a commercially viable level of thymidine production. Accordingly, there is a continuing need to enhance the production of thymidine by cells expressing dTMPase in order to make thymidine production by fermentation commercially viable, by lowering the production cost relative to the current chemical synthesis methods.
The biochemical pathway for pyrimidine deoxynucleotide production, for example, in
E. coli
is highly regulated at the levels of transcription and translation as well as at the protein level by mechanisms including attenuation, feedback inhibition and enzyme activation. Neuhard, J. and R. A. Kelln, Biosynthesis and Conversion of Pyrimidines, Chapter 35 [In] Neidhardt, F. C. et al. [eds] “
Escherichia coli
and Salmonella Cellular and Molecular Biology”, Second Edition, Vol. I, pp580-599, ASM Press, Washington D.C., 1996. The expression of dTMPase and elimination of thymidine breakdown by mutations in the deoA (thymidine phosphorylase), udp (uridine phosphorylase) and tdk (thymidine kinase) genes and therefore resulting expression products results in thymidine synthesis in
E. coli
but not at a commerically viable level.
SUMMARY OF THE INVENTION
The biosynthesis of purines and pyrimidines involves a common step of reducing a ribonucleoside diphosphate (in some species triphosphate) to its corresponding deoxy analog. In the overall process the reduction of the ribose moiety to 2-deoxyribose requires a pair of hydrogen atoms which are ultimately donated by NADPH and H
+
. However, the immediate electron donor is not NADPH but the reduced form of a heat stable protein called thioredoxin or glutaredoxin and at least one other unidentified source since the
E. coli
ribonucleotide reductase system still works in trxA (thioredoxin) grx (glutaredoxin) double mutants (Neuhard and Kelln, supra). The reducing equivalents of the reduced thioredoxin are transferred to ribonucleoside diphosphate reductase which carries out the reduction process. Manipulation of, for example, this step could prove useful in improving the commerical production of purine and pyrimidine deoxynucleosides.
It is an object of the present invention to provide novel DNA constructs e.g. vectors and genetically modified microorganisms comprising said vectors particularly for use in the production of recoverable amounts, especially commercially useful amounts, of pyrimidine and purine deoxynucleosides.
It is also an object of the present invention to provide processes which represent an improvement over McCandliss and Anderson described supra.
In accordance with one aspect of the present invention there is provided a DNA construct comprising a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene or a uridine kinase gene and/or a dCTP deaminase gene.
In one embodiment the DNA construct comprises a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene.
In another embodiment the DNA construct comprises a transcriptional unit which comprises a uridine kinase gene and/or a dCTP deaminase gene.
Preferably the DNA construct comprises a transcriptional unit which comprises a uridine kinase gene and a dCTP deaminase gene.
Most preferably the DNA construct comprises a transcriptional unit which comprises a ribonucleotide reductase gene and a thioredoxin gene and a uridine kinase gene and a dCTP deaminase gene.
In accordance with another aspect of the present invention there is provided a modified host cell comprising a DNA construct according to the invention.
In accordance with yet another aspect of the present invention there is provided a culture medium comprising the modified host cells of the invention and processes for the production of a purine or pyrimidine, for example thymidine comprising the use of said modified host cells.
In one embodiment the host cells comprise a DNA construct which construct comprises a transcription DNA unit (e.g. operon) which unit comprises DNA sequences encoding for ribonucleotide reductase and thioredoxin in which said reductase preferably displays less sensitivity to allosteric inhibition than a wild type host cell equivalent or counterpart wherein said cell further comprises one or more of the following features:
(a) a transcription unit (e.g. operon), preferably located on said DNA construct, comprising DNA sequences encoding for (and preferably heterologous with respect to host cell equivalent) thymidylate synthase;
(b) a transcription unit (e.g. operon), preferably located on said DNA construct, comprising DNA sequences encoding for uridine kinase and preferably dCTP deaminase; and
(c) repressed or absent Uracil DNA glycosylase activity.
In another embodiment the DNA construct for use in the production of recoverable amounts of pyrimidine and derivatives thereof, in particular pyrimidine deoxyribonucleosides such as thymidine, comprises a transcription unit (e.g. operon) which unit comprises (preferably heterologous) DNA sequences encoding for
Anderson David Martin
Liu Lin
Podkovyrov Sergey
Wang Baomin
Conger Michael M.
Prouty Rebecca E.
Ramirez Delia M.
SmithKline Beecham Corporation
LandOfFree
Vectors, cells and processes for pyrimidine... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Vectors, cells and processes for pyrimidine..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Vectors, cells and processes for pyrimidine... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3341672