Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-06-23
2004-11-23
Myers, Carla J. (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C536S024300, C536S024330, C530S350000, C435S069100, C435S252300
Reexamination Certificate
active
06822084
ABSTRACT:
BACKGROUND OF THE INVENTION
Certain products and by-products of naturally-occurring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed ‘fine chemicals’, include organic acids, both proteinogenic and non-proteinogenic amino acids, nucleotides and nucleosides, lipids and fatty acids, diols, carbohydrates, aromatic compounds, vitamins and cofactors, and enzymes. Their production is most conveniently performed through large-scale culture of bacteria developed to produce and secrete large quantities of a particular desired molecule. One particularly useful organism for this purpose is
Corynebacterium glutamicum
, a gram positive, nonpathogenic bacterium. Through strain selection, a number of mutant strains have been developed which produce an array of desirable compounds. However, selection of strains improved for the production of a particular molecule is a time-consuming and difficult process.
SUMMARY OF THE INVENTION
The invention provides novel bacterial nucleic acid molecules which have a variety of uses. These uses include the identification of microorganisms which can be used to produce fine chemicals, the modulation of fine chemical production in
C. glutamicum
or related bacteria, the typing or identification of
C. glutamicum
or related bacteria, as reference points for mapping the
C. glutamicum
genome, and as markers for transformation. These novel nucleic acid molecules encode proteins, referred to herein as stress, resistance and tolerance (SRT) proteins.
C. glutamicum
is a gram positive, aerobic bacterium which is commonly used in industry for the large-scale production of a variety of fine chemicals, and also for the degradation of hydrocarbons (such as in petroleum spills) and for the oxidation of terpenoids. The SRT nucleic acid molecules of the invention, therefore, can be used to identify microorganisms which can be used to produce fine chemicals, e.g, by fermentation processes. Modulation of the expression of the SRT nucleic acids of the invention, or modification of the sequence of the SRT nucleic acid molecules of the invention, can be used to modulate the production of one or more fine chemicals from a microorganism (e.g. to improve the yield or production of one or more fine chemicals from a
Corynebacterium
or
Brevibacterium
species).
The SRT nucleic acids of the invention may also be used to identify an organism as being
Corynebacterium glutamicum
or a close relative thereof, or to identify the presence of
C. glutamicum
or a relative thereof in a mixed population of microorganisms. The invention provides the nucleic acid sequences of a number of
C glutamicum
genes; by probing the extracted genomic DNA of a culture of a unique or mixed population of microorganisms under stringent conditions with a probe spanning a region of a
C. glutamicum
gene which is unique to this organism, one can ascertain whether this organism is present. Although
Corynebacterium glutamicum
itself is nonpathogenic, it is related to species pathogenic in humans, such as
Corynebacterium diphtheriae
(the causative agent of diphtheria); the detection of such organisms is of significant clinical relevance.
The SRT nucleic acid molecules of the invention may also serve as reference points for mapping of the
C. glutamicum
genome, or of genomes of related organisms. Similarly, these molecules, or variants or portions thereof, may serve as markers for genetically engineered
Corynebacterium
or
Brevibacterium
species.
The SRT proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, permitting
C. glutamicum
to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Given the availability of cloning vectors for use in
Corynebacterium glutamicum
, such as those disclosed in Sinskey et al., U.S. Pat. No. 4,649,119, and techniques for genetic manipulation of
C. glutamicum
and the related Brevibacterium species (e.g., lactofermentum) (Yoshihama et al,
J. Bacteriol.
162: 591-597 (1985); Katsumata et al.,
J. Bacteriol.
159: 306-311 (1984); and Santamaria et al,
J. Gen. Microbiol.
130: 2237-2246 (1984)), the nucleic acid molecules of the invention may be utilized in the genetic engineering of this organism to make it a better or more efficient producer of one or more fine chemicals, through the ability of these proteins to permit growth and multiplication of
C. glutamicum
(and also continuous production of one or more fine chemicals) under circumstances which would normally impede growth of the organism, such as those conditions frequently encountered during large-scale fermentative growth. For example, by overexpressing or engineering a heat-shock induced protease molecule such that it is optimized in activity, one may increase the ability of the bacterium to degrade incorrectly folded proteins when the bacterium is challenged with high temperatures. By having fewer misfolded (and possibly misregulated or nonfunctional) proteins to interfere with normal reaction mechanisms in the cell, the cell is increased in its ability to function normally in such a culture, which should in turn provide increased viability. This overall increase in number of cells having greater viability and activity in the culture should also result in an increase in yield, production, and/or efficiency of production of one or more desired fine chemicals, due at least to the relatively greater number of cells producing these chemicals in the culture.
This invention provides novel SRT nucleic acid molecules which encode SRT proteins which are capable of, for example, permitting
C. glutamicum
to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Nucleic acid molecules encoding an SRT protein are referred to herein as SRT nucleic acid molecules. In a preferred embodiment, the SRT protein participates in metabolic pathways permitting
C. glutamicum
to survive in a setting which is either chemically or environmentally hazardous to this microorganism. Examples of such proteins include those encoded by the genes set forth in Table 1.
Accordingly, one aspect of the invention pertains to isolated nucleic acid molecules (e.g., cDNAs, DNAs, or RNAs) comprising a nucleotide sequence encoding an SRT protein or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection or amplification of SRT-encoding nucleic acid (e.g., DNA or mRNA). In particularly preferred embodiments, the isolated nucleic acid molecule comprises one of the nucleotide sequences set forth in Appendix A or the coding region or a complement thereof of one of these nucleotide sequences. In other particularly preferred embodiments, the isolated nucleic acid molecule of the invention comprises a nucleotide sequence which hybridizes to or is at least about 50%, preferably at least about 60%, more preferably at least about 70%, 80% or 90%, and even more preferably at least about 95%, 96%, 97%, 98%, 99% or more homologous to a nucleotide sequence set forth in Appendix A, or a portion thereof. In other preferred embodiments, the isolated nucleic acid molecule encodes one of the amino acid sequences set forth in Appendix B. The preferred SRT proteins of the present invention also preferably possess at least one of the SRT activities described herein.
In another embodiment, the isolated nucleic acid molecule encodes a protein or portion thereof wherein the protein or portion thereof includes an amino acid sequence which is sufficiently homologous to an amino acid sequence of Appendix B, e.g., sufficiently homologous to an amino acid sequence of Appendix B such that the protein or portion thereof maintains an SRT activity. Preferably, the protein or portion thereof encoded by the nucleic acid molecule maintains the ability to increase the survival of
C. glutamicum
in a setting which is either chemically or envi
Haberhauer Gregor
Kim Hyung-Joon
Kroger Burkhard
Lee Heung-Shick
Pompejus Markus
BASF - Aktiengesellschaft
DiRocco Lisa M.
Hanley Elizabeth A.
Lahive & Cockfield LLP
Myers Carla J.
LandOfFree
Corynebacterium glutamicum genes encoding stress, resistance... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Corynebacterium glutamicum genes encoding stress, resistance..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Corynebacterium glutamicum genes encoding stress, resistance... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3346641