Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-06-23
2004-12-14
Horlick, Kenneth R. (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024320, C536S024330, C530S350000, C435S006120
Reexamination Certificate
active
06831165
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 the large-scale culture of bacteria developed to produce and secrete large quantities of one or more desired molecules. 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 homeostasis and adaptation (HA) 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 HA 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 HA nucleic acids of the invention, or modification of the sequence of the HA 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 HA 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 HA 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.
e.g.e.g. The HA proteins encoded by the novel nucleic acid molecules of the invention are capable of, for example, performing a function involved in the maintenance of homeostasis in
C. glutamicum
, or in the ability of this microorganism to adapt to different environmental conditions. 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. This improved production or efficiency of production of a fine chemical may be due to a direct effect of manipulation of a gene of the invention, or it may be due to an indirect effect of such manipulation.
There are a number of mechanisms by which the alteration of an HA protein of the invention may directly affect the yield, production, and/or efficiency of production of a fine chemical from a
C. glutamicum
strain incorporating such an altered protein. For example, by engineering enzymes which modify or degrade aromatic or aliphatic compounds such that these enzymes are increased or decreased in activity or number, it may be possible to modulate the production of one or more fine chemicals which are the modification or degradation products of these compounds. Similarly, enzymes involved in the metabolism of inorganic compounds provide key molecules (e.g. phosphorous, sulfur, and nitrogen molecules) for the biosynthesis of such fine chemicals as amino acids, vitamins, and nucleic acids. By altering the activity or number of these enzymes in
C. glutamicum
, it may be possible to increase the conversion of these inorganic compounds (or to use alternate inorganic compounds) to thus permit improved rates of incorporation of inorganic atoms into these fine chemicals. Genetic engineering of
C. glutamicum
enzymes involved in general cellular processes may also directly improve fine chemical production, since many of these enzymes directly modify fine chemicals (e.g., amino acids) or the enzymes which are involved in fine chemical synthesis or secretion. Modulation of the activity or number of cellular proteases may also have a direct effect on fine chemical production, since many proteases may degrade fine chemicals or enzymes involved in fine chemical production or breakdown.
Further, the aforementioned enzymes which participate in aromatic/aliphatic compound modification or degradation, general biocatalysis, inorganic compound metabolism or proteolysis are each themselves fine chemicals, desirable for their activity in various in vitro industrial applications. By altering the number of copies of the gene for one or more of these enzymes in
C. glutamicum
it may be possible to increase the number of these proteins produced by the cell, thereby increasing the potential yield or efficiency of production of these proteins from large-scale
C. glutamicum
or related bacterial cultures.
The alteration of an HA protein of the invention may also indirectly affect the yield, production, and/or efficiency of production of a fine chemical from a
C. glutamicum
strain incorporating such an altered protein. For example, by modulating the activity and/or number of those proteins involved in the construction or rearrangement of the cell wall, it may be possible to modify the structure of the cell wall itself such that the cell is able to better withstand the mechanical and other stresses present during large-scale fermentative-culture. Also, large-scale growth of
C. glutamicum
requires significant cell wall production. Modulation of the activity or number of cell wall biosynthetic or degradative enzymes may allow more rapid rates of cell wall biosynthesis, which in turn may permit increased growth rates of this microorganism in culture and thereby increase the number of cells producing the desired fine chemical.
By modifying the HA enzymes of the invention, one may also indirectly impact the yield, production, or efficiency of production of one or more fine chemicals from
C. glutamicum
. For example, many of the general enzymes in
C. glu
Haberhauer Gregor
Kröger Burkhard
Pompejus Markus
Schröder Hartwig
Zelder Oskar
BASF - Aktiengesellschaft
DiRocco Lisa M.
Hanley Elizabeth A.
Horlick Kenneth R.
Lahive & Cockfield LLP
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