Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-12-13
2003-03-25
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S375000
Reexamination Certificate
active
06537815
ABSTRACT:
TECHNICAL FIELD
This invention is related to the field of microbiology. Specifically, the invention relates to alteration of metabolic pathways in
E. coli
and other bacteria.
BACKGROUND ART
Bacteria grown in the laboratory have three stages of growth: the lag phase, in which little change in the number of viable cells occurs; the log phase, in which exponential increase in numbers occurs;.and the stationary phase, in which increase ceases. During the transition into stationary phase, bacteria acquire numerous new physiological properties which enhance their ability to compete and survive under suboptimal conditions. For reviews, see Kolter (1992)
ASM News
58:75-79; Matin (1991)
Mol. Microbiol.
5:3-10; Matin et al. (1989)
Ann. Rev. Microbiol.
43:293-316; and Siegele et al. (1992)
J. Bacteriol.
174:345-348. A few global regulatory factors mediate many of the extensive changes in gene expression that occur as
E. coli
enters the stationary phase. In
E. coli
, for example, the induction of several genes and operons requires a putative sigma factor known as rpoS or katF. Bohannon et al. (1991)
J. Bacteriol.
173:4482-4492; Lange et al. (1991)
Mol. Microbiol.
5:49-51; Matin (1991); and Schellhorn et al. (1992)
J. Bacteriol.
174:4769-4776.
One of the metabolic pathways transcriptionally activated in stationary phase mediates glycogen biosynthesis. The accumulation of glycogen in the early stationary phase reflects at least two levels of control: allosteric regulation of the committed step of the biochemical pathway; and enhanced expression of the structural genes for the pathway. Expression of this pathway does not require rpoS for expression. Bohannon et al. (1991).
The essential enzymes of the glycogen pathway are glgC (encoding ADPglucose pyrophosphorylase [EC 2.7.7.27]) and glgA (encoding glycogen synthase [EC 2.4.1.21]), which are apparently cotranscribed in an operon, glgCAY. Romeo et al. (1990)
Curr. Microbiol.
21:131-137; and Romeo et al. (1989)
J. Bacteriol.
171:2773-2782. The operon also includes the gene glgY or glgP, which encodes the catabolic enzyme glycogen phosphorylase [E.C 2.4.1.1]. Romeo et al. (1988)
Gene
70:363-376; and Yu et al. (1988)
J. Biol. Chem.
263:13706-13711. Four stationary-phase-induced transcripts have been mapped within the 0.5 kb upstream noncoding region of the glgC gene from
E. coli
, implying a complex transcriptional regulation of glgCA.
Located upstream of glgCAY is another operon, glgBX, also encoding genes involved in the glycogen pathway. The gene glgB encodes glycogen branching enzyme [EC 2.4.1.18] and is transcribed independently of glgCA. Baecker et al. (1986)
J. Biol. Chem.
261:8738-8743; Romeo et al. (1988); Preiss et al. (1989)
Adv. Microb. Physiol.
30:183-233; and Romeo et al. (1989).
The gene csrA or “carbon storage regulator” is a trans-acting factor which effects potent negative regulation of glycogen biosynthesis. Romeo et al. (1993a)
J. Bacteriol.
175:4744-4755; and Romeo et al. (1993b)
J. Bacteriol.
175:5740-5741. CsrA is a global regulator which controls numerous genes and enzymes of carbohydrate metabolism. In
E. coli
K-12, it exerts pleiotropic effects, acting as a negative regulator of glycogen biosynthesis, gluconeogenesis and glycogen catabolism, as a positive factor for glycolysis, and affecting cell surface properties. Romeo et al. (1993a); Liu et al. (1995)
J. Bacteriol.
177:2663-2672; Sabnis et al. (1995)
J. Biol. Chem.
270:29096-29104; and Yang et al. (1996)
J. Bacteriol.
178:1012-1017.
Csr is the third system discovered to be involved in the regulation of glgCA-mediated glycogen biosynthesis and the only one known to down-regulate the expression of this operon. The other two involve cyclic AMP (cAMP)/cAMP receptor protein and guanosine 3′-bisphosphate 5′-bisphosphate (ppGpp), which are positive regulators of glgCA. Romeo et al. (1990); Romeo et al. (1989); Bridger et al. (1978)
Can. J. Biochem.
56:403-406; Dietzler et al. (1979)
J. Biol. Chem.
254:8308-8317; Dietzler et al. (1977)
Biochem. Biophys. Res. Commun.
77:1459-1467; Leckie et al. (1983)
J. Biol. Chem.
258:3813-3824; Leckie et al. (1985)
J. Bacteriol.
161:133-140; and Taguchi et al. (1980)
J. Biochem.
88:379-387. The physiological role played by these three systems may be to establish an intrinsic metabolic capacity for glycogen synthesis in response to nutritional status. The effects of other regulatory factors, such as the allosteric effectors fructose-1,6-bisphosphate and AMP, may be superimposed upon this intrinsic metabolic capacity.
glgCA expression does not appear to be regulated by other global systems such as the nitrogen starvation system, mediated by NtrC and NtrA or &sgr;
54
; heat shock, mediated by &sgr;
32
; or the katF-dependent system. Preiss et al. (1989); Romeo et al. (1989); and Hengge-Aronis et al. (1992)
Mol. Microbiol.
6:1877-1886.
A homolog of csrA in the pathogenic Erwinia species is rsmA (repressor of stationary phase metabolites). Chatterjee et al. (1995)
Appl. Environ. Microbiol.
61:1959-1967; and Cui et al. (1995)
J. Bacteriol.
177:5108-5115. rsmA has a role in the expression of several virulence factors of soft rot disease of higher plants, including pectinase, cellulase and protease activities. rsmA may also modulate the production of the quorum-sensing metabolite N-(3-oxohexanoyl)-L-homoserine lactone. Homologs of this metabolite are secreted by numerous Gram-negative bacteria, where they activate the expression of a variety of genes in response to cell density, as reviewed in Fuqua et al. (1994)
J. Bacteriol.
176:269-275; and Swift et al. (1996)
TIBS
21:214-219. Widespread phylogenetic distribution of csrA homologs among eubacteria points to a broad significance and ancient origin for this regulatory system in this group of organisms. White et al. (1.996)
Gene
182:221-223; and Romeo (1996)
Res. Microbiol.
147:505-512.
The csrA gene product or protein (CsrA) is a 61 amino acid protein containing a conserved RNA-binding motif and apparently mediates its regulatory activity via a cis-acting region located close to or overlapping the glgC ribosome binding site. Liu et al. (1995). CsrA strongly inhibits glycogen accumulation and affects the ability of cells to utilize certain carbon sources for growth. The down-regulated expression of csrA and CsrA can be useful for enhancing expression of products produced by alternative pathways. Such products include, but are not limited to, antibiotics, metabolites, organic acids, amino acids and a wide variety of industrially important compounds produced in bacterial fermentation systems.
As an example, down-regulating csrA expression can be used to increase the production of aromatic amino acids (e.g., tyrosine, phenylalanine, and tryptophan). These amino acids, which are commercially produced using
E. coli
cultures, have numerous uses, including the production of aspartame (Nutrisweet™). The TRI-5 mutation in csrA (csrA::kan
R
) causes overexpression of the genes pckA [encoding phosphoenolpyruvate (PEP) carboxykinase] and pps (phosphoenolpyruvate synthase), thereby raising production of PEP. PEP is in turn a precursor of aromatic amino acids and other metabolic products.
Further “metabolic engineering” can lead to even greater yields of desired amino acids or other products. In addition to being an amino acid precursor, PEP is a precursor of glucose via gluconeogenesis. Glucose is, in turn, a precursor of glycogen. Gluconeogenesis and glycogen synthesis are elevated in csrA mutants and would compete for the synthesis of aromatic amino acids. Therefore, in order to further increase carbon flow into the desired products (e.g., amino acids), engineering of gluconeogenesis, glycogen biosynthesis and possibly other pathways is desirable. A mutation in fbp, which encodes fructose-1,6-bisphosphatase, prevents gluconeogenesis from proceeding beyond the synthesis of fructose-1,6-bisphosphate. A mutation in glgC (ADP-glucose pyrophosphorylase) or glgA (glycogen synthase)
McKelvey Terry
Morrison & Foerster / LLP
University of North Texas Health Science Center at Fort Worth
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