Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
1991-11-01
2003-11-18
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S069100, C435S320100, C435S115000, C435S190000, C435S252300, C435S252330, C435S243000, C530S350000, C530S324000, C530S326000, C536S023100, C536S023700
Reexamination Certificate
active
06649379
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is generally in the area of biochemistry and is specifically directed to production of the amino acid threonine.
Amino acids are often referred to as the building blocks of proteins. Amino acids also serve as sources of nitrogen and sulfur and can be catabolized to provide energy. There are twenty common amino acids all containing at least one carbon atom covalently bonded to a carboxyl group (COOH), an amino group (NH
3
), hydrogen (H) and a variable side chain (R).
Amino acids are necessary for the survival of all organisms. Some amino acids are synthesized by the organism while others are provided in the diet. Enzymes transform biomolecules into amino acids, degrade amino acids, and convert amino acids from one type to another. The absence or excess of an amino acid in humans can cause a clinical disorder such as Phenylketonuria, Cystinuria, Fanconi's syndrome or Hartnup disease. Treatment for these disorders currently involves dietary restrictions to reduce intake of the amino acids found in excess and supplementation of the deficient amino acids. The production of large quantities of purified amino acids is essential for scientific research involving amino acid metabolism and treatment of amino acid disorders.
The amino acid threonine has an uncharged polar R group containing a hydroxyl group. The synthesis of threonine proceeds from the substrate aspartate via the branched amino acid biosynthetic pathway as shown in FIG.
1
. Aspartate is synthesized from oxaloacetate, a product of glucose metabolism through the tricarboxylic acid cycle. Briefly, oxaloacetate is converted to L-aspartate by a transaminase. L-aspartate is converted to &bgr;-aspartylphosphate by aspartokinase which is dehydrogenated to L-aspartic-&bgr;-semialdehyde which is, in turn, dehydrogenated to L-homoserine by homoserine dehydrogenase encoded by the gene hom. Homoserine kinase encoded by the gene thrB converts L-homoserine to O-phospho-L-homoserine. Threonine synthase encoded by the gene thrC converts O-phospho-L-homoserine to the amino acid L-threonine.
Attempts have been made to produce threonine from bacteria. European Patent Application No. 82104088.8 entitled “Method for Producing L-Threonine by Fermentation” describes high yield producing strains of Corynebacterium produced by recombinant techniques. The antimetabolite &agr;-amino-&bgr;-hydroxy-valeric acid is used to screen strains for threonine over-production. Cells resistant to &agr;-amino-&bgr;-hydroxy-valeric acid toxicity are generally high producers of threonine. Genomic DNA from these resistant strains are inserted into Corynebacterium compatible plasmids and used to transform &agr;-amino-&bgr;-hydroxy-valeric acid sensitive strains to produce resistant clones. The gene or genes controlling resistance are not identified or characterized, and threonine production is only achieved with the isolated, resistant strain disclosed. The publication of Eikmanns et al.,
Appl. Microbiol. Biotechnol
., 34:617:622 (1991) similarly describes a mutant of the hom gene designated hom
fbr
, a homoserine dehydrogenase gene resistant to feedback inhibition by threonine. The hom
fbr
-thrB operon of
C. glutamicum
is expressed in corynebacterial strains for the production of threonine. However, neither the site nor the region of the mutation causing resistance to feedback inhibition is identified or characterized.
The threonine biosynthetic pathway has been studied extensively in bacteria such as the Gram-positive bacterium
Corynebacterium glutamicum
(
C. glutamicum
),
Escherichia coli
(
E. coli
) and
Bacillus subtilis
(
B. subtilis
). Although threonine is synthesized via the same reaction path shown in
FIG. 1
in all three bacteria, the genetic and biochemical organization responsible for the enzymes homoserine dehydrogenase, homoserine kinase, and threonine synthase differ in each organism.
In Corynebacterium such as
C. glutamicum
, and
C. flavum
, the homoserine dehydrogenase and homoserine kinase enzymes are encoded by a two-gene operon hom-thrB, as described by the publications of Follettie et al., Organization and regulation of the
Corynebacterium glutamicum
hom-thrB and thrC loci,
Mol. Microbiol
. 2:53-62 (1988) and Peoples et al., Nucleotide sequence and fine structural analysis of the
Corynebacterium glutamicum
hom-thrB operon,
Mol. Microbiol
. 2:63-72 (1988), and U.S. Ser. No. 07/062,552 filed Jun. 12, 1987. Transcription of the hom-thrB operon is repressed. by the amino acid methionine while the activity of homoserine dehydrogenase is allosterically inhibited by the amino acid end product threonine.
The
E. coli
threonine operon (thrABC) encodes four enzyme activities, namely a bi-functional polypeptide, aspartokinase-I-homoserine dehydrogenase-I, a monofunctional homoserine kinase and a threonine synthase. A second bi-functional protein, aspartokinase-II-homoserine dehydrogenase-II, is encoded by the metL gene. Expression of the thrABC operon is controlled by threonine-isoleucine mediated attenuation. Both of the activities encoded by the thrA gene, aspartokinase-I-homoserine dehydrogenase-I, are allosterically inhibited by the amino acid threonine.
The
B. subtilis
homoserine dehydrogenase, threonine synthase and homoserine kinase genes are closely linked in the order hom-thrC-thrB and most likely form an operon. The homoserine dehydrogenase enzyme is repressed by the amino acids threonine and methionine.
In all three bacteria, regulation of threonine synthesis is accomplished by end-product inhibition of the first enzyme in the threonine pathway, the enzyme homoserine dehydrogenase, encoded by the gene hom or thrA. The phenomenon of allosteric inhibition of the monofunctional homoserine dehydrogenase enzyme of
C. glutamicum
is characterized in the publication of Follettie et al.,
Mol. Microbiol
. 2:53-62 (1988). Threonine inhibits the enzyme with an inhibition rate constant (Ki) of 0.16 mM. Most likely, threonine inhibits the enzymatic activity of homoserine dehydrogenase by binding to a binding site on the enzyme.
Peoples et al.,
Mol. Microbiol
. 2:63-72 (1988), have sequenced the hom gene of
C. glutamicum
which encodes homoserine dehydrogenase and from this sequence have determined an amino acid sequence encoding a 43,000 dalton polypeptide. The
C. glutamicum
homoserine dehydrogenase exhibits 27% and 31% homology with the
E. coli
and
B. subtilis
homoserine dehydrogenase amino acid sequences respectively.
Attempts have been made to utilize the genes encoding the enzymes involved in threonine biosynthesis to achieve threonine over-production. Morinaga et al.,
Agric. Biol. Chem
. 51:93-100 (1987) describe transformation of bacterial cells with a plasmid containing both the gene for homoserine kinase from a threonine-producing mutant bacterial strain and the gene for homoserine dehydrogenase. Miwa et al.
Agric. Biol. Chem
. 48:2233-2237 (1984) describe a recombinant
E. coli
strain transformed with a recombinant plasmid containing the threonine operon (thrA, thrB and thrC,) of
E. coli
. Nakamori et al.,
Chem. Abstracts
102:216318g (1985) transform
Brevibacterium lactofermentum
with a recombinant plasmid containing the gene for homoserine kinase. Nakamori et al.,
Agric. Biol. Chem
. 51:87-91 (1987) transform
Brevibacterium lactofermentum
with a recombinant plasmid containing the gene for homoserine dehydrogenase from B. lactofermentum M-15, a threonine and lysine-producing mutant. Takagi et al.,
Chem. Abstracts
106:48643w (1987) transform coryneform bacteria with a recombinant plasmid containing homoserine kinase-encoding genes. The problems with these methods of producing threonine is that the mutations are not characterized, and the resulting plasmids are inherently unstable, resulting in transformed bacteria that are genetically fragile.
What is needed is a method of producing threonine that involves a characterized structural mutation. A mutation of the homoserine dehydrogenase gene that prevents end-product inhibition by threonine should result in
Archer John A. C.
Follettie Maximillian T.
Sinskey Anthony J.
Guzo David
Holland & Knight LLP
Massachusetts Institute of Technology
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