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
1995-04-12
2001-01-30
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252320, C435S252330, C435S252800, C435S252100, C435S440000, C435S477000, C435S487000, C435S843000, C435S849000
Reexamination Certificate
active
06180373
ABSTRACT:
The invention relates to microorganisms for the production of tryptophan and to a process for the preparation thereof.
It is known that tryptophan metabolism takes place by a single biosynthetic pathway in all micro-organisms hitherto investigated (Somerville, R. L., Herrmann, R. M., 1983, Aminoacids, Biosynthesis and Genetic Regulation, Addison-Wesley Publishing Company, U.S.A.: 301-322 and 351-378; Aida et al., 1986, Bio-technology of amino acid production, progress in industrial microbiology Vol. 24, Elsevier Science Publishers, Amsterdam: 188-206). Tryptophan metabolism, its linkage to serine metabolism, and the genes coding for the principal enzymes are depicted in FIG.
1
.
Known processes for tryptophan production are based on the expression of a mutated trpE gene which codes for a tryptophan-insensitive anthranilate synthase together with the other genes of the trp operon on a suitable autonomously replicable vector. Owing to the relatively high copy number of the genes, there is increased expression of the trp genes and correspondingly an increased amount of the individual enzymes of tryptophan metabolism. This results in overproduction of tryptophan.
Examples of such processes are described for a number of organisms: for example for
E. coli
: EP 0 293 207, U.S. Pat. No. 4,371,614, for bacillus U.S. Pat. No. 4,588,687, for corynebacterium and brevibacterium EP 0 338 474. A number of problems of process control arise in these processes. There may be instability and loss of the vector or a slowing of growth of the producer strain.
EP-A-0 401 735 (Applicant: Kyowa Hakko Kogyo Co.) describes a process for the production of L-tryptophan with the aid of corynebacterium or brevibacterium strains which contain recombinant plasmids. These plasmids harbour the genetic information for synthesizing the enzymes DAHP synthase, anthranilate synthase, indole-3-glycerol-P synthase, tryptophan synthase and phosphoglycerate dehydrogenase. Feedback-resistant anthranilate synthase alleles are used.
It is furthermore known to increase tryptophan production in strains with deregulated tryptophan metabolism by introducing a plurality of serA, or serA, B, C, wild-type genes. Thus, Chemical Abstracts CA 111 (1989) 16 86 88q and CA 111 (1989) 16 86 89r describe the use of bacillus strains which overexpress respectively the serA wild-type allele and all wild-type genes of serine metabolism (serA, serB and serC) on plasmids for the production of tryptophan WO-A-87/01130 describes the use of serA, serB and serC wild-type alleles for the production of tryptophan in
E. coli.
Increasing the tryptophan yield by preventing serine degradation in the cell is disclosed in EP-A-0 149 539 (Applicant: Stauffer Chemical Company). This patent application describes
E. coli
K12 mutants in which the serine-degrading enzyme serine deaminase (sda) is destroyed. It also describes the use of strains of this type for the production of amino acids. Example VIII describes the use of a strain of this type for the overproduction of tryptophan from anthranilate. The explanation for the improved tryptophan yield compared with a strain with intact serine deaminase in the European patent application is that, in microorganisms in which the reserve of tryptophan precursors is very high, serine or the serine biosynthesis capacity is rate-limiting for the production of tryptophan.
The object of the invention was to provide microorganisms which produce increased amounts of tryptophan and to provide processes which make it possible to prepare microorganisms of this type.
The object is achieved by strains of micro-organisms which are characterised in that they have a deregulated tryptophan metabolism and a serine metabolism which is deregulated at least by one feedback-resistant serA allele.
For the purpose of the present invention, feedback-resistant serA alleles means mutants of the serA gene which code for a phosphoglycerate dehydrogenase with a serine sensitivity which is less than that of the corresponding wild-type phosphoglycerate dehydrogenase of the particular microorganism.
The combination according to the invention of at least one feedback-resistant serA allele with a micro-organism with deregulated tryptophan metabolism results in an increase in the tryptophan yield by, astonishingly, up to 2.6-fold compared with the yield achievable with the same microorganism without the feedback-resistant serA allele under culturing conditions which are otherwise the same.
The increased production of tryptophan by the strains according to the invention is unexpected and surprising because feedback-resistant serA alleles show an effect only at a high intracellular serine level (Tosa T, Pizer L. J., 1971, journal of Bacteriology Vol. 106: 972-982; Winicov J., Pizer L. J., 1974, Journal of Biological Chemistry Vol. 249: 1348-1355). According to the state of the art (for example EP-A-0 149 539), however, microorganisms with deregulated tryptophan metabolism have a low serine level. This is why no increase in tryptophan production is to be expected by the introduction, according to the invention, of a feedback-resistant serA allele into microorganisms with deregulated tryptophan metabolism.
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Somerville, R.L., Herrmann, K.M., 1983, Aminoacids, Biosynthesis and Genetic Regulation, Adelison-Wesley Publishing Company, USA: 301-322 and 351-378.
Aida et al., 1986, Biotechnology of amino acid production, progress in industrial microbiology vol. 24, Elsevier Science Publishers, Amsterdam: 188-206.
Tosa T., Pizer L.J., 1971, Journal of Bacteriology vol. 106, No. 3, 972-982; “Biochemical Bases for the Antimetabolite Action of L-Serine Hydroxamate”.
Winicov I., Pizer L.J., 1974, Journal of Biological Chemistry vol. 249: 1348-1355, “The Mechanism of End Product Inhibition of Serine Biosynthesis”.
Miller J.H., 1972, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, USA: 113-185 Introduction to Unit III.
Bauerle R. et al., 1987, Methods in Enzymology, vol. 142: 366-386 “Anthranilate Synthase-Anthranilate Phosphoribosyltransferase Complex and Subunits ofSalmonella typhimurium”.
Caliqiuiri M.G., Bauerle R., 1991, J. of Biol. Chem. vol. 266: No. 13; pp. 8328-8335; “Indentification of Amino Acid Residues Involved in Feedback Regulation of the Anthranilate Synthase Complex fromSalomonella typhimurium”.
Matsui K. et al., 1987, J. Bact. vol. 169: 5330-5332; “Two Single-Base-Pair Substitutions Causing Desensitization to Tryptophan Feedback Inhibition of Anthranilate Synthase and Enhanced Expression of Tryptophan Genes ofBrevibacterium lactofermentum”.
Sarkar G., Sommer, S.S., 1990, Bio Techniques 8: 404-407 “The “Megaprimer” Method of Site-Directed Mutagenesis”.
Ausubel F.M. et al., 1987, Current Protocols in Molecular Biology, Greene Publishing Associates, “Mutagenesis of Cloned DNA”.
Smith M., 1985, Ann. Rev., Genet., 19: 423-462, “In Vitro Mutagenesis”.
Morse D.E., Yanofsky C., J. Mol. Biol. 44, 1969, 185-193; “Amber Mutants of the trp Regulatory Gene”.
Junetsu Ito and Irving P. Crawford; Genetics 52, 1965, 1303-1316, “Regulation of the Enzymes of the Tryptophan Pathway inEscherichia coli”.
Theodore K. Gartner and Monica Riley; J. Bact. 89, No. 2, 1965, 313-318 (erroneously cited as: J. Bact. 85, 1965, 680-685) “Isolation of Mutants Affecting Tryptophanase Production inEscherichia coli”.
Karen L. Tobey and Gregory A. Grant, 1986, J. Biol. Chem. vol. 261, No. 26, pp. 1
Backman Keith
Leinfelder Walfred
Wich G{umlaut over (u)}nter
Collard & Roe P.C.
Consortium f{umlaut over (u)}r elektrochemische Industrie GmbH
Mosher Mary E.
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