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
2001-08-24
2003-05-13
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
C435S252330
Reexamination Certificate
active
06562601
ABSTRACT:
FIELD OF THE INVENTION
The invention provides a new process for the fermentative preparation of L-threonine with Enterobacteriaceae.
PRIOR ART
L-Threonine is used in animal nutrition, in human medicine and in the pharmaceuticals industry.
It is known that L-threonine can be prepared by fermentation of strains of the Enterobacteriaceae family, in particular
Escherichia coli.
Because of the great importance of this amino acid, work is constantly being undertaken to improve the preparation processes. Improvements to the process can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form, by e.g. ion exchange chromatography, or the intrinsic output properties, i.e. those of genetic origin, of the microorganism itself.
It is known from the prior art, such as is described, for example, in U.S. Pat. No. 5,538,873 and in EP-B-0593792 or by Okamoto et al. (Bioscience, Biotechnology, and Biochemistry 61 (11), 1877—1882, 1997), that threonine is prepared by fermentation in the batch process (batch) or feed process (fed batch).
OBJECT OF THE INVENTION
The inventors had the object of providing new measures for improved fermentative preparation of L-threonine.
SUMMARY OF THE INVENTION
The invention provides a fermentation process, which is characterized in that
a) an L-threonine-producing microorganism of the Enterobacteriaceae family is cultured by the feed process (fed batch) in a known manner, subsequently
b) a portion of the fermentation broth is separated off, 1 to 90 vol. %, in particular 1 to 50 vol. %, preferably 1 to 25 vol. % and particularly preferably 5 to 50 vol. % of the total volume of the fermentation broth remaining in the fermentation tank, subsequently
c) the remaining fermentation broth is topped up with growth medium and, preferably after a growth phase, a further fermentation is carried out by the feed process (fed batch) mentioned,
d) steps b) and c) are optionally carried out several times, and
e) the L-threonine is isolated from the fermentation broths collected.
DETAILED DESCRIPTION OF THE INVENTION
The microorganisms with which the process according to the invention can be carried out can prepare L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, or from glycerol and ethanol, the preparation from glucose, sucrose or molasses being preferred. They are representatives of Enterobacteriaceae, in particular of the genera Escherichia, Serratia and Providencia. Of the genus Escherichia the species
Escherichia coli
and of the genus Serratia the species Serratia marcescens are to be mentioned in particular.
Suitable L-threonine-producing strains of the genus Escherichia, in particular of the species
Escherichia coli ,
are, for example
Escherichia coli
TF427
Escherichia coli
H-4225
Escherichia coli
H-4226
Escherichia coli
H-4257
Escherichia coli
H-4258
Escherichia coli
H-4435
Escherichia coli
H-4436
Escherichia coli
H-4578
Escherichia coli
H-7256
Escherichia coli
H-7263
Escherichia coli
H-7293
Escherichia coli
H-7294
Escherichia coli
H-7700
Escherichia coli
H-7729
Escherichia coli
H-8309
Escherichia coli
H-8311
Escherichia coli
H-9244
Escherichia coli
KY10935
Escherichia coli
EL1003
Escherichia coli
VNIIgenetika MG-442
Escherichia coli
VNIIgenetika VL334/pYN7
Escherichia coli
VNIIgenetika M1
Escherichia coli
VNIIgenetika 472T23
Escherichia coli
VNIIgenetika TDH-
Escherichia coli
BKIIM B-3996
Escherichia coli
BKIIM B-5318
Escherichia coli
B-3996-C43
Escherichia coli
B-3996-C80
Escherichia coli
B-3996/pTWV-pps
Escherichia coli
B-3996(pMW::THY)
Escherichia coli
B-3996/pBP5
Escherichia coli
Ferm BP-3756
Escherichia coli
Ferm BP-4072
Escherichia coli
Ferm BP-1411
Escherichia coli
kat 13
Escherichia coli
KCCM-10132
Escherichia coli
KCCM-10133.
Suitable L-threonine-producing strains of the genus Serratia, in particular of the species
Serratia marcescens,
are, for example
Serratia marcescens
HNr21
Serratia marcescens
TLr156
Serratia marcescens
T2000
Strains from the Enterobacteriaceae family which produce L-threonine preferably have, inter alia, one or more genetic or phenotypic features chosen from the group consisting of: resistance to &agr;-amino-&bgr;-hydroxyvaleric acid, resistance to thialysine, resistance to ethionine, resistance to &agr;-methylserine, resistance to diaminosuccinic acid, resistance to a-aminobutyric acid, resistance to borrelidin, resistance to rifampicin, resistance to valine analogues, such as, for example, valine hydroxamate, resistance to purine analogues, such as, for example, 6-dimethylaminopurine, a need for L-methionine, optionally a partial and compensatable need for L-isoleucine, a need for meso-diaminopimelic acid, auxotrophy in respect of threonine-containing dipeptides, resistance to L-threonine, resistance to L-homoserine, resistance to L-lysine, resistance to L-methionine, resistance to L-glutamic acid, resistance to L-aspartate, resistance to L-leucine, resistance to L-phenylalanine, resistance to L-serine, resistance to L-cysteine, resistance to L-valine, sensitivity to fluoropyruvate, defective threonine dehydrogenase, optionally an ability for sucrose utilization, enhancement of the threonine operon, enhancement of homoserine dehydrogenase I-aspartate kinase I, preferably of the feed back resistant form, enhancement of homoserine kinase, enhancement of threonine synthase, enhancement of aspartate kinase, optionally of the feed back resistant form, enhancement of aspartate semialdehyde dehydrogenase, enhancement of phosphoenol pyruvate carboxylase, optionally of the feed back resistant form, enhancement of phosphoenol pyruvate synthase, enhancement of transhydrogenase, enhancement of the RhtB gene product, enhancement of the RhtC gene product, enhancement of the YfiK gene product, enhancement of a pyruvate carboxylase, and attenuation of acetic acid formation.
Thus, for example, the strain 472T23 (U.S Pat. No. 5,631,157) has, inter alia, an enhanced, “feed back” resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain B-3996 (U.S. Pat. No. 5,175,107) has, inter alia, an enhanced, “feed back” resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine deaminase, an attenuated threonine dehydrogenase, a resistance to at least 5 g/l L-threonine and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain kat-13 (U.S. Pat. No. 5,939,307) has, inter alia, an enhanced, “feed back” resistant aspartate kinase I-homoserine dehydrogenase I, an attenuated threonine dehydrogenase, resistance to borrelidin and the ability to utilize sucrose as a source of carbon.
Thus, for example, the strain KCCM-10132 (WO 00/09660) has a resistance to &agr;-methylserine, a resistance to diaminosuccinic acid, sensitivity to fluoropyruvate, a resistance to L-glutamic acid and a resistance to at least 7% L-threonine. The strain is also in need of the amino acids L-methionine and L-isoleucine.
The term “enhancement” in this connection describes the increase in the intracellular activity of one or more enzymes in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or allele or of the genes or alleles, using a potent promoter or using a gene or allele which codes for a corresponding enzyme having a high activity, and optionally combining these measures.
By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on the starting microorganism.
The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (p
Hermann Thomas
Rieping Mechthild
Degussa - AG
Saidha Tekchand
Walicka Malgorzata A.
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