Process for the fermentative preparation of L-threonine

Details Process for the fermentative preparation of L-threonine Process for the fermentative preparation of L-threonine

2001-03-08

2003-10-07

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C435S069100, C435S183000, C435S252300, C435S252320, C435S320100, C530S350000, C536S023200

Reexamination Certificate

active

06630332

ABSTRACT:

This invention relates to the new amino acid sequence of the malate:quinone oxidoreductase enzyme protein (Mqo) of Enterobacteriaceae and to a process for the fermentative preparation of L-threonine using Enterobacteriaceae in which the mqo gene is enhanced.
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 Enterobacteriaceae, in particular
Escherichia coli
and
Serratia marcescens
. Because of their great importance, 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 of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites, such as e.g. the threonine analogue &agr;-amino-&bgr;-hydroxyvaleric acid (AHV), or are auxotrophic for metabolites of regulatory importance and produce L-threonine are obtained in this manner.
Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Enterobacteriaceae strains which produce L-threonine, by amplifying individual threonine biosynthesis genes and investigating the effect on the L-threonine production.
OBJECT OF THE INVENTION
The inventors had the object of providing new measures for improved fermentative preparation of L-threonine.
DESCRIPTION OF THE INVENTION
The invention provides a polypeptide from Enterobacteriaceae with malate:quinone oxidoreductase (Mqo) activity (E.C. 1.1.99.16) chosen from the group consisting of
a) polypeptide with the amino acid sequence shown in SEQ ID NO. 2, or
b) polypeptide which is at least 70%, preferably at least 80%, particularly preferably at least 90 to 95% identical to the amino acid sequence shown in SEQ ID NO. 2, or
c) polypeptide according to SEQ ID NO. 2, including deletion, insertion or exchange of one or more amino acids, or
d) polypeptide according to SEQ ID NO. 2, including N- or C-terminal lengthening by one or more amino acids,
the total length of the polypeptide according to b), c) or d) being at least 514 and at most 544, preferably at least 519 and at most 539, in a preferred form at least 524 and at most 534, particularly preferably at least 527 and at most 531 amino acid radicals.
The invention furthermore provides a polynucleotide from Enterobacteriaceae which codes for a polypeptide with malate:quinone oxidoreductase (Mqo) activity (E.C. 1.1.99.16), chosen from the group consisting of
a) DNA which contains the nucleotide sequence corresponding to nucleobases 7 to 1593 of SEQ ID NO. 1, or
b) DNA according to a) corresponding to the degeneration of the genetic code, or
c) DNA according to a) containing sense mutations of neutral function, or
d) DNA which is at least 70%, preferably at least 80%, particularly preferably at least 90 to 95% identical to that mentioned in a) or b), or
e) polynucleotide which hybridizes with the DNA according to a), b), c) or d).
The invention also provides
a DNA which is capable of replication and codes for the polypeptide shown in SEQ ID NO. 2,
a vector containing the mqo gene corresponding to nucleobases 7 to 1593 of SEQ ID NO. 1, in particular plasmid pMW218mqo shown in FIG.
1
.
“Polynucleotide” in general relates to polyribonucleotides and polydeoxyribonucleotides, it being possible for these to be non-modified RNA or DNA or modified RNA or DNA.
“Polypeptides” is understood as meaning peptides or proteins which comprise two or more amino acids bonded via peptide bonds.
The polypeptides according to the invention include the polypeptides according to SEQ ID NO. 2, which have malate:quinone oxidoreductase activity, and also those which are at least 70%, preferably at least 80% and in particular at least 90% to 95% identical to the polypeptide according to SEQ ID NO. 2 and have the activity mentioned.
Finally, the invention provides a process for the fermentative preparation of L-threonine using Enterobacteriaceae which in particular already produce L-threonine and in which the nucleotide sequence(s) which code(s) for the mqo gene are enhanced, in particular over-expressed.
In particular, the process is a process for the preparation of L-threonine, which comprises carrying out the following steps:
a) fermentation of microorganisms of the family Enterobacteriaceae in which at least the mqo gene is enhanced (over-expressed), optionally in combination with further genes,
b) concentration of the L-threonine in the medium or in the cells of the microorganisms of the family Enterobacteriaceae, and
c) isolation of the L-threonine.
The term “enhancement” in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, using a potent promoter or a gene which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures.
The microorganisms which the present invention provides can prepare L-threonine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, or from glycerol and ethanol. They are representatives of Enterobacteriaceae, in particular of the genera Escherichia and Serratia. Of the genus Escherichia the species
E. 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
E. coli,
are, for example
Escherichia coli
TF427
Escherichia coli
H4578
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-6
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
kat 13
Escherichia coli
KCCM-10132
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
The nucleotide sequence of the chromosome of
E. coli
is known and is available in databanks accessible to the public, such as, for example, the databank of the European Molecular Biology Laboratories (EMBL, Heidelberg, Germany). Examples of such sequences deposited are the entries accessible under number AE000310 or D90850.
In the work on the present invention it was possible to identify the mqo gene, which codes for malate:quinone oxidoreductase, of
E. coli
(SEQ ID NO. 1) and the amino acid sequence of the Mqo enzyme protein formed (SEQ ID NO. 2).
It has furthermore been possible to isolate two further new malate:quinone oxidreductase [sic] proteins, designated protein B and C, which have the N-terminal amino acid sequence shown in SEQ ID No. 11 and 12. These are also provided by the invention.
It has been found that Enterobacteriaceae produce L-threonine in an improved manner after over-expression of the mqo gene, which codes for malate:quinone oxidoreductase (E.C. 1.1.99.16).
According to the invention, it is also possible to use a DNA section which contains the DNA sequence of the gene of the malate:quinone oxidoreductase given in the databank of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA) with accession number P33940.
Alleles of the mqo gene which result from the degeneracy of the genetic code or due to “sense mutations” of neutral funct

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