Note: Descriptions are shown in the official language in which they were submitted.
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Production Of L-amino Acids By Fermentation
Using Coryneform Bacteria
The invention provides a process for the production of L-
amino acids, especially L-lysine, by fermentation using
coryneform bacteria in which the mqo gene is amplified.
L-amino acids, especially L-lysine, are used in the feeding
of animals, in human medicine and in the pharmaceuticals
industry.
It is known that those amino acids are produced by
fermenting strains of coryneform bacteria, especially
Corynebacterium glutamicum. Because of their great
importance, work is continually being done to improve the
production processes. Improvements to the process may
concern measures relating to the fermentation process, such
as, for example, stirring and oxygen supply, or the
composition of the nutrient media, such as, for example,
the sugar concentration during the fermentation, or
working-up to the product form by, for example, ion-
exchange chromatography, or the intrinsic performance
properties of the microorganism itself.
To improve the performance properties of those micro-
organisms, methods of mutagenesis, selection and mutant
selection are used. In that manner there are obtained
strains that are resistant to antimetabolites, such as, for
example, the lysine analogue S-(2-aminoethyl)-cysteine, or
are auxotrophic for amino acids which are important in
terms of regulation, and produce L-amino acids.
For some years, methods of recombinant DNA technology have
also been used to improve the L-amino-acid-producing
strains of Corynebacterium gl_utamicum by ampii.fyi.ng
individual genes of amino acid biosynthesis and studying
the effect on the production of L-amino acids. General
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990012 BT/AL
2
articles on that subject will be found inter alia in
Kinoshita ("Glutamic Acid Bacteria", in: Biology of
Industrial Microorganisms, Demain and Solomon (eds.),
Benjamin Cummings, London, UK, 1985, 115-142), Hilliger
(BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6, 261-272
(1994)), Jetten and Sinskey (Critical Reviews in
Biotechnology 15, 73-103 (1995)) and Sahm et a1. (Annuals
of the New York Academy of Science 782, 25-39 (1996)).
L-amino acids, especially L-lysine, axe used in the feeding
of animals, in human medicine and in the pharmaceuticals
industry. There is, therefore, a general interest in making
available new, improved processes for the production of
those compounds.
The inventors have set themselves the task of making
available new measures for the improved production of
L-amino acids, especially L-lysine, by fermentation.
Any mention of L-lysine or lysine hereinbelow is to be
understood as meaning not only the base but also the salts,
such as, for example, lysine monohydrochloride or lysine
sulfate.
The invention provides a process for the production by
fermentation, of L-amino acids, especially L-lysine, using
coryneform bacteria which, especially, already produce th.t~
desired amino acid and in which the nucleotide sequence
coding for the enzyme malate:quinone oxidoreductase (mqo
gene) is amplified, especially overexpressed.
The term "amplification" in this connection describes the
increase in the intracellular activity of one or more
enzymes in a microorganism which are coded by the
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corresponding DNA, by, for example, increasing the copy
number of the gene or genes, using a strong promoter or
using a gene that codes for a corresponding enzyme having a
high degree of activity, and optionally combining those
measures.
The microorganisms provided by the present invention can
produce L-amino acids, especially L-lysine, from glucose,
saccharose, lactose, fructose, maltose, molasses, starch,
cellulose or from glycerol and ethanol. They are
representatives of coryneform bacteria, especially of the
genus Corynebacterium. In the genus Corynebacterium,
special mention may be made of the species Corynebacterium
glutamicum, which is known to those skilled in the art for
its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, especially
of the species Corynebacterium glutamicum, are the known
wild type strains
Corynebacterium glutamicum ATCC13032
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and L-amino-acid-producing, especially L-lysine-producing,
mutants and strains produced therefrom, such as, for
example,
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708
Brevibacterium lactofermentum FERM-P 1712
Brevibacterium flavum FERM-P 6463 and
Brevibacterium flavum FERM-P 6964.
The inventors have found that coryneform bacteria produce
L-amino acids, especially L-lysine, in an improved manner
after overexpressi.on of malate:quinone oxidoreductase.
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The mqo gene codes for the enzyme malate:quinone oxido-
reductase (EC 1.1.99.16), which catalyses the oxidation of
malate to oxalacetate with transfer of the electrons to
ubiquinone-1 (Molenaar et al., European Journal of Bio-
chemistry 254, 395-403 (1998)). The nucleotide sequence of
the mqo gene of Corynebacterium glutamicum has likewise
been determined by Molenaar et al. (European Journal of
Biochemistry 254, 395-403 (1998)) and is generally
available at the nucleotide sequence databank of the
National Center for Biotechnology Information (NCBI,
Behesda, MD, USA) under Accession Number AJ 22 4946.
The mqo gene of C. glutamicum described by Molenaar et al.
(European Journal of Biochemistry 254, 395-403 (1998)) can
be used according to the invention. It is also possible to
use alleles of the mqo gene which result from the
degeneracy of the genetic code or by function-neutral sense
mutations.
In order to achieve an overexpression, the copy number of
the corresponding genes can be increased, or the promoter
and regulation region, which is located in front of the
structural gene, can be mutated. Expression cassettes,
which are inserted in front of the structural gene, have
the same effect. By means of inducible promoters it is
additionally possible to increase the expression in the
course of the production of L-lysine by fermentation. The
expression is likewise improved by measures for lengthening
the life of the m-RNA. The enzyme activity is also
increased by preventing the degradation of the enzyme
protein. The genes or gene constructs can either be present
in plasmids with different copy numbers or be integrated
and amplified in the chromosome. Alternatively,
overexpression of the genes in question can also be
achieved by changing the composition of the media and the
manner in which culturing is carried out.
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The person skilled in the art will find instructions there-
for inter alia in Martin et a1. (Bio/Technology 5, 137-146
(1987)), in Guerrero et al. (Gene 138, 35-41 (1994)),
Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)),
5 in Eikmanns et a1. (Gene 102, 93-98 (1991)), in European
Patent Specification EP-B 0 472 869, in US Patent
4,601,893, in Schwarzer and Puhler (Bio/Technology 9, 84-87
(1991)), in Reinscheid et al. (Applied and Environmental
Microbiology 60, 126-132 (1994)), in LaBarre et a1.
(Journal of Bacteriology 175, 1001-1007 (1993)), in Patent
Application WO 96/15246, in Malumbres et al. (Gene 134,
15-24 (1993)) (sic) ~in Jensen and Hammer (Biotechnology and
Bioengineering 58, 191-195 (1998)), in Makrides
(Microbiological Reviews 60:512-538 (1996)) and in known
textbooks of genetics and molecular biology.
An example of a plasmid with the aid of which malate:
quinone oxidoreductase can be overexpressed is pRMl7
(Molenaar et al., 1998, European Journal of Biochemistry
254, 395-403). Plasmid pRMl7 is based on the shuttle vector
pJCl, which is described in Cremer et al. (Molecular and
General Genetics 220, 478-480).
In addition, it may be advantageous for the production of
L-amino acids to overexpress one or more enzymes of the
corresponding biosynthesis pathway as well as malate:
quinone oxidoreductase. Thus, for example, in the
production of L-lysine
~ the dapA gene coding for dihydrodipicolinate synthase can
be overexpressed at the same time (EP-B 0 197 335), or
~ a DNA fragment mediating S-(2-aminoethyl)-cysteine
resistance can be amplified at the same time
(EP-A 0 088 166).
It may also be advantageous for the production of I~-amino
acids to exclude undesired secondary reactions in addition
to the overexpression of malate:quinone oxidoreductase
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(Nakayama: "Breeding of Amino Acid Producing Micro-
organisms", in: Overproduction of Microbial Products,
Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982).
The microorganisms produced according to the invention may
be cultivated continuously or discontinuously in the batch
process or in the fed batch or repeated fed batch process
for the purposes of the production of L-amino acids. A
summary of known cultivation methods is described in the
textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die
Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart,
1991)) or in the textbook by Storhas (Bioreaktoren and
periphere Einrichtungen (Vieweg Verlag, Braunschweig/-
Wiesbaden, 1994)).
The culture medium to be used must meet the requirements of
the strains in question in a suitable manner. Descriptions
of culture media for various microorganisms are contained
in the handbook "Manual of Methods for General
Bacteriology" of the American Society for Bacteriology
(Washington D.C., USA, 1981). There may be used as the
carbon source sugars and carbohydrates, such as, for
example, glucose, saccharose, lactose, fructose, maltose,
molasses, starch and cellulose, oils and fats, such as, for
example, soybean oil, sunflower oil, groundnut oil and
coconut fat, fatty acids, such as, for example, palmitic
acid, stearic acid and linoleic acid, alcohols, such as,
for example, glycerol and ethanol, and organic acids, such
as, for example, acetic acid. Those substances may be used
individually or in the form of a mixture. There may be used
as the nitrogen source organic nitrogen-containing
compounds, such as peptones, yeast extract, meat extract,
malt extract, corn steep liquor, soybean flour and urea, or
inorganic compounds, such as ammonium sulfate, ammonium
chloride, ammonium phosphate, ammonium carbonate and
ammonium nitrate. 'Ihe nitrogen sources may be used
individually or in the form of a mixture. There may be used
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as the phosphorus source potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding sodium-
containing salts. The culture medium must also contain
salts of metals, such as, for example, magnesium sulfate or
iron sulfate, which are necessary for growth. Finally,
essential growth substances such as amino acids and
vitamins may be used in addition to the above-mentioned
substances. Moreover, suitable pre-stages may be added to
the culture medium. The mentioned substances may be added
to the culture in the form of a single batch or may be fed
in in a suitable manner during the cultivation.
In order to control the pH of the culture, basic compounds,
such as sodium hydroxide, potassium hydroxide, ammonia, or
acid compounds, such as phosphoric acid or sulfuric acid,
are used in a suitable manner. For controlling the
development of foam, antifoams, such as, for example, fatty
acid polyglycol esters, can be used. In order to maintain
the stability of plasmids, suitable substances having a
selective action, for example antibiotics, may be added to
the medium. In order to maintain aerobic conditions, oxygen
or oxygen-containing gas mixtures, such as, for example,
air, are introduced into the culture. The temperature of
.the culture is normally from 20°C to 45°C and preferably
from 25°C to 40°C. Culturing is continued until a maximum
of the desired L-amino acid has formed. That aim is
normally achieved within a period of from 10 hours to
160 hours.
Analysis of L-amino acids may be carried out by anion-
exchange chromatography with subsequent ninhydrin
derivatisation, as described by Spackman et al. (Analytical
Chemistry, 30, (1958), 1190).
The Corynebacterium glutamicum strain DM22/pRMl7 was
deposited at the Deutsche Sammlung von Mikroorgani_smen and
Zellkulturen (Braunschweig, Germany) under number DSM12711
i.n accordance with the Budapest Treaty.
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The process according to the invention is used for the
production of L-amino acids, especially L-aspartic acid,
L-asparagine, L-homoserine, L-threonine, L-isoleucine and
L-methionine, by fermentation using coryneform bacteria,
especially for the production of L-lysine.
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Examples
The present invention is explained in greater detail below
by means of Examples.
To that end, tests were carried out using the L-lysine-
producing Corynebacterium glutamicum strain DSM5715 (EP-B-
0 435 132), in which the advantageousness of the claimed
process becomes clear:
Example 1
Production of L-lysine producers containing amplified
malate:quinone oxidoreductase
The strain DSM5715 was transformed as in Liebl et a1. (FEMS
Microbiology Letters 65, 299-304 (1989)) with the plasmid
pRMl7 (Molenaar et al., 1998, European Journal of Bio-
chemistry 254 (395-403)). Selection of the transformants
was carried out on LBHIS agar to which 25 mg/1 of kanamycin
had been added. LBHIS agar consists of LB medium (Sambrook
et al. (Molecular Cloning a Laboratory Manual (1989) Cold
Spring Harbour Laboratories)) to which there have been
added 37 g/1 of brain heart bouillon from Merck (Darmstadt,
Germany), 0.5 M sorbitol and 15 g/1 of agar-agar. In that
manner, the strain DSM5715/pRMl7 was formed. The strain
DSM5715/pJCl was produced in the same manner.
Example 2
Production of L-lysine
The strains DSM5715/pRMl7 and DSM5715/pJCl were first
incubated on brain-heart agar, to which kanamycin (25 mg/1)
had been added, for 24 hours at 33°C. For cultivation in
liquid medium, CgIII medium (Kase & Nakayama, Agricultural
and Biological Chemistry 36 (9) 1611-1621 (1972)), to which
kanamycin (25 mg/1) had additionally been added, was used.
To that end, 10 ml of medium, which were contained in
100 ml Erlenmeyer flasks with 4 baffles, were inoculated
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with an inoculum of the strain and the culture was
incubated for 16 hours at 240 rpm and 30°C. The culture was
subsequently used further as a pre-culture.
The production or test medium used was MM medium, to which
5 kanamycin (25 mg/1) had additionally been added.
In the process using strain DSM5715, the corresponding
media did not contain kanamycin.
The composition and preparation of the MM medium was as
follows:
10 Corn Steep Liquor (CSL) 5 g/1
3-morpholino-propanesulfonic acid (MOPS) 20 g/1
glucose 50 g/1
(autoclaved separately)
Salts:
(NH4)2504) 25 g/1
KH2P04 0.1 g/1
MgS04*7H20 1.0 g/1
CaCl2*2H20 10 mg/1
FeS04*7H20 10 mg/1
MnS04*H20 5.0 mg/1
biotin 0.3 mg/1 (sterilised by filtration)
thiamine*HC1 0.2 mg/1 (sterilised by filtration)
CaC03 25 g/1
leucine 0.1 g/1
CSL, MOPS and the salt solution were adjusted to pH 7 using
ammonia water and autoclaved. The sterile substrate and
vitamin solutions and the dry autoclaved CaC03 were then
added.
Cultivation was carried out in 100 ml Erlenmeyer flasks
with baffles, which had been charged with 10 ml of the
above-described production medium. The cultures were so
inoculated with the pre-cult=ure that the optical density at
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the start was 0.1. Cultivation was carried out at 33°C and
80o relative humidity.
After incubation for 72 hours, the optical density of the
culture suspension and the concentration of L-lysine that
had formed were determined. The optical density was
determined using an LP2W photometer from Dr. Lange (Berlin,
Germany) at a measuring wavelength of 660 nm (sic) L-lysine
was determined using an amino acid analyser from Eppendorf-
BioTronik (Hamburg, Germany) by ion-exchange chromatography
and post-column reaction with ninhydrin detection. The
result of the test is shown in Table 1.
Table 1
--
Strain OD L-lysine
g/1
DSM5715 10.1 16.4
DSM5715/pJCl 9.9 16.5
DSM5715/pRMl7 10.2 17.8
