Note: Descriptions are shown in the official language in which they were submitted.
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Process for the fermentative production of L-amino acids
using coryneform bacteria
The present invention provides a process for the
fermentative production of L-amino acids using coryneform
bacteria, in which the glutamate dehydrogenase gene is
amplified.
Prior art
L-Amino acids are used in animal nutrition, human medicine
and the pharmaceuticals industry.
L-Amino acids are produced by fermentation using strains of
coryneform bacteria which produce L-amino acids, in
particular using Corynebacterium glutamicum. Due to the
significance of this group of products, efforts are
constantly being made to improve the production process.
Improvements to the process may relate to measures
concerning fermentation technology, for example stirring
and oxygen supply, or to the composition of the nutrient
media, such as for example sugar concentration during
fermentation, or to working up of the product by, for
example, ion exchange chromatography, or to the intrinsic
performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are
improved using methods of mutagenesis, selection and mutant
selection. In this manner, strains are obtained which are
resistant to antimetabolites, such as for example the
lysine analogue S-(2-aminoethyl)cysteine, or are
auxotrophic for regulatorily significant amino acids and
produce L-amino acids.
For some years, methods of recombinant DNA technology have
also been used to improve strains of Corynebacterium
glutamicum which produce L-amino acids by amplifying
individual biosynthesis genes and investigating the effect
on L-amino acid production. Review articles on this subject
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may 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)), Jetten and
Sinskey (Critical Reviews in Biotechnology 15, 73-lb3
(1995)) and Sahm et al. (Annuals of the New York Academy of
Science 782, 25-39 (1996)).
The enzyme glutamate dehydrogenase catalyses the reductive
amination of oc-ketoglutaric acid to yield glutamic acid.
French published patent application 2 575 492 describes a
DNA fragment from Corynebacterium melassecola 801 which
bears a glutamate dehydrogenase gene. It is possibly used
therein to increase glutamic acid production in the
fermentation of Corynebacterium melassecola. The nucleotide
sequence of the glutamate dehydrogenase gene of
Corynebacterium glutamicum ATCC13032 has been described by
Bormann et al. (Molecular Microbiology 6, 317-326 (1992)).
The nucleotide sequence of the glutamate dehydrogenase gene
of Peptostreptococcus asaccharolyticus is stated in
Snedecor et a1. (Journal of Bacteriology 193, 6162-6167
(1991) ) .
Object of the invention
The inventors set themselves the object of providing novel
measures for the improved fermentative production of other
L-amino acids.
Description of the invention
L-Amino acids are used in animal nutrition, human medicine
and the pharmaceuticals industry. There is accordingly
general interest in providing improved processes for the
production of L-amino acids.
When L-amino acids are mentioned below, they are taken to
mean the protein-forming amino acids L-lysine, L-threonine,
L-isoleucine, L-valine, L-proline, L-tryptophan and
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optionally the salts thereof and also L-homoserine, in
particular L-lysine, L-threonine and L-tryptophan.
The present invention provides a process for the
fermentative production of L-amino acids using coryneform
bacteria, which in particular already produce the
corresponding L-amino acids and in which the nucleotide
sequence coding for the enzyme glutamate dehydrogenase is
amplified, in particular overexpressed.
Preferred embodiments are stated in the claims.
In this connection, the term "amplification" describes the
increase in the intracellular activity of one or more
enzymes in a microorganism, which enzymes are coded by the
corresponding DNA, for example by increasing the copy
number of the gene or genes, by using a strong promoter or
a gene which codes for a corresponding enzyme having
elevated activity and optionally by combining these
measures.
The microorganisms provided by the present invention are
capable of producing L-amino acids from glucose, sucrose,
lactose, fructose, maltose, molasses, starch, cellulose or
from glycerol and ethanol. The microorganisms may comprise
representatives of the coryneform bacteria in particular of
the genus Corynebacterium. Within the genus
Corynebacterium, Corynebacterium glutamicum may in
particular be mentioned, which is known in specialist
circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in
particular 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 ATCC1406'7
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Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and mutants or strains produced therefrom, such as for
example
the L-lysine producing strains
Corynebacterium glutamicum FERM-P 1709
Brevibacterium flavum FERM-P 1708 and
Brevibacterium lactofermentum FERM-P 1712,
or the L-threonine producing strains
Corynebacterium glutamicum FERM-P 5835
Brevibacterium flavum FERM-P 4164 and
Brevibacterium lactofermentum FERM-P 4180,
or the L-isoleucine producing strains
Corynebacterium glutamicum FERM-P 756
Brevibacterium flavum FERM-P 759 and
Brevibacterium lactofermentum FERM-P 4192
or the L-valine producing strains
Brevibacterium flavum FERM-P 512 and
Brevibacterium lactofermentum FERM-P 1845,
and the L-tryptophan producing strains
Corynebacterium glutamicum FERM-BP 478
Brevibacterium flavum FERM-BP 475 and
Brevibacterium lactofermentum FERM-P 7127.
The inventors discovered that, after overexpression of
L-glutamate dehydrogenase, coryneform bacteria produce
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L-amino acids in an improved manner, wherein L-glutamic
acid is not claimed here.
The glutamate dehydrogenase gene of C. glutamicum described
by Bormann et al. (Molecular Microbiology 6, 317-326
5 (1992)) may be used according to the invention. The
glutamate dehydrogenase gene from other microorganisms,
such as for example that from Peptostreptococcus
asaccharolyticus, which has been described by Snedecor et
a1. (Journal of Bacteriology 173, 6162-6167 (1991)), is
also suitable. Alleles of the stated genes arising from the
degeneracy of the genetic code or from functionally neutral
sense mutations may also be used.
Overexpression may be achieved by increasing the copy
number of the corresponding genes, or the promoter and
regulation region located upstream from the structural gene
may be mutated. Expression cassettes incorporated upstream
from the structural gene act in the same manner. It is
additionally possible to increase expression during
fermentative L-amino acid production by means of inducible
promoters. Expression is also improved by measures to
extend the lifetime of the mRNA. Enzyme activity is
moreover amplified by preventing degradation of the enzyme
protein. The genes or gene constructs may either be present
in plasmids in a variable copy number or be integrated in
the chromosome and amplified. Alternatively, overexpression
of the genes concerned may also be achieved by modifying
the composition of the nutrient media and culture
conditions.
The person skilled .in the art will find guidance in this
connection inter alia in Martin et a1. (Bio/Technology 5,
137-146 (1987)), in Guerrero et a1. (Gene 138, 35-41
(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430
(1988)), in Eikmanns et aI. (Gene 102, 93-98 (1991)), in
European patent EPS 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
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60, 126-132 (1994)), in LaBarre et al. (Journal of
Bacteriology 175, 1001-1007 (1993)), in patent application
WO 96/15246, 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.
Examples of plasmids by means of which glutamate
dehydrogenase may be overexpressed are pEKl.9gdh-1 and
pEKExpgdh, which are present in strains
ATCC13032/pEKl.9gdh-1 and DHSa/pEKExpgdh. Plasmid
pEKl.9gdh-1 is a shuttle vector, which contains the NADP-
dependent glutamate dehydrogenase gene of C. glutamicum.
Plasmid pEKExpgdh is a shuttle vector, which contains the
NADP-dependent glutamate dehydrogenase [gene] of
Peptostreptococcus asaccharolyticus.
It may additionally be advantageous for the production of
the corresponding L-amino acids to overexpress one or more
enzymes of the particular amino acid biosynthesis pathway
as well as glutamate dehydrogenase. Thus, for example
~ the dapA gene which codes for dihydrodipicolinate
synthase may additionally be overexpressed in order to
improve L-lysine producing coryneform bacteria (EP-B
0197335),
~ the gene which codes for acetohydroxy acid synthase may
additionally be overexpressed in order to improve L-
valine producing coryneform bacteria (EP-B 0356739),
~ the gene which codes for anthranilic acid phosphoribosyl
transferase may additionally be overexpressed in order to
improve L-tryptophan producing coryneform bacteria (EP-B
0124048),
~ the gene which codes for homoserine dehydrogenase may
additionally be overexpressed in order to improve
coryneform bacteria which produce L-homoserine or
L-threonine or L-isoleucine (EP-A 0131171).
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It may furthermore be advantageous for the production of
the corresponding L-amino acid to switch off unwanted
secondary reactions in addition to overexpressing glutamate
dehydrogenase (Nakayama: "Breeding of Amino Acid Producing
Micro-organisms", in: Overproduction of Microbial Products,
Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982).
For the purposes of L-amino acid production, the
microorganisms according to the invention may be cultivated
continuously or discontinuously using the batch process or
the fed batch process or repeated fed batch process. A
summary of known cultivation methods is given 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 adequately satisfy the
requirements of the particular strains. Culture media for
various microorganisms are described in "Manual of Methods
for General Bacteriology" from American Society for
Bacteriology (Washington D.C., USA, 1981). Carbon sources
which may be used include sugars and carbohydrates, such as
for example glucose, sucrose, lactose, fructose, maltose,
molasses, starch and cellulose, oils and fats, such as for
example soya oil, sunflower oil, peanut oil and coconut
oil, 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. These substances may be used
individually or as a mixture. Nitrogen sources which may be
used comprise organic compounds containing nitrogen, such
as peptone, yeast extract, meat extract, malt extract, corn
steep liquor, soya flour and urea or inorganic compounds,
such as ammonium sulfate, ammonium chloride, ammonium
phosphate, ammonium carbonate and ammonium nitrate. The
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nitrogen sources may be used individually or as a mixture.
Phosphorus sources which may be used are potassium
dihydrogen phosphate or dipotassium hydrogen phosphate or
the corresponding salts containing sodium. The ..culture
medium must furthermore contain metal salts, such as for
example magnesium sulfate or iron sulfate, which are
necessary for growth. Finally, essential growth-promoting
substances such as amino acids and vitamins may also be
used in addition to the above-stated substances. Suitable
precursors may furthermore be added to the culture medium.
The stated feed substances may be added to the culture as a
single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia, or acidic compounds, such as phosphoric
acid or sulfuric acid, are used appropriately to control
the pH of the culture. Antifoaming agents, such as for
example fatty acid polyglycol esters, may be used to
control foaming. Suitable selectively acting substances,
such as for example antibiotics, may be added to the medium
in order to maintain plasmid stability. Oxygen or gas
mixtures containing oxygen, such as for example air, are
introduced into the culture in order to maintain aerobic
conditions. The temperature of the culture is normally from
20°C to 45°C and preferably from 25°C to 40°C. The
culture
is continued until a maximum quantity of the desired
L-amino acid has been formed. This objective is normally
achieved within 10 hours to 160 hours.
L-Amino acids may be analysed automatically using anion
exchange chromatography with subsequent ninhydrin
derivatisation, as described by Spackman et al. (Analytical
Chemistry, 30, 1190 (1958)).
The following microorganisms have been deposited with
Deutschen Sammlung fur Mikrorganismen and Zellkulturen
(DSMZ, Braunschweig, Germany) in accordance with the
3S Budapest Treaty:
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Corynebacterium glutamicum strain ATCC13032/pEKl.9gdh-1 as
DSM 12614.
Escherichia coli K12 strain DHSa/pEKExpgdh as DSM 12613.
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Examples
The present invention is illustrated in greater detail by
the following practical examples.
To this end, testing was performed with amino acid
5 producing strains, in which the superiority of the claimed
process is demonstrated:
a) the L-lysine producing strain Corynebacterium
glutamicum DSM5715, (EP-B- 0435 132) and
b) the L-threonine and L-isoleucine producing strain
10 Brevibacterium flavum DSM5399 (EP-B- 0385 940) and
c) the L-valine producing, isoleucine-requiring strain
ATCC130320i1vA, which has been deposited as DSM12455
with Deutschen Sammlung fur Mikroorganismen and
Zellkulturen in Braunschweig (Germany) in accordance
with the Budapest Treaty.
Example 1
Production of L-amino acid producers with amplified
glutamate dehydrogenase
Plasmid pEKl.9gdh-1 corresponds to the plasmid pEKl.9gdh
described by Bormann et a1. (Molecular Microbiology 6, 317-
326 (1992)). It was isolated from ATCC13032/pEKl.9gdh-1.
The known plasmid pEKExpgdh (Marx et al., Metabolic
Engineering 1, 35-48 (1999)), which bears the glutamate
dehydrogenase gene of Peptostreptococcus asaccharolyticus
(Snedecor et al., Journal of Bacteriology 173, 6162-6167
(1991)) was isolated in the same manner from E. coli strain
DHSa/pEKExpgdh.
Strains DSM5715, DSM5399 and ATCC13032~i1vA were
transformed with plasmid pEKl.9gdh-1 as described by Liebl
et a1. (FEMS Microbiology Letters 65, 299-304 (1989)). The
transformants were selected on brain/heart agar from Merck
(Darmstadt, Germany) which had been supplemented with
50 mg/1 of kanamycin. In this manner, strains
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DSM5715/pEKl.9gdh-1, DSM5399/pEKl.9gdh-1 and
ATCC13032~i1vA/pEKl.9gdh-1 were obtained. Strain DSM5715
was transformed in the same manner with plasmid pEKExpgdh
and strain DSM5715/pEKExpgdh obtained.
Example 2
Production of L-lysine
Strain DSM5715/pEKl.9gdh-1 was precultivated in complex
medium 2TY consisting of 16 g/1 of tryptone, 10 g/1 of
yeast extract and 5 g/1 of NaCl. To this end, 60 ml of
medium 2TY, contained in a 500 ml Erlenmeyer flask with 2
flow spoilers, were inoculated with an inoculating loop of
the strain and the culture incubated for 12 hours at 150
rpm and 30°C.
In order to inoculate 60 ml of production medium, contained
in a 500 ml Erlenmeyer flask with 2 flow spoilers, the
preculture was centrifuged for 10 minutes at 5000 rpm in a
Sepatech Minifuge RF (Heraeus, Hanau, Germany) centrifuge.
The supernatant was discarded and the pellet resuspended in
1 ml of production medium. An aliquot of this cell
suspension was added to the production medium, such that an
OD600 of approx. 2.0 was obtained. The production medium
used was medium CGXII with a pH of 7.0 (Table 1) described
by Keilhauer et a1. (Journal of Bacteriology 175, 5595-5603
(1993)) supplemented with 20 g/1 of glucose, 350 mg/1 of
leucine and 50 mg/1 of kanamycin monosulfate. The cultures
were incubated for 72 hours at 30°C and 150 rpm.
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Table 1
Component Concentration per
litre
(NHa) ZISOQ 20 g
Urea 5 g
KH2 P04 1 g
KZHP04 1 g
MgS04 ~ 7Hz0 0.25 g
3-Morpholinopropane- 42 g
sulfonic acid
FeS04 ~ 7H20 10 mg
MnS04 ~ H20 10 mg
ZnS04 ~ 7Hz0 1 mg
CuS04 0.2 mg
NiCl2 ~ 6H20 0.02 mg
CaCl2 10 mg
Protocatechuic acid 0.03 mg
Biotin 200 ~,g
Optical density (OD) (Biochrom Novaspec 4049, LKB
Instrument GmbH, Grafelfing, Germany) was then determined
S at a measuring wavelength of 600 nm, as was the
concentration of L-lysine formed using an amino acid
analyser from Eppendorf-BioTronik (Hamburg, Germany) by ion
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exchange chromatography and post-column reaction with
ninhydrin detection. Table 2 shows the result of the test.
Table 2
Strain OD L-lysine
g/1
DSM5715 16.5 4.5
DSM5715/pEKl.9gdh-1 19.4 6.2
Example 3
Production of L-threonine and L-isoleucine
Strain DSM5399/pEKl.9gdh-1 was precultivated in complete
medium CgIII (Kase & Nakayama, Agricultural and Biological
Chemistry 36 (9) 1611- 1621 (1972)) with 50~g/ml of
kanamycin. To this end, 10 ml of medium CgIII, contained in
a 100 ml Erlenmeyer flask with 4 flow spoilers, were
inoculated with an inoculating loop of the strain and the
culture incubated for 16 hours at 240 rpm and 30°C.
In order to inoculate 10 ml of production medium, contained
in a 100m1 Erlenmeyer flask with 4 flow spoilers, the OD
(660 nm) of the preculture was determined. The main culture
was inoculated to an OD of 0.1. The production medium used
was the medium CgXII described by Keilhauer et al. (Journal
of Bacteriology 1993, 175: 5595 - 5603). The composition of
the medium is shown in Example 2. 4% of glucose and 50 mg/1
of kanamycin sulfate were added. The cells were incubated
for 48 hours at 33°C, 250 rpm and 80% atmospheric humidity.
Optical density at 660 nm was then determined, as was the
concentration of the L-threonine and L--isoleucine formed as
stated in Example 2. Table 3 shows the result of the test.
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Table 3
Strain OD L-threonine L-
g/1 isoleucine
DSM5399 10.5 1.77 1.05
DSM5399/pEKl,9gdh-1 11.0 2.26 1.44
Example 4
Production of L-valine
Strain ATCC130320i1vA/pEKl.9gdh-1 was precultivated in
complete medium CgIII (Kase & Nakayama, Agricultural and
Biological Chemistry 36 (9) 1611- 1621 (1972)) with 50~g/ml
of kanamycin. To this end, 50 ml of medium CgIII, contained
in a 500 ml Erlenmeyer flask with 4 flow spoilers, were
inoculated with an inoculating loop of the strain and the
culture incubated for 16 hours at 140 rpm and 30°C.
In order to inoculate 60 ml of production medium, contained
in a 500 ml Erlenmeyer flask with 4 flow spoilers, the OD
(660 nm) of the preculture was determined. The main culture
was centrifuged and the supernatant discarded. The pellet
was resuspended in 5 ml of production medium and the main
culture inoculated to an OD of 0.3. The production medium
used was medium CgXII (Keilhauer et al., Journal of
Bacteriology 1993 175: 5595 - 5603) as described in Example
3 (with 4% glucose). The cells were incubated for 48 hours
at 30°C, 150 rpm.
Optical density at 660 nm was then determined, as was the
concentration of L-valine formed as described as stated in
Example 2. Table 4 shows the result of the test.
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Table 4
Strain OD L-valine
g/1
ATCC130320i1vA 18.5 0.29
ATCC130320i1vA /pEKl.9gdh-1 17.6 0.45
Example 5
Production of L-lysine, L-valine and L-alanine
5 Strain DSM5715/pEKExpgdh was precultivated in complex
medium 2TY consisting of 16 g/1 of tryptone, 10 g/1 of
yeast extract and 5 g/1 of NaCl. To this end, 60 ml of
medium 2TY, contained in a 500 ml Erlenmeyer flask with 2
flow spoilers, were inoculated with an inoculating loop of
10 the strain and the culture incubated for 12 hours at
150 rpm and 30°C.
In order to inoculate 60 ml of production medium, contained
in a 500 ml Erlenmeyer flask with 2 flow spoilers, the
preculture was centrifuged for 10 minutes at 5000 rpm in a'
15 Sepatech Minifuge RF (Heraeus, Hanau, Germany) centrifuge.
The supernatant was discarded and the pellet resuspended in
1 ml of production medium. An aliquot of this cell
suspension was added to the production medium such that an
OD600 of approx. 0.4 was obtained. The production medium
used was medium CGC (table 5) described by Schrumpf et a1.
(Journal of Bacteriology 173, 4510-4516 (1991)),
supplemented with 25 g/1 of glucose, 350 mg/1 of leucine,
42 g/1 of 3-morpholinopropanesulfonic acid and 50 mg/1 of
kanamycin monosulfate at pH 7. The cultures were incubated
for 30 hours at 30°C and 150 rpm.
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Table 5
Component Concentration
per litre
(NH4) 5 g
zS04
Urea 5 g
KH2 0 . 5 g
PO4
K2HP04 0.5 g
MgS04 7H20 0.25 g
~
FeS04 7H20 10 mg
~
MnS04 H20 10 mg
~
ZnS04 7H20 1 mg
~
CuS04 0.2 mg
NiCl2 6H20 0.02 mg
~
CaClz 2H20 10 mg
~
Biotin 200 ~.g
Optical density (OD) ( Biochrom Novaspec 4049, LKB
Instrument GmbH, Grafelfing, Germany) was then determined
at a measuring wavelength of 600 nm, as was the
concentration of L-alanine, L-lysine and L-valine formed
using an amino acid analyser from Eppendorf-BioTronik
(Hamburg, Germany) by ion exchange chromatography and post-
column reaction with ninhydrin detection. Table 6 shows the
result of the test.
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Table 6
Strain OD L- L-lysine L-valine
Alanine g/1 g/1
DSM5715 29.4 Traces 1.6 0.1
DSM5715/pEKExpgdh 19.4 0.6 2.1 0.6
