Note: Descriptions are shown in the official language in which they were submitted.
CA 02386539 2002-04-04
WO,Ol/27307 PCT/EP00/09720
Process for fermentatively producing L-cysteine or L
cysteine derivatives
The invention relates to a process for producing
L-cysteine or L-cysteine derivatives by fermenting
microorganisms, and to microorganisms which are
suitable for the process.
The amino acid L-cysteine is of economic importance.
For example, it is used as a foodstuff additive (in
particular in the baking industry), as a substance
which is employed in cosmetics, and as a starting
material for producing pharmacological active compounds
(in particular N-acetylcysteine and S
carboxymethylcysteine).
L-cysteine derivatives are all S-containing metabolites
which, in their synthesis, are derived from cysteine,
that is cystine, methionine, glutathione, biotin,
thiazolidines, thiamine, lipoic acid and coenzyme A,
for example.
In bacteria, cysteine biosynthesis is regulated at two
levels (Fig. 1):
1. At the level of enzyme activity, serine acetyl-
transferase (product of the cysE gene) is subject
to end product inhibition by L-cysteine. This
means that an accumulation of L-cysteine Leads
directly to inhibition of the first specific
reaction of cysteine biosynthesis and further
synthesis is stopped.
2. At the level of transcription, the regulatory
protein CysB (encoded by the cysB gene) functions
as a transcription activator and ensures that the
provision of reduced sulphur is regulated. CysB
CA 02386539 2005-12-23
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requires N-acetylserine, which is formed in the
cell from 0-acetylserine when insufficient reduced
sulphur is available for the 0-acetylserine
sulphhydrylase reaction, as an inducer.
Consequently, all the genes which are connected
with the uptake, reduction and incorporation of
sulphur are under the control of CysB. These genes
are the operons cysPTWAM, cysDNC and cysJIH and
also the cysK gene. Whereas acetylserine acts as
an inducer for CysB, sulphide and thiosulphate
exhibit a negative effect as so-called
"antiinducers", since their presence indicates the
availability of SH groups.
The state of the art with regard to obtaining L-
cysteine and L-cysteine derivatives is discussed
in detail in WO 97/15673 (corresponds to US Patent
6,218,168). WO 97/15673 itself described a fermentative
production process which uses feedback-resistant serine
acetyltransferases. The application also discloses that
it is possible to obtain a further increase in cysteine
yield by additionally deregulating the regulatory
protein CysB at the gene level such that the gene is
constitutively expressed.
The patent application EP 885962 A1 (corresponds to
the US Patent 5,972,663)
discloses microorganisms which are suitable for
fermentatively producing L-cysteine, L-cystine, N
acetyl serine and thiazolidine derivatives and which
are characterized in that they overexpress at least one
gene which encodes a protein which is directly suitable
for secreting antibiotics, or other substances which
are toxic for the microorganisms, out of the cell.
CA 02386539 2002-04-04
_ 3 _
CysB belongs to the family of LysR type transcription
regulators (LTTR), more than 100 representatives of
which are already known (Schell M.A., 1993, Annu. Rev.
Microbiol. 47: 597-626). They are distinguished by the
fact that they possess an N-terminal DNA-binding
domain, having a helix-turn-helix motif, and a C-
terminal inducer-binding domain. Detailed DNA-binding
studies are, available for CysB. Furthermore, CysB is
the first LTTR protein for which a crystal structure is
also available (Tyrell et al., 1997, Structure 5:1017-
1032). As a rule, LTTR proteins act as positive gene
regulators which are dependent on the presence of an
inducer molecule. It has previously been assumed that
only highly active CysB variants, which are independent
of effector molecules (so-called constitut.ively active
variants), allow an increase in cysteine production
since such forms display a constantly high degree of
gene activation (Nakamori S. et al., 1998, Appl. Env.
Microbiol. 64:1607-1611).
Examples of constitutively active forms of CysB have
been described in the case of Salmonella typhimurium.
These variants exhibit a high degree of activity, with
this activity being completely independent of the
inducer N-acetylserine and the negative effect of
thiosulphate and sulphide (Colyer T. E., K:redich N. M.,
1994, Mol. Microbiol. 13: 797-805).
The present invention relates to a microorganism strain
which is suitable for fermentatively producing L-
cysteine or L-cysteine derivatives and possesses a
deregulated cysteine metabolism, with this deregulation
of the cysteine metabolism not being based on a change
in CysB activity, characterized in that the strain
additionally possesses an increased CysB activity, with
CA 02386539 2005-12-23
the CysB activity having a regulatory pattern which is
typical of a wild-type CysB.
In accordance with one embodiment of the present invention
there is provided an isolated microorganism strain that is
transformed with a polynucleotide encoding the transcription
regulator protein CysB, for fermentatively producing a
substance selected from the group consisting of L-cysteine
and L-cysteine derivatives, the microorganism having a
deregulated cysteine metabolism, with this deregulation of
the cysteine metabolism not being based on a change in CysB
activity, and the strain additionally having an increased
CysB activity as compared to an unmodified microorganism
strain, with the CysB activity having a regulatory pattern
such that CysB activity is induced by the presence of N-
acetylserine and reduced by the presence of sulphide or
thiosulphide.
In accordance with another embodiment of the present
invention there is provided an isolated microorganism strain
that is transformed with a polynucleotide encoding the
transcription regulator protein CysB for fermentatively
producing a substance selected from the group consisting of
L-cysteine and L-cysteine derivatives comprising a
deregulated cysteine metabolism, with this deregulation of
the cysteine metabolism not being based on a change in CysB
activity, and in which strain homologous or heterologous
cysB genes, which encode CysB having a regulatory pattern
such that CysB activity is induced by the presence of N-
acetylserine and reduced by the presence of sulphide or
thiosulphide, are expressed to increased extent compared to
an unmodified microorganism.
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Microorganism strains which possess a deregulated cysteine
metabolism in which this deregulation is not due to an
alteration in CysB activity are known. These are strains
which possess modified cysE alleles, as described, for
example, in WO 97/15673 or Nakamori S. et al., 1998, Appl.
Env. Microbiol. 64: 1607-1611, or strains in which efflux
genes have been inserted, as described, for example, in EP
0885962 A1 (corresponds to U.S. Patent 5,972,663), or
strains which are isolated using nonspecific mutagenesis
methods combined with methods for screening for cysteine
overproduction or decreased cysteine breakdown, as
described, for example, in WO 97/15673 or in Nakamori S. et
al., 1998, Appl. Env. Microbiol. 64:1607-1611.
Within the meaning of the invention, CysB activity is
increased when this activity is at least 10'o higher than
that in the wild-type strain.
Preference is given to the CysB activity being at least 250
higher.
Particular preference is given to the CysB activity being at
least SOo higher.
An Escherichia coli strain (MC4100::AKZL300) which is
suitable for determining the CysB activity is described in
Example 2. This strain was deposited in the DSMZ (Deutsche
Sammlung fur Mikroorganismen and Zellkulturen
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[German collection of microorganisms and cell cultures]
GmbH, D-38142 Braunschweig) under number DSM 12886 on
23.6.99 in accordance with the Budapest Treaty. In
addition, each respective cysB-containing construct is
5 introduced into strain MC4100::?~KZL300 in a manner
known per se.
The CysB activity exhibits a regulatory pattern, in
dependence on the S source, which is typical of a wild-
10 type CysB, when the CysB activity of cells which are
grown with thiosulphate is less than 750 of that of
cells which are grown with sulphate and the CysB
activity of cells which are grown with cystine is less
than 20~ of that of cells which are grown with sulphate
15 (see Example 2).
Microorganism strains according to the invention
secrete L-cysteine or an L-cysteine derivative in
greater amounts than does a microorganism strain whose
20 cysteine metabolism is deregulated without its CysB
activity being increased.
Consequently, a microorganism strain according to the
invention is a microorganism strain which possesses a
25 deregulated cysteine metabolism, with this deregulation
of the cysteine metabolism not being based on a change
in CysB activity, and in which homologous or
heterologous cysB genes, which encode CysB having a
regulatory pattern which is typical of wild-type CysB,
30 are expressed to an increased extent.
Preference is given to the Escherichia coli strains
being strains which possess a deregulated cysteine
metabolism, with this deregulation of cysteine
35 metabolism not being based on a change in CysB
CA 02386539 2002-04-04
. ~ _ 6 _
activity, and in which a wild-type cysB gene is
overexpressed.
Particular preference is given to the Escherichia coli
strains being strains which possess a deregulated
cysteine metabolism, with this deregulation of cysteine
metabolism not being based on a change in. CysB
activity, and in which the copy number of the
Escherichia coli wild-type cysB gene is increased and
this gene is overexpressed.
Surprisingly, it was not found, as postulated in the
abovementioned disclosures of the prior art, that
cysteine production is increased by using cysB alleles
which encode constitutively active CysB regulatory
proteins, but that, quite on the contrary, cysteine
production is increased by increasing the expression of
a wild-type cysB gene.
This finding is also surprising and unexpected since
there is no previously known example in which increased
expression of a regulatory protein from the LysR-type
transcription regulator family enables a metabolite to
be overproduced.
The invention consequently also relates to the use of
a regulatory protein from the LysR-type transcription
regulator family for overproducing a metabolite and
also to a process for overproducing a metabolite,
characterized in that a regulatory gene from the LysR-
type transcription regulator family is overexpressed in
a microorganism and brings about an increased
production of the metabolite in the microorganism.
The CysB activity in a microorganism can be increased,
CA 02386539 2002-04-04
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while at the same time retaining the typical regulatory
pattern, by, far example:
1. increasing the copy number of a cysB gene, which
encodes CysB having a regulatory pattern which is
5 typical of wild-type CysB, in the microorganism
under the control of a promoter, or
2. increasing the expression of a cysB gene, which
encodes CysB having a regulatory pattern which is
typical of wild-type CysB, by replacing the
10 promoter which regulates the expression of the
wild-type cysB gene with a stronger promoter.
cysB genes are known from Escherichia coli, Salmonella
typhimurium, Klebsiella aerogenes, Haemophilus
15 influenzae, Pseudomonas aeruginosa and Thiocapsa
roseopersicina. cysB genes are preferably understood as
being genes whose gene products exhibit an identity of
at least 40~ with the Escherichia coli CysB protein.
The homology values refer to results which are obtained
20 with the "Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wisconsin" computer
program. In this connection, the database is searched
with the "blast" subprogram using standard parameters.
25 In addition, cysB genes are those alleles of wild-type
cysB genes the sequence of whose gene products is
altered without the regulatory pattern which is typical
of wild-type CysB thereby being lost. CysB variants
containing conservative amino acid substitutions are an
30 example of this.
A microorganism according to the invention can be
prepared, for example, by using methods which are known
per se to increase the copy number of the wild-type
35 cysB gene or of a cysB gene which encodes CysB having
CA 02386539 2005-12-23
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20
a regulatory pattern which is typical of wild-type CysB, or
by using methods which are known per se to increase the
expression of the wild-type cysB gene, or of a cysB gene
which encodes a CysB having a regulatory pattern which is
typical of wild-type CysB, in a microorganism strain which
possesses a deregulated cysteine metabolism, with this
deregulation of the cysteine metabolism not being based on a
change in CysB activity.
In accordance with one embodiment of the present invention
there is provided a process for preparing a microorganism
for fermentatively producing a substance selected from the
group consisting of L-cysteine and L-cysteine derivatives
comprising in a microorganism strain possessing a
deregulated cysteine metabolism, increasing the copy number
of a wild-type cysB gene, or of a cysB gene which encodes
CysB having a regulatory pattern which is typical of wild-
type CysB, or bringing about an increased expression of the
wild-type cysB gene, or of a cysB gene which encodes a CysB
having a regulatory pattern which is typical of wild-type
CysB.
In that which follows, the term "CysB" denotes both "wild-
type CysB" and "CysB variant having a regulatory pattern
which is typical of a wild-type CysB". In that which
follows, the term "cysB gene" denotes "wild-type cysB gene"
and also "cysB gene which encodes CysB having a regulatory
pattern which is typical of wild-type CysB".
Methods which are known to a skilled person can be used to
increase the copy number of the cysB gene in a
microorganism. Thus, the cysB gene can, for example, be
cloned into plasmid vectors which are present in multiple
CA 02386539 2005-12-23
- 8a -
copies per cell (e.g. pUCl9, pBR322 and pACYC184) and
introduced into a microorganism which possesses a
deregulated cysteine metabolism. Alternatively, the cysB
gene can be integrated several times into the chromosome of
a microorganism which possesses a deregulated cysteine
metabolism. Integration methods which can be used are the
known systems employing temperate bacteriophages or
integrated plasmids, or else integration by way of
homologous recombination (e.g. Hamilton et al., 1989, J.
Bacteriol. 171: 4617-4622).
Preference is given to increasing the copy number by
30
CA 02386539 2002-04-04
. i _ g _
cloning a cysB gene into plasmid vectors under the
control of a promoter. Particular preference is given
to increasing the copy number by cloning a cysB gene
into pACYC derivatives such as pACYC184-LH (deposited
in the Deutsche Sammlung fur Mikroorganismen and
Zellkulturen, Braunschweig, under number DSM 10172 on
18.8.95 in accordance with the Budapest Treaty).
The natural promoter and operator region of the gene
can serve as the control region for expressing a
plasmid-encoded cysB gene.
However, the expression of a cysB gene can also be
increased using other promoters. Appropriate promoter
systems are known to a skilled person (Makrides S.C.,
1996, Microbiol. Rev. 60: 512-538). These constructs
can be used on plasmids or chromosomally in a manner
known per se.
For example, a cysB gene is cloned into plasmid vectors
by being specifically amplified by means of the
polymerase chain reaction using specific primers which
encompass the complete cysB gene including the promoter
and operator sequence and then being ligated to vector
DNA fragments.
The preferred vectors used for cloning a cysB gene are
plasmids which already contain genetic elements for
deregulating the cysteine metabolism, for example a
cysEX gene (W097/15673) and an efflux gene (EP 0885962
Al). Such vectors enable a microorganism strain
according to the invention to be prepared from any
arbitrary microorganism strain since such a vector also
deregulates the metabolism of cysteine in a
microorganism.
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- l~ -
The invention consequently also relates to a plasmid which
is characterized in that it possesses genetic elements for
deregulating cysteine metabolism, with these genetic
elements not bringing about any change in the CysB activity,
and also contains a cysB gene under the control of a
promoter.
One embodiment of the invention provides plasmid having
genetic elements for deregulating cysteine metabolism, with
these genetic elements not bringing about any change in CysB
activity, and also containing a cysB gene under the control
of a promoter, with the gene having a regulatory pattern
such that CysB activity is induced by the presence of N-
acetylserine and reduced by the presence of sulphide or
thiosulphide.
The cysB-containing plasmids are introduced into bacteria
using a current transformation method (e. g. electro-
poration), and plasmid-harbouring clones are selected, for
example by means of antibiotic resistance.
The invention consequently also relates to a process for
preparing a microorganism strain according to the invention,
characterized in that a plasmid according to the invention
is introduced into a microorganism strain.
A microorganism strain according to the invention is used to
produce cysteine or cysteine derivatives in a fermenter in
accordance with methods which are known per se. The C
sources employed can, for example, be glucose or lactose, or
other sugars, while the N source employed can be ammonium or
protein hydrolyzate. The S source employed can, for
example, be sulphide, sulphite, sulphates or thiosulphate.
CA 02386539 2005-12-23
- 10a -
L-cysteine which is formed during fermentation can be
oxidized to give difficulty soluble cysteine or be condensed
with aldehydes or ketones to give thiazolidines (e. g. with
pyruvic acid to give 2-methylthiazolidine-2,9-dicarboyxlic
acid) .
The invention consequently also relates to a process for
producing L-cysteine or L-cysteine derivatives
30
CA 02386539 2002-04-04
' - 11 -
which is characterized in that a microorganism strain
according to the invention is employed in the
fermentation in a manner known per se and the L
cysteine or L-cysteine derivative is separated off from
the fermentation mixture.
The following examples serve to clarify the invention.
Example 1: Cloning the v~ild-type cysB gene and the
cysB(T149M) allele
The Escherichia coli wild-type cysB gene was cloned
using the polymerase chain reaction (PCR). The specific
oligonucleotide primers cysBP1 (SEQ. ID.NO: 1) and
cysBP2 (SEQ. ID. NO: 2) (20 pmol per mixture) were used
to amplify a genomic DNA fragment of 3107 base pairs in
length which encompasses the wild-type cysB gene,
together with flanking regions, and possesses terminal
EcoRI and Sall restriction cleavage sites,
respectively.
5'-GTT ACG AGA TCG AAG AGG-3' (phosphorothioate bond at
the 3'-end) (SEQ. ID.NO: 1)
5'-GTC ACC GAG TGG TCA ATG-3' (phosphorothioate bond at
the 3'-end) (SEQ. ID.NO: 2)
The PCR reaction was carried out using the Boehringer
(Mannheim, Germany) Pwo DNA polymerase and employing
10 ng of genomic DNA as the template. The programme
comprised 29 cycles with an annealing temperature of
56°C (30 seconds per cycle), an extension temperature
of 72°C (60 seconds per cycle) and a denaturing
temperature of 94°C (30 seconds per cycle). The DNA
fragment was then treated with the restriction enzymes
CA 02386539 2005-12-23
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EcoRI and SalI and purified by way of a preparative gel
electrophoresis and the Geneclean method (Geneclean° Kit
BI0101 P.O. Box 2284, La Jolla, California, USA, 92038
2284). This fragment was ligated into the Bio-Rad
Laboratories (Hercules, California, USA) phagemid
vector pTZl9U, which had been cut with EcoRI/SalI and
treated with phosphatase, thereby resulting in the
plasmid pTZl9U-cysB (Fig. 2). Following transformation,
positive clones were identified by means of restriction
analysis.
In order to construct a constitutively active cysB
allele, codon 147 of the wild-type cysB gene was
mutated (in analogy with the mutation in the Salmonella
typhimurium wild-type cysB gene described in Colyer T.
E., Kredich N. M., 1994, Mol. Microbiol. 13: 797-805)
into a methionine codon using the "Mutagene In Vitro
Mutagenesis" kit from Bio-Rad Laboratories (Hercules,
California, USA). The oligonucleotide CysBMut4 (SEQ.
ID. N0. 3) was used in this context. The underlined
bases indicate the difference from the wild-type
sequence.
S'-TTC GCT ATC GCC ATG GAA GCG CTG CAT-3' (SEQ. ID. NO.
3)
For activity tests (see Example 2), the two cysB
alleles were cloned, as EcoRI/SalI fragments, after
treatment with Klenow, into the EcI136II-cut,
phosphatase-treated vector pACYCI84-LH.
Example 2: Determining CysB activity in vivo
A reporter gene assay was selected for measuring the
activity of CysB. For this, the control region of the
CA 02386539 2002-04-04
- - 13 -
cysK gene was fused to the lacZ gene, which encodes the
enzyme (3-galactosidase. When this fusion is integrated
into strains which lack an endogenous a-galactosidase,
this enzyme is formed in dependence on CysB activity
S and consequently provides, in the form of ~i-
galactosidase activity, an indirect measure of CysB
activity. The system described by Simons et al. (Simons
R. W. et al., 1987, Gene 53: 85-96) was used for
constructing the fusion. The promoter region of the
cysK gene, including the first fifteen codons, was
first of all amplified by means of the polymerase chain
reaction using the oligonucleotide primers cysKP1 (SEQ.
ID. NO: 4) and cysKP3 (SEQ. ID. NO: 5) and 10 ng of
Escherichia coli chromosomal DNA and Pwo polymerase.
5'-CCG GAA TTC CCG TTG CCG TTT GTG GCG-3' (SEQ. ID. N0:
4)
5'-CGC GGA TCC GTG TGA CCG ATA GTC AGC-3' (SEQ. ID. NO:
5)
The conditions corresponded to those which were
described in Example 1. The resulting 317 base pair
product was digested with the restriction enzymes EcoRI
and BamHI, in accordance with the manufacturer's
instructions, and purified by means of preparative gel
electrophoresis and the Geneclean method. The product
was then ligated to the vector pRS552 (deposited in the
Deutsche Sammlung fur Mikroorganismen and Zellkulturen,
Braunschweig, under number DSM 13034 on 14.9.99 in
accordance with the Budapest Treaty), which had
likewise been digested with EcoRI-BamHI and treated
with phosphatase. The strain MC4100 (ATCC 35695) was
transformed with the ligation mixture using
electroporation, and positive clones were identified
f
CA 02386539 2002-04-04
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with the aid of restriction analyses. These positive
clones contain a translational cysK-lacZ fusion. The
resulting plasmid was recombined with the bacteriophage
ARS45 (deposited in the Deutsche Sammlung fur
Mikroorganismen and Zellkulturen, Braunschweig, under
number DSM 13035 on 14.9.99 in accordance with the
Budapest Treaty), following the instructions of Simons
et al., and a homogeneous lysate of the recombinant
phage, which was designated ~KZL300, was prepared. The
~lac strain MC4100 was infected with this phage and
lysogenic clones (MC4100::J~KZL300), which it was now
possible to use for measuring CysB activity, were
identified by kanamycin selection.
Tn order to compare the effect of a multicopy cysB gene
cloned into pACYC184-LH and of a cysB(T149M) gene,
MC4100::?~KZL300 was transformed with the corresponding
plasmids, i.e. pACYC-cysB and pACYC-cysB(T149M).
The strains were cultured in VB minimal medium (3.5 g
of Na(NH4)HP04/l; 10 g of KHzP04/1; 2 g of citrate x
Hz0/1; 0.078 g of MgClz/1; pH adjusted to 6.5 with NaOH,
5 g of glucose/1; 5 mg of vitamin B1/1) containing
different sulphur sources (in each case 1 mM sulphur),
with 15 mg of tetracycline being added per litre. (3
Galactosidase activity was determined in accordance
with the method described by Miller (Miller J. H.,
1972, Experiments in Molecular Genetics, Cold Spring
Harbor, New York, 352-355).
The results are shown in Table 1. In the case of the
control (MC4100::AKZL300/pACYC184-LH), the regulatory
pattern of the CysB activity, in dependence on the S
source, is found to be that which is typical of a wild-
type CysB; the CysB activity of cells which are grown
with thiosulphate is less than 75~ of that when grown
CA 02386539 2002-04-04
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with sulphate, and the CysB activity of cells which are
grown with cystine is less than 200 of that when grown
with sulphate. This pattern is retained, at a level of
activity which is raised overall, in the presence of
multiple copies of a wild-type cysB gene (example
MC4100::AKZL300/pACYC-cysB, in accordance with the
invention). By contrast, the cysB(T149M) allele leads
to a constitutively high level of activity with loss of
the typical regulatory pattern.
Table 1
Determination of the CysB activity, in the form of ~i-
galactosidase activity, of strains harbouring a
chromosomal cysK-lacZ fusion
Strain MC4100:: MC4100:: MC4100::
AKZL300 1~KZL300 AKZL300
Plasmid pACYC184-LH pACYC-cysB pACYC-
cysB(T149M)
Genotype cysB cysB cysB(T149M)
single copy multicopy multicopy
S source: (3-galactosidase
activity in
Miller units
Cystine 26 341 3791
Sulphate 1906 2708 3824
Thiosulphate 726 1094 3595
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- Example 3: Constructing plasmids according to the
invention
Organisms according to the invention are characterized
by a deregulated cysteine metabolism and, for example,
by a cysB gene which is present in multiple copies. The
plasmid (pACYC184-cysEX-GAPDH-ORF306) was chosen as the
basic construct for preparing these organisms. This
plasmid contains the feedback-resistant cysE allele and
the efflux gene as elements for deregulating cysteine
metabolism. It is described in detail in patent
application EP 0885962 A1 (Example 2D). A cysB fragment
was inserted into this construct, which had been
digested with the restriction enzyme SnaBI and treated
with phosphatase, between the cysEX allele and the
efflux gene. The cysB fragment was obtained from
plasmid pTZl9U-cysB (Fig. 2) by restricting with the
enzymes EcoRI and BstXI and subsequently smoothing the
DNA ends with Klenow enzyme. The plasmid is designated
pHC34.
An organism according to the invention is obtained by
transforming the Escherichia coli strain W3110 (ATCC
27325; Bachmann B. J., 1996, in: Neidhardt F. C. (ed.)
Escherichia coli and Salmonella: cellular and molecular
biology, American Society for Microbiology, Washingivn
D.C., Chapter 133). The transformation was carried out
using electroporation. For transformation, 0.1 ug of
plasmid DNA was added to a thick cell suspension in an
ice-cold 10~ solution of glycerol and the suspension
was then subjected to an electric pulse at. 2500 V, 200
Ohms and 12.5 uF. After the mixture had been
transferred into sterile LB medium (1o tryptone, 0.5~
yeast extract, 1~ NaCl) and incubated at 30°C for one
hour, plasmid-harbouring clones were selected on LB
CA 02386539 2002-04-04
. . _ 1'j _
agar plates containing 15 ug tetracycline/mi.
In order to compare the effect of the wild-type cysB
gene with that of the constitutive cysB(T149M) allele,
an analogous construct (pHC30) was prepared and
likewise introduced into the strain W3100. In addition,
the organism W3110, which is described in EP 885962 A1,
was used, transformed with the plasmid pACYC184/cysEX-
GAPDH-orf306, as the basic construct for the comparison
and, at the same time, for delimiting from the prior
art.
Since W3110 is a wild-type strain, all the cysteine
production effects are to be attributed to plasmid
encoded genes.
Example 4: Cysteine production using microorganisms in
accordance with the invention
In order to detect cysteine production, the
microorganisms described in Example 3 were cultured in
fermenters in the fed batch mode with continuous
feeding of glucose and thiosulphate. The device
employed was a Braun Biotech (Melsungen, Germany)
~ Biostat M appliance having a maximum culture volume of
2 1.
20 ml of LB medium (10 g of tryptone/1, 5 g of yeast
extract/l, 10 g of NaCl/1), which additionally
contained 15 mg of tetracycline/l, were inoculated, as
a preculture, and incubated at 30°C and 150 rpm in a
shaker. After 7 hours, the entire mixture was
transferred into 100 ml of SM1 medium (12 g of KZHPO9/1;
3 g of KHzP04/1; 5 g of (NH4)2S04/l; 0.3 g of MgS04 X
7Hz0/l; 0.015 g of CaCl2 X 2HZ0/1; 0.002 g of FeS04 X
7H20/l; 1 g of Na3 citrate X 2H20/l; 0.1 g of NaCl/l;
CA 02386539 2002-04-04
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1 ml of trace element solution~,~consisting of 0.15 g of i
Na.2Mo04 X 2H20/ 1; 2 . 5 g of NaB03 / 1; 0 . 7 g of CoClz X
6HZ0/1; 0.25 g of CuS04 X 5HZ0/l; 1.6 g of MnCl2 X
4H20/1; 0.3 g of ZnS04 X 7H20/1) which was supplemented
with 5 g of glucose/1; 0.5 mg of vitamin Bl/1 and 15
mg of tetracycline/l. Subsequent incubation took place
at 30°C for 17 hours at 150 rpm.
This preculture (optical density of approx. 3 at
600 nm) was used to inoculate the fermenter containing
900 ml of fermentation medium (15 g of glucose/1; 10 g
of trypton/1; 5 g of yeast extract/l; 5 g of
(NH4)zS04/l; 1.5 g of KHzP04/1; 0.5 g of NaCI/l; 0.3 g of
MgS04 X 7Hz0/1; 0.015 g of CaCl2 X 2H20/1; 0.075 g of
FeS04 X 7H20/1; 1 g of Na3 citrate X 2Hz0/1 and 1 ml of
trace element solution, see above, per litre, 5 mg of
vitamin B1/1 and 15 mg of tetracycline/1, adjusted to
pH 7.0 with 25~ ammonia). During the fermentation, the
temperature was set at 30°C and the pH was kept
constant at a value of 7.0 by metering in 25~ ammonia.
The culture was gassed, at 1.5 vol/vol/min, with
sterilized compressed air and stirred with a stirring
device at a rotational speed of 200 rpm. After the
oxygen saturation had decreased to a value of 50~, the
rotational speed was increased, using a control device,
to a value of 1200 rpm in order to maintain 50~ oxygen
saturation.
After 2 hours, a 30o solution of Na thiosulphate was
metered in at a rate of 3 ml/h. Glucose was fed in from
a 56o stock solution as soon as the content in the
fermenter, which was initially 15 g/l, had fallen to
approx. 5-10 g/l. The glucose was fed in at a flow rate
of 8-14 ml/h, with an attempt being made to maintain a
constant glucose concentration of approx. 5-10 g/1. The
CA 02386539 2002-04-04
. , _ 19 -
glucose was determined using a YSI (Yellow Springs,
Ohio, USA) glucose analyser.
The production of L-cysteine was monitored
colorimetrically using the Gaitonde test (Gaitonde,
M.K. (1967), Biochem. J. 104, 627-633). In this
connection, account has to be taken of the fact that
the test does not discriminate between L-cysteine and
the condensation product of L-cysteine and pyruvate (2-
methylthiazolidine-2,4-dicarboxylic acid) which is
described in EP 0885962 A1. Difficultly soluble
cystine, which is formed from L-cysteine by oxidation,
was likewise detected as L-cysteine, in dilute solution
at pH 8.0, after dissolving in 8~ hydrochloric acid and
subsequently reducing with dithiothreitol (DTT).
Table 2 shows the production course of a fermentation
of an organism harbouring the basic construct
pACYC184/cysEX-GAPDH-ORF306, which is described in EP
0885962 A1, as compared with that of a fermentation of
an organism harbouring a plasmid pHC34 according to the
invention and of a fermentation of an organism
harbouring a corresponding construct containing the
cysB(T149M) allele, which encodes a constitutively
active CysB gene product. It is clear that the use,
according to the invention, of the wild-type cysB gene
exerts a positive effect on productive performance
whereas the constitutive allele cysB(T149M) exhibits a
negative effect.
Table 2
Production of L-cysteine using the pHC34 constuct
according to the invention and using control
constructs.
CA 02386539 2002-04-04
r - 20 -
Plasmid pACYC184/cys pHC34 pHC30
construct E X-GAPDH-
ORF306
Genotype cysEX cysEX c:ysEX
orf306 cysB cysB(T149M)
orf306 orf306
Fermentatio yield of L-cysteine
in g/1
n time '
24 h 6.3 8.0 + 2.6 2.0
*
48 h 10.2 + 7.0* 10.0 + 3.4
12.6*
* the values which are given with an asterisk are
values for L-cysteine which is present in oxidized form
as difficultly soluble cystine.
CA 02386539 2002-11-27
SBQUENCE LISTING
<110> CONSORTIUM FUR ELERTROCHEMISCHE INDUSTRIE GHBH
<120> PROCESS FOR FERMENTATIVELY PRODUCING L-CYSTEINE OR L~CYSTEINE
DERIVATIVES
<130> 1546-348
<140> 2,386,539
<141> October 5, 2000
<150> PCT/EP00/09720
<151> October 5, 2000
<150> DE 199 49 579.3
<151> October 14, 1999
<160> 5
<170> Patentln version 3.1
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR
<400> 1
gttacgagat cgaagagg 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR
<400> 2
gtcaccgagt ggtcaatg 18
CA 02386539 2002-11-27
<210> 3
<211> 27
<212> DNA
<213> Artificial sequence
<220>
<223> Oligonuclaotide for in vitro mutagenonsis
<400> 3
ttcgctatcg ccatggaagc gctgcat 27
<210> 4
<211> 27 .
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR
<400> 4
ccggaattcc cgttgccgtt tgtggcg 27
<210> 5
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer for PCR
<400> 5
cgcggatccg tgtgaccgat agtcagc 27
