Process for preparing O-acetylserine, L-cysteine and...

Details Process for preparing O-acetylserine, L-cysteine and... Process for preparing O-acetylserine, L-cysteine and...

1998-05-13

2001-04-17

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S193000, C435S320100, C536S023200, C536S023700, C530S350000

Reexamination Certificate

active

06218168

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for preparing O-acetylserine, L-cysteine and sulfur-containing compounds derived therefrom.
2. The Prior Art
L-cysteine and its derivatives are employed in the pharmaceutical sphere (treatment of bronchial diseases), in the cosmetics sector (as constituents of hair shampoos and permanent wave lotions) and in the foodstuffs sphere (as antioxidants, as flavor enhancers and as adjuvants in the working of the dough). L-cysteine has hitherto been obtained by extraction from keratin-containing material, such as hair, bristles, horns, hooves and feathers, or by the enzymic transformation of precursors. An overproduction of L-cysteine by microorganisms is very desirable since L-cysteine is not only an economically interesting compound but, in addition, as is evident from
FIGS. 1-3
, constitutes an important intermediate in the synthesis of glutathione, methionine and biotin.
In all organisms, L-cysteine occupies a key position in sulfur metabolism and is used in the synthesis of proteins, glutathione, biotin, methionine and other sulfur-containing metabolites. Furthermore, L-cysteine acts as a precursor in the biosynthesis of coenzyme A; in addition to this, L-cysteine can readily be oxidized to cystine. A close connection exists between the biosynthesis of L-cysteine and that of other amino acids such as L-serine, glycine and L-methionine.
The synthesis of L-cysteine (
FIG. 4
) has been investigated in detail in prokaryotes, in particular bacteria (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965; Kredich, N. M., 1987, Biosynthesis of Cysteine. In: Neidhardt F. C., Ingraham, J. L., Magasanik, B., Low. K. B., Schaechter, M., Umbarger, H. E. (eds)
Escherichia coli
and
Salmonella typhimurium:
cellular and molecular biology, Vol. 1. American Society for Microbiology, Washington D.C., 419-428). The key reaction consists of the transfer of an acetyl group to serine in order to produce O-acetylserine 1), followed by the substitution of the acetyl group by the SH group, resulting in the synthesis of L-cysteine 2).
1) L-serine+acetyl-coenzyme A→O-acetylserine+coenzyme A
2) O-acetylserine+H
2
S→L-cysteine+acetate
In microorganisms and in plants, O-acetylserine, and not serine, acts as the immediate precursor of the carbon skeleton of L-cysteine (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965). The reaction in which the acetyl group is transferred in order to produce an activated form of L-serine is catalyzed by the serine acetyltransferase (EC 2.3.1.30) which is encoded by the cysE gene and is subject to strict control by the end product L-cysteine. The gene for serine acetyltransferase has already been cloned and the amino acid sequence which is deduced from the DNA sequence is known (Denk, D. and Böck, A. 1987, J. Gen. Microbiol. 133: 515-525).
The formation of L-cysteine itself is catalyzed by two O-acetylserine sulfhydrylase isoenzymes (EC 4.2.99.8), encoded by the genes cysK (O-acetylserine sulfhydrylase A) and cysM (O-acetylserine sulfhydrylase B), a reaction in which O-acetylserine functions as a &bgr;-alanyl donor and H
2
S as a &bgr;-alanyl acceptor (Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241: 4955-4965), with the O-acetylserine sulfhydrylase A making the major contribution to the cysteine synthesis. In addition, O-acetylserine sulfhydrylase B (cysM) is able to utilize thiosulfate as a sulfur source (Sirko, A. et al., 1987, J. Gen. Microbiol. 133: 2719-2725). The O-acetylserine sulfhydrylase B catalyzes the reaction between O-acetylserine and thiosulfate to form S-sulfocysteine, which can then be converted to cysteine (Nakamura, T., et al, 1983, J. Bacteriol. 156, 656-662).
The end product inhibition by L-cysteine of the wild-type form of serine acetyltransferase is a physiologically important factor in the kinetic regulation of cysteine biosynthesis (Kredich, N. M. 1971, J. Biol. Chem. 246, 3474-3484; Kredich, N. M. and G. M. Tomkins 1966, J. Biol. Chem. 241, 4955-4965). The activity of the wild-type form of serine acetyltransferase is inhibited by cysteine. This inhibition has been investigated kinetically and was found to have a competitive character. An inhibitor constant K
i
=1.1×10
−6
M was determined in the presence of 0.1 mM acetyl-coenzyme A and 1 mM L-serine (Kredich, N. M. 1971 and Tomkins G. M. 1966, J. Biol. Chem. 241, 4955-4965).
An example is known from the literature of it being possible to isolate a cysteine-prototrophic revertant, whose serine acetyltransferase activity exhibits an end product inhibition by L-cysteine which is only weakly pronounced due to an amino acid substitution in the coding region, by chemically mutagenizing a cysteine-auxotrophic strain with ethyl methanesulfonate (Denk, D., Böck, A., 1987, J. Gen. Microbiol. 133: 515-525). According to the literature reference mentioned, the feedback resistance of this mutant is elevated 10-fold. Consequently, the K
i
of this mutant is approx. 0.01 mM, when comparison is made with the wild-type form.
SUMMARY OF THE INVENTION
The present invention relates to serine acetyltransferases which exhibit a sensitivity to the inhibitor L-cysteine which is reduced in comparison to the wild-type enzyme and whose protein sequence exhibits at least one mutation or deletion when compared with the wild-type sequence, wherein the mutation is in the sequence region from the amino acid in position 97 up to and including the amino acid in position 273, or the deletion is in the carboxyterminal sequence region from the amino acid in position 227 onwards, with position 1 being the start methionine in
FIG. 5
(SEQ ID NO: 1) and with the protein sequence having the mutation of Met to Ile in position 256 being excluded.
It has been found, surprisingly, that the novel amino acid substitutions and/or amino acid deletions of the carboxyl terminus of the serine acetyltransferase lead to a diminution in the cysteine sensitivity while at the same time allowing adequate enzymic activity to be retained.
The novel serine acetyltransferases preferably have an inhibitor constant, K
i
, of from 0.005 to 2.3 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, where serine acetyltransferases having at least one mutation preferably possess an inhibitor constant, K
i
, of from 0.015 to 2.3 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA, while serine acetyltransferases having at least one carboxyterminal deletion preferably exhibit an inhibitor constant, K
i
, of from 0.005 to 0.03 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA.
The inhibitor constant (K
i
) towards L-cysteine of the particularly preferred enzyme mutants are between 0.02 and 2.3 mM in the presence of 1 mM L-serine and 0.1 mM acetyl-CoA.
Novel serine acetyltransferases exhibit an activity which is adequate for the growth of the microorganisms which contain them.
Preferably, the protein sequence of a novel serine acetyltransferase contains the amino acid substitution of at least one of the cysE mutants specified in Tab. 1a or 1b.
TABLE 1a
Feedback-resistant cysE alleles possessing
single or multiple amino acid changesin the coding region
Nucleotide
Amino Acid
spec. act.
cysE mutant
substitution (No.)
substitution (No.)
K
i
(&mgr;M)
&mgr;mol/min × mg
cysEII
GGC—>AGC (934)
Gly238—>Ser238
10
0.068
cysEIII
GGT—>GAT (716)
Gly165—>Asp165
10
0.030
cysEIV
GCT—>GTT (932)
Ala237—>Val237
40
0.170
GGC—>AGC (934)
Gly238—>Ser238
cysEV
GCT—>GTT (932)
Ala237—>Val237
GGC—>AGC (934)
Gly238—>Ser238
10
0.246
ATG—>ATA (990)
Met256—>Ile256
cysEVI
GGC—>AGC (934)
Gly238—>Ser238
10
0.075
ATG—>ATA (990)
Met256—>Ile256
cysEVII
GCT—>GTT (932)
Ala237—>Val237
10
0.253
cysEVIII
ATG—>ATA (990)
Met256—>Ile256
30
0.160
GCT—>GTT (932)
Ala237—>Val237
cysEX
ACG—>GCG (721)
Thr167—>Ala167
50
0.156
cysEXI
ACG—>GCG (721)
Thr167—>Ala167
700
0.117
GGT—>AGT (955)
Gly245—>Ser245
cysEXII
AAA—>CA

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