WO2002061106A2

WO2002061106A2 - Method for producing cysteine, cystine and glutathione by fermentation

Info

Publication number: WO2002061106A2
Application number: PCT/EP2002/001122
Authority: WO
Inventors: Markus Wirtz; Rüdiger HELL
Original assignee: Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung
Priority date: 2001-02-02
Filing date: 2002-02-04
Publication date: 2002-08-08
Also published as: DE10104722A1; AU2002244700A1; WO2002061106A3

Abstract

The invention relates to a method for detecting genes and DNA- sequences which code for the enzyme serine acetyltransferase (SAT) and are suitable for the production of cysteine by fermentation. The invention relates to a method for producing compounds containing sulphur such as cysteine, cystine and glutathione in bacteria and other host organisms by overexpression of SAT genes, wherein the host organism is disturbed in their own glutathoine synthesis.

Description

Process for the fermentative production of cysteine, cystine and glutathione

The present invention relates to a method for finding genes and DNA sequences which code for the enzyme serine acetyltransferase (SAT) and are suitable for the fermentative production of cysteine. The invention further relates to a method for producing sulfur-containing compounds such as cysteine, cystine and glutathione in bacteria and other host organisms by means of overexpression of SAT genes, the host organism being disturbed in its own glutathione synthesis.

The sulfur-containing amino acid cysteine is the end product of assimilatory sulfate reduction in bacteria, fungi and plants. In addition to its role in proteins, cysteine acts essentially and ubiquitously in all organisms as a donor of reduced sulfur for the biosynthesis of other compounds such as methionine, Fe / S clusters and various vitamins (e.g. biotin). Direct relatives of cysteine are its oxidation product cystine and the tripeptide glutathione (γ-glutamylcysteinylglycine). In addition, cysteine, glutathione and related thiol compounds have important functions in the defense against stress from humans and animals. Cysteine also partially compensates for the need for the essential amino acid methionine.

The system biosynthesis takes place in bacteria and in plants in a two-stage process. In the first step, serine acetyltransferase (SAT; EC 2.3.1.30) ensures the formation of the activated thioester O-acetylserine (OAS) from serine and acetyl-coenzyme A. In the second step, free sulfide is enzymatically catalysed by O-acetylserine (thiol ) Lyase (OAS-TL; EC 4.2.99.8) inserted in O-acetylserine to obtain cysteine and acetate. SAT represents the speed-limiting component, the activity of this enzyme being found only in connection with O-acetylserine (thiol) lyase in the cysteine synthase complex. OAS-TL is present in large excess due to the activity of SAT-free homodimers (Kredich et al. (1969) J. Biol. Chem. 244, 2428-2439; Droux et al. (1998) Eur. J. Biochem. 255 , 235-245; Saito (2000) Curr. Opin. Biol. 3, 188-195). Cysteine is almost the only compound where reduced sulfur enters cell metabolism, although sulfur is needed for the biosynthesis of essential compounds, including methionine, biotin, and Fe / S clusters.

The production of sulfur-containing compounds such as cysteine, cystine and glutathione is therefore of great biotechnological interest for pharmacological processes and for nutritional supplements.

Cysteine can be obtained by chemical synthesis or by extraction from animal

Sources such as keratin are made or extracted. The large-scale production of cysteine in microorganisms is mainly influenced by regulatory mechanisms of the cys regulon (Kredich (1996) In Escherichia coli and Salmonella tyhpimurium. Cellular and molecular biology (Neidhardt et al., Eds.), Pp. 514 - 527. ASM Press, Washington DC, USA) and its toxicity at higher levels

Concentrations (Harris (1983) J. Bacteriol. 145, 115-132; Soerensen and Pedersen (1991) J. Bacteriol. 173, 5244-5246) are more difficult. Various approaches have been taken to circumvent these restrictions. A high yield of cysteine in the culture medium can be achieved by overexpressing an export pump for various metabolites of the cysteine metabolism, as has been shown for the -fe-D gene product of E. coli (Daßler et al. (2000) Mol. Microbiol 36, 1101-1112).

The inhibition of constitutively expressed E. coli SAT (CYSE protein, encoded by the cy-sE gene) by cysteine is essential for the function of the cys regulon. The inhibition of SAT by cysteine has an inhibition constant (K;) of approximately 10 ^"6 M in Salmonella typhimurium and E. coli (Kredich et al. (1969) vide supra; Kredich (1996) vide supra) and thus effectively controls the flow rate reduced sulfur in a feedback loop (end product inhibition). Attempts have been made to address this Mechanism to be overcome by screening for cysteine-insensitive serine acetyltransferases from E. coli by means of random mutagenesis (Denk and Bock (1987) J. Gen. Microbiol. 133, 515-525; Takagi et al. (1999) FEBS Lett. 452, 323-327). Alternatively, targeted mutagenesis was used, whereby amino acid positions within the SAT enzyme were exchanged that are close to the positions obtained from the random screening (Nakamori et al. (1998) J. Appl. Environ. Microbiol. 64, 1607-1611 ). Although no kinetic inhibition constants were determined in the course of these investigations, the authors were able to identify mutants with a 15-fold less sensitive feedback inhibition and to achieve the accumulation of considerable amounts of cysteine and cystine in the growth medium. However, further improvements based on amino acid exchanges hardly seem possible, since neither the three-dimensional structure of the SAT is known, nor is sufficient sequence data from cysteine-insensitive SAT forms available from other organisms.

In an alternative approach, naturally occurring SAT proteins are used, which are found in higher plants and are structurally closely related to the bacterial enzyme. Plants generally contain at least three core-coded SAT isoforms, which are found in the plastids, the cytosol and in the

Mitochondria are localized. These SAT enzymes differ considerably in their feedback sensitivity to cysteine, which could be shown by heterologous expression in E. coli (Noji et al. (1998) J. Biol. Chem. 273, 32739 - 32745; h oue et al . (1999) Eur. J. Biochem. 266, 220-227; Saito (2000) vide supra). Despite the relatively large differences in cysteine sensitivity, all SAT enzymes are able to form an active cysteine synthase complex with bacterial OAS-TL (Droux et al. (1998), Eur. J. Biochem. 255, 235 - 245). Various determinants for cysteine inhibition were mapped from E. coli in analogy to SAT (Noji et al. (1998) vide supra; Inoue et al. (1999) vide supra). A first report shows that the heterologous expression of SAT encoding cDNA clones from Arabidopsis thaliana (Bogdanova et al. (1995) FEBS Lett. 358, 43 - 47; Murillo et al. (1995) Cell. Mol. Biol. Res. 41, 425 - 433) in an efficient accumulation of Cysteine in the growth medium of E. coli results (Tagaki et al. (1999) FEMS Microbiol. Lett. 179, 453-459).

In view of the need for efficient processes for the production of cysteine, it is an object of the present invention to provide new feedback-insensitive SAT genes and isofoπnen which are used in high-performance processes for the production of large amounts of cysteine and related sulfur-containing compounds in bacteria and other organisms can be used.

Further tasks are the provision of a method for the selection of cysteine-insensitive SAT genes and the provision of a method for the production of cysteine, cystine and glutathione in microorganisms.

The solutions to these problems are given in the attached independent claims. Preferred embodiments are described in the subclaims. Further tasks and solutions result from the following description.

Surprisingly, it has been possible to demonstrate a new successful screening approach for cDNA clones which code for cysteine-insensitive SAT isoforms. A new E. co / z strain is used in which the cysE gene, which codes for the bacterial SAT, is inactivated by mutation. By means of functional complementation, SAT genes with less pronounced feedback inhibition can be selected using such a SAT-free E. co z mutant. The invention therefore relates to a method for finding DNA sequences which code for cysteine-insensitive serine acetyltransferases, characterized by the use of a cy5E ^" bacterial strain for functional complementation. The bacterial strain is preferably an Escherichia co / z- Tribe. The method according to the invention is thus particularly useful for identifying naturally occurring, cysteine-insensitive S ATs from plants, microorganisms or other sources which are suitable for microbial fermentation of cysteine.

In the context of this invention, the term “cysteine-insensitive serine acetyl transferase” is understood to mean a serine acetyl transferase which has an iso value (I ₅ o = half-maximum inhibition) of at least 30 μM, preferably of at least 35, 40 μM and particularly preferably of at least 45 uM, 50 uM, and most preferably of at least 55 uM, 60 uM.

The I ₀ value is defined here as the cysteine concentration at which, under otherwise optimal conditions, there is a 50% inhibition of the SAT activity. The I ₅₀ value is determined by determining the SAT activity with increasing cysteine

Concentrations determined. By plotting the activity against the cysteine concentration, a hyperbolic function is obtained, from which the I ₅₀ value is determined by curve fitting or manually. The inhibitor constant Ki, regardless of the type of inhibitor, is initially defined as the dissociation constant of an enzyme-inhibitor complex (Ki = [E] [I] / [EI], according to AL Lehninger, Verlag Chemie 1977) and has no direct relationship to I ₅₀ - Value. In principle, however, a low Kr value correlated with a low I ₅ o-value for an inhibitor.

The term "functional complementation" means that a certain enzyme activity of a bacterial strain deficient in this enzyme activity restores the enzyme activity in the bacterial strain by expression of a heterologous gene which codes for this enzyme activity under the control of a suitable bacterial promoter. The restoration of the enzyme activity permits the prototrophic growth of the otherwise auxotrophic, deficient bacterial strain under suitable conditions (in the context of the present invention in the absence of cysteine).

As already mentioned above, the bacterial cysE gene codes for the bacterial serine acetyltransferase; the DNA sequence of the cysE gene is described in the prior art, e.g. in Denk and Bock (1987) J. Gen. Microbiol. 133, 515-525.

The preparation of bacterial strains according to the invention which are suitable for screening for cys-insensitive SAT genes by means of functional complementation due to the inactivation in the cysE gene is described in the examples. These bacterial strains are referred to as SAT-deficient or SAT-free in the context of this invention.

The techniques required to inactivate bacterial genes by mutation are known to those skilled in the art, e.g. from the standard work by Sambrook et al. (1989) Molecular cloning: Laboratory Manual, 2nd edition, Coldspring Harbor Laboratory Press, Coldspring Harbor, New York. In addition, SAT-free bacterial strains are available from strain collections, e.g. the E. co / z strains EC1801 and JM39, available from the E. coli Genetic Stock Center, Yale University, New Haven, CT, USA.

As explained in the examples below, the generation of the SAT-free E. co / t mutant MW1 followed a standard microbiological method (Hamilton et al. (1989) J. Bacteriol. 171, 4617-4622). This and similar methods for hairdressing mutagenesis by homologous recombination have been described many times (e.g. Walkenhorst et al. (1995) Microbiol. Res. 150, 347-361; Link et al. (1997) J. Bacteriol. 179, 6228 - 6237; Selbischka et al. (1993) Appl. Microbiol. Biotechnol. 38, 615-618). The biological principle is described in standard works (HA Nash, In: Escherichia coli and Salmonella. Cellular and Molecular Biology, Vol. IFC Neidhardt et al., Eds., ASM Press, Washington DC, 1996, pages 2363-2376).

For the production of a genetically stable SAT-deficient strain, the mutagenesis described in the attached examples is carried out by homologous

Recombination such as B. Hamilton et al. (1989, vide supra).

The screening for genes by means of functional complementation is very much dependent on the genetic stability of the host organism, that is, the stability of that for

Selection of mutation used. This applies in particular to the heterologous complementation between distant taxa and the case that due to the pronounced genomic complexity of the donor organism, e.g. a higher plant, many clones have to be screened. In some cases, the SAT-deficient bacterial strains described in the prior art are not suitable for the method according to the invention due to their genetic instability. On the one hand, a large number of clones from the plant cDNA banks have to be screened, on the other hand, the inhibition test for cysteine sensitivity can only be carried out without an endogenous SAT background, since bacterial SAT activities are extremely sensitive to cysteine.

The SAT-deficient strains produced in the context of this invention are distinguished by the fact that they show practically no reversion of the phenotype. These genetically extremely stable strains are particularly useful for functional complementation for the discovery of genes which code for cysteine-insensitive SATs.

The strain MW1 described in the examples, which shows no reversion whatsoever, can also be used to remove existing bacterial strains with MW1 transduce. In this way, further cysteine auxotrophic strains are produced which are distinguished by the desired genetic stability.

The functional identity of the genotypes was verified in the context of the invention i) biochemically by enzyme tests, ii) genetically by DNA-DNA hybridization and iii) physiologically by complementation with known plant cDNAs.

The screening by means of functional complementation is familiar to the person skilled in the art and e.g. described in Howarth et al. (1997) Biochim. Biophys. Acta 1350, 123-127; Noji et al. (1994) Mol. Gen. Genet. 244, 57-66, and Setya et al. (1996) Proc. Natl. Acad. Be. USA 93, 13383-13388.

Finally, screening for functional complementation requires a suitable cDNA library from the organism from which cysteine-insensitive SAT gene sequences are to be isolated. The preparation of cDNA libraries is also familiar to the person skilled in the art and is possible using routine molecular biological methods. In addition, cDNA libraries are now commercially available from almost any organism, e.g. from companies like Stratagene, La Jolla, USA.

The invention also relates to genes which code for cysteine-insensitive SAT enzymes and isoforms and which are isolated on the basis of functional complementation by means of the screening method described above.

In a preferred embodiment, these SAT gene sequences are sequences from plants, in particular from Nicotiana tabacum.

The cDNA sequences of the Nicotiana tabacum SAT genes 1, 4 and 7, which are added as sequences, are particularly preferred: cDNA sequence of SAT 1 as SEQ ID NO. 1, the derived amino acid sequence as SEQ ID NO. 2; the cDNA sequence of SAT 4 as SEQ ID NO. 3, the amino acid sequence derived therefrom as SEQ ID NO. 4; the cDNA sequence of SAT 7 as SEQ ID NO. 5 and the amino acid sequence derived therefrom as SEQ ID NO. 6th

The invention thus also relates to a DNA sequence which codes for a protein with the enzymatic activity of a serine acetyltransferase from Nicotiana tabacum, selected from the group consisting of: a) DNA sequences which comprise a nucleotide sequence which comprises the sequence shown in SEQ ID NO. 2, SEQ TD NO. 4 or SEQ ID NO. 6 encode specified amino acid sequence or fragments thereof, b) DNA sequences which the SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 5 specified nucleotide sequence or parts thereof, c) DNA sequences which comprise a nucleotide sequence which hybridizes with a complementary strand of the nucleotide sequence of a) or b) or parts of this nucleotide sequence, d) DNA sequences which comprise a nucleotide sequence is degenerate to a nucleotide sequence of c), or comprise parts of this nucleotide sequence, e) DNA sequences which are a derivative, analog or fragment of a nucleotide sequence of a), b), c) or d).

In the context of the present invention, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Coldspring Harbor, Laboratory Press, Coldspring Harbor, New York.

The SAT nucleic acid molecules which can be used in the context of the invention also comprise fragments, derivatives and allelic variants of the DNA sequences described above which code for SAT or a biologically, ie enzymatically active, fragment thereof. Fragments are parts of the nucleic acid molecules understood that are long enough to encode a polypeptide or protein with the enzymatic activity of a SAT or a comparable enzymatic activity. In this context, the term derivative means that the sequences of these molecules differ from the sequences of the abovementioned nucleic acid molecules at one or more positions and have a high degree of homology to these sequences. Homology means a sequence identity of at least 70%, preferably 80%, in particular an identity of at least 85% and 90%, preferably at least 92% and particularly preferably at least 95%, 98%, 99%, or that the homologous sequence under stringent conditions , which are known to the person skilled in the art, hybridized with the above-mentioned SAT sequences. The deviation from the nucleic acid molecules described above may have resulted from deletion, addition, substitution, insertion or recombination. Homology also means that there is functional and / or structural equivalence between the nucleic acid molecules concerned and the proteins encoded by them.

The invention also relates to sequences from other plants coding for SAT which are identified by the sequence shown in SEQ ID 1, 3 and 5 nucleic acid sequences shown in the coding region show an identity of at least 80%, 85%, 90% and in particular at least 94%, 96%. These are particularly preferably DNA sequences which have the sequence shown in SEQ ID No. 3 (SAT 4 from tobacco) shown nucleic acid sequence in the coding region have an identity of at least 80%>, 85%), 90% o and in particular at least 94%, 96%.

The degree of sequence identity of a nucleic acid molecule with the im

Sequence listing sequences can be determined using conventional algorithms. For example, the program for determining sequence identity is suitable here, which is available at http://www.ncbi.nhn.nih.gov/BLAST (on this page, for example, the link "Standard nucleotide-nucleotide BLAST [blastn]"). Figure 2 shows a comparison of the SAT sequences from tobacco isolated in the context of this application with five known SAT sequences from Arabidopsis thaliana (in the MIPS genome nomenclature) and the known SAT sequence from E. coli. From this amino acid alignment it can be seen that the cysteine-insensitive SAT 4 from tobacco, which could be isolated in the context of this invention, interestingly differs from the known SAT sequences in two positions: i) an insertion of two residues, a cysteine and a serine, at position 63 and 64 within the amino acid sequence of the SAT 4; and ii) a three amino acid deletion near the C-terminus; both places are highlighted in Figure 2.

The invention thus also relates to DNA sequences which code for SAT and which have a deletion or insertion corresponding to the deletion or insertion marked in FIG. 2, or both features, the position of course varying relative to SAT 4 from tobacco. Particularly preferably, in addition to the two features mentioned, which distinguish them from known SAT genes, the SAT sequences additionally have a strong homology to the entire coding sequence of SAT 4 (SEQ ID No. 3).

The insertion mentioned does not necessarily have to include two amino acid residues, but only one residue or more than two residues can be inserted in comparison to other SAT sequences. The insertion is within the conserved motif: LF / L / MY E / DL / IFXXV / T / A / IDLXAF / V / AK / RXRDPACI / L / NSY / F (where X is any amino acid), see Figure 2 , corresponds to the motif between position 63 and position 98 of the SAT gene from E. coli.

The deletion mentioned does not necessarily have to include three amino acid residues, it can also be only one residue, two residues or more than three residues in the Deleted unlike other SAT sequences. The deletion is within the last 20 C-terminal amino acids, especially in the conserved motif:

C / G / SL / E / MXMD / K / EH / QXS / A / EXXXE / FW / FS / RD / HN / Y (where X is any amino acid), see Figure 2, corresponds to the motif between position 259 and position 274 of the E. coli SAT gene.

In any case, the person skilled in the art can easily determine by means of a sequence comparison, as shown in FIG. 2, whether a SAT sequence he has isolated by means of the method according to the invention is one of the features observed for SAT 4 from tobacco, that is to say the deletion and / or insertion, at a position corresponding to the position within SAT 4. The identical amino acids need not necessarily be deleted or inserted, the decisive factor is whether there is a deletion and / or insertion in the corresponding positions at all.

The discovery of plant SAT sequences which have a strong homology to the sequences from tobacco disclosed in this application and are therefore particularly suitable for producing cysteine in microorganisms due to a pronounced cysteine insensitivity can, on the one hand, by means of functional complementation, as here described. On the other hand, it can also be done using traditional methods such as hybridization or PCR. The sequences disclosed in the attached sequence listing or parts thereof can be used as a hybridization probe. Likewise, the person skilled in the art can of course use the disclosed sequences to design suitable PCR primers and use them for the amplification of SAT-coding sequences.

The invention also relates to a method for producing sulfur-containing compounds, in particular cysteine, cystine and glutathione, in host organisms, in particular microorganisms, by overexpressing the Screening method described above by means of functional complementation found cysteine-insensitive SAT genes or other SAT genes with the desired cysteine insensitivity in host organisms.

Figure 3 shows the accumulation of cysteine in E. co / z ^' cultures that express the SAT sequences from tobacco. The sequences SAT1, SAT4 and SAT7 from tobacco were expressed in the strain MW1. Cell density (black dots) and cysteine levels in the culture medium were determined during growth as indicated. C600 is the wild type.

Expression of the SAT4 from tobacco resulted in total cysteine contents of up to 300 mg / 1 culture medium.

The invention thus also relates to a process for the fermentative production of cysteine, in which cysteine contents in the growth medium of the bacteria are achieved which are at least 20 mg / 1, preferably at least 50 mg / 1 and particularly preferably at least 100, 200 mg / 1 culture medium , In comparison to the wild-type strain, an increase in the cysteine content in the growth medium of at least 3-fold, preferably at least 5-fold and particularly preferably of at least 10-fold, 20-fold is achieved by means of the method according to the invention expressing a cysteine-insensitive SAT.

In a preferred embodiment, the microorganism which is used for the overexpression of the cysteine-insensitive SAT gene sequences is a glutathione-deficient strain. The glutathione-deficient bacterial strain is preferably the E. co / z ^' strain JTG10 with the chromosomal marker gshA20 :: Tnl0kan, which is available, for example, from the E. coli Genetic Stock Center, Yale University, New Haven, CT, USA, can be obtained (CGSC # 6926) and was originally described by Greenberg & Demple (1986, J. Bacteriol. 168, 1026). Another suitable glutathione-deficient bacterial strain is, for example, the E. co / t strain 821, ID # 9836 with the mutation gshA2 (Apontoweil and Behrends (1975) Mol. Gen. Genet. 141, 91-95), which is also from the E coli Genetic Stock Center, Yale University, New Haven, CT, USA.

The use of a glutathione deficient strain has the advantage that the absence of glutathione as a control agent for the accumulation of cysteine in the bacterial cell results in the secretion of cysteine from the bacterial cell into the growth medium. This effect leads to a considerable increase in the total cysteine accumulation.

The use of a glutathione-deficient strain to produce cysteine has not previously been considered in the prior art. The overproduction of cysteine in a glutathione-deficient strain, which was observed for the first time in the context of this invention, can also be regarded as surprising, since glutathione and others. is an important protection against oxidative stress in bacteria and the successful overproduction of cysteine by a glutathione-deficient strain in an aerobic culture could not be expected.

The invention thus relates to a process for the production of cysteine, cystine and glutathione in microorganisms, in particular bacteria, characterized in that a DNA sequence which codes for a cysteine-insensitive serine acetyltransferase is expressed in the microorganism, and it is present in the microorganism is a glutathione-deficient cell.

The DNA sequences for SAT expressed in the glutathione-deficient microorganism are generally selected so that they show the desired overproduction of cysteine in the microorganism. cystine and glutathione arise as a result of cysteine overproduction or can be formed preferentially through targeted measures.

In a preferred embodiment, it is a SAT gene which is identified by means of functional complementation using the inventive screening method described above. These are preferably plant SAT genes, particularly preferably from Nicotiana tabacum.

The sequences disclosed in this application code for vegetable SAT enzymes which are distinguished from SAT proteins from the literature by greatly reduced sensitivity to cysteine. With the method according to the invention for the detection of cysteine-insensitive SAT sequences, further suitable coding sequences, in particular from plants, can be identified and their suitability for the overproduction of cysteine in microorganisms can be examined. With the aid of the screening method according to the invention, even more suitable SAT genes can be found, that is to say genes which code for protein and which show an even more pronounced cysteine insensitivity. If cDNA clones are isolated as described here, the coded SAT proteins can be expressed in bacteria by standard methods and introduced into existing strains to improve cysteine production, preference being given to using glutathione-deficient strains.

In a further preferred embodiment, the microorganism is a glutathione-deficient bacterial strain, in particular a bacterial strain which is inactivated by mutation in the first glutathione synthesis step (gshl ^~ ).

The particularly preferred strain JTG10 contains a gshA gene which is inactivated by transposon TnlOkan and which codes for the first enzyme of glutathione synthesis, γ-glutamylcysteine synthetase. Any inactivation of this gene leads to complete or partial reduction of the enzyme activity, is suitable for the cysteine production according to the invention.

The expression of the cysteine-insensitive SAT genes in a glutathione-deficient bacterial strain leads to the fact that the cysteine formed is discharged from the cell and accumulates in high concentrations in the surrounding culture medium as cysteine and cystine. The enriched sulfur-containing compounds can then be purified using standard procedures. The purification of cysteine from aqueous solutions is e.g. in JP56140966A2 (Preparation of purified cysteine) and JP61057549 (Method of purifying cysteine). Principal methods for the purification of amino acid with cation exchange chromatography are e.g. described by T. G. Cooper (Biochemical Working Methods, W. de Gryuter Verlag, Berlin, 1980).

The techniques required for the production of a microorganism suitable for the fermentative production of cysteine, cystine and glutathione, such as the transformation of bacteria with plasmids, the selection of transformed bacteria, the cultivation of bacteria in suitable growth media, are well known and described to the person skilled in the art, e.g. in Sambrook et al. (1989) vide supra.

The same applies to the production of bacterial expression vectors using conventional techniques; suitable expression vectors for the overexpression of heterologous genes in bacteria are also commercially available, e.g. from the company Qiagen, Hilden Germany.

The use of the synthesized and accumulated cysteine lies particularly in the supplementation of food for humans and animals. Furthermore, derivatives such as N-acetylcysteine, which have pharmaceutical and cosmetic use, can be easily produced from this. The invention is explained below in the following examples, which serve only to illustrate the invention and are in no way to be understood as a limitation.

Examples

General cloning and PCR procedures were carried out according to Sambrook et al. (1989, vide supra). DNA sequences were obtained with a 373A sequencer (Perkin-Elmer, Boston, USA).

The cDNA library from N. tabacum cv used. Samsun was created in λ-ZAPII according to specifications and with materials from the manufacturer, Stratagene. In vivo excision was also carried out according to the manufacturer's instructions (Stratagene) in order to obtain recombinant plasmids from the phage bank. These can be used to transform the SAT-deficient E.co/t standard.

Southern blot analysis of E. coli genomic DNA was performed as described in Hell et al. (1994, FΕBS Lett. 351, 257-262), using the 2.2 kb c sE fragment given below as a hybridization probe.

Production of a cys--r bacterial strain

The insertional inactivation of the wild-type cysE gene from E. coli C600 (thr leu thi lac (λ) -Pl + F '; Clontech, Heidelberg, Germany) was carried out as described by Hamilton et al. (1989) J. Bacteriol. 171, 4617-4622. This should create an S AT-deficient strain that has increased stability compared to currently available strains. The wild-type cysE gene was including its flanking regions using PCR with genomic DNA from strain C600 and the primers

ECS 155 5'-CGTGGATCCTTAGGCGATCAAATTCC-3 'and ECS156 5'-GGGGAGTCGACGGCGCTGTATGTACTCCCT-3'

amplified.

The PCR protocol was as follows:

In 50 ul reaction volume were:

20 pmol of the two primers, 10 ng genomic DNA, 1 U Taq polymerase (Promega, Heidelberg, Germany), polymerase buffer from Promega according to the manufacturer's instructions. After 5 minutes at 94 ° C there were 35 cycles of 30 seconds at 94 ° C, 30 seconds at 58 ° C, 60 seconds at 72 ° C, followed by 10 minutes at 72 ° C.

The resulting 2.2 kb DNA fragment was ligated into the Sall and BamHI restriction sites of linearized pUC18. Within this plasmid, the cysE gene was cut with Clal at position 522, based on the open reading frame, and inactivated by inserting a 2.2 kb Clal / Clal fragment from p AC YC 184-Gm. This fragment from the plasmid pACYC184-Gm (Chang and Cohen (1978) J. Bacteriol. 134, 1141-1156) carries a gentamycin resistance gene; the resulting pUC plasmid was named pUC18cysΕ-Gm. A maximum of 174 amino acids of the E. co / z-SAT enzyme can thus be translated in this plasmid. The cysE-Gm cassette was cut out with Sall and BamHI and ligated as a 4.4 kb fragment into the same sites of the plasmid pMAK705, which carries a kanamycin resistance gene and a temperature-sensitive origin of replication (pHOl; Hashimoto-Gotoh and Sekiguchi (1977 ) J. Bacteriol. 131, 405-412). The resulting plasmid pMW1 was used for gene exchange using homologous recombination. For this purpose strain C600 transformed with pMWl to select kanamycin resistant colonies at 44 ° C (non-permissive temperature). Cointegrates were first purified from the plasmid by culturing at 30 ° C (permissive temperature) followed by non-permissive conditions and the resulting cysteine auxotrophic strain was named MW1 (fhr leu thi lac (λ) -PH-F 'cysE GmX) , In this way, the inactivated cysE gene was introduced into the genome of E. coli C600 via homologous recombination.

The complementation and auxotrophy tests were carried out on solid M9 minimal medium, supplemented with IPTG (1 mM), ampicillin (100 μg / ml), gentamycin (50 μg / ml), thiamine (0.1 mM) and 1 mM all amino acids except methionine and cysteine. Individual plasmids or the cDNA library from tobacco were introduced into strain MW1 by electroporation (BioRad, Munich, Germany) and colonies were selected by incubation at 37 ° C. for up to 48 hours.

Protein expression and enzyme tests

Expression of SAT enzymes was induced in all constructs with isopropyl thiogalactoside in full medium (LB) or minimal medium (M9) supplemented with ampicillin or gentamycin or both (Bogdanova et al. (1995) vide supra; Wirtz et al. ( 2000) hi Brunold et al., Eds, Sulfur Nutrition and Sulfur Assimilation in Higher Plants: Molecular, Biochemical and Physiological Aspects. P. Haupt Bern, pages 297-298). The cells were harvested and disrupted by ultrasound treatment (Sonicator Bandelin, Berlin, Germany), then the soluble supernatant (10 min at 30,000 xg) was desalted by gel filtration on a PD 10 column (Amersham, Freiburg, Germany) and at -80 ° C stored. The protein contents were determined according to Bradford (1976). The SAT activity of purified fractions and crude fractions was determined in 250 μl, containing 50 mM Tris HC1, pH 7.5, 0.2 mM acetyl-CoA, 2 mM dithiothreitol and 5 mM serine in the presence or absence of varying cysteine concentrations were examined. The absorption at 232 nm was documented for several minutes according to Kredich and Becker (1971, h Meihods in Enzymology (Tabor H. and Tabor CW, eds) pages 459-469, Acadamic Press, New York, USA). SAT activities were carried out according to Nakamura et al. (1987, Plant Cell Physiol 28, 885-891).

Quantification of the thiols

For the quantification of the thiols, the content of cysteine and glutathione in the medium or in the cells after extraction with 0.1 N HCl was determined in a 1: 5 ratio. After reduction with dithiothreitol, the sulfhydryl groups were derivatized with monobromobimane (Calbiochem, Darmstadt, Germany). The separation, detection and quantification of the fluorescent adducts was carried out by means of HPLC (reversed-phase column Waters Nova-Pak C18, 4.6 x 250 mm; Waters HPLC system; Hell and Bergmann (1990) Planta 180, 630 -612) ,

Identification of cDNA clones encoding cysteine-insensitive SAT by complementation of MW1

Electroporation-competent cells from MW1 were transformed with a plasmid cDNA library (obtained by in vivo excision of the λZAPII library) from Nicotiana tabacum var. SNN. The cells were regenerated in 1 ml of LB

Complete medium at 37 °, 220 rpm for 1 hour. The volume was plated onto 2 petri dishes containing the M9 minimal medium (Sambrook et al. (1989), vide supra) with 100 μg / ml ampicillin, 50 μg / ml gentamycin and 1 mM isopropylthiogalactoside and 1 mM each of all proteinogenic amino acids except cysteine and methionine , The dishes were incubated at 37 ° C for 24 hours. The received Colonies were then cultivated further and used to check the cysteine sensitivity of the selected SAT or plasmid isolation.

The cysteine insensitivity was checked in two ways:

1. Selected colonies were grown in 4 ml LB medium. An aliquot corresponding to an optical density at 600 nm of 0.05 was used to inoculate a shake culture with 20 ml Cl medium (minimal medium according to Nakamori et al. (1998) Appl. Environm. Microbiol. 64, 1607-1611, supplemented with 0, 1 mM thiamine and 1 mM threonine and leucine are used as the only amino acids, and after 48 hours the cysteine accumulation in the medium is determined as a measure of the cysteine sensitivity of the respective plasmid-encoded vegetable SAT.

2. Selected colonies were grown in 200 ml LB full medium (Sambrook et al., (1989), vide supra) with 100 μg / ml ampicillin as described above. The soluble protein fraction was isolated as described above after ultrasound treatment and the SAT activity was determined in the standard test according to Kredich and Becker (1971, vide supra) with 10 μM cysteine compared to the absence of cysteine.

The plasmids of selected colonies were again transformed into MWl as above and checked for growth in the absence of cysteine. Growth of all plated cells was interpreted as positive functional complementation. The cDNA inserts of the plasmids in question were then subjected to DNA sequencing.

Production of cysteine, cystine and glutathione in the E. co z strain MWl

For the production of cysteine, cystine and glutathione in MWl, 200 ml shake cultures with M9 medium, supplemented with IPTG (ImM), ampicillin (100 μg / ml) and the 18 proteinogenic amino acids in addition to cysteine and methionine with an inoculum of 0.05 OD from MWl were inoculated with a plasmid with a SAT-encoding cDNA insert. The formation of cysteine and glutathione was followed in the course of the culture. The values after 18 hours of growth are shown in Table 2. Cystine was created as an oxidation product of cysteine in the medium and was not determined separately.

The production of cysteine and cystine in the glutathione-deficient strain JTG10 was carried out as well as the production of the sulfur-containing compound in MWl.

The screening for genes by means of functional complementation is strongly dependent on the stability of the corresponding mutation in the host organism. This is particularly the case with heterologous complementation between remote taxa and the need for a large number of transformants if the

Organism from which the gene complementing the mutation originates is genetically very complex. The mutant E. co / z strain MW1 described above, which was produced for the efficient screening of SAT cDNAs, was the already known cysE-E. Co / z ^' mutants EC1801 and JM39 consider that MWI showed no reversion of the phenotype. This means that the screening for SAT genes from heterologous sources can be carried out under any conditions without being affected by undesired genetic events. The functional identity of the genotype was verified (i) biochemically using enzyme assays, (ii) genetically using DNA-DNA hybridization and (iü) physiologically using complementation with known cDNA clones from plants.

The MW1 strain showed no detectable SAT activity (see Table 1). Table 1: SAT enzymatic activity in crude extracts from E. coli wild-type strain C600 and mutant MWl

The position of the homologous recombination was confirmed by hybridizing genomic DNA of the wild-type strain C600 with the cysE gene as a probe. Restriction digestion with ΕcoRV, which cuts outside of cysE, and with StuI, which cuts inside the cysE gene, gave a 4.6 kb signal or 7.4 kb and 1.0 kb signals, which the prediction based on the Map of the E. coli

Genome at 81.44 min. equivalent. The insertion of the 2.2 kb gentamycin cassette would enlarge the ΕcoRV genomic fragment to 6.8 kb. In contrast, an internal ΕcoRV interface in the cassette leads to signals of 4.7 kb and 2.1 kb in the DNA-DNA hybridization, which confirms the position, orientation and identity of the insertion. Apart from the gentamic resistance, the specific identity of the MWl strain was demonstrated by complementation with a cDNA coding for SAT from Arabidopsis thaliana (Wirtz, diploma thesis 1998, Ruhr University Bochum, investigation of cysteine biosynthesis by mutagenesis of plant serine acetyltransferase) on minimal medium without cysteine , The SAT deficiency in the MWl strain was characterized by low and medium strength

Raten Expression rates of SAT A functionally complemented using vectors with different copy numbers.

Plant cysteine synthesis was thus implemented in E. coli without any endogenous background. - 24 '

In terms of their sensitivity to cysteine, SAT7 and SAT1 from tobacco correspond to the SAT-A from E. coli with an I ₅₀ value of approximately 45 μM (Noji et al. (1998) vide supra), the values of SAT7 and SAT1 being clear lie above the inhibition constant of the bacterial SAT (Ki 1.1 μM, Kredich et al. (1969) vide supra). SAT4 is essentially msensitive to cysteine, ie an I ₅₀ value cannot be measured for SAT4 (see Figure 1). Such complete insensitivity to cysteine has never been described for a cloned or biochemically isolated SAT from any organism.

Production of cysteine and glutathione

Table 2: Accumulation of cysteine in the growth medium

E. cotϊ cells with or without pBS-SK plasmids with tobacco cDNA clones for SAT were cultivated in supplemented M9 minimal medium without a reduced sulfur source; cysteine levels were determined after 18 hours of growth, as determined from inoculation of the culture.

Table 2:

nd = undetectable

The GSH content in the medium of C600 and MWl is essentially in the order of magnitude of the cysteine content. It can be assumed that cystine is also present in the medium since the medium is continuously aerated by shaking, which promotes cysteine oxidation.

The cysteine content in the medium is approximately 10-fold higher for MW1, which expresses the SAT cDNA clones from tobacco, compared to C600, which carries the wild-type cs-E gene. A further 10-fold increase in the secretion of cysteine is achieved by expression of the tobacco SAT in the E. co z strain JTG10. The lack of glutathione as a control agent for the accumulation of cysteine in the bacterial cell thus results in the secretion of the cysteine. The combination of feedback-insensitive SAT enzymes with glutathione-deficient cell lines allows the production of cysteine in amounts 3 to 10 times higher than has been achieved in the prior art using other strategies (Daßler et al. (2000) vide supra ; Takagi et al. (1999) vide supra; Nakamori et al. (1998) vide supra).

Illustration

Figure 1 shows the inhibition of plant SAT enzymes by cysteine. SAT1, SAT4 and SAT7 from tobacco were expressed in the MW1 strain and examined for their SAT activity with increasing cysteine concentrations. Sequence protocol

SEQ ID NO. 1 cDNA sequence from SAT 1 from Nicotiana tabacum

GCGGCCGCCTTTCTCTTTGTTTATCTCTCTCCTCCCTCGCCGCCACATATTC TCCTACACACATTTCGCTTCAATGTCCACTAATTTCCTCGGATCACCACCA CCCCTTTTCAAGAATGTAATCTCTCCTTGTAATAAACTCTCCACTTTCACA ATAAGAGCTTGTTTACATTCTTGTGAGCCCAAAATTGATGATCATATCTA CAACAACTACACTAAATACTGCACTCCCAATTTCCCAAATCATGTTTCTC AGACACCCATTTCAGAAAAACAGCCAAAAACCAACAAGAACCATACAAT TTTGGACAATTTTGCAAAAGATGATGATTTGTGGCTAAAAATGCAAAAAG AGGCAAGGTTAGATATTGAGCAAGAACCCCTTTTGTCAAATTACTATAAA AATTCAATCTTGGCTCATGATTCTATAGAAAGTGCTTTAGCTAACCATCTT TCAATGAAATTGAGCAATTTGAGTATTTCTAGTGAAACTCTATATGATCT TTTCATGGGGGTGCTCACAGAGGATCAAGAATTGATTTTTGATGTTAATG CTGATTTGATAGCTGTTAAAGAAAGAGATCCAGCTTGTATTAGTTATATA CATTGTTTCTTGAATTTTAAAGGGTTTTTAGCATGTCAAGCACATAGAAT AGCACATAAGTTATGGTCTAAAGGGAGAAAGATTTTAGCTTTAGTAATAC AAAATAGAGTATGTGAAGTTTTTGCTGTGGATATTCATCCTGGAGCAAGA ATTGGTAGAGGAATATTATTAGATCATGCAACTGGAGTTGTAATTGGTGA GACAGCAATTATAGGAAATAATGTGTCAATTTTACATAATGTAACATTAG GTGGAACCGGAAAAATGTGTGGTGATAGACATCCAAAAATTGGTGATGG TGTATTAATAGGTGCAGGGACTTGTGTTCTTGGAAATGTTAGAATTGAAA ATGGTGCTAAAATTGGAGCTGGTTCTGT TGTGTTAATGGAAGTTCCTGCT AGAACAACTGCTGTTGGAAATCCAGCTAGATTGATTGGTGGGAAAGCAA ATCCAATTAAGCTTGATAAAATTCCTAGTTTGCCTATGGATCATACTTCAT ATTTATCTGAGTGGTCTGATTATGGGTTGGGTGGTGGGTGGTGGGT SEQ ID NO. 2

Amino acid sequence of SAT 1 from Nicotiana tabacum

RPPFSLFISLLPRRHIFSYTHFASMSTNFLGSPPPLFK-> WISPCNKLSTFTIRACL HSCEPKIDDHiYNNYTKYCTPNFPNHNSQTPISEKQPKTNK-> ΗTILDl ^ AKDD DLWLKMQKEARLDIEQEPLLSNYYKNSILAHDSIESALANHLSMKLSNLSISS ETLYDLFMGVLTEDQELIFDNNADLIANKERDPACISYIHCFLNFKGFLACQA H ^ iAHKLWSKGRKILALNIQNRNCENFANDLHPGARIGRGILLDHATGNNIGE TAIIGΝΝNSILHΝNTLGGTGKMCGDRHPKIGDGNLIGAGTCNLGΝVRIEΝGA KIGAGS LJVIEVPARTTAVGΝPARLIGGKAΝPIKLDKIPSLPMDHTSYLSEW SDYVI

SEQ ID ΝO. 3 cDΝA sequence from SAT 4 from Nicotiana tabacum

GCGGCCGCAACTCCTCCTACAAATCCACTTTCTCGCGATCCAAACAAGCC CCAAATCGACAATCATGTCTATAACTACGTTAAATTCTGTCGACCCAGTT TCCCTGAGCTTGTTTCTTGCGCACCCATTCCTGAAAAGAACTCCAAAATC GGTCGTAACGAAGAGGAAGACGATTTGTGGCTAAAAATGAAAGATGAGG CTAGATCAGACATTGATCAAGAACCCATTTTGTCTACTTACTACATAACT TCAATCTTGGCTCATGATTCTATGGAAAGGGCTTTAGCTAATCATCTTTCA ATGAAATTGAGTAATTCAAGTCTTCCTAGCAGCACTTTGTATGATCTTTTC CTAGGGGTGCTCACAGAGGATTGCTCACAAGATATAATTAAAGCTGTTAT AGCTGATTTAAGGGCAGTTAAAGAAAGGGACCCAGCTTGTATTAGTTATG TACACTGTTTCTTGAATTTTAAAGGGTTTTTAGCATGTCAAGCTCATAGGA TTGCACATAAATTATGGTCAAATGGTAGGCAAATTTTGGCACTTTTGATA CAAAACAGGGTATCTGAAGTTTTTGCTGTCGACATACATCCTGGTGCTAA AATTGGTAAAGGGATTTTACTTGATCATGCTACTGGAGTTGTCGTTGGTG AAACTGCTGTGATTGGAAATAATGTGTCAATTTTGCATAACGTGACATTG GGTGGAACTGGCAAAATATCTGGGGATAGACATCCTAAAATTGGTGATG GGGTTTTAATTGGTGCTGGAACTTGTGTTCTTGGAAATGTTATAATTGAA GATGGAGCTAAAATTGGGGCAGGGTCCGTGGTGCTGAAGAAAGTTCCGG CGAGGACTACCGCCGTTGGGAATCCGGCGAGGTTGCTCGGAGGGAAGGA AAATCCAAAGAAACTTGATAAGATTCCTAGTTTGACCATGGACCATACAT ATGAGTGGTCTGATTATGTAATTTAGAGTAATAACAACTTTTACTTTGTTT ACTACTGTTTTAGGTTTTTATTAGATTAAGTGAAACGAAGGGAATTCTTG GCCGTAACCGATAAAGTTGCTGCTATGTGATTAAGAAGGTTACGAAGTTC CGAACTGTAANAAACAACCTTTTGCAAAAAAATACNGGTAAGAATTGCG TACAATAAACCCTTGNGGTCTGACCCTTNCTCAATCCCCCCCCATAGCCG GGGAGTTTTATTGCACCCAAAAANGTCATTTTTTANTAAAATTAAGTGGA GATGCCCCTCGAGGAATTTTAGTTGCAGGGAGAATATTTCCTTGAGTGGA GATGTTGTACAAGCCATTTACTTCTATGGTAACTGGTTTATATTAAGAGA TTATTGTACTAGATTTCTTGCTAGAGTAAACGGTTCAAATGCAATCTGAC TAAGATTGAGGCGGCCGC

SEQ ID NO. 4 amino acid sequence of SAT 4 from Nicotiana tabacum

AAATPPTNPLSRDPNIO'QroNHVYNYViα ^ C ^ SFPELVSCAPIPEKNSKIGRN EEEDDLWLKMKDEARSDroQEPILSTYYITSILAHDSMERALA-> fflLSMKLSN SSLPSSTLYDLFLGVLTEDCSQDI - KAVIADLRAVKERDPACISYVHCFLNFKG FLACQAHRIAHKLWSNGRQILALLIQNRVSEVFAVDIHPGAKIGKGILLDHAT GVVVGETAVIGNNVSILHNVTLGGTGKISGDRHPKIGDGVLIGAGTCVLGNV IffiDGAKIGAGSVVLKKVPARTTAVGNPARLLGGKENPKKLDK-TPSLTMDHT YEWSDYVI SEQ ID NO. 5 cDNA sequence of SAT 7 from Nicotiana tabacum

CGCGGCCGCCGACGTTATCCGATATGCCAGCCGGAGAATACCGCAATGC CACACCGGCGACACCACATCCACCGACAGACACGGCGGAAGAATCCACA TGGCTATGGACACAAATCAAAGCCGAAGCTCGGCGCGACGCCGAAGCCG AGCCGGCATTAGCCAGCTACTTATACTCAACTATACTCTCTCACTCTTCGC TTGAACGTTCGCTCTCTTTCCATTTGGGAAACAAGCTTTGTTCTTCCACGC TCTTATCCACACTCCTTTATGATCTGTTTCTCAATACTTTCTCTAATGAAC CTGAGCTACGCGCCGCCGCTTCCGCTGACCTACTCGCTGCTCGTTACCGG GACCCTGCTTGTGTTTCATTCTCTCATTGTTTGCTTAACTACAAAGGTTTC CTTGCTTGTCAGGCACATCGAGTAGCCCACAAGCTTTGGACTCAATCCCG AAGGCCACTTGCTCTGGCACTTCAATCCCGAATCTCTGATGTTTTTGCTGT TGACATTCATCCAGCTGCCAAGATCGGTAAAGGTATCCTCTTTGATCATG C AAC AGGAGTGGTGGTTGGCGAGACTGC AGTTATTGGAAACAACGTGTC AATTCTTCACCATGTAACCTTAGGAGGGACTGGTAAGATTGGTGGTGACC GGCACCCTAAGATTGGGGATGGTGTGCTCATAGGTGCAGGTGCCACAAT ATTGGGCAACGTGAGGATTGGTGAGGGGGCCAAGATTGGCGCTGGATCA GTGGTATTGATTGACGTGCCACCACGGACAACTGCAGTTGGGAATCCAGC AAGGTTGGTTGGAGGGAAGGAACAACCAACTAAGCATGAGGAATGTCCC GGAGAGAGTATGGATCATACATCTTTCATTTCTGAATGGTCTGATTACAT CATATGACTTGCATCCCTCATGTATGCTATATCTGCAAAGTGAACAAGAA GTCGTCTACGAACCTAGCAGAGAGGACAAA GGTTCAATCTTAACGCTACT GACTGTTAAACATGCTCTTTGTGCTAGTCCAACAGTCCAAAGTGCAAAGA ACTTATATCTATCTTTTTTTTTCCCAATAGTCTTTCTGTTTTTCTACATTTA CATGCTGTCTGAGAGGGTTGAAGGCTCTTGTTTTTATGCAGAGATTTCTG CTTGGAGTCGCCTTACAGCAAGTTCCCTTTGATGGCACAAATATATAAGT AGTATGTATTATGAGTTCAGTATCTACTCGTTGCTGGTCTTCCTAGGCCTT AAAAACTGGTGAAAATTAACTGTACGAAGTACTGGCCTATTATTATTGGT ATCATTATTGCTGCGGGCC SEQ ID NO. 6

Amino acid sequence of SAT 7 from Nicotiana tabacum

PJPTLSDMPAGEY1 ^ ATPATPHPPTDTAEESTWLWTQIKAEARRDAEAEPAL ASYLYSTILSHSSLERSLSFHLGNKLCSSTLLSTLLYDLFLNTFSNEPELRAAA SADLLAA-RYRDPACVSFSHCLLNYKGFLACQAHRVAHKLWTQSRRPLALAL QSRISDVFAVDΓHPAAKIGKGILFDHATGVVVGETAVIGNNVSILHHVTLGGT GKIGGDRHPKIGDGVLIGAGATILGNVRIGEGAKIGAGSVVLIDVPPRTTAVG NPARLVGGKEQPTKHEECPGESMDHTSFISEWSDYΠ

Claims

PAT EN T AN SPRÜCHE

1. Process for the preparation and production of sulfur-containing compounds such as cysteine, cystine and glutathione in a microorganism which contains a heterologous DNA sequence coding for a cysteine-insensitive serine acetyltransferase and is deficient in glutathione.

2. The method according to claim 2, wherein the DNA sequence is of plant origin.

3. The method according to claim 1 or 2, wherein the microorganism is an E. coli strain.

4. The method according to any one of the preceding claims, wherein the

Microorganism is inactivated in the first glutathione synthesis step.

5. The method according to any one of the preceding claims, wherein the DNA sequence is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, a sequence coding for a SAT, which leads to

SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 5 has an identity of at least 80% within the coding region.

6. The method according to any one of the preceding claims, wherein the DNA sequence in a method for finding DNA sequences which are for

Encoding proteins with the enzymatic activity of a cysteine-insensitive serine acetyltransferase has been found, in which the discovery is carried out by expression of the DNA sequences and the associated functional complementation in a bacterial strain whose endogenous serine acetyltransferase activity is disrupted.

7. The method according to claim 6, wherein the bacterial strain whose SAT activity is disturbed is an Escherichia eo / t bacterial strain which has a mutation in the cysE gene (cysE).

8. The method according to any one of the preceding claims, wherein the cysteine-insensitive serine acetyltransferase has an I ₅ o value of at least 30 μM.

9. Use of cysteine-insensitive serine acetyltransferase genes, which code for serine acetyltransferases and which have an iso value of at least 30 μM, for the production and production of sulfur-containing compounds such as cysteine, cystine and glutathione in glutathione-deficient microorganisms.

10. Use according to claim 9, wherein the serine acetyltransferase gene is of plant origin.

11. A method for the discovery of DNA sequences which code for proteins with the enzymatic activity of a cysteine-insensitive serine acetyltransferase, characterized in that the discovery is carried out by expression of the DNA sequences and the associated functional complementation in a bacterial strain whose endogenous Serine acetyltransferase activity is disrupted.

12. The method according to claim 11, wherein the bacterial strain is an Escherichia co / z bacterial strain and has a mutation in the cysE gene (cysET).

13. The method according to claim 11 or 12, wherein it is vegetable DNA sequences.

14. The method according to any one of claims 11 to 13, wherein the cysteine-insensitive serine acetyltransferase has an iso value of at least 30 μM.

15. DNA sequence which codes for a protein with the enzymatic activity of a serine acetyltransferase from Nicotiana tabacum, selected from the group consisting of: a) DNA sequences which comprise a nucleotide sequence which corresponds to that described in SEQ TD NO. 2, SEQ ID NO. 4 or SEQ ID NO. 6 encode specified amino acid sequence or fragments thereof, b) DNA sequences which the SEQ ID NO. 1, SEQ ID NO. 3 or SEQ ID NO. 5 specified nucleotide sequence or parts thereof, c) DNA sequences which comprise a nucleotide sequence which hybridizes with a complementary strand of the nucleotide sequence of a) or b) or parts of this nucleotide sequence, d) DNA sequences which comprise a nucleotide sequence is degenerate to a nucleotide sequence of c), or parts thereof

Nucleotide sequence comprise, e) DNA sequences which represent a derivative, analog or fragment of a nucleotide sequence of a), b), c) or d).