PROCESS FOR THE PRODUCTION OF L-CYSTEINE ACID

Description

[0001] The invention relates to a process for the production of L-cysteic acid, wherein O-acetyl-L-serine (OAS) is reacted with at least one enzyme selected from the class of O-acetyl-L-serine sulfhydrylases (OAS sulfhydrylases, EC 4.2.99.8) in the presence of a salt of sulfurous acid, wherein the OAS sulfhydrylase is CysM and the biotransformation is carried out under active pH control and the OAS concentration in the mixture is at least 10 g / L. This biotransformation provides L-cysteic acid. L-cysteic acid can be used, for example, in fish farming (Nakamura et al., Fisheries Science (2021) 87: 353-363) or in the cosmetics sector (US4053630), e.g. as an ingredient in Regu ®< -Slim (DSM) for skin care. In peptide chemistry, L-cysteic acid is used as a water-soluble protecting group. L-cysteic acid can also be converted to taurine by decarboxylation.L-cysteic acid ((R)-2-amino-3-sulfopropionic acid, 3-sulfo-L-alanine, CAS 498-40-8) can be produced chemically, e.g., by oxidation of cysteine ​​with chlorine in alcoholic solution (Tao et al. Amino Acids (2004) 27: 149-151), with bromine in HCl or iodine-HCl in DMSO, or by oxidative cleavage of cystine with performic acid. Furthermore, L-cysteic acid can also be produced by oxidation of L-cysteine ​​sulfinic acid. The known processes for the chemical production of L-cysteic acid, which are not considered sustainable, use environmentally hazardous chemicals and are poorly accepted by consumers, particularly for applications in the food, cosmetics, and pharmaceutical sectors. Therefore, there is a need for a more environmentally friendly and sustainable production process, for which a biotechnological process is a suitable option.

[0002] L-Cysteic acid is a non-proteinogenic L-amino acid that can be found naturally as an oxidation product of the proteinogenic amino acid L-cysteine, for example, in sheep's wool. Cysteic acid is also an intermediate in the biosynthesis of coenzyme M (CoM, 2-mercaptoethanesulfonic acid, CAS 3375-50-6) in methanogenic archaebacteria.

[0003] The state of the art provides processes for the production of non-proteinogenic amino acids, e.g., by direct fermentation of microorganisms deregulated in cysteine ​​metabolism (EP 1 191 106 B1) or by OAS sulfhydrylase-catalyzed biotransformation of OAS (EP 1 247 869 B1). These processes are based on an OAS sulfhydrylase catalyzing the reaction of OAS with a nucleophile to form a non-proteinogenic amino acid according to the general formula (1): (1) OAS + nucleophile -> non-proteinogenic amino acid + acetate

[0004] OAS is used in cysteine ​​metabolism, e.g. Escherichia colias a biosynthetic precursor of L-cysteine. The latter is formed by substitution of the acetate group at the beta position with a thiol residue. This reaction, known as beta-substitution, is catalyzed by enzymes of the OAS sulfhydrylase class (EC 4.2.99.8). Thus, OAS is the actual substrate (also referred to as the reactant) of the OAS sulfhydrylase reaction, and the nucleophile is the variable cosubstrate.

[0005] In EP 1 247 869 B1, a variety of different nucleophiles were investigated for their suitability as nucleophiles for the OAS sulfhydrylase (e.g., CysM)-catalyzed reaction with OAS, including selenides, selenol, azides, cyanides, azoles, and isoxazolinones. In addition, sulfur compounds from the group of thiosulfates and thiols of the general formula HSR, where the radical R was a monovalent substituted or unsubstituted alkyl, alkoxy, aryl, or heteroaryl radical, were also investigated.

[0006] Non-proteinogenic amino acids such as S-phenyl-L-cysteine, which are not used naturally as building blocks for protein biosynthesis, were produced. None of the disclosed nucleophiles enables the production of L-cysteine ​​acid.

[0007] Joo et al. (2018), J. Agric. Food Chem. 66: 13454 - 13463, describe a Metabolic Engineering Approach to the production of taurine in the bacterium Corynebacterium glutamicum.In the strain, the genes for L-cysteine ​​acid synthase, cysteine ​​dioxygenase, and L-cysteine ​​sulfinic acid decarboxylase were heterologously expressed to achieve taurine production. Figure 2 of Joo et al. (2018), J. Agric. Food Chem. 66: 13454–13463, describes various metabolic pathways to taurine, including a pathway ("L-cysteine ​​sulfonic acid pathway") that, starting from O-phospho-L-serine, leads to taurine via L-cysteine ​​acid and, in principle, would also be suitable for the production of L-cysteine ​​acid. However, the figure also shows that no known biosynthetic pathway leads from OAS to L-cysteine ​​acid, but only to L-cysteine.

[0008] Tevatia et al., Algal Research (2015) 9: 21-26, describe the natural production of taurine in microalgae, where L-cysteic acid was also detected as an intermediate. As described in Fig. 1a) of Tevatia et al., Algal Research (2015) 9: 21-26, a biosynthetic pathway leads from L-serine to L-cysteic acid ("cysteate" in Fig. 1a). None of the described biosynthetic pathways leads to L-cysteic acid via OAS. The intracellular L-cysteic acid content detected in microalgae was very low and accompanied by several byproducts such as methionine, cysteine, cysteine ​​sulfinic acid, hypotaurine, and taurine that make purification difficult, thus making microalgae cultivation unsuitable for the production of L-cysteic acid.

[0009] US 2019 / 0062757 A1 (KnipBio) describes in a Metabolic EngineeringApproach heterologous production strains for the production of taurine, whereby these strains are also said to be suitable for the production of L-cysteic acid. Figures 4 to 9 and 12 of US 2019 / 0062757 A1 describe various biosynthesis pathways to taurine that contain L-cysteic acid as an intermediate and would therefore, in principle, be suitable for the production of L-cysteic acid. None of these biosynthesis pathways originates from OAS. Furthermore, only yields for hypotaurine and taurine were given, which were very low at a maximum of 419 ng / ml. Yields for the production of L-cysteic acid were not mentioned. It must be assumed that higher yields cannot be achieved for L-cysteic acid. Metabolic Engineering This approach is therefore not suitable for the biotechnological production of L-cysteine ​​acid.

[0010] Ono et al. (Free Radical Biology and Medicine 106, pp. 69-79, 2017) revealed that the OAS sulfhydrylases CysM and CysK from Salmonella entericumLT2 analogous to the enzymes from E. coli (see Maier, Nature Biotechnology 21, pp. 422-427, 2003, Table 1) are able to form cysteine ​​from the substrates OAS and sulfide in high yields of over 70%. Ono further reveals that CysM and CysK are Salmonella entericum LT2 can also form the cysteine ​​derivative L-cysteine ​​acid (cysteine ​​sulfonate, salt of cysteic acid) from OAS in the presence of Na 2 SO 3 under otherwise identical reaction conditions in small amounts, ie in a molar yield of less than 0.2%.

[0011] The state of the art therefore discloses only chemical processes and no economical biotechnological process for the production of L-cysteic acid that would be suitable for industrial use.

[0012] The object of the present invention was to provide a biotechnological process for the production of L-cysteic acid by biotransformation.

[0013] The problem was solved by a process for the production of L-cysteic acid, wherein O-acetyl-L-serine (OAS) is reacted with at least one enzyme selected from the class of O-acetyl-L-serine sulfhydrylases (OAS sulfhydrylases, EC 4.2.99.8) in the presence of a salt of sulfurous acid, wherein the OAS sulfhydrylase is CysM and the biotransformation is carried out under active pH control and the OAS concentration in the mixture is at least 10 g / L.

[0014] This process provides L-cysteine ​​acid produced by biotransformation.

[0015] The advantage of the process according to the invention is that it is a sustainable and technically feasible biotransformation process for the production of L-cysteic acid. Environmentally hazardous chemicals can be dispensed with. No raw materials of fossil origin are consumed and no toxic chemical waste and / or exhaust gases are generated. The production process of the present invention is therefore environmentally friendly and sustainable. Furthermore, the process requires neither extreme reaction conditions nor special equipment and is therefore easily implemented industrially. A further advantage of the process is that natural L-cysteic acid can be produced in this way, which is in increasing demand. It has surprisingly been found that salts of sulfurous acid (hereinafter referred to as sulfites or SO 3 2-<) are suitable as nucleophiles in reaction (1) and enable the synthesis of L-cysteic acid in a reaction according to equation (2).(2) OAS + SO 3 2-< -> L-cysteic acid + acetate.

[0016] In the context of the present invention, manufacturing processes are distinguished as follows: 1. Chemical processes 2. Biotechnological processes: a) by metabolic engineering Metabolic Engineering (also “Pathwaydesign.” In contrast to biotransformation, biotransformation is a method of biotechnology in which the metabolic pathways of an organism are modified by optimizing or modifying genetic and regulatory processes. By supplementing the genome with enzyme genes, new or modified enzymes can be introduced into an organism, or genes of endogenous enzymes can be expressed in an increased or reduced form, thereby establishing new metabolic pathways in an organism or strengthening or weakening existing metabolic pathways. Metabolic Engineeringis that the organism produces a metabolite either newly or a cellular metabolite with increased yield. Metabolic Engineering-Process does not use any starting materials specific to the metabolite, such as an enzyme substrate, such as OAS in the present invention, but merely a nutrient medium, also referred to as a culture medium, which is required for the growth of the organism in question and is composed of a C source (e.g. glucose), an N source (e.g. an ammonium salt or a complex amino acid mixture such as peptone or yeast extract) and other salts required for growth. Such nutrient media are known to the person skilled in the art from microbiological practice. b) by biotransformation Biotransformation is defined as the conversion of one or more reactants into a product under enzymatic catalysis, wherein the enzyme substrate is added to a reaction mixture with the enzyme.In the reaction mixture, the added enzyme substrate, such as OAS in the present invention, is enzymatically converted (in the present invention by CysM, an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of a salt of sulfurous acid), according to equation (2). The starting material(s) can originate from chemical or biotechnological production. The OAS used in the process according to the invention can originate, for example, from chemical synthesis or from biotechnological production by fermentation of a production strain. The enzyme used for the enzymatic catalysis originates either from biotechnological production by cultivating a production strain, e.g. by fermentation, or biological material containing the enzyme is used (e.g. plants, fungi, algae, animal organs). The biomass can originate from the cultivation of the production strain orThe biological material can be used directly, or the enzyme can be isolated from it, depending on the requirements of the biotransformation. The CysM enzyme used in the process according to the invention is obtained biotechnologically by fermentation of a production strain.

[0017] A natural production process is defined as a biotechnological production method that does not use genetically modified organisms (GMOs) or products (educts, enzymes) derived from GMOs. In the present invention, a natural production process for L-cysteic acid exists if OAS as the organic reactant and the OAS sulfhydrylase CysM were not produced using GMOs and were not produced chemically. Sulfite as the cosubstrate in equation (2) is an inorganic compound and essentially the product of the dissolution of SO2 in water, according to equations (4) to (8), which does not correspond to an (irreversible) chemical synthesis, but rather to the reversible hydration of the gas SO2 and the pH-dependent dissociation of the hydrate H2SO3.

[0018] According to a statement (ref.: 6790-10-02 of 1991) of the Central Commission for Biological Safety (ZKBS), self-cloning within the meaning of Section 3 No. 3 Sentence 4 of the Genetic Engineering Act (GenTG) is a process in which genetically identical or different forms of only one species, including its viruses and plasmids, serve as donor and recipient organisms.

[0019] In the context of the present invention, a reaction mixture is defined as a mixture of reactant (starting material), enzyme and optionally other reactants, in which the reactant is converted into a product.

[0020] The yield of the reaction within the meaning of the invention is defined as the amount of reactant used that is converted to the product under the reaction conditions. The yield can be expressed as an absolute amount (g or mmol), as a volume yield (concentration) in absolute, volume-related amounts of product (mM or g / L), or as a relative yield of product as a percentage of the reactant used (taking into account the molecular weights of the reactant and the product), also referred to as the percentage yield.

[0021] Fermentation is a process step for the production (cultivation) of cell cultures on an industrial scale, in which a preferably microbial production strain is induced to grow under defined conditions of culture medium, temperature, pH, oxygen supply, and medium mixing. The goal of fermentation, depending on the configuration (genetic makeup) of the production strain, is the production of a protein / enzyme or a metabolite, each with the highest possible yield for further use. The components of the process according to the invention, OAS and OAS sulfhydrylase CysM, can be produced by fermentation.The end product of fermentation is a fermenter broth consisting of the biomass of the cells of the production strain (fermenter cells) and the fermentation medium (fermentation supernatant) freed of biomass, which is formed during the fermentation from the culture medium and the metabolic products secreted by the fermenter cells. The target products of fermentation can be found in the fermenter cells or in the fermentation medium. For example, OAS is found in the fermentation medium, while the enzyme OAS sulfhydrylase is found in the fermenter cells.

[0022] The open reading frame (ORF, synonymous with cds, coding sequence) is the region of DNA or RNA that begins with a start codon and ends with a stop codon and encodes the amino acid sequence of a protein. The ORF is also called the coding region or structural gene.

[0023] A gene is the DNA segment that contains all the basic information needed to produce biologically active RNA. A gene contains the DNA segment from which a single-stranded RNA copy is produced through transcription, as well as the expression signals involved in regulating this copying process. Expression signals include, for example, at least a promoter, a transcription start site, a translation start site, and a ribosome binding site (RBS). Further expression signals can include a terminator and one or more operators.

[0024] A gene construct is a DNA molecule in which a gene is linked to other genetic elements (e.g., promoter, terminator, selection marker, origin of replication). A gene construct within the scope of the invention is a circular DNA molecule and is referred to as a plasmid, vector, or expression vector. The genetic elements of the gene construct cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the gene.

[0025] L-cysteic acid from the inventive biotransformation of OAS with a salt of sulfurous acid can either be used directly without further processing steps or enriched or purified using known methods. The degree of enrichment depends on the subsequent use. Such methods are known to the person skilled in the art from processes for isolating amino acids. They include, for example, filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0026] In a preferred embodiment, the process is characterized in that L-cysteic acid is extracted from the reaction mixture. Separation of the particulate biomass, e.g., by centrifugation, is particularly preferred.

[0027] In a further preferred embodiment, the process is characterized in that the L-cysteic acid produced in the process according to the invention is used directly, ie the reaction mixture containing L-cysteic acid is used without further processing, purification, or isolation steps such as filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization. OAS sulfhydrylases have been isolated from a wide variety of plants and microorganisms. E. coliFor example, two OAS sulfhydrylase enzymes exist, designated CysK and CysM. The corresponding genes are also known and are designated cysK and cysM, respectively. CysM OAS sulfhydrylases within the meaning of the present invention are characterized by the fact that they can catalyze the synthesis of the proteinogenic amino acid L-cysteine ​​from OAS according to equation (3), with sulfide serving as the nucleophile in this case. (3) OAS + S 2-< -> L-cysteine ​​+ acetate

[0028] Although both enzymes have a very similar reaction mechanism and are involved in the biosynthesis of L-cysteine, CysM, in contrast to CysK, has a variable substrate spectrum with respect to the nucleophile, which can react with OAS according to equation (1).

[0029] For example, CysM, unlike CysK, is known to catalyze the reaction of OAS with thiosulfate to form S-sulfocysteine ​​(CAS number 1637-71-4). This reaction plays an important role in the growth of bacteria with thiosulfate as the sole sulfur source.

[0030] Furthermore, the use of CysM for the production of non-proteinogenic amino acids is known from EP 1 247 869 B1 (Wacker).

[0031] Preferably, the method is characterized in that the OAS sulfhydrylase CysM is a bacterial enzyme, particularly preferably CysM of the strain E. coli is.

[0032] Sulfurous acid forms a multitude of chemical species coexisting in reversible equilibria, the respective suitability of which as nucleophiles in the biotransformation according to the invention was not foreseeable. Thus, it is known that sulfurous acid (H 2 SO 3 ) is the aqueous solution of gaseous SO 2 and, as a dibasic acid, exists in different equilibria depending on the pH of the aqueous solution, the species of which also have different suitability as nucleophiles. The following equilibria (4) to (8) are known: (4) SO 2 (gaseous) <-> SO 2 (dissolved) (5) SO 2 (dissolved) + H 2 O <-> H 2 SO 3 (6) H 2 SO 3 <-> HSO 3 -< + H +< (7) HSO 3 -< <-> SO 3 2-< + H +< (8) 2 HSO 3 -< <-> S 2 O 5 2-< + H 2 O

[0033] Sulfurous acid and its salts are used as preservatives in the food industry because of their antimicrobial effects. This means that sulfurous acid and its salts can kill microorganisms by inactivating the enzymes necessary for the viability of the microorganism. Therefore, the skilled person would expect that the CysM enzyme would also be inactivated when using sulfurous acid or its salts, and that L-cysteic acid would not be accessible using the process disclosed in EP 1 247 869 B1.

[0034] For the reasons mentioned, it was surprising to the person skilled in the art that L-cysteic acid can be produced in a biotransformation using sulfite and OAS. In principle, all conceivable salts of sulfurous acid are suitable for the reaction. The process is preferably characterized in that Na 2 SO 3 , K 2 SO 3 , (NH 4 ) 2 SO 3 , NaHSO 3 (or its anhydride Na 2 S 2 O 5 ) or KHSO 3 is used as the salt of a sulfurous acid. Particular preference is given to using Na 2 SO 3 , NaHSO 3 (or its anhydride Na 2 S 2 O 5 ) and (NH 4 ) 2 SO 3 and especially preferably Na 2 SO 3 and NaHSO 3 (or its anhydride Na 2 S 2 O 5 ) as the salt of a sulfurous acid.

[0035] It is conceivable to use gaseous sulfur dioxide, the anhydride of sulfurous acid, which can be introduced into the reaction mixture, where it hydrates to the sulfurous acid H 2 SO 3 and, depending on the pH, is in equilibrium with the deprotonated forms HSO 3 -< and SO 3 2-<.

[0036] A prerequisite for the process is the availability of OAS. Possible methods for producing OAS include chemical processes, e.g., by acetylation of L-serine, which is expensive due to the high price of L-serine, or the production of the racemate O-acetyl-D / L-serine, which can be used directly, or OAS is obtained beforehand from the racemate, e.g., by racemate resolution. During direct acetylation, N-acetyl-L-serine (NAS) can be formed as a by-product, e.g., by non-selective acetylation at the hydroxy or amino group of L-serine or the well-known rearrangement of OAS to NAS at neutral to alkaline pH values ​​(Tai et al. (1995), Biochemistry 34: 12311-12322), which reduces yields or requires the prior introduction of a protecting group at the amino group of L-serine. For this reason, direct acetylation of L-serine is not practical for an economical process.

[0037] The biotechnological production of OAS is also known, as disclosed, for example, in EP 1 233 067 B1. This involves the use of organisms that exhibit deregulated cysteine ​​metabolism and therefore produce high levels of OAS. This provides cost-effective production systems for OAS production.

[0038] In a preferred embodiment, the process is characterized in that OAS originates from fermentative production. Fermentative production can be carried out using both GMOs and non-GMO organisms.

[0039] In a particularly preferred embodiment, the process is characterized in that OAS is produced fermentatively with the aid of microorganisms which are not GMOs, wherein it is particularly preferred that OAS is produced fermentatively with the aid of the strain E. coliW3110 / pACYCcysEX-GAPDH-ORF306. The latter particularly preferred embodiment is disclosed in Example 1.

[0040] The process of the present invention for producing L-cysteic acid is preferably characterized by being a natural production process. This means that not only are no GMOs used in the process, but both the starting material OAS and the enzyme OAS sulfhydrylase are naturally produced, i.e., they are neither produced with GMOs nor chemically.

[0041] This particularly preferred embodiment is disclosed in the examples of the invention, which describe a natural production process for L-cysteic acid in which both OAS and the OAS sulfhydrylase CysM are naturally produced. Both the OAS-producing strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) as well as the CysM production strain E. coliDH5α / pFL145 (Example 2) originate from self-cloning and are not classified as GMOs. The fact that both OAS and OAS sulfhydrylase can be produced without the use of GMOs is a particular advantage of the invention, as it discloses a natural production process for L-cysteic acid, which is now attracting great interest due to its potential applications in animal feed and cosmetics.

[0042] The skilled person can use isotope analysis to determine whether a substance, such as OAS, that they intend to use as a starting material in the process originates from chemical or fermentative production. An isotope analysis method suitable for distinguishing between the two is described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527 and is based on determining the isotope ratios for, for example, C or N, which differ depending on whether a product originates from chemical (petroleum-based) or fermentative (plant-based raw materials) production.

[0043] An advantage of the present invention is that an OAS-containing fermenter broth, such as that obtained from a fermentation conducted according to EP 1 233 067, can be used directly in the process according to the invention as an OAS source after separation of the particulate biomass, e.g., by centrifugation, without further processing, purification, or isolation steps such as extraction, adsorption, ion exchange chromatography, precipitation, or crystallization. This procedure is particularly economical and avoids the isolation of an unstable compound.

[0044] A fermentative process for the production of OAS is disclosed in EP 1 233 067 B1 and described in Example 1 of the present invention, wherein the strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 is used. This strain is deposited under the Budapest Treaty with the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig) under number DSM 13495.

[0045] The process is preferably characterized in that the OAS sulfhydrylase CysM originates from fermentative production, particularly preferably is produced fermentatively with the aid of microorganisms which are not GMOs, and particularly preferably with the aid of a E. coli tribe, including in particular preferably with the help of the tribe E. coli DH5α / pFL145 is produced.

[0046] A fermentative biotechnological production of CysM with the strain E. coli DH5α / pFL145 is disclosed in Example 2. The production strain consists of a host strain, as in this case E. coliDH5α and a gene construct suitable for the expression of OAS sulfhydrylase, preferably the gene construct pFL145. The host strain and gene construct, as well as the preparation of the production strain, are described in EP 1 247 869 B1 (Wacker). The production strain is deposited under the Budapest Treaty at the DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Brauschweig) under the number DSM 14088.

[0047] The OAS sulfhydrylase CysM obtained by fermentation can be used in the process according to the invention either as fermenter broth that has not been further processed or as a cell suspension after re-isolation of the cells from the fermenter broth, e.g. by centrifugation. Furthermore, the OAS sulfhydrylase can be used in the form of a cell homogenate after mechanical disruption of the cell suspension or in the form of chemically permeabilized cells (e.g. by chloroform) or as a cell extract after separation of particulate components from the cell homogenate or as e.g. chromatographically purified enzyme. Preference is given to using the OAS sulfhydrylase as fermenter broth that has not been further processed, as a cell suspension after re-isolation of the cells from the fermenter broth or as a cell homogenate after mechanical disruption of the cell suspension or in the form of chemically permeabilized cells (e.g. by chloroform).

[0048] It is particularly preferred to use the OAS sulfhydrylase as a cell suspension after re-isolation of the cells from the fermenter broth or as a cell homogenate.

[0049] In a particularly preferred embodiment, the process is characterized in that the cells of the production strain isolated and resuspended from the fermenter broth are used as OAS sulfhydrylase.

[0050] In a particularly preferred embodiment, the process for producing L-cysteic acid is characterized in that both the OAS sulfhydrylase and OAS are produced by fermentation.

[0051] OAS, the starting material in the biotransformation process according to the invention, isomerizes to N-acetyl-L-serine at pH values ​​above approximately 7 and is then no longer suitable for reaction with sulfite to form L-cysteic acid. The mechanism of the reaction was investigated in Tai et al. (1995), Biochemistry 34: 12311-12322 and is based on an intramolecular, nucleophilic attack of the deprotonated amino group at the carbonyl carbon of the acyl residue. This reaction is suppressed with decreasing pH, so that the compound is stable, for example, at pH 4.0.

[0052] The biotransformation process according to the invention is thus characterized in that the reaction of OAS to L-cysteic acid is carried out under pH conditions which minimize the isomerization of OAS to N-acetyl-L-serine.

[0053] Preferably, the process is characterized in that the reaction is carried out at a pH value which is at least 5.5 and ≤7.5, particularly preferably ≤7.0 and especially preferably ≤6.5.

[0054] In a further preferred embodiment of the biotransformation process, the substrate OAS is metered into the reaction mixture of OAS sulfhydrylase and sulfite in a so-called feed process (Example 5). In this case, a pH of ≤6.5, more preferably ≤6.0, and especially preferably ≤5.5 is preferably adjusted in the added OAS, which suppresses isomerization to N-acetyl-L-serine. At the same time, the pH in the reaction mixture is adjusted to favor the reaction to L-cysteic acid. According to equation (2), acetic acid is released in stoichiometric amounts during the reaction of OAS to L-cysteic acid, which can lead to a reduction in the pH of the mixture during the course of the reaction. Since a pH that is too low impairs the activity of OAS sulfhydrylase, an excessive drop in the pH must be prevented.This can be done passively by adding a suitable highly concentrated buffer to the mixture or, according to the invention, actively by using a measuring and control unit.

[0055] According to the invention, the active pH control is carried out by a measuring and control unit, as disclosed in Example 5, which, in case of a deviation of the pH value from the target value, resets the desired pH value by adding an alkali or acid (so-called pH-stat method).

[0056] The reaction temperature is preferably chosen between 5°C and 70°C. A reaction temperature between 10°C and 60°C is preferred, particularly preferably between 15°C and 50°C, and especially preferably between 20°C and 40°C.

[0057] The process for producing L-cysteic acid is preferably carried out in an aqueous environment, i.e. water is preferably used as the solvent for the reaction.

[0058] The process according to the invention for producing L-cysteic acid can be operated discontinuously or continuously. In discontinuous operation (batch operation), all reactants are added to the batch during the reaction, and the batch is processed after completion of the reaction. In continuous operation, OAS, OAS sulfhydrylase, and a salt of sulfurous acid are continuously added during the reaction, and a solution containing the product L-cysteic acid is simultaneously withdrawn from the batch. A steady state is established in which the reactants are added such that they can react to form the product L-cysteic acid during the residence time in the reaction vessel. A process for the continuous production of unnatural amino acids is disclosed, for example, in EP 1 247 869 B1 (Wacker).

[0059] The process according to the invention for producing L-cysteic acid is preferably carried out in a discontinuous manner.

[0060] Preferably, the process is characterized in that the concentration of the salt of sulfurous acid is at least in equimolar concentration, particularly preferably in at least 1.5-fold molar excess, particularly preferably in at least 2-fold molar excess and furthermore preferably in at least 5-fold molar excess to OAS.

[0061] The OAS concentration in the mixture is 10 g / L and preferably at least 40 g / L.

[0062] In the biotransformation of OAS, the molar yield of L-cysteic acid based on the molar amount of OAS used is preferably at least 60%, particularly preferably at least 70% and especially preferably at least 80%.

[0063] The invention is further illustrated by the following examples: Example 1: Production of OAS

[0064] The strain disclosed in EP 1 233 067 B1 (Wacker) was used E. coliW3110 / pACYC-cysEX-GAPDH-ORF306, deposited under the Budapest Treaty with the DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Braunschweig) under the number DSM 13495. OAS was produced by fermentation as described in EP 1 233 067 B1. At the end of fermentation, a pH of 4.5 was adjusted with 21% (v / v) phosphoric acid to stabilize OAS. The cells were separated by centrifugation for 10 min at 4000 rpm (Heraeus Megafuge 1.0 R). The OAS content in the fermentation supernatant, determined by HPLC, was 15.3 g / L.

[0065] HPLC analysis of OAS and L-cysteic acid: For the quantitative determination of the compounds analyzed in the examples, an HPLC method calibrated for OAS and L-cysteic acid was used. All reference substances used for calibration were commercially available (Sigma-Aldrich). An Agilent HPLC instrument, Model 1260 Infinity II, was used. It was equipped with a pre-column derivatization with o-phthalaldehyde (OPA derivatization) from the same manufacturer, a process known from the analysis of amino acids. The HPLC instrument was equipped with a fluorescence detector for the detection of the OPA-derivatized products OAS and L-cysteic acid. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. Furthermore, an Accucore™< aQ column from Thermo Scientific™<, length 100 mM, inner diameter 4.6 mm, particle size 2.6 µm, heated to 40°C in the column oven was used.

[0066] Mobile phase A: 25 mM Na phosphate, pH 6.0. Mobile phase B: methanol. The separation was performed in gradient mode: 0–25 min, 10% mobile phase B to 60% mobile phase B, followed by 2 min of 60% mobile phase B to 100% mobile phase B, followed by another 2 min of 100% mobile phase B, at a flow rate of 0.5 ml / min. Retention time of L-cysteic acid: 3.2 min. Retention time of OAS: 17.0 min. Example 2: Production of the enzyme CysM

[0067] The strain disclosed in EP 1 247 869 B1 (Wacker) was used E. coli DH5α / pFL145, deposited under the Budapest Treaty at the DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Brauschweig) under the number DSM 14088. CysM enzyme was produced by both shake flask cultivation and fermentation. A) Cultivation in shake flask: From the strain E. coliA preculture of DH5α / pFL145 was established in LBamp medium (10 g / L tryptone (GIBCO™), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 100 mg / L ampicillin (Sigma-Aldrich)) and grown overnight at 37°C and 120 rpm. A 25 ml preculture was used as the inoculum for a main culture of 250 ml LBamp medium (1 L baffled Erlenmeyer flask). The main culture was shaken at 30°C and 110 rpm. After 4 h, a cell density OD 600 of 1.0 / ml was reached (OD 600 : photometric determination of the cell density / ml cell suspension by determining the absorbance at 600 nm; Genesys™< 10S UV-Vis spectrophotometer from Thermo Scientific™< ). The inducer tetracycline (Sigma-Aldrich, 3 mg / L final concentration) was then added, and cultivation continued for another 20 h at 30°C and 110 rpm. After completion of cultivation, the cell density OD 600 was 3 / ml. B) Fermentative production of CysM with the strain E. coliDH5α / pFL145 is disclosed in EP 1 247 869 B1. The cells from the fermentation were separated by centrifugation at 4000 rpm for 10 min (Heraeus Megafuge 1.0 R) and suspended in KPi6.5 buffer (0.1 M potassium phosphate, pH 6.5) so that the cell density was OD 600 90 / ml.

[0068] Cells from shake flask culture or fermentation were isolated for further use by centrifugation (10 min, 15,000 rpm, Sorvall Centrifuge RC5C equipped with an SS34 rotor). For further use to prepare a cell homogenate, as described below, the cell pellet was resuspended in KPi6.5 buffer as a cell suspension. To prepare the cell suspension, sufficient KPi6.5 buffer was used to achieve a cell density of OD 600 of 30 / ml: for example, 50 ml of cells from shake flask culture with an OD 600 of 3 / ml were centrifuged and resuspended in 5 ml of KPi6.5 buffer (10-fold concentration), or 1 ml of cells from fermentation with an OD 600 of 90 / ml were resuspended in 3 ml of KPi6.5 buffer (3-fold dilution).

[0069] In this way, the cells of the strain isolated and resuspended from the fermenter broth E. coliDH5α / pFL145, which were subsequently used as OAS sulfhydrylase CysM in the process according to the invention.

[0070] The FastPrep-24™<5G cell homogenizer from MP Biomedicals was used to prepare a cell homogenate. 1 ml of cell suspension in KPi6.5 buffer with a cell density of OD 600 30 / ml was disrupted in 1.5 ml tubes pre-filled by the manufacturer with glass beads ("Lysing Matrix B") (3 × 20 sec at a shaking frequency of 6000 rpm with a 30-sec break between each interval). The resulting cell homogenate was used directly as OAS sulfhydrylase (CysM enzyme) in the method according to the invention or used to prepare a cell extract.

[0071] To prepare a cell extract, the resulting cell homogenate was centrifuged (10 min 15000 rpm, Sorvall centrifuge RC5C, equipped with an SS34 rotor) and the supernatant was labeled cell extract and used as OAS sulfhydrylase (CysM enzyme) in the method according to the invention or further used to determine the CysM enzyme activity.

[0072] The protein content of the cell extract was determined using a Qubit 3.0 fluorometer from Thermo Fisher Scientific using the Qubit® Protein Assay Kit according to the manufacturer's instructions. The protein content of the cell extract from the shake flask culture was 5.3 mg / ml. The protein content of the cell extract from the fermentation was 4.0 mg / ml.

[0073] CysM enzyme activity was determined as described in EP 1 247 869 B1 (Wacker). For this purpose, OAS (Sigma-Aldrich) was used in the presence of Na 2 S and cell extract from the culture of the strain E. coliDH5α / pFL145 was incubated at 37°C. The assay mixture (0.4 ml final volume) in KPi6.5 buffer contained 10 mM OAS (added from a 200 mM stock solution in 500 mM sodium succinate buffer pH 5.5), 10 mM sodium sulfide (Na 2 S), and 5 µl of cell extract containing CysM. The cysteine ​​produced in the CysM reaction was determined with ninhydrin (Sigma-Aldrich) according to Gaitonde (1967), Biochem. J. 104: 627-633. The CysM enzyme activity in the cell extract from the culture of the strain E. coliThe DH5α / pFL145 concentration in the shake flask was 57.1 U / ml. Since the cells from the shake flask culture (OD 600 of 3 / ml) were concentrated 10-fold to prepare the cell extract, the enzyme activity in the cells from the shake flask culture was 5.7 U / ml. The CysM enzyme activity in the cell extract after fermentation of the E. coli DH5α / pFL145 strain was 58.1 U / ml. Since the cells from the fermentation (OD 600 of 90 / ml) were diluted to an OD 600 of 30 / ml to prepare the cell extract, the enzyme activity in the concentrated (OD 600 of 90 / ml) cell suspension of the fermenter cells was 174.4 U / ml.

[0074] The specific CysM enzyme activity of the cell extract from the culture of the strain E. coli DH5α / pFL145 in the shake flask was 10.8 U / mg protein. The specific CysM enzyme activity of the cell extract after fermentation of the strain E. coliDH5α / pFL145 was 14.5 U / mg. Assuming that CysM activity was completely released from the cells during cell extract preparation, the CysM enzyme activity determined in the cell extracts was equated with the enzyme activity contained in CysM cell suspensions in the following examples.

[0075] 1 U / ml CysM enzyme activity is defined as the production of 1 µmol cysteine / min from OAS and Na 2 S under test conditions in 1 ml of cell extract (volume activity). The specific CysM enzyme activity in U / mg protein is obtained by dividing the volume activity of the cell extract (U / ml) by the protein concentration of the cell extract (mg / ml) and is defined as the CysM enzyme activity in U per 1 mg of protein in the cell extract. Example 3: Production of L-cysteic acid from commercially available OAS and Na 2 SO 3 using CysM produced in shake flask culture

[0076] Two approaches were carried out in parallel: Step 1:8.25 ml of NaPi6.5 buffer (50 mM Na phosphate, pH 6.5) were placed in a 100 ml Erlenmeyer flask, and 1 ml of a 0.2 M solution of Na 2 SO 3 in NaPi6.5 buffer, 0.4 ml of CysM cell extract from the shake flask culture (from Example 2A) with an activity of 57.1 U / ml (2.3 U / ml final concentration in the mixture), and 350 µl of a 0.2 M solution of OAS x HCl (Sigma-Aldrich) in 0.5 M Na succinate, pH 5.5, were added successively. The mixture volume was 10 ml. Step 2: The mixture (comparison mixture without Na 2 SO 3 ) had the same composition as mixture 1. Instead of the Na 2 SO 3 solution, mixture 2 contained 1 ml of NaPi6.5 buffer.

[0077] Both batches were incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 1 h and 3 h, 1 ml of each batch was incubated for 5 min at 80°C to stop the reaction, centrifuged, and the supernatant analyzed by HPLC. The amount of L-cysteic acid detected by HPLC is shown in Table 1. Table 1: Amount of L-cysteic acid detected by HPLC as a function of reaction time using commercially available OAS, Na 2 SO 3 and a CysM-containing cell extract. Time [h] Answer 1 with Na 2 SO 3 L-cysteine ​​acid [mg / L] Ansatz 2 without Na 2 SO 3 L-Cysteinic acid [mg / L] 0 0, 0 0,0 1 78, 0 0,0 3 95, 8 0,0 Example 4: Production of L-cysteic acid from OAS-containing fermentation supernatant and Na 2 SO 3 using CysM produced in shake flask culture

[0078] In a 100 ml Erlenmeyer flask, 1 ml of cell culture supernatant from the fermentation of the strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 with an OAS content of 15.3 g / L (from Example 1) was added, and 6 ml of NaPi6.5 buffer, 1 ml of a 1 M solution of Na2SO3 in NaPi6.5 buffer, and 2 ml of CysM cell suspension from the shake flask culture (from Example 2A, cell density OD600 30 / ml; 57.1 U / ml CysM enzyme activity) were added successively. The reaction volume was 10 ml. The CysM enzyme activity in the reaction was 11.4 U / ml. The reaction was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 2 h, 1 ml of the reaction mixture was incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed by HPLC for OAS and L-cysteic acid content. The time course of the reaction is summarized in Table 2. Table 2: Amount of L-cysteic acid and OAS detected by HPLC using an OAS-containing cell culture supernatant, Na 2 SO 3 and a CysM-containing cell suspension. Time [h] OAS [mg / L] L-Cysteinic acid [mg / L] 0 1530, 0 0, 0 2 0, 0 1473,3 Example 5: Preparative production of L-cysteine ​​acid by biotransformation of OAS at constant pH

[0079] A double-walled 0.5 L thermostattable glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and heated to 37°C. 50 ml of CysM-containing cell suspension in KPi6.5 buffer (OD 600 90 / ml, 8720 U CysM enzyme activity) from the fermentation of strain DH5α / pFL145 (from Example 2B) and 6.6 ml of a 400 g / L solution of Na2S2O5 (13.9 mmol, molecular weight 190.1 g / mol) in KPi6.5 buffer were added. In dissolved form, this corresponded to 27.8 mmol of NaHSO3 (1.78-fold molar excess over the 15.6 mmol of OAS added later). The mixture was stirred with a magnetic stirrer. The mixture was further equipped with a pH electrode (Mettler Toledo) connected to a pH control unit (Titrator TitroLine alpha, Schott) operated in pH-stat mode according to the manufacturer's instructions.Under pH-stat conditions, the pH in the reaction vessel was kept constant at the set pH 6.5 throughout the reaction run by adding 2 M NaOH from a burette connected to the control unit. 150 ml of OAS-containing cell culture supernatant (OAS content 15.3 g / L, 2.3 g; 15.64 mmol) from the fermentation of strain . E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) was added to the mixture from a receiver via a pump (Watson Marlow peristaltic pump 101U / R) at a flow rate of 0.35 ml / min.

[0080] The reaction time was 19 h. Since the reaction was carried out in an open reaction vessel, the reaction volume was 185 ml due to evaporation after the reaction. At 0.5 h, 3 h, and 19 h after the start of the reaction, 1 ml aliquots of the reaction were taken, and the L-cysteic acid content was analyzed by HPLC. The time course of the formation of L-cysteic acid is summarized in Table 3. After 19 h of reaction, the L-cysteic acid content in the reaction was 12,970 mg / L (76.65 mM), which, with a reaction volume of 185 ml, corresponded to an absolute molar yield of 14.18 mmol of L-cysteic acid. Based on the amount of OAS used (15.64 mmol), this corresponded to a yield of 90.1%. Table 3: Amount of L-cysteine ​​acid detected by HPLC as a function of reaction time using an OAS-containing fermentation supernatant, NaHSO 3 and a cell suspension of CysM-containing fermenter cells Time [h] L-Cysteinic acid [mg / L] L-Cysteinic acid [mM] 0,5 758, 0 4,47 3 4244, 0 25,08 19 12970, 0 76, 65

Claims

1. Process for producing L-cysteic acid, wherein O-acetyl-L-serine (OAS) is converted using at least one enzyme selected from the class of O-acetyl-L-serine sulfhydrylases (OAS sulfhydrylases, EC 4.2.99.8) in the presence of a salt of sulfurous acid, wherein the OAS sulfhydrylase is CysM and the biotransformation is carried out under active pH control and the OAS concentration in the batch is at least 10 g / L.

2. Process according to Claim 1, characterized in that the OAS sulfhydrylase is a bacterial enzyme.

3. Process according to one or both of Claims 1 and 2, characterized in that the OAS sulfhydrylase is CysM from the strain E. coli.

4. Process according to one or more of Claims 1 to 3, characterized in that the OAS sulfhydrylase stems from fermentative production.

5. Process according to one or more of Claims 1 to 4, characterized in that the OAS sulfhydrylase is produced fermentatively with the aid of microorganisms that are not genetically modified organisms (GMOs).

6. Process according to one or more of Claims 1 to 5, characterized in that the OAS sulfhydrylase is produced fermentatively with the aid of the strain E. coli DH5α / pFL145.

7. Process according to one or more of Claims 1 to 6, characterized in that OAS stems from fermentative production.

8. Process according to one or more of Claims 1 to 7, characterized in that OAS is produced fermentatively with the aid of microorganisms that are not GMOs.

9. Process according to one or more of Claims 1 to 8, characterized in that OAS is produced fermentatively with the aid of the strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306.

10. Process according to one or more of Claims 1 to 9, characterized in that the process is a natural production process, a natural production process being defined by the fact that no GMOs are used in the process and the reactant OAS and the enzyme OAS sulfhydrylase stem from natural production, i.e., are not produced using GMOs and are not produced chemically.

11. Process according to one or more of Claims 1 to 10, characterized in that the salt of a sulfurous acid used is Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 or its anhydride Na2S2O5 or KHSO3.

12. Process according to one or more of Claims 1 to 11, characterized in that the concentration of the salt of sulfurous acid is at least in equimolar concentration to OAS.

13. Process according to one or more of Claims 1 to 12, characterized in that the reaction is carried out at a pH of at least 5.5 and of less than 7.5.

14. Process according to one or more of Claims 1 to 13, characterized in that L-cysteic acid is enriched from the reaction batch.