Method for producing L-cysteic acid

Abstract

The present invention relates to a method for producing L-cysteic acid, in which 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 sulfite. This economical method for preparing L-cysteic acid is environmentally friendly, sustainable and technically easy to implement. Another advantage of this method is that it allows the preparation of natural L-cysteic acid, which is increasingly in demand.

Description

[Technical Field]

[0001] The present invention relates to a method 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 sulfhydrase (OAS sulfhydrase, EC4.2.99.8) in the presence of a sulfite, and L-cysteic acid is obtained as a result of this in vivo conversion. [Background technology]

[0002] L-cysteic acid can be used, for example, in fish farming (Nakamura et al., Fisheries Science (2021) 87:353-363) or in the cosmetics field (US4053630), such as as an ingredient in Regu(R)-Slim(DSM) for skincare. In peptide chemistry, L-cysteic acid is used as a water-soluble protecting group. Furthermore, L-cysteic acid can be converted to taurine by decarboxylation.

[0003] L-cysteic acid ((R)-2-amino-3-sulfopropanoic acid, 3-sulfo-L-alanine, CAS 498-40-8) can be produced chemically, for example, by oxidation of cysteine ​​with chlorine in an alcohol solution (Tao et al., Amino Acids (2004), 27:149-151), by oxidation of cystine 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-cysteinesulfinic acid. Known methods for the chemical production of L-cysteic acid are not considered sustainable and use environmentally harmful chemicals, making them unacceptable to consumers, particularly in the food, cosmetic, and pharmaceutical sectors. Therefore, there is a need for more environmentally friendly and sustainable production methods, one option being biotechnology.

[0004] L-cysteic acid is a non-proteinogenic L-amino acid that can be found in nature, for example in sheep's wool, as an oxidation product of the proteinogenic amino acid L-cysteine. Cysteic acid is also an intermediate in the biosynthesis of the coenzyme M (CoM, 2-mercaptoethanesulfonic acid, CAS 3375-50-6) by methanogenic archaea.

[0005] Prior art provides methods for producing non-proteinogenic amino acids, for example, by direct fermentation of microorganisms with deregulated cysteine ​​metabolism (EP1191106B1) or by the biocatalytic conversion of OAS by OAS sulfhydrilase (EP1247869B1). These methods are based on the principle that, according to general formula (1), OAS sulfhydrilase catalyzes the reaction between OAS and a nucleophile to form non-proteinogenic amino acids.

[0006] (1) OAS + nucleophile -> non-proteinogenic amino acid + acetate In cysteine ​​metabolism in Escherichia coli and other bacteria, OAS acts as a precursor for L-cysteine ​​biosynthesis. The latter is formed by substituting the β-position acetate group with a thiol group. This reaction, called beta substitution, is catalyzed by enzymes of the OAS sulfhydrilase class (EC 4.2.99.8). Therefore, OAS is the actual substrate (also called the reactant) of the OAS sulfhydrilase reaction, and the nucleophile is a variable auxiliary substrate.

[0007] In EP1247869B1, numerous different nucleophiles, including selenides, selenols, azides, cyanides, azoles, and isoxazolinones, were tested for their suitability as nucleophiles in OAS sulfhydrylase-catalyzed (e.g., CysM catalyst) reactions with OAS. Furthermore, sulfur compounds from the group of thiosulfates and thiols of the general formula HSR were tested, where the group R is a monovalent substituted or unsubstituted alkyl, alkoxy, aryl, or heteroaryl group.

[0008] The compounds produced were non-proteinogenic amino acids such as S-phenyl-L-cysteine, which are not used in nature as building blocks for protein biosynthesis. None of the disclosed nucleophiles enable the production of L-cysteic acid.

[0009] Joo et al. (2018), J.Agric.Food Chem. 66:13454-13463 describes a metabolic engineering approach for taurine production in the bacterium Corynebacterium glutamicum. To achieve taurine production, the genes for L-cysteine ​​synthase, cysteine ​​dioxygenase, and L-cysteine ​​sulfinate decarboxylase were heterologously expressed in this strain. Figure 2 of Joo et al. (2018), J.Agric.Food Chem. 66:13454-13463 describes various metabolic pathways to taurine, including a pathway that starts from O-phospho-L-serine and yields taurine via L-cysteine ​​("L-cysteine ​​sulfonate pathway"), which is also suitable in principle for the production of L-cysteine. However, the figure also shows that there is no known biosynthetic pathway from OAS to L-cysteic acid and then to L-cysteine ​​alone.

[0010] Tevatia et al., Algal Research (2015) 9:21-26, described the spontaneous production of taurine in microalgae, and L-cysteic acid was also detected as an intermediate. As described in Figure 1a) of Tevatia et al., Algal Research (2015) 9:21-26, the biosynthetic pathway leads from L-serine to L-cysteic acid ("cyste" in Figure 1a). None of the described biosynthetic pathways result in L-cysteic acid via OAS. The intracellular content of L-cysteic acid detected in microalgae was very low and accompanied by several by-products that made post-processing more difficult, such as methionine, cysteine, cysteinesulfinic acid, hypotaurine, and taurine, so the growth of microalgae is not suitable for the production of L-cysteic acid.

[0011] In the metabolic engineering approach, US2019 / 0062757A1 (KnipBio) describes a heterologous production strain for taurine production, which is also intended to be suitable for the production of L-cysteic acid. Figures 4 to 9 and Figure 12 of US2019 / 0062757A1 describe various biosynthetic pathways to taurine containing L-cysteic acid as an intermediate, and thus are suitable in principle for the production of L-cysteic acid. None of these biosynthetic pathways start from OAS. Furthermore, only the yields of hypotaurine and taurine were reported, which were very low, up to 419 ng / ml. No mention is made of the yield for the production of L-cysteic acid. It has to be assumed that a higher yield cannot be achieved for L-cysteic acid. Therefore, this metabolic engineering approach is not suitable for the biotechnological production of L-cysteic acid.

[0012] Ono et al. (Free Radical Biology and Medicine 106, pp. 69-79, 2017) have disclosed that OAS sulfhydrases CysM and CysK derived from Salmonella entericum LT2 can form cysteine ​​from the substrate OAS and sulfides in high yields exceeding 70%, similar to enzymes derived from Escherichia coli (Maier, Nature Biotechnology 21, pp. 422-427, 2003, see Table 1). Ono further discloses that CysM and CysK derived from Salmonella entericum LT2 can form cysteine ​​from Na 2 SO 3 In the presence of [substance name], under otherwise identical reaction conditions, it is disclosed that cysteine ​​derivatives L-cysteic acid (cysteine ​​sulfonate, cysteine ​​salt) can be further formed from OAS in low amounts, i.e., in a molar yield of less than 0.2%.

[0013] Therefore, the prior art only discloses chemical methods and does not disclose an economically feasible biotechnology method for producing L-cysteic acid suitable for industrial use.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0015]

Non-Patent Document 1

[0016] An object of the present invention is to provide a biotechnology method for producing L-cysteic acid by in vivo conversion. [Means for Solving the Problems]

[0017] This object is achieved by a method for producing L-cysteic acid, in which O-acetyl-L-serine (OAS) is converted using at least one enzyme selected from the class of O-acetyl-L-serine sulfhydrylase (OAS sulfhydrylase, EC 4.2.99.8) in the presence of sulfite. This method provides L-cysteic acid produced by in vivo conversion.

[0018] The advantage of the method according to the present invention is that it is a sustainable and technically feasible biotransformation method for producing L-cysteic acid. It eliminates the use of environmentally harmful chemicals. No fossil fuels are consumed, and no toxic chemical waste and / or exhaust gases are generated. Therefore, the production method of the present invention is environmentally friendly and sustainable. Furthermore, this method is technically easy to implement as it does not require any extreme reaction conditions or special equipment. Another advantage of this method is that it allows for the production of naturally occurring L-cysteic acid, which is in increasing demand.

[0019] Surprisingly, sulfites (hereinafter referred to as sulfites or SO3) 2- It was found that (called) is suitable as a nucleophile in reaction (1), and enables the synthesis of L-cysteic acid in a previously unknown reaction according to formula (2).

[0020] (2) OAS+SO3 2- -> L-cysteic acid + acetate [Modes for carrying out the invention]

[0021] In the context of the present invention, the manufacturing methods are distinguished as follows:

[0022] 1.Chemical method 2. Biotechnological methods: a) By metabolic engineering Metabolic engineering (also known as "pathway design") is a biotechnology method in which, in contrast to biotransformation, an organism's metabolic pathways are modified through the optimization or alteration of genes and regulatory processes. Novel or modified enzymes can be introduced into an organism by supplementing the genome with the enzyme's gene, or by expressing the gene for an endogenous enzyme at an enhanced or attenuated level, thereby establishing a new metabolic pathway in the organism or enhancing or attenuating an existing one. The goal of metabolic engineering is for the organism to produce either a new metabolite or an endogenous metabolite in high yield. Metabolic engineering methods do not use enzyme substrates, such as metabolite-specific starting materials, such as OAS in this invention. Instead, they use only nutrient media, also called growth media, which are necessary for the growth of the organism in question and consist of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts or complex amino acid mixtures such as peptone or yeast extract), and other salts necessary for growth. Such nutrient media are known to those skilled in the art from microbiological practice.

[0023] b) By biotransformation Bioconversion is defined as the conversion of one or more reactants to a product under enzyme catalysis, and the enzyme substrate is added to the reaction batch along with the enzyme. In the reaction batch, the added enzyme substrate, for example, OAS of the present invention, is converted enzymatically according to formula (2) (in the present invention, by an enzyme selected from the class of OAS sulfhydrase (EC4.2.99.8) in the presence of sulfite). The reactants may be derived from chemical or biotechnological production. The OAS used in the method according to the present invention can be obtained, for example, from chemical synthesis or biotechnological production by fermentation of a production strain. The enzyme used in the enzyme catalysis is derived from the growth of a production strain, for example from biotechnological production by fermentation, or biological materials containing the enzyme are used (e.g., plants, fungi, algae, animal organs). Biomass or biological materials from the growth of a production strain can be used directly, or the enzyme is isolated therefrom depending on the requirements of bioconversion. The CysM enzyme used in the method according to the present invention is derived from biotechnological production by fermentation of a production strain.

[0024] A natural production method is defined as a biotechnology production method that does not use genetically modified organisms (GMOs) or products (reactants, enzymes) from production using GMOs. In this invention, the method for producing L-cysteic acid is a natural production method when OAS and OAS sulfhydrase as organic reactants are not produced using GMOs and are not produced chemically. The sulfite as an auxiliary substrate in formula (2) is an inorganic compound, basically a product obtained by dissolving SO2 in water according to formulas (4) to (8), which does not correspond to (irreversible) chemical synthesis, but corresponds to the reversible hydration of gaseous SO2 and the pH-dependent dissociation of the hydrate H2SO3.

[0025] Self-cloning in the sense of Section 3, Sentence 4 of §3 of the German Genetic Engineering Code (Gentechnikgesetz, GenTG) is a method in which only one species of genetically identical or different forms, including its virus and plasmid, is used as the donor and recipient organisms, according to a statement issued by the Central Commission for Biological Safety (ZKBS, German Central Committee for Biological Safety) (Reference: 6790-10-02, 1991).

[0026] In the context of the present invention, a reaction batch is defined as a mixture of reactants (starting materials), an enzyme, and optionally other reactants, the reactants being converted into a product.

[0027] In the sense of the present invention, the yield of a reaction is defined as the amount of reactants used that are converted into a product under the reaction conditions. The yield can be expressed as an absolute amount (g or mmol), as a volume yield (concentration) as the absolute amount of product per unit volume (mM or g / L), or as a relative yield of the product as a percentage of the reactants used (taking into account the molecular weights of the reactants and products), also known as a yield percentage.

[0028] Fermentation is a method and process for the industrial-scale production (cultivation) of cell cultures, preferably involving the growth of a microbial strain under specified conditions of culture medium, temperature, pH, oxygen supply, and medium mixture. Depending on the composition (genetic composition) of the production strain, the purpose of fermentation is to produce proteins / enzymes or metabolites in the highest possible yield for further use. The process components according to the present invention, OAS and OAS sulfhydrilase, can be produced by fermentation. The final product of fermentation is fermenter broth, consisting of the biomass of the production strain cells (fermenter cells) and the fermentation medium (fermentation supernatant) formed from metabolites removed from the biomass and secreted by the growth medium and fermenter cells during fermentation. The target product of fermentation can be present in the fermenter cells or the fermentation medium. For example, OAS is found in the fermentation medium, and the enzyme OAS sulfhydrilase is found in the fermenter cells.

[0029] An open reading frame (ORF, cds, or coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon, encoding the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0030] A gene refers to a portion of DNA that contains all the basic information necessary to produce biologically active RNA. A gene includes the portion of DNA from which a single-stranded RNA copy is generated by transcription, and expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, transcription start, translation start, and ribosome-binding sites (RBS). Terminators and one or more operators are additional possible expression signals.

[0031] A gene construct refers to a DNA molecule in which a gene is linked to other genetic elements (e.g., promoters, terminators, selection markers, origins of replication). In the context of this invention, a gene construct is a circular DNA molecule and is called a plasmid, vector, or expression vector. The genetic elements of a gene construct induce extrachromosomal inheritance during cell growth, leading to the production of the protein encoded by the gene.

[0032] L-cysteic acid obtained from the biotransformation of OAS with sulfites according to the present invention can be used directly without further post-treatment steps, or it can be concentrated or purified by known methods. The degree of concentration here depends on further use. Such methods are known to those skilled in the art from methods for isolating amino acids. Examples include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0033] In a preferred embodiment, the method concentrates L-cysteic acid from the reaction batch. For example, removal of particulate biomass by centrifugation is particularly preferred.

[0034] In a more preferred embodiment, the method further uses the L-cysteic acid produced by the method according to the present invention directly, i.e., the reaction batch containing L-cysteic acid is further used without further post-treatment, purification, or isolation steps including filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0035] OAS sulfhydrases have been isolated from a wide variety of plants and microorganisms. For example, Escherichia coli (E. coli) contains two OAS sulfhydrase enzymes, known as CysK and CysM. The associated genes are also publicly known and are called cysK and cysM, respectively.

[0036] Within the scope of the present invention, OAS sulfhydrases can catalyze the synthesis of the proteogenic amino acid L-cysteine ​​from OAS according to formula (3), where the nucleophile used is a sulfide. Therefore, both CysM-related enzymes and CysK-related enzymes are OAS sulfhydrases within the scope of the present invention.

[0037] (3) OAS+S 2- -> L-cysteine ​​+ acetate Both enzymes have very similar reaction mechanisms and are involved in the biosynthesis of L-cysteine. However, unlike CysK, CysM has a variable substrate spectrum with respect to nucleophiles that can react with OAS according to equation (1).

[0038] For example, it is known that, unlike CysK, CysM can catalyze the reaction of OAS with thiosulfate to form S-sulfocysteine (CAS number 1637-71-4). This reaction plays an important role in bacterial growth using thiosulfate as the sole sulfur source.

[0039] Furthermore, EP1247869B1 (Wacker) discloses the use of CysM for the production of non-proteinogenic amino acids.

[0040] Preferably, the OAS sulfhydrylase is a bacterial enzyme, particularly preferably CysM, and particularly preferably CysM derived from an E. coli strain.

[0041] Sulfurous acid forms a number of chemical species that coexist in a reversible equilibrium state, and its respective suitability as a nucleophile in the in vivo conversion according to the present invention could not be predicted. Therefore, sulfurous acid (H2SO3) is an aqueous solution of gaseous SO2 and, as a dibasic acid, exists in different equilibrium states depending on the pH of the aqueous solution, and it is known that the species in this equilibrium state also vary in their suitability as nucleophiles. The following equilibrium equations (4)-(8) are known.

[0042] (4) SO2 (gaseous) <-> SO2 (dissolved) (5) SO2 (dissolved) + H2O <-> H2SO3 (6) H2SO3 <-> HSO3 - + H + (7) HSO3 - <-> SO3 2- + H + (8) 2HSO3 - <-> S2O5 2- + H2O Sulfites and their salts are used as preservatives in the food industry because they exhibit antimicrobial effects. This means that sulfites and their salts can kill microorganisms, which is due to the inactivation of enzymes necessary for microbial survival. Therefore, those skilled in the art will expect that the CysM enzyme will also be inactivated when using sulfites or their salts, and that L-cysteic acid cannot be prepared by the method disclosed in EP1247869B1.

[0043] For the reasons mentioned above, it was surprising to those skilled in the art that L-cysteic acid could be produced by using sulfite and OAS in biotransformation.

[0044] In principle, all conceivable sulfites are suitable for the reaction. Preferably, the sulfite used in this method is Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its anhydrous form Na2S2O5), or KHSO3. Particularly preferred are Na2SO3, NaHSO3 (or its anhydrous form Na2S2O5), and (NH4)2SO3, with Na2SO3 and NaHSO3 (or its anhydrous form Na2S2O5) being particularly preferred.

[0045] It is conceivable to use gaseous sulfur dioxide, anhydrous sulfurous acid, which can be introduced into the reaction batch, hydrated to sulfurous acid H2SO3, and then deprotonated into HSO3 depending on the pH. - and SO3 2- It is in equilibrium.

[0046] This method requires the availability of OAS. For example, a chemical method for producing OAS by acetylation of L-serine, which is expensive due to the high price of L-serine, or the production of a racemic O-acetyl-D / L-serine that can be used directly, or the possibility that OAS can be obtained in advance from a racemic mixture, for example by resolution. In the case of direct acetylation, N-acetyl-L-serine (NAS) may be formed as a byproduct, for example, by non-selective acetylation of the hydroxyl or amino group of L-serine, or by a known rearrangement of OAS to NAS at neutral to alkaline pH values ​​(Tai et al. (1995), Biochemistry 34:12311-12322), which reduces the yield or requires prior introduction of a protecting group to the amino group of L-serine. Therefore, direct acetylation of L-serine is not practical as an economically viable method.

[0047] Biotechnological production of OAS is also known, as disclosed in EP1233067B1, for example. This involves the use of organisms that exhibit unregulated cysteine ​​metabolism and thus provide high levels of OAS. As a result, cost-effective production systems for producing OAS are available.

[0048] In a preferred embodiment, the method concentrates OAS from fermentation production. Fermentation production can be carried out using GMOs or non-GMO organisms.

[0049] In a particularly preferred embodiment, the method is characterized in that the OAS is produced fermentatively with the help of a non-GMO microorganism, in which case it is particularly preferred that the OAS is produced fermentatively with the help of Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306. The last specifically preferred embodiment is disclosed in Example 1.

[0050] The method for producing L-cysteic acid according to the present invention is preferably a natural production method. This means that not only are there no GMOs used in this method, but both the reactant OAS and the enzyme OAS sulfhydrase are derived from natural production, i.e., they are not produced using GMOs and are not produced chemically.

[0051] A particularly preferred embodiment is disclosed in the examples of the present invention, which describe a method for the natural production of L-cysteic acid in which both OAS and OAS sulfhydrilase CysM are naturally produced. The OAS-producing strain Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) and the CysM-producing strain Escherichia coli (E. coli) DH5α / pFL145 (Example 2) are both derived from autocloning and are not classified as GMOs. Since the present invention discloses a method for the natural production of L-cysteic acid, which is of great interest for possible applications in the supply sector and cosmetics, the fact that both OAS and OAS sulfhydrilase can be produced without the use of GMOs is a particular advantage of the present invention.

[0052] Those skilled in the art can use isotope analysis to determine whether a substance intended for use as a reactant in this method, such as OAS, originates from chemical production or fermentation production. Distinguishing isotope analysis methods are described, for example, by Sieper et al., Rapid Commun. Mass Spectrom (2006) 20:2521-2527, and are based on determining, for example, carbon or nitrogen isotope ratios, which vary depending on whether the product originates from chemical (petroleum-based) production or fermentation production (plant-based raw materials).

[0053] An advantage of the present invention is that OAS-containing fermenter broth, such as that obtained from fermentation carried out according to EP1233067B1, can be used directly as a source of OAS by the method according to the present invention after the removal of particulate biomass by centrifugation without further post-treatment, purification, or isolation steps, such as extraction, adsorption, ion exchange chromatography, precipitation, and crystallization. This procedure is particularly economical and avoids the isolation of unstable compounds.

[0054] A fermentation method for producing OAS, using the Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306, is disclosed in EP1233067B1 and Example 1 of the present invention. This strain is deposited with DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig) under the number DSM 13495 in accordance with the Budapest Convention.

[0055] This method preferably involves the production of OAS sulfhydrase (preferably containing CysM) fermentatively, derived from fermentation production, particularly preferably with the help of non-GMO microorganisms, and especially preferably with the help of Escherichia coli (E. coli) strains, including E. coli DH5α / pFL145.

[0056] Example 2 discloses a procedure for fermentation biotechnology production of CysM using the E. coli (DH5α) / pFL145 strain. The production strain consists of a host strain, in this case E. coli (DH5α), and a gene construct suitable for the expression of OAS sulfhydrase, preferably the gene construct pFL145. The host strain, gene construct, and production strain are described in EP1247869B1 (Wacker). This production strain is deposited with DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 14088 in accordance with the Budapest Convention.

[0057] In the method according to the present invention, the OAS sulfhydrase obtained by fermentation can be used as a fermenter broth without further post-treatment, or as a cell suspension after re-isolation of cells from the fermenter broth by, for example, centrifugation. Furthermore, the OAS sulfhydrase can be used in the form of cell homogenates after mechanical disruption of the cell suspension, or in the form of chemically permeable cells (e.g., chloroform), or as a cell extract after removing particulate components from the cell homogenates, or as an enzyme purified by, for example, chromatography.

[0058] The use of OAS sulfhydrase is preferred as a fermenter broth without further post-processing, as a cell suspension after re-isolation of cells from the fermenter broth, as a cell homogenate after mechanical disruption of the cell suspension, or in the form of chemically permeable cells (e.g., chloroform).

[0059] The use of OAS sulfhydrase as a cell suspension or cell homogenate after re-isolation of cells from fermenter broth is particularly preferred.

[0060] In a particularly preferred embodiment, cells of a production strain isolated and resuspended from fermenter broth are used as OAS sulfhydrase.

[0061] In a particularly preferred embodiment, the method for producing L-cysteic acid involves the generation of both OAS sulfhydrase and OAS by fermentation.

[0062] In the biotransformation method according to the present invention, OAS, used as a reactant, isomerizes to N-acetyl-L-serine at a pH of approximately 7, and subsequently becomes unsuitable for reaction with sulfite, forming L-cysteic acid. The reaction mechanism was studied by Tai et al. (1995), Biochemistry 34:12311-12322, and involves intramolecular nucleophilic attack by a deprotonated amino group on the carbonyl carbon of the acyl group. This reaction is inhibited as the pH decreases, so the compound is stable at, for example, pH 4.0.

[0063] Therefore, the bioconversion method according to the present invention is distinguished by the fact that the reaction of OAS to form L-cysteic acid is carried out under pH conditions that minimize the isomerization of OAS to N-acetyl-L-serine.

[0064] Preferably, the reaction is carried out at a pH value of at least 5.5, ≤7.5, particularly preferably ≤7.0, and most preferably ≤6.5.

[0065] In a more preferred embodiment of the bioconversion method, the substrate OAS is metered and supplied to a reaction batch consisting of OAS sulfhydrase and sulfite in a so-called supply step (Example 5). The metered and supplied OAS is set to a pH that inhibits isomerization to N-acetyl-L-serine, preferably pH ≤ 6.5, particularly preferably pH ≤ 6.0, and most preferably pH ≤ 5.5. At the same time, the pH in the reaction batch is adjusted to promote the reaction and form L-cysteic acid.

[0066] According to equation (2), the reaction of OAS to form L-cysteic acid releases a stoichiometric amount of acetic acid, which can lead to a decrease in the pH of the batch as the reaction progresses. Since excessively low pH affects the activity of OAS sulfhydrase, it is necessary to prevent an excessive decrease in pH. This can be done passively by providing an appropriate high-concentration buffer in the batch, or actively by a measurement and control unit.

[0067] As disclosed in Example 5, active pH control by a measurement and control unit is preferred, in which the desired pH is restored by measuring and adding an alkaline solution or acid when the pH deviates from the target value (so-called pH stat method).

[0068] The reaction temperature is preferably selected between 5 and 70°C. A reaction temperature of 10 to 60°C is preferred, 15 to 50°C is particularly preferred, and 20 to 40°C is particularly preferred.

[0069] The method for producing L-cysteic acid is preferably carried out in an aqueous environment, that is, the solvent used in the reaction is preferably water.

[0070] The method according to the present invention for producing L-cysteic acid can be carried out in discontinuous or continuous operation. In discontinuous operation (batch operation), all reactants are added to a batch during the reaction process, and the batch is post-processed after the reaction is complete. In continuous operation, OAS, OAS sulfhydrylase, and sulfite are continuously metered in during the reaction, and the solution containing the product L-cysteic acid is simultaneously removed from the batch. What is established is a steady state in which the reactants are metered and supplied so that they can react completely during the residence time in the reaction vessel to form the product L-cysteic acid. A method for the continuous production of non-natural amino acids is disclosed, for example, in EP1247869B1 (Wacker).

[0071] Discontinuous operation is preferred in the method according to the present invention for producing L-cysteic acid.

[0072] Preferably, in this method, the concentration of sulfite is at least equimolar relative to OAS, particularly preferably at least 1.5 times molar excess, particularly preferably at least 2 times molar excess, and even more preferably at least 5 times molar excess.

[0073] The OAS concentration in the batch is preferably at least 1 g / L, particularly preferably at least 10 g / L, and most preferably at least 40 g / L.

[0074] In the bioconversion 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 particularly preferably at least 80%.

[0075] The present invention will be further described by the following embodiments, but will not be limited by these embodiments. [Examples]

[0076] [ Example 1 ] OAS generation We used the Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306, disclosed in EP1233067B1 (Wacker) and deposited with DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 13495 in accordance with the Budapest Convention. OAS was produced by fermentation as described in EP1233067B1. At the end of fermentation, the OAS was stabilized by setting the pH to 4.5 using 21% (v / v) phosphate. Cells were removed by centrifugation at 4000 rpm for 10 minutes (Heraeus Megafuge 1.0 R). The HPLC-measured content of OAS in the fermentation supernatant was 15.3 g / L.

[0077] HPLC analysis of OAS and L-cysteic acid: For the quantitative analysis of the compounds analyzed in the examples, HPLC methods calibrated for OAS and L-cysteic acid were used, respectively. All reference substances used for calibration were commercially available (Sigma-Aldrich). As known from the analysis of amino acids, an Agilent 1260 Infinity II HPLC system with units from the same manufacturer was used for pre-column derivatization using o-phthalidaldehyde (OPA derivatization). For the detection of the OPA derivatization products OAS and L-cysteic acid, the HPLC system was equipped with a fluorescence detector. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. A Thermo Scientific® Accucore® aQ column (100 m in length) was thermally equilibrated at 40°C in a column oven. m (with an inner diameter of 4.6 mm and a particle size of 2.6 μm) was also used.

[0078] Eluent A: 25 mM sodium phosphate, pH 6.0. Eluent B: methanol. Separation was performed in gradient mode, from 10% eluent B to 60% eluent B over 0-25 minutes, followed by from 60% eluent B to 100% eluent B over 2 minutes, and then to 100% eluent B at a flow rate of 0.5 ml / min for another 2 minutes. Retention time for L-cysteic acid: 3.2 minutes. Retention time for OAS: 17.0 minutes.

[0079] [ Example 2 ] Manufacturing of the enzyme CysM We used the Escherichia coli (E. coli) strain DH5α / pFL145, disclosed in EP1247869B1 (Wacker) and deposited with DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 14088 in accordance with the Budapest Convention. The CysM enzyme was produced by both growth and fermentation in a shaking flask.

[0080] A) Growth in a shaking flask: A pre-culture of Escherichia coli (E. coli) strain DH5α / pFL145 was prepared 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)) (grow overnight at 37°C and 120 rpm). 25 ml of the pre-culture was used as inoculum for the main culture in 250 ml of LBamp medium (1 L Erlenmeyer flask with baffles). The main culture was shaken at 30°C and 110 rpm. After 4 hours, the cell density was 1.0 / ml (OD). 600 Reached (OD) 600 Photometric measurement of cell density per 1 ml of cell suspension by measuring absorbance at 600 nm (Genesys® 10S UV-Vis spectrophotometer, Thermo Scientific®). Subsequently, the inducer tetracycline (Sigma-Aldrich, final concentration 3 mg / L) was added, and proliferation was continued for a further 20 hours at 30°C and 110 rpm. At the end of proliferation, the cell density OD 600 The concentration was 3 / ml.

[0081] B) Fermentation production of CysM using Escherichia coli (E. coli) strain DH5α / pFL145 is disclosed in EP1247869B1. Cells from fermentation were removed by centrifugation at 4000 rpm for 10 minutes (Heraeus Megafuge 1.0 R), and the cell density OD was determined. 600 The solution was suspended in KPi6.5 buffer (0.1 MK phosphate, pH 6.5) to a concentration of 90 / ml.

[0082] Cells from shaking flask growth or fermentation were isolated for further use by centrifugation (15,000 rpm for 10 minutes, Sorvall RC5C centrifuge with SS34 rotor). For further use in the preparation of cell homogenates, the cell pellet was resuspended in KPi6.5 buffer as a cell suspension, as described below. The cell suspension was prepared with a cell density of OD. 600 It was prepared by using a sufficient amount of KPi6.5 buffer to achieve a concentration of 30 / ml. For example, 3 / ml of OD 600 50 ml of cells from a shaking flask growth system are centrifuged and resuspended in 5 ml of KPi6.5 buffer (10x concentration), or 90 / ml of OD. 600 1 ml of cells from fermentation containing [the specified substance] were resuspended in 3 ml of KPi6.5 buffer (3-fold dilution).

[0083] This resulted in the generation of cells of the E. coli (DH5α / pFL145) strain isolated from the fermenter broth and resuspended, which were used as OAS sulfhydrase CysM in the method according to the present invention.

[0084] To prepare the cell homogenates, we used the FastPrep-24® 5G cell homogenizer from MP Biomedicals. The cell density was 30 / ml. 600A 1 ml cell suspension in KPi6.5 buffer containing glass beads ("lysate matrix B") was disrupted in a 1.5 ml tube assembled by the manufacturer (shaking at a frequency of 6000 rpm for 3 × 20 seconds with a 30-second pause between each interval). The resulting cell homogenate was used directly as OAS sulfhydrylase (CysM enzyme) in the method according to the present invention, or used in the preparation of cell extracts.

[0085] To prepare the cell extract, the obtained cell homogenate was centrifuged (15,000 rpm for 10 minutes in a Sorvall RC5C centrifuge equipped with an SS34 rotor), and the supernatant was referred to as the cell extract, which was used as OAS sulfhydrylase (CysM enzyme) in the method according to the present invention, or further used for the measurement of CysM enzyme activity.

[0086] The protein content of cell extracts was determined using the "Qubit(R) Protein Assay Kit" according to the manufacturer's instructions and a Thermo Fisher Scientific Qubit 3.0 Fluorometer. The protein content of cell extracts from shaking flask growth was 5.3 mg / ml. The protein content of cell extracts from fermentation was 4.0 mg / ml.

[0087] CysM enzyme activity was determined as described in EP1247869B1 (Wacker). For this purpose, OAS (Sigma-Aldrich) was incubated at 37°C in the presence of Na2S and cell extracts from E. coli (DH5α / pFL145) growth. The assay in KPi6.5 buffer (final volume 0.4 ml) contained 10 mM OAS (added from a 200 mM stock solution in 500 mM sodium succinate buffer pH 5.5), 10 mM sodium sulfide (Na2S), and 5 μl of CysM-containing cell extract. The cysteine ​​produced in the CysM reaction was measured using ninhydrin (Sigma-Aldrich) according to the method of Gaitonde (1967), Biochem. J. 104:627-633. The CysM enzyme activity in cell extracts from E. coli (DH5α / pFL145) grown in a shaking flask was 57.1 U / ml. 600 Since the enzyme was concentrated 10-fold for the preparation of cell extracts, the enzyme activity in cells grown in shaking flasks was 5.7 U / ml. The CysM enzyme activity in cell extracts after fermentation of Escherichia coli (E. coli) strain DH5α / pFL145 was 58.1 U / ml. 600 ) is 30 / ml OD for the preparation of cell extracts 600 Since it was diluted, the fermenter cells were concentrated (90 / ml OD 600 The enzyme activity in the cell suspension was 174.4 U / ml.

[0088] The specific CysM enzyme activity of cell extracts from E. coli (DH5α / pFL145) strains grown in a shaking flask was 10.8 U per mg of protein. The specific CysM enzyme activity of cell extracts from E. coli (DH5α / pFL145) strains after fermentation was 14.5 U / mg. Assuming that CysM activity was completely released from the cells during the preparation of the cell extracts, the CysM enzyme activity measured in the cell extracts was equivalent to the enzyme activity present in the CysM cell suspensions in the following examples.

[0089] CysM enzyme activity at 1 U / ml is defined as the cysteine ​​production (volume activity) of 1 μmol / min from OAS and Na2S in 1 ml of cell extract under assay conditions. The specific CysM enzyme activity in U per 1 mg of protein is obtained by dividing the volume activity (U / ml) of the cell extract by the protein concentration (mg / ml) of the cell extract, and is defined as the CysM enzyme activity in U based on 1 mg of protein in the cell extract.

[0090] [ Example 3 ] Production of L-cysteic acid from commercially available OAS and Na2SO3 using CysM produced by shaking flask culture. We ran two batches in parallel: Batch 1: In a 100 ml Erlenmeyer flask, 8.25 ml of NaPi6.5 buffer (50 mM sodium phosphate, pH 6.5) was initially added. Then, 1 ml of 0.2 M solution of Na2SO3 in NaPi6.5 buffer, 0.4 ml of CysM cell extract from shaking flask growth (from Example 2A) with an activity of 57.1 U / ml (final concentration in the batch: 2.3 U / ml), and 350 μl of 0.2 M solution of OAS × HCl (Sigma-Aldrich) in 0.5 M sodium succinate, pH 5.5 were added sequentially. The batch volume was 10 ml.

[0091] Batch 2: Batch 2 (a comparison batch without Na2SO3) had the same composition as Batch 1. Instead of the Na2SO3 solution, 1 ml of NaPi6.5 buffer was added to Batch 2.

[0092] Both batches were incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 1 hour and 3 hours, 1 ml of each batch was incubated at 80°C for 5 minutes to stop the reaction, then centrifuged, and the supernatant was analyzed by HPLC. The amount of L-cysteic acid detected by HPLC is shown in Table 1.

[0093] Table 1: L-cysteic acid levels detected by HPLC based on reaction time using commercially available OAS, Na2SO3, and CysM-containing cell extracts.

[0094] [Table 1]

[0095] [ Example 4 ] Production of L-cysteic acid from OAS-containing culture supernatant and Na2SO3 using CysM produced by shaking flask culture. In a 100 ml Erlenmeyer flask, first load 1 ml of cell culture supernatant from the fermentation of Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306 (from Example 1) having an OAS content of 15.3 g / L, then add 6 ml of NaPi6.5 buffer, 1 ml of 1 M solution of Na2SO3 in NaPi6.5 buffer, and 2 ml of CysM cell suspension from shaking flask growth (from Example 2A, cell density OD 30 / ml). 600 CysM enzyme activity (57.1 U / ml) was added sequentially. The batch volume was 10 ml. The CysM enzyme activity in the batch was 11.4 U / ml. The batch was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 2 hours, a 1 ml batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC for OAS and L-cysteic acid content. The reaction course over time is summarized in Table 2.

[0096] Table 2: L-cysteic acid and OAS detected by HPLC using OAS-containing cell culture supernatant, Na2SO3, and CysM-containing cell suspension.

[0097] [Table 2]

[0098] [ Example 5 ] Preparation and production of L-cysteic acid by bioconversion of OAS at a constant pH. A 0.5L thermostatic double-walled glass container (Diehm) was connected to a thermostat (Lauda) via a hose connection and the temperature was adjusted to 37°C.

[0099] 50 ml of CysM-containing cell suspension in KPi6.5 buffer from fermentation of DH5α / pFL145 strain (from Example 2B) (90 / ml OD) 600 Initially, 6.6 ml (13.9 mmol, molecular weight 190.1 g / mol) of a 400 g / L solution of Na2S2O5 in KPi6.5 buffer (CysM enzyme activity 8720 U) was added. In dissolved form, this corresponded to 27.8 mmol of NaHSO3 (1.78 molar excess compared to the 15.6 mmol of OAS to be weighed later). The batch was stirred with a magnetic stirrer. The batch was also equipped with a pH electrode (Mettler Toledo), which was connected to a pH control unit (TitroLine alpha titrator, Schott) and 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 entire reaction by weighing and 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 Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) was metered and supplied to a batch from a reservoir via a pump (Watson Marlow 101U / R peristaltic pump) at a flow rate of 0.35 ml / min.

[0100] The reaction time was 19 hours. Since the batch was carried out in an open reaction vessel, the batch volume was 185 ml after the reaction was complete due to evaporation. At 0.5 hours, 3 hours, and 19 hours after the start of the reaction, 1 ml aliquots of the batch were removed, and the L-cysteic acid content was analyzed by HPLC. The formation of L-cysteic acid over time is summarized in Table 3. After 19 hours of reaction, the L-cysteic acid content in the batch was 12970 mg / L (76.65 mM), which corresponds to an absolute molar yield of 14.18 mmol of L-cysteic acid per 185 ml batch volume. Based on the use of 15.64 mmol of OAS, this corresponds to a yield of 90.1%.

[0101] Table 3: L-cysteic acid detected by HPLC based on reaction time using OAS-containing fermentation supernatant, NaHSO3, and cysM-containing fermenter cell suspensions.

[0102] [Table 3]

Claims

1. A method for producing L-cysteic acid, A method comprising converting O-acetyl-L-serine (OAS) in the presence of a sulfite using at least one enzyme selected from the class of O-acetyl-L-serine sulfhydrases (OAS sulfhydrase, EC 4.2.99.8), wherein the OAS sulfhydrase is CysM, the in vivo conversion is performed under active pH control, and the OAS concentration in the batch is at least 10 g / L.

2. The method according to claim 1, wherein OAS sulfhydrase is a bacterial enzyme.

3. The method according to claim 1 or 2, wherein the OAS sulfhydrase is CysM derived from the Escherichia coli (E. coli) strain.

4. The method according to any one of claims 1 to 3, wherein the OAS sulfhydrase is derived from fermentation production.

5. The method according to any one of claims 1 to 4, wherein OAS sulfhydrase is produced fermentatively with the help of a non-GMO microorganism.

6. The method according to any one of claims 1 to 5, wherein OAS sulfhydrase is fermentatively produced with the help of Escherichia coli (E. coli) strain DH5α / pFL145.

7. The method according to any one of claims 1 to 6, wherein the OAS is derived from fermentation production.

8. The method according to any one of claims 1 to 7, wherein OAS is produced fermentatively with the help of non-GMO microorganisms.

9. The method according to any one of claims 1 to 8, wherein OAS is produced fermentatively with the help of Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306.

10. The method according to any one of claims 1 to 9, wherein the method is a naturally occurring method, and a naturally occurring method is defined by the fact that no genetically modified organism (GMO) is used in the method, and the reactant OAS and the enzyme OAS sulfhydrase are naturally occurring, i.e., not produced using a GMO and not produced chemically.

11. The salt of sulfurous acid used is Na ２ SO ３ , K ２ SO ３ , (NH ４ ) ２ SO ３ , NaHSO₃ (or its anhydride Na ２ S ２ O ５ ) or KHSO ３ , The method according to any one of claims 1 to 10.

12. The method according to any one of claims 1 to 11, wherein the concentration of sulfite is at least equimolar relative to OAS.

13. The method according to any one of claims 1 to 12, wherein the reaction is carried out at a pH of at least 5.5 and less than 7.

5.

14. The method according to any one of claims 1 to 13, wherein L-cysteic acid is concentrated from the reaction batch.

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