Method for the enzymatic oxidation of sulfinic acids to sulfonic acids

Abstract

The present invention relates to a method for the enzymatic oxidation of sulfinic acids of formula HN-CH(R)-CH-SOH to sulfonic acids of formula HN-CH(R)-CH-SOH by means of an enzyme selected from the class of H0-generating oxidases in the presence of a substrate for said enzyme, in particular to a method for the enzymatic oxidation of L-cysteine ​​sulfinic acid to L-cysteic acid and hypotaurine to taurine.

Description

[Technical field]

[0001] The present invention relates to a method for the enzymatic oxidation of sulfinic acids of formula HN-CH(R)-CH-SOH to sulfonic acids of formula HN-CH(R)-CH-SOH by means of an enzyme selected from the class of H0-generating oxidases in the presence of a substrate for said enzyme, in particular to a method for the enzymatic oxidation of L-cysteine ​​sulfinic acid to L-cysteic acid and hypotaurine to taurine. [Background technology]

[0002] Sulfinic acids are a class of compounds containing organically bonded sulfur and oxygen of the general structure RS(=O)-OH, where R is an organic radical. The parent sulfinic acid has the structure HS(=O)-OH and is tautomeric to sulfoxylic acid HO-S-OH. The salts of sulfinic acids are sulfinates.

[0003] Sulfonic acids are organosulfur compounds with the general structure R-SO2-OH, where R is an organic radical. - and their salts and esters having R1-SO2-O-R2 (R1, R2 in each case being an organic group) are known as sulfonates.

[0004] Conversion of sulfinic acids to sulfonic acids is chemically possible, for example, by oxidation with peroxide (see, for example, Chauvin and Pratt, Angew. Chem. Int. Ed. (2017) 56: 6255-6259). The possibility of enzymatic oxidation of sulfinic acids to sulfonic acids, although not of interest in the chemical industry, is of interest for biotechnological processes to produce naturally occurring sulfonic acids from the corresponding sulfinic acids.

[0005] Examples of naturally occurring sulfinic acids include L-cysteine ​​sulfinic acid and hypotaurine. Examples of naturally occurring sulfonic acids include L-cysteic acid and taurine.

[0006] Taurine (2-aminoethanesulfonic acid, CAS number 107-35-7) is an aminosulfonic acid that occurs naturally in nature as a decomposition product of the amino acids cysteine ​​and methionine. Taurine is economically important; it is, for example, an ingredient in energy drinks, and is also used, for example, in pet food for cats, or in fish farming (Salze and Davis, Aquaculture (2015) 437: 215-229). However, taurine is also thought to have health-promoting effects (Ripps and Shen, Molecular Vision (2012) 18: 2673-2686).

[0007] Currently, commercial taurine is produced chemically. One known process, for example that of the Changshu Yudong Chemical Factory, starts with ethylene and then ethyleneimine to taurine. Due to the consumer-driven trend away from chemically produced ingredients, biotechnological processes for the production of taurine are increasingly being investigated.

[0008] In nature, taurine is found almost exclusively in the animal kingdom, with only a few examples in plants, algae or bacteria. There are various biosynthetic pathways leading to taurine (see, for example, KEGG Pathway Database: “Taurine and hypotaurine metabolism”), starting, among others, from L-cysteine. The most important synthetic steps leading to taurine from L-cysteine ​​are shown in formulas (1)-(5). (1) L-cysteine ​​+ O2 → L-cysteine ​​sulfinic acid (2) L-cysteine ​​sulfinic acid + 1 / 2O2 → L-cysteic acid (3) L-cysteine ​​sulfinic acid → hypotaurine + CO2 (4) Hypotaurine + 1 / 2O2 → Taurine (5) L-cysteic acid → taurine + CO2

[0009] (1): In the first step, L-cysteine ​​is oxidized to L-cysteine ​​sulfinic acid (3-sulfinoalanine, CAS number 207121-48-0) by the enzyme cysteine ​​dioxygenase (CDO, EC 1.13.11.20).

[0010] (3) and (5): Cysteine ​​sulfinic acid decarboxylase (CSAD, EC 4.1.1.29) decarboxylates L-cysteine ​​sulfinic acid to hypotaurine (2-aminoethanesulfinic acid, CAS number 300-84-5), and a similar reaction can decarboxylate L-cysteic acid to taurine.

[0011] (2) and (4): The oxidation of cysteine ​​sulfinic acid to cysteic acid and the oxidation of hypotaurine to taurine have not yet been clearly elucidated.

[0012] A disadvantage of known biotechnological processes for producing taurine is that hypotaurine is generally the main product, and when taurine is the product, it is necessary to use chemical processes for the oxidation of hypotaurine.

[0013] Honjoh et al. (2010), Amino Acids 38: 1173-1183, described a genetically engineered yeast strain heterologously expressing CDO and CSAD genes from common carp (Cyprinus carpio). The major product was hypotaurine, with a smaller proportion of taurine. Both products accumulated intracellularly. Thus, cell disruption was required for analysis of the products. Hypotaurine in the cell extracts could be chemically oxidized to taurine for analytical purposes by treatment with H2O2. No steps were described detailing how hypotaurine could be converted to taurine in a non-chemical process for further use.

[0014] WO17 / 213142A1 (Ajinomoto) describes a taurine-producing strain obtained by heterologous expression of cysteine ​​dioxygenase and L-cysteine ​​sulfinic acid decarboxylase in a strain that naturally produces cysteine. The main product was hypotaurine with a maximum yield of 450 μM, which could be converted to taurine only in low yield by subsequent chemical treatment with alkali.

[0015] US2019-0062757A1 (KnipBio) describes heterologous production strains for the production of taurine or its precursor substances, the disclosed production strains have a mostly heterogeneous production profile and achieve the highest yield of hypotaurine. Moreover, the achieved yields were very low, with a maximum of 419 ng / ml hypotaurine and undetectable taurine.

[0016] In addition to low yields, the biotechnological approaches to producing taurine disclosed in the prior art suffer from an inconsistent product spectrum, with hypotaurine occurring as the major product and taurine only as a by-product.

[0017] An approach to the enzymatic oxidation of hypotaurine was reported by Veeravalli et al. (2020), bioRxiv Preprint Server doi: https: / / doi.org / 10.1101 / 750273They explain that mammalian flavin-dependent monooxygenase 1 (FMO1) is the enzyme that converts hypotaurine to taurine. The oxidation proceeds according to equation (6): (6) Hypotaurine + NAD(P)H + O2 → taurine + NAD(P) + H2O.

[0018] It is unclear whether the mammalian enzyme FMO1 for converting hypotaurine to taurine according to equation (6) is also suitable for the oxidation of other sulfinic acids of the formula H2N-CH(R)-CH2-SO2H.

[0019] For the oxidation of hypotaurine, FMO1 uses the cofactors NADH or NADPH in stoichiometric amounts. The use of these commercially expensive cofactors makes their technical use uneconomical. Furthermore, for the FMO1 identified in mammals, no production process suitable for industrial production is known. Therefore, there is no economic justification for enzymatic oxidation using the FMO1 enzyme. Summary of the Invention [Problem to be solved by the invention]

[0020] It is therefore an object of the present invention to provide an economically advantageous biotechnological process for the oxidation of sulfinic acids to sulfonic acids, in particular L-cysteine ​​sulfinic acid to L-cysteic acid and hypotaurine to taurine. [Means for solving the problem]

[0021] The object is achieved by a method for enzymatically oxidizing a sulfinic acid of formula H2N-CH(R)-CH2-SO2H by an enzyme selected from the class of H2O2-generating oxidases in the presence of a substrate for said enzyme to a sulfonic acid of formula H2N-CH(R)-CH2-SO3. Preferably, the method of the invention is characterized in that the sulfinic acid is an aminoalkylsulfinic acid, more preferably a 2-aminoalkylsulfinic acid. Preferably, the method of the invention is characterized in that the sulfonic acid is an aminoalkylsulfonic acid, more preferably a 2-aminoalkylsulfonic acid. More preferably, the sulfinic acid is an aminoalkylsulfinic acid and the sulfonic acid is an aminoalkylsulfonic acid.

[0022] The group R can be any group, preferably hydrogen, an organic group, a linear group, a branched group, a cyclic group, a saturated or unsaturated group, an aromatic group or a heteroaromatic group, with or without a substituent. This means that the group R can be substituted or unsubstituted. Preferred substituents are -CN, -NCO, -NR2, -COOH, -COOR, -halogen, -(meth)acryloyl, -epoxy, -SH, -OH, -CONR2, -OR, -CO-R, -COO-R, -OCO-R or -OCOO-R, -SR, -NR-, -N=R, -N=NR or -P=R. Preferably, saturated or unsaturated groups with C1-C4 alkyl, more preferably C1-C4 alkyl, vinyl, in particular methyl or ethyl, especially methyl, are used. R is preferably selected from R=H or R=CO2H, i.e. the sulfinic acid is preferably hypotaurine or cysteine ​​sulfinic acid. It is particularly preferred that R=H.

[0023] The present invention therefore also relates to the enzymatic oxidation of L-cysteine ​​sulfinic acid to L-cysteic acid and to the enzymatic oxidation of hypotaurine to taurine as process steps in the biotechnological production of taurine.

[0024] A substrate for an enzyme selected from the class of H2O2-generating oxidases is understood to mean a substance that can be oxidized by an oxidase. The oxidase reacts with its oxidizable substrate according to equation (7). (7) Substrate + O2→ Substrate (ox) +H2O2

[0025] The overall reaction catalyzed by H2O2-generating oxidase proceeds according to equation (8): (8) Substrate+O2+H2N-CH(R)-CH2-SO2H → substrate (ox) +H2O2+H2N-CH(R)-CH2-SO2H → substrate (ox) +H2O+H2N-CH(R)-CH2-SO3H.

[0026] Suitable H2O2-generating oxidases can be found in the "KEGG Enzymes" database under the search term "oxidase" as a subset of the enzymes listed therein. However, among the many H2O2-generating oxidases, only those that use inexpensive substrates to generate H2O2 by oxidation of the substrate according to equation (7) have the potential for industrial application.

[0027] Examples of suitable H2O2 generating oxidases include, but are not limited to, glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase (EC 1.1.3.10), L-lactate oxidase (EC 1.1.3.2), aryl alcohol oxidase (EC 1.1.3.7), galactose oxidase (EC 1.1.3.9), L-sorbose oxidase (EC 1.1.3.10), and the like. oxidase (EC 1.1.3.11), aldehyde oxidase (EC 1.2.3.1), pyruvate oxidase (EC 1.2.3.3 or EC 1.2.3.6), oxalate oxidase (EC 1.2.3.4), glyoxylate oxidase (EC 1.2.3.5), L-amino acid oxidase (EC 1.4.3.2), D-amino acid oxidase (EC 1.4.3.3 or EC 1.4.3.1), sulfite oxidase (EC 1.8.3.1) and thiol oxidase (EC 1.8.3.2).

[0028] Particularly preferred H2O2 generating oxidases are glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase (EC 1.1.3.10) and L-lactate oxidase (EC 1.1.3.2).

[0029] Thus, preferably, the method of the invention is characterized in that the combination of the H2O2-generating oxidase and its oxidizable substrate is selected from glucose oxidase / glucose, alcohol oxidase / methanol, alcohol oxidase / ethanol, secondary alcohol oxidase / isopropanol and L-lactate oxidase / lactate.

[0030] In a preferred embodiment, the method of the invention is characterized in that the H2O2 generating oxidase is an alcohol oxidase and the substrate present is a primary alcohol, more preferably methanol, i.e. the H2O2 generating oxidase is an enzyme of the KEGG database class EC 1.1.3.13 designated as alcohol oxidase.

[0031] Alcohols are compounds of the general formula R-OH, which have one or more hydroxyl groups and do not contain other functional groups of greater importance. Alcohols are distinguished by the number of carbon and hydrogen atoms on the carbon atom of the functional group to which the hydroxyl group is attached. In the case of primary alcohols, in addition to the carbon atom, two hydrogen atoms are attached to said carbon atom, giving the general formula RCH2OH. Furthermore, primary alcohols can be converted to aldehydes by oxidation. For example, ethanol becomes the aldehyde ethanal by removing two hydrogen atoms.

[0032] Preferred primary alcohols include methanol and ethanol, and more preferably the primary alcohol is methanol.

[0033] R in the alcohol is an alkyl, alkenyl, or alkynyl group, but not an aryl, acyl, or heteroatom.

[0034] In another preferred embodiment, the method of the invention is characterized in that the H2O2 generating oxidase is glucose oxidase and the substrate present is glucose, i.e. the H2O2 generating oxidase is an enzyme of the class EC 1.1.3.4 of the KEGG database designated as glucose oxidase.

[0035] A prerequisite for carrying out this process industrially is the availability of an H2O2-generating oxidase and an inexpensive oxidizable substrate.

[0036] H2O2-generating oxidases can be produced, for example, by culturing a suitable production strain that naturally produces the oxidase concerned (homologous production) or as a result of the expression of a suitable recombinant gene construct in the host organism (heterologous production). Furthermore, various H2O2-generating oxidases are commercially available, which may positively affect the economics of the process. Examples of commercially available oxidases are the alcohol oxidase AOX from the yeast Pichia pastoris (available, for example, from Sigma-Aldrich) or the glucose oxidase GOX from the fungus Aspergillus niger (available, for example, from Sigma-Aldrich). Another example of a commercially available glucose oxidase is, for example, the enzyme commercially available under the trade name Gluzyme® (Novozymes), which is used in the bakery industry. The H2O2-generating oxidase is preferably produced by fermentation.

[0037] Furthermore, cheap availability of the substrate for the H2O2-generating oxidase is important for the economic viability of the process. In the case of AOX alcohol oxidase, the substrate is a primary alcohol, preferably selected from methanol or ethanol. The AOX enzyme produces H2O2 from methanol according to reaction (9): (9) Methanol + O2 → formaldehyde and H2O2.

[0038] In the case of glucose oxidase GOX, the substrate is D-glucose. The GOX enzyme produces H2O2 with glucose according to reaction (10): (10) D-glucose + O2 → D-glucono-1,5-lactone + H2O2.

[0039] For the purposes of this invention, a process is defined as a multi-step, consecutive operation in which reactants (starting materials) are converted into products in a defined order, via intermediates determined by the reaction conditions, in one or more successive reaction batches.

[0040] For the purposes of this invention, a biotechnological process is defined as the use of enzymes, cells or whole organisms in industrial applications for the production of compounds, such as the production of taurine from hypotaurine using glucose oxidase. In contrast to biotechnological processes, processes featuring chemical process steps are available.

[0041] A batch or reaction batch is defined as a mixture of reactants (starting materials), enzymes, and optionally other reactants, where the reactants are converted into products under defined conditions. The batch or reaction batch of the present invention comprises at least one sulfinic acid of formula H2N-CH(R)-CH2-SO2H, at least one enzyme selected from the class of H2O2-generating oxidases, a substrate oxidizable by the oxidase, and atmospheric oxygen O2.

[0042] Biotransformation is defined as the conversion of reactants to products under enzyme catalysis. The method of the present invention is a biotransformation.

[0043] The yield of the reaction can be expressed as a volumetric yield, the absolute amount of product per unit volume (mM or g / L), or as a relative yield of the product as a proportion of the reactants used (taking into account the molecular weights of the reactants and products), also called percent yield. In the context of the present invention, the molar yield expressed as a percentage refers to the total molar amount of sulfonic acid of formula H2N-CH(R)-CH2-SO3H after enzymatic oxidation by the H2O2-generating oxidase in the presence of its substrate, relative to the total molar amount of sulfinic acid of formula H2N-CH(R)-CH2-SO2 and H2N-CH(R)-CH2-SO3H used in the reaction.

[0044] Fermentation is a process step for the production of cultured cells in which a producer microbial strain is grown under defined conditions of medium, temperature, pH, oxygen supply and medium mixing. Depending on the composition of the producer strain, the aim of fermentation is in each case to produce the highest possible yield of proteins / enzymes and / or metabolic products for further use in the process.

[0045] Enzyme activity is expressed in U / ml, with 1 U / ml defined as the conversion of 1 μmol of substrate / min in a 1 ml test batch under the test conditions.

[0046] An open reading frame (ORF, synonymous with cds or coding sequence) is a region of DNA or RNA beginning with a start codon and ending with a stop codon that codes for the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0047] A gene or expression unit refers to a section of DNA that contains all the basic information to produce biologically active RNA. A gene includes a section of DNA that is transcribed to produce a single-stranded RNA copy, and expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, a transcription start point, a translation start point, and a ribosome binding site. Terminators and one or more operators are additional possible expression signals.

[0048] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for protein synthesis. mRNA provides assembly instructions for a specific protein in the cell. The mRNA molecule conveys the message required for protein synthesis from the genetic information (DNA) to the ribosomes responsible for protein synthesis. In the cell, mRNA molecules are formed as a transcript of a segment of DNA that corresponds to a gene. The genetic information stored in DNA remains unchanged by this process.

[0049] Eukaryotic genes are mostly known as mosaic genes and, unlike prokaryotic genes, also contain non-coding parts known as introns (intragenic regions). Coding sequences known as exons (expressed regions) are the portions of DNA of eukaryotic genes that are transcribed into RNA and then translated by ribosomes into the amino acid sequence of a protein. After transcription from DNA to RNA, introns are spliced ​​out of the primary transcript. The protein-coding RNA from which the introns have been removed is called messenger RNA (mRNA), or "mature" mRNA. It undergoes further modifications such as capping and polyadenylation. The coding region of the mature mRNA is then translated into a protein sequence. When eukaryotic genes containing exon / intron structures are expressed in prokaryotes, it is necessary to reverse translate the protein sequence or coding region of the mature mRNA into DNA without introns, since processing of the exon / intron structure does not take place in prokaryotes. In the context of the present invention, when referring to a protein sequence or a gene sequence derived from an mRNA, it is precisely this process of reverse translation that is meant. The optimisation of the sequence, ie the adaptation to the corresponding prokaryotic codon usage (codon optimisation), preferably takes place simultaneously with the back-translation of the mRNA sequence into a DNA sequence.

[0050] An operon is defined as a high-level expression unit in which multiple genes are transcribed under the control of a single promoter, but each is translated from its own ribosome binding site.

[0051] In the context of the present invention, a genetic construct is a circular DNA molecule (plasmid, vector) in which at least one gene is linked to further genetic elements (e.g., promoter, ribosome binding site (RBS), terminator, selection marker, transcription start point). The genetic elements of the genetic construct cause their extrachromosomal inheritance during cell proliferation to produce the protein encoded by the gene.

[0052] As disclosed in Examples 1 and 2 of the present invention, the H2O2-generating oxidase, in combination with its oxidizable substrate, is suitable for oxidizing sulfinic acids, such as L-cysteine ​​sulfinic acid and hypotaurine, to the corresponding sulfonic acids, L-cysteic acid and taurine, respectively. This observation was novel and surprising. According to the prior art, H2O2 is in principle suitable for the oxidation of sulfinic acids to sulfonic acids, although it has been mostly investigated only on an analytical scale (see, for example, Chauvin and Pratt, Angew. Chem. Int. Ed. (2017) 56: 6255-6259). However, for a quantitative reaction, H2O2 must be used in at least stoichiometric amounts according to equation (8), which means that for the production of relatively large amounts of sulfonic acids, a correspondingly large amount of H2O2 is required. Therefore, although H2O2-generating oxidases have been known for a long time, those skilled in the art could not predict which of the amounts of H2O2 produced by the alcohol oxidase reaction and the glucose oxidase reaction would be sufficient to oxidize a relatively large amount of sulfinic acid.

[0053] Thus, the method of the present invention has the advantage that it is not only a biotechnological method but also an economically favorable method since it can be carried out on an industrial scale and does not require costly cofactors.

[0054] As disclosed for the first time in Example 3 and as was not expected from the prior art, surprisingly, by using glucose oxidase / glucose, an almost quantitative oxidation of e.g. hypotaurine to taurine was achieved at a concentration suitable for chemical synthesis, i.e. 20 g / L. The present invention therefore provides a biotechnological process suitable for the ever-increasing demand for the sustainable production of sulfonic acids such as taurine for use in the food, animal feed or pharmaceutical sectors, while avoiding chemical synthesis steps.

[0055] Preferably, the method of the invention is characterized in that the sulfinic acid is 2-aminoethanesulfinic acid (hypotaurine) and the sulfonic acid formed is 2-aminoethanesulfonic acid (taurine).

[0056] The enzymatic process of the invention for oxidizing sulfinic acids to sulfonic acids is preferably carried out at temperatures between 15°C and 80°C, more preferably between 20°C and 60°C, and particularly preferably between 25°C and 50°C.

[0057] The pH at which the method of the present invention is carried out depends on the enzymatic properties of the H2O2-generating oxidase. As described in the Examples, the reaction of alcohol oxidase was carried out at pH 7.5, and the reaction of glucose oxidase was carried out at pH 5.5. The pH range in which the reaction is preferably carried out is pH 3.0 to pH 8.5, more preferably pH 4.0 to pH 8.0, and particularly preferably pH 4.5 to pH 7.5.

[0058] The method of the invention is carried out with atmospheric oxygen supplied by passive inflow, for example caused by mixing the reaction batch on an incubation shaker, or by active inflow, such as caused by the movement of compressed air.

[0059] Alongside parameters such as temperature, pH and oxygen inflow, the dosage of the H2O2-generating oxidase in the method of the invention determines the conversion of the reaction and is selected so that the reaction occurs in the shortest possible time with the lowest possible dosage of H2O2-generating oxidase. The dosage of the H2O2-generating oxidase, for example glucose oxidase, is preferably 1 U / ml to 200 U / ml, more preferably 2 U / ml to 150 U / ml, particularly preferably 4 U / ml to 100 U / ml.

[0060] The substrate oxidizable by the H2O2-generating oxidase is preferably used in the method of the invention in an amount at least equimolar relative to the molar content of sulfinic acid (the molar ratio of sulfinic acid to oxidizable substrate is preferably at least 1:1). Particularly preferred is a molar ratio of sulfinic acid to oxidizable substrate of at least 1:2, particularly preferred of at least 1:5.

[0061] The reaction time depends on the dosage of the H2O2-generating oxidase, and is preferably 72 hours or less, more preferably 48 hours or less, and particularly preferably 24 hours or less.

[0062] The solvent used in the reaction is preferably water.

[0063] The concentration of sulfinic acid in the batch is preferably at least 1 g / l, more preferably at least 10 g / l, particularly preferably at least 20 g / l.

[0064] The molar conversion of sulfinic acid to sulfonic acid in the biotransformation of the present invention is preferably at least 60%, more preferably at least 80%, and especially preferably at least 90%.

[0065] The process of the invention for the oxidation of sulfinic acids can be carried out discontinuously or continuously. In discontinuous operation (batch operation), all reactants are added to a batch during the course of the reaction, and the batch is worked up after the reaction is completed.

[0066] In a continuous operation, the oxidase enzyme is first loaded in the form of a stationary phase, for example in a membrane reactor or immobilized on a support, and the substrate as a mobile phase containing the sulfinic acid and the substrate oxidizable by the H2O2-generating oxidase is brought into contact with the stationary phase. The contact time between the mobile phase and the stationary phase is preferably set so that the sulfinic acid can react to form the sulfonic acid product.

[0067] Discontinuous (batch) operation is preferred.

[0068] The sulfonic acids derived from the enzymatic oxidation of the present invention can be used further directly without further work-up steps or can be concentrated or purified by known methods, the degree of concentration then depending on the subsequent use.

[0069] In a preferred embodiment, the process of the invention is characterized in that the sulfonic acid, more preferably taurine, is isolated from the reaction batch.

[0070] Methods for isolating sulfonic acids, in particular taurine, are known to the skilled artisan, for example from methods for isolating amino acids, for example filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization.

[0071] The method of the present invention discloses a novel and unexpected pathway for the enzymatic oxidation of sulfinic acids to the corresponding sulfonic acids and is particularly suitable for the biotechnological production of L-cysteic acid and taurine.

[0072] In the method of the invention, the progress of the reaction is monitored by measuring the content of sulfinic acids (reactants) and sulfonic acids (products) at the beginning, at various times during the reaction, and at the end of the reaction. The content of sulfinic acids of the formula H2N-CH(R)-CH2-SO2H, such as the content of hypotaurine, or the content of sulfonic acids of the formula H2N-CH(R)-CH2-SO3H, such as the content of taurine, in a solution or cell culture can be determined as follows:

[0073] If the concentration of the sulfinic acid reactant at the start of the reaction is at least 0.1 g / L and corresponds to an equivalent amount of sulfonic acid product at the end of the reaction with complete conversion, as determined by HPLC, the content can be quantified directly from an aliquot of the batch. This can be done by taking a 1 ml aliquot of the batch, incubating it at 80° C. for 5 min, then removing all solid components, for example by centrifuging for 5 min at maximum speed in a tabletop centrifuge, and quantifying the supernatant by HPLC calibrated for the relevant sulfinic or sulfonic acid. If the concentration of the sulfinic acid reactant at the start of the reaction is less than 0.1 g / L, the sample can be pre-concentrated to increase the accuracy of the measurement of both the sulfinic acid reactant and the sulfonic acid product, for example by evaporating the sample and redissolving it in an appropriate volume of H2O (for example, 10% of the sample volume corresponding to a 10-fold concentration).

[0074] When a culture broth containing sulfinic acid is used in the method of the present invention, it can be used directly for the quantification. Alternatively, the cells can be first removed from the culture broth, for example by centrifugation or filtration, and the resulting sulfinic acid-containing cell culture supernatant can be used in the method of the present invention. It is also possible to isolate the sulfinic acid from the cell culture supernatant by known methods, and use the purified sulfinic acid in the method of the present invention.

[0075] The following biotransformation assays can be used to demonstrate the methods of the invention and determine transformation.

[0076] A biotransformation batch is produced using a sulfinic acid-containing solution or culture, such as a hypotaurine-containing culture broth, preferably a fermentation broth, or the corresponding cell culture supernatant, or a purified or commercially available sulfinic acid such as hypotaurine. A biotransformation batch on a laboratory scale has a batch volume of 10 ml and contains at least 0.1 g / L of sulfinic acid (from culture / culture broth / fermentation broth, from centrifuged culture supernatant, or as purified material and quantification, or as a dosed commercial product thereof, as described above). The batch volume can be larger depending on the production scale, for example at least 1 L on a preparative scale. An H2O2-generating oxidase, preferably AOX or GOX, and a substrate oxidizable by the oxidase are added such that the dosage of the enzyme activity is at least 1 U / ml. When AOX is used, at least 1% (v / v) methanol is added as a substrate for the AOX enzyme, i.e. at least 0.1 ml per 10 ml of reaction batch. As a substrate for the GOX enzyme, glucose is added to the reaction batch in a dosage of at least 10 g / L. The biotransformation batch is supplied with atmospheric oxygen, for example passively via mixing by an incubation shaker or stirrer, in both cases with the introduction of air, or actively by the introduction of compressed air or pure oxygen, with or without mixing. When AOX is used, the pH of the reaction is 7.5. When GOX is used, the pH of the reaction is 5.5. The pH of the batch can be kept constant, for example by equipping it with a pH electrode connected to a pH control unit that meters the corrective agent (alkali or acid) from a burette ("pH stat" method). The reaction temperature is 25°C (AOX) or 30°C (GOX). The progress of the reaction can be monitored through the consumption of the reactant hypotaurine and the formation of the product taurine, which can be quantitatively determined, for example by HPLC. The end time of the reaction is chosen depending on the progress of the reaction. The selected end point of the reaction is preferably when the molar yield of sulfonic acid of formula H2N-CH(R)-CH2-SO3H, such as taurine, is at least 60%, more preferably at least 80%, particularly preferably at least 90%.The sulfonic acid of formula H2N-CH(R)-CH2-SO3H formed in the reaction batch is then available for further use.

[0077] The sulfinic acid may be derived from chemical or fermentative production, and the sulfinic acid used in the method of the present invention is preferably derived from fermentative production. More preferably, the method of the present invention is characterized in that the sulfinic acid is hypotaurine, which is derived from fermentative production. In the method of the present invention, when a sulfinic acid of the formula H2N-CH(R)-CH2-SO2H, such as hypotaurine, is derived from fermentative production and a sulfonic acid of the formula H2N-CH(R)-CH2-SO3H, such as taurine, is formed therefrom, it is of great advantage to provide a biotechnological method for producing a sulfonic acid of the formula H2N-CH(R)-CH2-SO3H, such as taurine, according to the present invention. This method does not include a chemical reaction step.

[0078] Suitable for the production of sulfinic acids of the formula H2N-CH(R)-CH2-SO2H, such as hypotaurine, are strains of bacteria (e.g. E. coli, Corynebacterium glutamicum, Pantoea ananatis, Bacillus subtilis), algae (e.g. Chlamydomonas reinhardtii), yeasts (e.g. Saccharomyces cerevisiae, Yarrowia lipolytica) or fungi (e.g. Aspergillus niger).

[0079] When the sulfinic acid of formula H2N-CH(R)-CH2-SO2H is hypotaurine, a microbial strain suitable for hypotaurine production is used. A microbial strain suitable for hypotaurine production is characterized in that it contains a metabolic pathway leading to hypotaurine according to one of the metabolic pathways identified in the KEGG pathway database "Taurine and hypotaurine metabolism". Since the biosynthesis of hypotaurine of formulas (1) and (3) starts from L-cysteine, a microbial strain also suitable for cysteine ​​production is preferred. As described, for example, in Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73: 48-54, cysteine ​​production in wild-type microorganisms is tightly regulated, which means that, as documented in the prior art, a microbial strain with a regulated cysteine ​​biosynthetic pathway is not suitable for the production of commercially viable amounts of hypotaurine. Therefore, a microbial strain suitable for hypotaurine production with a deregulated cysteine ​​biosynthetic pathway is preferred.

[0080] Microbial strains having a deregulated cysteine ​​biosynthetic pathway and therefore suitable for cysteine ​​production are characterized in that they exhibit at least one of the following modifications: a) The microbial strain is characterized by a modified serA gene encoding a 3-phosphoglycerate dehydrogenase (SerA) in which feedback inhibition by L-serine is reduced by at least 2-fold compared to the corresponding wild-type enzyme, the enzymatic activity of SerA being capable of being determined photometrically, for example via the oxidation of NADH dependent on the SerA substrate 3-phosphohydroxypyruvate, as described, for example, in McKitrick and Pizer, J. Bacteriol. (1980) 141: 235-245. In particularly preferred mutants of 3-phosphoglycerate dehydrogenase (serA), feedback inhibition by L-serine is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment by at least 50-fold, compared to the corresponding wild-type enzyme. and / or b) the microbial strain comprises a modified cysE gene encoding a serine-O-acetyl-transferase (CysE) in which feedback inhibition by cysteine ​​is reduced by at least two-fold compared to the corresponding wild-type enzyme (e.g. as described in EP0858510B1 or Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611), the enzymatic activity of CysE can be determined photometrically, for example via the consumption of acetyl-CoA, a substrate for CysE, which reacts with L-serine to give O-acetyl-L-serine, as described, for example, in Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611). In particularly preferred mutants of serine-O-acetyltransferase (cysE), feedback inhibition by cysteine ​​is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment by at least 50-fold, compared to the corresponding wild-type enzyme. and / or c) In a microbial strain, the excretion of cysteine ​​outside the cell is increased by at least 2-fold as a result of overexpression of the excretion gene compared to the corresponding wild-type cell, and the excretion of cysteine ​​can be determined by photometric measurement of the cysteine ​​content of the cells (including cysteine, cystine and the adduct (R)-2-methylthiazolidine-2,4-dicarboxylic acid formed from cysteine ​​and pyruvate) according to Gaitonde, Biochem. J (1967) 104: 627-633, as described, for example, in EP 0 885 962 B1. Overexpression of the efflux gene preferably results in at least a 5-fold, more preferably at least a 10-fold, particularly preferably at least a 20-fold increased excretion of cysteine ​​from the cells compared to wild-type cells. The efflux genes are preferably derived from ydeD (see EP0885962B1), yfiK (see EP1382684B1), cydDC (see WO2004 / 113373A1), bcr (see US2005-221453AA) and emrAB (see US2005-221453AA) from E. coli, or corresponding homologous genes from different microorganisms.

[0081] A suitable microbial strain for the production of hypotaurine is used for the production of hypotaurine, preferably by fermentation, according to the prior art. A hypotaurine-containing culture broth, preferably a fermentation broth, is formed. The content of hypotaurine and taurine, if present as by-products, can be quantified from the culture broth. This is achieved by culturing a culture broth containing hypotaurine at a cell density OD of at least 1.0 / ml. 600 This can be done by taking a 1 ml aliquot of the culture broth having 1 / ml of erythrocyte sedimentation, incubating this at 80° C. for 5 minutes, then removing all solid components, e.g. by centrifugation for 5 minutes in a tabletop centrifuge, and quantifying the supernatant by HPLC calibrated for hypotaurine and taurine, e.g. as described for hypotaurine and taurine in Example 1.

[0082] Those skilled in the art can use isotopic analysis to determine whether a substance such as hypotaurine that is desired to be used as sulfinic acid in the method is derived from chemical or fermentative production. Isotopic analysis methods that can be differentiated are described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527, and are based on, for example, the determination of carbon or nitrogen isotope ratios, depending on whether the product is derived from chemical (petroleum-based) or natural (plant-based) production. The method for producing hypotaurine described in Example 5 is considered to be a natural (plant-based) production method, since the glucose used to culture the production strain is derived from plant-based production according to the prior art.

[0083] A homologous gene, protein or homologous sequence should be understood to mean that the DNA or amino acid sequence of the gene, DNA or protein portion is at least 70% identical, preferably at least 80% identical, more preferably at least 90% identical.

[0084] The degree of DNA identity is http: / / blast.ncbi.nlm.nih.gov / and determined by the "nucleotide blast" program, which is based on the blastn algorithm. Default parameters serve as algorithm parameters for the alignment of two or more nucleotide sequences. Default general parameters are: max target sequences=100; short query="automatically adjust parameters for short input sequences"; expected threshold=10; word size=28; automatically adjust parameters for short input sequences=0. Corresponding default scoring parameters are: match / mismatch score=1, -2; gap cost=linear.

[0085] Protein sequences are compared using the "protein blast" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. Default parameters serve as algorithm parameters for alignment of two or more protein sequences. Default general parameters are: max target sequences = 100; short query = "automatically adjust parameters for short input sequences"; expected threshold = 10; word size = 3; automatically adjust parameters for short input sequences = 0. Default scoring parameters are: gap cost = presence: 11 extension: 1; composition adjustment = conditional composition score matrix adjustment.

[0086] In a preferred embodiment, the method of the invention is characterized in that the sulfinic acid is hypotaurine and that this hypotaurine is derived from bacterial production, i.e. is produced using a bacterial production strain, preferably a strain of the species Escherichia coli.

[0087] A preferred bacterial production strain is also characterized by being suitable for cysteine ​​production.Preferred strains suitable for cysteine ​​production are E. coli K12 W3110×pCys and E. coli K12 W3110-ppsA-MHI×pCys strains disclosed in the Examples.As disclosed in Example 5 of the present invention, heterologous expression of the CDO gene encoding cysteine ​​dioxygenase and the CSAD gene encoding cysteine ​​sulfinic acid decarboxylase in the strain W3110×pCys-CDOrn-CSADhs derived from the strain W3110×pCys and the strain W3110-ppsA-MHI×pCys-CDOrn-CSADhs derived from the strain W3110-ppsA-MHI×pCys results in the production of hypotaurine and only a small amount of taurine.

[0088] The microbial strain designated ppsA-MHI is characterized by the lack of the coding sequence of the Wt-ppsA gene and its replacement by the ppsA-MHIcds of SEQ ID NO:9, which encodes a protein having the amino acid sequence of SEQ ID NO:10.

[0089] ppsA refers to a gene encoding a protein having an enzymatic activity of the enzyme class designated as EC 2.7.9.2 in the KEGG database. The corresponding protein is also called PpsA or phosphoenolpyruvate synthase (PEP synthase) or synonymously phosphoenolpyruvate-HO dikinase. The enzyme class is defined as being capable of producing pyruvate from phosphoenolpyruvate in a reversible reaction according to the following equation: (11) Phosphoenolpyruvate + phosphate + AMP ←→ pyruvate + H2O + ATP

[0090] As disclosed in Example 6, hypotaurine from such batches can be completely converted to taurine in an unknown manner by using glucose oxidase together with D-glucose. Thus, the method of the invention allows the biotechnological production of taurine by combining the biosynthetic production of hypotaurine with its enzymatic oxidation to taurine.

[0091] In developing and simplifying the method, it is also conceivable to express H2O2-generating oxidase in the hypotaurine-producing strain. Genes of suitable H2O2-generating oxidases can be identified in databases such as NCBI (National Center for Biotechnology Information). An example of a heterologous glucose oxidase produced in E. coli is the GOX gene from Penicillium amagasakiense (Witt et al., App. Environ. Microbiol (1998) 64: 1405-1411).

[0092] A biotechnological method for producing taurine is preferred, in which in a first step hypotaurine is produced by culturing a production strain and in a second step the formed hypotaurine is enzymatically oxidized to taurine without further processing, with the enzymatic oxidation of hypotaurine to taurine by glucose oxidase / D-glucose being particularly preferred. In the examples of the present invention, the biotechnological production of hypotaurine (Example 5) and its enzymatic oxidation to taurine (Example 6) are disclosed.

[0093] In a preferred embodiment, the method of the invention is characterized in that the sulfinic acid is hypotaurine and that this hypotaurine is of bacterial production, i.e. is produced using a bacterial production strain with a deregulated cysteine ​​biosynthetic pathway, the microbial strain with a deregulated cysteine ​​biosynthetic pathway being as defined above.

[0094] More preferably, cysteine ​​is first produced, which is further reacted to cysteine ​​sulfinic acid via the CDO enzyme present in the production strain according to equation (1) (CDO reaction) and to hypotaurine via the CSAD enzyme also present in the production strain according to equation (3) (CSAD reaction).

[0095] Particularly preferably, the method is characterized in that the sulfinic acid is hypotaurine produced by a bacterial production strain and that the production strain is one of the strains E. coli K12 W3110×pCYS-CDOrn-CSADhs or E. coli K12 W3110-ppsA-MHI×pCYS-CDOrn-CSADhs, more preferably the strain E. coli K12 W3110-ppsA-MHI×pCYS-CDOrn-CSADhs.

[0096] The present invention therefore also demonstrates production strains for the biotechnological production of hypotaurine. Particularly preferred starting strains are the strain E. coli K12 W3110×pCys or one of the strains E. coli K12 W3110-ppsA-MHI×pCys suitable for cysteine ​​production. As described in Example 4 of the present invention, the vector pCys (Figure 1) is extended with the genes of cysteine ​​dioxygenase (CDorn) from Rattus norvegicus, SEQ ID NO: 1, which encodes a protein with the amino acid sequence of SEQ ID NO: 2, and L-cysteine ​​sulfinic acid decarboxylase (CSADhs) from homo sapiens, SEQ ID NO: 3, nt 1 to nt 1509, which has the amino acid sequence of SEQ ID NO: 4. This results in the vector pCys-CDOrn-CSADhs suitable for the production of hypotaurine (Figure 2).

[0097] The CDO gene used in the present invention is not limited to the species Rattus norvegicus. Any CDO gene is suitable whose mRNA-derived gene product (protein) of formula (1) is suitable for oxidizing L-cysteine ​​to L-cysteine ​​sulfinic acid. Such proteins can be found, for example, in the NCBI database by searching for CDO proteins in the subdatabase "protein" using the search term "cysteine ​​dioxygenase". Other CDO proteins known from the prior art, but are not limited thereto, are preferably CDO proteins from Homo sapiens (human), Cyprinus carpio (carp) or Synechococcus (algae), or proteins having an amino acid sequence 70% identical thereto, more preferably proteins having an amino acid sequence 80% identical thereto, and particularly preferably the amino acid sequence of the CDO protein has the sequence specified in SEQ ID NO:2 and is encoded by the DNA sequence specified in SEQ ID NO:1.

[0098] Likewise, the CSAD gene used is not limited to the species Homo sapiens. Any CSAD gene is suitable, whose gene product (protein) from the mRNA of formula (3) is suitable for decarboxylating L-cysteine ​​sulfinic acid to hypotaurine. Such proteins can be found, for example, in the NCBI database, by searching for CSAD proteins in the subdatabase "protein" with the search term "cysteine ​​sulfinic acid decarboxylase". Other CSAD proteins known from the prior art, but without being limited thereto, are preferably CSAD proteins from Rattus norvegicus (rat), Cyprinus carpio (carp), Synechococcus (algae) or E. coli, or proteins with 70% identical amino acid sequences thereto, more preferably proteins with 80% identical amino acid sequences thereto, and particularly preferably, the amino acid sequence of the CSAD protein has the sequence specified in SEQ ID NO: 4 and is encoded by the DNA sequence specified in SEQ ID NO: 3 from nt 1-1509.

[0099] A genetic construct comprising the CDO and CSAD genes, for example and particularly preferably the construct pCys-CDOrn-CSADhs, can be produced according to the prior art using recombinant DNA techniques known to those skilled in the art, as detailed for illustrative purposes in Example 4.

[0100] This is done by cloning the CDO gene encoding cysteine ​​dioxygenase and the CSAD gene encoding cysteine ​​sulfinic acid decarboxylase into a vector suitable for this purpose. In principle, any vector is suitable which allows the CDO and CSAD genes to be expressed in the production strain.

[0101] To generate a gene construct containing CDO and CSAD genes, the CDO and CSAD genes can be cloned into a vector as separate expression units, each with its own promoter and optionally a terminator, or can be cloned into any desired arrangement as an operon under the control of a single promoter. Also possible is an operon structure in which the CDO and CSAD genes, each with its own ribosome binding site, are cloned after one of the genes already present in the vector and expressed under the control of the promoter of that gene. Furthermore, all other possible combinations of expression of the CDO and CSAD genes under their own promoters and in the form of an operon are possible.

[0102] It is also conceivable that the CDO and CSAD genes are cloned into a separate vector as a separate expression unit independent of the vector ensuring deregulated cysteine ​​biosynthesis, e.g. pCys, or integrated into the genome of a host organism, such as E. coli.

[0103] The preferred form of expression of the CDO and CSAD genes is in the form of an artificial operon, each with its own ribosome binding site (RBS) and without its own promoter.

[0104] Particularly preferably, the vector is pCys-CDOrn-CSADhs, in which the CDO and CSAD genes are cloned after the serA317 gene present in pCys, so that their expression occurs under the control of the serA317 promoter according to the following sequence of expression units: serA promoter → (serA317-cds) → RBS-(CDOrn-cds) → RBS-(CSADhs-cds)-rrnB terminator. It is also possible for the expression units to be in a configuration in which the CSADcd is placed before the CDOcd.

[0105] pCys-CDOrn-CSADhs has the additional feature of ensuring deregulation of cysteine ​​biosynthesis by including expression units for the feedback-resistant variant serA317 of 3-phosphoglycerate dehydrogenase (serA gene), the feedback-resistant variant CysEX of serine O-acetyltransferase (cysE gene) and the ydeD gene of the cysteine ​​excretion protein. The present invention relates to vectors in which at least one, preferably two and more preferably three of the genes serA317, cysEX and ydeD are present, the relevant genes can be present in the vector in any desired selection and arrangement.

[0106] Once CDO and CSAD have been cloned into the vector, this is transformed into a suitable host strain in a known manner. A suitable host strain is any microorganism that is accessible to recombinant DNA techniques and is suitable for the fermentative production of recombinant proteins. Preferred host strains are strains of the Escherichia coli species, more preferably E. coli K12 W3110 or E. coli K12 W3110-ppsA-MHI. Particularly preferred are strains resulting from transformation such as W3110×pCys-CDOrn-CSADhs or W3110-ppsA-MHI×pCys-CDOrn-CSADhs, which are suitable for the production of hypotaurine, as described in Example 5.

[0107] The production of hypotaurine, particularly preferably using one of the strains E. coli K12 W3110×pCys-CDOrn-CSADhs or E. coli K12 W3110-ppsA-MHI×pCys-CDOrn-CSADhs, can be achieved by culturing in shake flasks (Example 5) or by fermenting the strains on a production scale.

[0108] In the method of the invention, the hypotaurine formed is converted to taurine by enzymatic oxidation by incubating the hypotaurine-containing cell culture with an H2O2-generating oxidase in the presence of its substrate, for example glucose oxidase in the presence of glucose (see Example 6).

[0109] In a preferred embodiment, the method of the invention is characterized in that the sulfinic acid is cysteine ​​sulfinic acid and the sulfonic acid formed is cysteic acid.

[0110] Preferably, the method of the invention provides a molar yield of sulfonic acid of formula H2N-CH(R)-CH2-SO3H of at least 60%, more preferably at least 80%, in particular at least 90%, based on the molar addition concentrations used of sulfinic acid of formula H2N-CH(R)-CH2-SO2H and sulfonic acid of formula H2N-CH(R)-CH2-SO3H. The amount of sulfinic acid used is preferably at least 1 mM (109.2 mg / L), more preferably at least 10 mM (1.1 g / L), particularly preferably at least 100 mM (10.9 g / L).

[0111] To determine the molar yield value, the molar content of sulfinic and sulfonic acids used in the enzymatic oxidation is determined at the beginning and end of the reaction by HPLC, as known in the prior art. For example, the determination can be carried out as described in Example 1, based on a molecular weight of hypotaurine of 109.2 g / mol and a molecular weight of taurine of 125.2 g / mol. The molar content of sulfonic acids in the batch at the end of the reaction relative to the total molar content of sulfinic and sulfonic acids at the start of the reaction gives the molar yield of sulfonic acids in percent. [Brief description of the drawings]

[0112] The figure shows the plasmids used in the examples. [Figure 1] pCys [Diagram 2] pCys-CDOrn-CSADhs [Diagram 3] pKD46 [Figure 4] pKan-SacB

[0113] Abbreviations used in the figure: bla: ampicillin (β-lactamase) resistance gene kanR: kanamycin resistance gene araC: araC gene (repressor gene) ParaC: promoter of the araC gene ParaB: promoter of the araB gene Gam: lambda phage Gam recombination gene Bet: lambda phage Bet recombination gene Exo: lambda phage Exo recombination gene ORI101: Temperature-sensitive origin of replication RepA: gene for plasmid replication protein A sacB: levansucrase gene pr-f: primer binding site f (forward) pr-r: primer binding site r (reverse) OriC: origin of replication C TetR: tetracycline resistance gene P15AORI: Origin of replication serA317:serA (the 3-phosphoglycerate dehydrogenase gene encoding amino acids 1 to 317) cds cysEX:cysE (serine O-acetyltransferase gene, feedback resistant) cds ORF306: ydeD (cysteine ​​excretion gene) cds ScaI: Restriction enzyme ScaI cleavage site PpuMI: Restriction enzyme PpuMI cleavage site CDOrn:CDO (cysteine ​​dioxygenase) R. norvegicus cds CSADhs: CSAD (cysteine ​​sulfinic acid decarboxylase) H. sapiens cds RBS: ribosome binding site

[0114] The following examples serve to further illustrate the invention without limiting it. EXAMPLES

[0115] Example 1: Oxidation of hypotaurine and cysteine ​​sulfinic acid by alcohol oxidase (AOX) A) Oxidation of cysteine ​​sulfinic acid to cysteic acid by AOX: The reaction was investigated in two parallel batches, i.e. with and without AOX. 12 mg of L-cysteine ​​sulfinic acid monohydrate (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.9 ml of 100 mM sodium phosphate at pH 7.5 and 0.1 ml of methanol was added (final concentration 1% v / v). To start the reaction, 30 μl of a commercial solution of AOX from Pichia pastoris (Sigma-Aldrich) in 100 mM sodium phosphate at pH 7.5 was placed in one batch. According to the manufacturer's information on enzyme activity, the AOX activity in the batch was 5 U / ml. The second batch without AOX (comparison batch) was treated with 30 μl of 100 mM sodium phosphate at pH 7.5. The batches were shaken (Infors incubator shaker) at 25 °C and 140 rpm. At the beginning of the reaction and after 5 hours from the start of the reaction, 1 ml was taken from each batch, incubated at 80° C. for 5 minutes, centrifuged at 13,000 rpm for 5 minutes (Heraeus™ Fresco™ 21 centrifuge) and the supernatants were analyzed by HPLC. The contents of L-cysteine ​​sulfinic acid and L-cysteic acid in the batches with and without AOX at the beginning (0 hours) and after 5 hours of reaction time are summarized in Table 1.

[0116] [Table 1]

[0117] B) Oxidation of hypotaurine to taurine by AOX: The reaction was investigated in two parallel batches, i.e. with and without AOX. 12 mg hypotaurine (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.9 ml 100 mM sodium phosphate at pH 7.5 and 0.1 ml methanol was added (final concentration 1% v / v). To start the reaction, 30 μl of a commercial solution of AOX from Pichia pastoris (Sigma-Aldrich) in 100 mM sodium phosphate at pH 7.5 was placed in one batch. According to the manufacturer's information on enzyme activity, the AOX activity in the batch was 5 U / ml. The batch without AOX (comparison batch) was treated with 30 μl 100 mM sodium phosphate at pH 7.5. The batches were shaken (Infors incubator shaker) at 25 °C and 140 rpm. At the beginning of the reaction and after 5 hours from the start of the reaction, 1 ml was taken from each batch, incubated at 80° C. for 5 minutes, centrifuged at 13,000 rpm for 5 minutes (Heraeus™ Fresco™ 21 centrifuge) and the supernatant was analyzed by HPLC. The hypotaurine and taurine contents in the batches with and without alcohol oxidase (AOX) at the beginning (0 hours) and after 5 hours of reaction time are summarized in Table 2.

[0118] [Table 2]

[0119] HPLC Analysis of L-Cysteine ​​Sulfinic Acid, L-Cysteic Acid, Hypotaurine and Taurine: The compounds quantitatively analyzed in the examples were quantified using HPLC methods calibrated for L-cysteinesulfinic acid, L-cysteic acid, hypotaurine and taurine, 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 was used, equipped with a unit for pre-column derivatization with o-phthaldialdehyde (OPA derivatization) from the same manufacturer. The HPLC system was equipped with a fluorescence detector to detect the OPA derivatization products of L-cysteinesulfinic acid, L-cysteic acid, hypotaurine and taurine. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. A Thermo Scientific Accucore aQ column with a length of 100 mm, an internal diameter of 4.6 mm and a particle size of 2.6 μm was also used, thermally equilibrated at 40° C. in a column oven. Eluent A: 25 mM sodium phosphate, pH 6.0 Eluent B: Methanol

[0120] The separation was performed in gradient mode: 10% eluent B to 60% eluent B in 0-25 min, followed by 60% eluent B to 100% eluent B in 2 min, followed by 100% eluent B in another 2 min, at a flow rate of 0.5 ml / min. Retention time of L-cysteic acid: 3.2 min. Retention time of L-cysteine ​​sulfinic acid: 4.1 min. Retention time of taurine: 14.8 min. Retention time of hypotaurine: 15.7 min.

[0121] Example 2: Oxidation of hypotaurine and cysteine ​​sulfinic acid by glucose oxidase (GOX) A) Oxidation of cysteine ​​sulfinic acid to cysteic acid by GOX: The reaction was investigated in two parallel batches, i.e. with and without GOX. 12 mg of L-cysteine ​​sulfinic acid monohydrate (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.5 ml of 100 mM sodium acetate at pH 5.5 and 0.5 ml of a 200 g / L glucose solution in the same buffer was added. To start the reaction, 50 μl of a commercial solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM sodium acetate at pH 5.5 was placed in one batch. According to the manufacturer's information, the GOX activity in the batch was 5 U / ml. The batch without GOX (comparison batch) was treated with 50 μl of 100 mM sodium acetate at pH 5.5. The batches were shaken (Infors incubator shaker) at 30 °C and 140 rpm. At the start of the reaction and 5 hours after the start of the reaction, 1 ml was taken from each batch, incubated at 80° C. for 5 min, centrifuged at 13,000 rpm for 5 min (Heraeus™ Fresco™ 21 centrifuge) and the supernatants were analyzed by HPLC as described above.

[0122] The contents of L-cysteine ​​sulfinic acid and L-cysteic acid in the batches with and without GOX at the start (0 h) and after 5 h reaction time are summarized in Table 3.

[0123] [Table 3]

[0124] B) Oxidation of hypotaurine to taurine by GOX: The reaction was investigated in two parallel batches, i.e. with and without GOX. 12 mg hypotaurine (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.5 ml of 100 mM sodium acetate at pH 5.5 and 0.5 ml of a 200 g / L glucose solution in the same buffer was added. To start the reaction, 50 μl of a commercial solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM sodium acetate at pH 5.5 solution was placed in one batch. According to the manufacturer's information, the GOX activity in the batch was 5 U / ml. The batch without GOX (comparison batch) was treated with 50 μl of 100 mM sodium acetate at pH 5.5. The batches were shaken (Infors incubator shaker) at 30 °C and 140 rpm. At the start of the reaction and 5 hours after the start of the reaction, 1 ml was taken from each batch, incubated at 80° C. for 5 min, centrifuged at 13,000 rpm for 5 min (Heraeus™ Fresco™ 21 centrifuge) and the supernatants were analyzed by HPLC as described above.

[0125] The hypotaurine and taurine contents in the batches with and without GOX at the start (0 h) and after 5 h of reaction time are summarized in Table 4.

[0126] [Table 4]

[0127] Example 3: Preliminary oxidation of hypotaurine to taurine by GOX The reaction was investigated in two parallel batches, i.e. with different doses of the enzyme GOX, the concentration of the hypotaurine substrate to be oxidized was 20 g / L in each case.

[0128] For batch 1, 200 mg of hypotaurine (Sigma-Aldrich) was weighed into a 100 ml Erlenmeyer flask and dissolved in 6.95 ml of 100 mM sodium acetate, pH 5.5, and 3 ml of a 200 g / L glucose solution in the same buffer was added. To start the reaction, 50 μl of a commercial solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM sodium acetate, pH 5.5 was added. According to the manufacturer's information on enzyme activity, the GOX activity in the batch was 5 U / ml.

[0129] For batch 2, 200 mg of hypotaurine (Sigma-Aldrich) was weighed into a 100 ml Erlenmeyer flask and dissolved in 6.5 ml of 100 mM sodium acetate, pH 5.5, and 3 ml of a 200 g / L glucose solution in the same buffer was added. To start the reaction, 500 μl of a commercial solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM sodium acetate, pH 5.5 was added. According to the manufacturer's information on enzyme activity, the GOX activity in the batch was 50 U / ml.

[0130] Batches 1 and 2 were shaken at 30° C. and 140 rpm (Infors incubator shaker). 3, 6 and 24 hours after the start of the reaction, 1 ml aliquots of each batch were taken, incubated at 80° C. for 5 min, centrifuged at 13,000 rpm for 5 min (Heraeus™ Fresco™ 21 centrifuge) and the supernatants were analyzed by HPLC as described above. The progress of the reactions over time is summarized in Table 5.

[0131] [Table 5]

[0132] Example 4: Construction of hypotaurine producing strains E. coli K12 W3110×pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI×pCys-CDOrn-CSADhs Cysteine ​​dioxygenase CDOrn: CDOrn: The amino acid sequence of cysteine ​​dioxygenase from Rattus norvegicus is disclosed in the NCBI (National Center for Biotechnology Information) database under sequence ID: AAH70509.1. The amino acid sequence was used to derive a DNA sequence that was codon-optimized for expression in E. coli (publicly available Eurofins Genomics GENEius software) and was synthetically generated (Eurofins Genomics). This DNA sequence, designated CDOrn, is disclosed in SEQ ID NO:1 and encodes a protein with the amino acid sequence of SEQ ID NO:2.

[0133] Cysteine ​​sulfinic acid decarboxylase CSADhs: The amino acid sequence of cysteine ​​sulfinic acid decarboxylase (CSADhs) from Homo sapiens is disclosed in the NCBI (National Center for Biotechnology Information) database as sequence ID: XP_016861786.1. The amino acid sequence was used to derive a DNA sequence that was codon-optimized for expression in E. coli (publicly available Eurofins Genomics GENEius software). This DNA sequence, designated CSADhs, is disclosed in SEQ ID NO:3 at nt 1 to 1509 and encodes a protein having the amino acid sequence of SEQ ID NO:4. The DNA sequence of the E. coli rrnB terminator (nt 1510 to 1842 of SEQ ID NO:3) was joined to nt 1509. The DNA sequence of the rrnB terminator is disclosed in Orosz et al., Eur. J. Biochem. (1991) 201: 653-659. The DNA disclosed in SEQ ID NO:3 consisting of the CSADhs cds and the rrnB terminator was synthetically produced (Eurofins Genomics) and named CSADhs-rrnB.

[0134] Construction of vector pCys-CDOrn-CSADhs: - vector pCys (Figure 1) refers to the plasmid pACYC184-cysEX-GAPDH-ORF306-serA317, which is a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306 disclosed in EP0885962B1. Plasmid pACYC184-cysEX-GAPDH-ORF306 contains not only the origin of replication and the tetracycline resistance gene (parent vector pACYC184), but also the cysEX allele encoding a serine O-acetyltransferase with reduced feedback inhibition by cysteine, and the efflux gene ydeD (ORF306), the expression of which is controlled by the constitutive GAPDH promoter.

[0135] To obtain pACYC184-cysEX-GAPDH-ORF306-serA317, the serA317 gene fragment encoding the N-terminal 317 amino acids of the SerA protein from E. coli and disclosed in Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184 (referred to therein as "NSD:317") was cloned after the ydeD (ORF306) excretion gene in pACYC184-cysEX-GAPDH-ORF306. SerA317 encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. Expression of serA317 is regulated by the serA promoter.

[0136] As a result of deregulated biosynthesis, E. coli cells transformed with pCys produce the induction product cysteine ​​sulfinic acid and the starting product hypotaurine, cysteine.

[0137] - Vector pCys-CDOrn-CSADhs (Figure 2): pCys was digested with ScaI and PpuMI, and the resulting 6.1 kb vector fragment was isolated by preparative agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

[0138] CDOrn DNA was amplified from synthetic DNA (CDOrn) of SEQ ID NO:1 by PCR (Phusion™ High Fidelity DNA polymerase, Thermo Scientific™) using primers cdorn-1f (SEQ ID NO:5) and csadhs-2r (SEQ ID NO:6) and isolated as a 0.6 kb fragment.

[0139] Primer cdorn-1f included 28 nt starting from the 5′ end overlapping with the 3′ end of the 6.1 kb pCys ScaI / PpuMI vector fragment (nt 1 to 28 of SEQ ID NO:5) obtained by ScaI digestion, the ribosome binding site (RBS) (nt 31 to 36 of SEQ ID NO:5), and the first 22 nt of the CDOrn cds (nt 44 to 65 of SEQ ID NO:5).

[0140] Primer csadhs-2r contained 22 nt starting from the 5′ end overlapping the start of the CSADhs cds (nt 1–22 of SEQ ID NO:6), followed by a ribosome binding site (nt 30–35 of SEQ ID NO:6) and the last 20 nt of the CDOrn cds (nt 38–57 of SEQ ID NO:6) in reverse complement form.

[0141] - The DNA fragment was amplified from synthetic DNA of SEQ ID NO:3 (CSADhs-rrnB) by PCR ("Phusion™ High Fidelity" DNA polymerase, Thermo Scientific™) using CSADhs-rrnB DNA: primers csadhs-3f (SEQ ID NO:7) and glf-2r (SEQ ID NO:8) and isolated as a 1.8 kb fragment.

[0142] Primer csadhs-3f included 20 nt starting from the 5′ end and overlapping with the 3′ end of the CDOrn cds (nt 1 to 20 of SEQ ID NO:7), a ribosome binding site (nt 23 to 28 of SEQ ID NO:7), and the first 22 nt of the CSADhs cds (nt 36 to 57 of SEQ ID NO:7).

[0143] Primer glf-2r started from the 5' end and contained 33 nt overlapping the 5' end of the 6.1 kb pCys ScaI / PpuMI vector fragment obtained by PpuMI digestion (nt 1 to 33 of SEQ ID NO:8), followed by the last 22 nt of the rrnB terminator (nt 34 to 55 of SEQ ID NO:8) in reverse complement form.

[0144] - Vector pCys-CDOrn-CSADhs: the 6.1 kb pCys ScaI / PpuMI vector fragment, the 0.6 kb CDOrn PCR product (CDOrn DNA) and the 1.8 kb CSADhs-rrnB PCR product (CSADhs-rrnBDNA) were ligated using the NEBuilder® Cloning Kit (NEB New England Biolabs) according to the manufacturer's instructions.

[0145] E. coli NEB®10-beta cells (NEB New England Biolabs) were then transformed with the ligation mixture. Clones from the transformation were selected in LBtet, which contained 10 g / L tryptone (Gibco™), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 15 g / L agar, and 15 mg / L tetracycline (Sigma-Aldrich). Single clones from the transformation were analyzed by culturing in LBtet liquid medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 mg / L tetracycline) and isolating the vector from the cell pellet in the culture. The correct 8.5 kb vector was named pCys-CDOrn-CSADhs (Figure 2).

[0146] -E.coli W3110 strain: The strain used was E. coli K12 W3110 (commercially available from DSMZ: Deutsche Sammmlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures] under strain number DSM5911).

[0147] -E. coli W3110-ppsA-MHI strain: The strain used was E. coli K12 W3110-ppsA-MHI. E. coli K12 W3110-ppsA-MHI is characterized by a mutant ppsA gene ppsA-MHI (SEQ ID NO: 9) encoding a protein having the protein sequence of SEQ ID NO: 10 and the enzyme activity of PEP synthase (enzyme class designated as EC2.7.9.2 in the KEGG database). E. coli K12 W3110-ppsA-MHI was produced by using a combination of lambda-Red recombination and counterselection screening for gene modification known to those skilled in the art (see, for example, Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245). To replace the ppsAWT gene of E. coli K12 W3110 with ppsA-MHI, the following procedure was performed. 1) E. coli K12 W3110 was transformed with the plasmid pKD46 (FIG. 3, disclosed in the "GenBank" gene database under the accession number AY048746.1) and the E. coli W3110×pKD46 strain was isolated (ampicillin selection). 2) From plasmid pKan-sacB (Figure 4), the 3.2 kb Kan-sacB cassette was isolated by PCR using primers pps-9f (SEQ ID NO: 11, which binds to the site designated "pr-f" in Figure 4), and pps-10r (SEQ ID NO: 12, which binds to the site designated "pr-r" in Figure 4). Plasmid pKan-sacB contained expression cassettes for both the kanamycin (Kan) resistance gene and the sacB gene encoding the enzyme levansucrase. The E. coli kanamycin resistance gene (Kan), which encodes aminoglycoside phosphotransferase, is disclosed in the NCBI database under accession number SH02_03400. The B. subtilis sacB gene is disclosed in the NCBI database under accession number 936413. Primer pps-9f contains the first 30 nt of the ppsA WT gene (identical to nt 1-30 of SEQ ID NO:9) and connected thereto 20 nt of the site designated "pr-f" in Figure 4. Primer pps-10r contains the last 30 nt of the ppsA WT gene (identical to nt 2350-2379 of SEQ ID NO:9) and connected thereto 20 nt of the site designated "pr-r" in Figure 4 in reverse complement form. E. coli W3110×pKD46 was transformed with the 3.2 kb Kan-sacB cassette, and kanamycin-resistant clones were isolated. 4) The resulting clones were plated on LBSC plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar, and 15 mg / L kanamycin). Clones with the integrated sacB gene produced toxic levan from sucrose, which resulted in growth inhibition (sucrose sensitivity). A kanamycin-resistant, sucrose-sensitive clone was selected and named W3110-ppsA::Kan-sacB×pKD46. 5) W3110-ppsA::Kan-sacB×pKD46 was transformed with DNA of the ppsA-MHI gene (SEQ ID NO: 9, synthetically produced from Eurofins Genomics), and clones were selected on LBS plates (10 g / L tryptone, 5 g / L yeast extract, 7% sucrose, 1.5% agar) without kanamycin. Only clones that no longer contained an active sacB gene were able to grow on LBS plates. These clones were plated on LBkan plates (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 1.5% agar, 15 mg / L kanamycin) to select clones that also did not contain an active Kan gene and whose growth was inhibited in the presence of kanamycin. 6) A clone showing positive growth in the presence of sucrose and negative growth in the presence of kanamycin was selected, the ppsAMHI gene was isolated from the genomic DNA of the strain by PCR, and DNA sequencing (Eurofins Genomics) confirmed that the ppsAMHI gene having the DNA sequence disclosed in SEQ ID NO: 9 was integrated and encoded a protein corresponding to the sequence from SEQ ID NO: 10. After removal of the plasmid pKD46 by incubation at 42° C., the strain was named E. coli W3110-ppsA-MHI.

[0148] - Production strains: Plasmid DNA of the vector pCys-CDOrn-CSADhs was transformed into the strains E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI. For comparison, E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI were transformed with the vector pCys. Transformants were selected with LBtet. One clone was isolated in each case. The strains were named E. coli K12 W3110xpCys-CDOrn-CSADhs and E. coli K12 W3110xpCys, respectively, and E. coli K12 W3110-ppsA-MHIxpCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHIxpCys, respectively. E. coli K12 W3110xpCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHIxpCys-CDOrn-CSADhs were used as production strains for the production of hypotaurine.

[0149] Example 5: Production of hypotaurine in shake flasks Precultures in LBtet liquid medium were made from E. coli K12 W3110xpCys-CDOrn-CSADhs, E. coli K12 W3110xpCys, E. coli K12 W3110-ppsA-MHIxpCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHIxpCys (grown overnight at 37° C., 120 rpm).

[0150] Main culture: 0.5 ml of preculture was placed in a 300 ml conical flask (with baffles) containing 30 ml of SM1-Ac medium containing 15 g / L glucose, 2 g / L Na2S2O3·5H2O, 0.1 g / L L-isoleucine, 0.1 g / L D,L-methionine, 0.1 g / L L-threonine, 5 mg / L vitamin B, and 15 mg / L tetracycline.

[0151] SM1-Ac medium composition: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L NH4 acetate, 0.3 g / L MgSO4·7H2O, 0.015 g / L CaCl2·2H2O, 0.002 g / L FeSO4·7H2O, 1 g / L trisodium citrate dihydrate, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0152] Composition of trace element solution: 0.15 g / L Na2MoO4·2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2·6H2O, 0.25 g / L CuSO4·5H2O, 1.6 g / L MnCl2·4H2O, 0.3 g / L ZnSO4·7H2O.

[0153] The culture was incubated for 24 hours at 30° C. and 140 rpm in an incubator shaker (Infors). After 24 hours, a 1 ml sample was taken and the cell density OD was measured using a Thermo Scientific™ Genesys™ 10S UV / Visible spectrophotometer. 600 The amount of hypotaurine and taurine in the culture supernatants was measured by HPLC (cell density OD of the cultures, measured photometrically at 600 nm) and the amount of hypotaurine and taurine was measured by HPLC. The hypotaurine content in the culture supernatants, measured by HPLC, was 157.3 mg / L (cell density OD of the cultures, measured photometrically at 600 nm) for E. coli K12 W3110×pCys-CDOrn-CSADhs. 600 The taurine content in the culture supernatant was 52.8 mg / L.

[0154] E. coli K12 W3110-ppsA-MHIxpCys-CDOrn-CSADhs (cell density OD 600 The hypotaurine content in the culture supernatant of the cultured medium was measured by HPLC to be 1059.2 mg / L, and the taurine content was 304.1 mg / L.

[0155] The two comparative strains E. coli K12 W3110xpCys (cell density OD 600 / ml: 6.1 / mL) and E. coli K12 W3110-ppsA-MHIxpCys (cell density of culture OD 600 / ml:7.5 / ml), neither hypotaurine nor taurine was detectable.

[0156] Example 6: Oxidation of Hypotaurine to Taurine from Shake Flask Cultures 10 ml batches of each from shake flask cultures of E. coli W3110×pCys-CDOrn-CSADhs and E. coli W3110-ppsA-MHI×pCys-CDOrn-CSADhs (Example 5) were centrifuged (Heraeus™ Megafuge® 1.0R) at 4000 rpm for 10 minutes and 9.4 ml of each supernatant was transferred to a 100 ml conical flask and adjusted to pH 5.5 with 0.7 M NaOH. To this, 0.5 ml of a 200 g / L solution of glucose in HO (final concentration in the mixture 10 g / L) and 100 μl of a 1 U / μl stock solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM sodium acetate at pH 5.5 (final concentration 10 U / ml) were added and the batches (volume 10 ml) were incubated at 30 °C and 140 rpm in an incubator shaker (Infors). At the start of the incubation and 2 h after the start, 1 ml aliquots of each batch were taken, incubated at 80 °C for 5 min, centrifuged at 13,000 rpm for 5 min (Heraeus™ Fresco™ 21 centrifuge) and the supernatants were analyzed by HPLC.

[0157] As summarized in Table 6, hypotaurine present in the shake flask culture (Example 5) - 157.3 mg / L for the W3110×pCys-CDOrn-CSADhs strain and 1059.2 mg / L for the W3110-ppsA-MHI×pCys-CDOrn-CSADhs strain - was completely consumed, forming taurine at concentrations of 212.8 mg / L and 1355.6 mg / L, respectively. The molar yields were determined taking into account the difference in molecular weight (109.2 g / mol for hypotaurine and 125.2 g / mol for taurine). As shown in Table 6, in the case of the W3110×pCys-CDOrn-CSADhs strain, the combined content of hypotaurine (1.4 mM) and taurine (0.4 mM) at the start of the reaction was 1.8 mM. Two hours after the start of the reaction, the taurine content was 1.7 mM and hypotaurine was no longer detectable. The molar yield of taurine from the enzymatic oxidation of the hypotaurine / taurine product mixture, i.e., based on the total hypotaurine and taurine input (1.4 + 0.4 = 1.8 mM) from the shake flask culture, was 94.4%. For the W3110-ppsA-MHI × pCys-CDOrn-CSADhs strain, the combined hypotaurine (9.7 mM) and taurine (2.4 mM) content at the start of the reaction was 12.1 mM. Two hours after the start of the reaction, the taurine content was 10.8 mM and hypotaurine was no longer detectable. The molar yield of taurine from the enzymatic oxidation of the hypotaurine / taurine product mixture, i.e., based on the total input of hypotaurine and taurine (9.7 + 2.4 = 12.1 mM) from the shake flask culture, was 89.3%.

[0158] [Table 6]

Claims

1. Formula H ２ N-CH(R)-CH ２ -SO ２ The sulfinic acid of H ２ O ２ By an enzyme selected from the class of product oxidases, in the presence of a substrate for said enzyme, the product is produced by the reaction of formula H ２ N-CH(R)-CH ２ -SO ３ 1. A method for the enzymatic oxidation of H to a sulfonic acid, wherein the group R is selected from the group consisting of hydrogen, and organic, linear, branched, cyclic, saturated, unsaturated, aromatic and heteroaromatic groups, with or without substituents, said substituents being -CN, -NCO, -COOH, -halogen, -(meth)acryloyl, -epoxy, -SH or -OH.

2. The H ２ O ２ 2. The method of claim 1, wherein the product oxidase is an alcohol oxidase and the substrate present is a primary alcohol.

3. The H ２ O ２ 2. The method of claim 1, wherein the product oxidase is glucose oxidase and the substrate present is glucose.

4. The method of any one of claims 1 to 3, wherein the sulfinic acid is an aminoalkylsulfinic acid and the sulfonic acid is an aminoalkylsulfonic acid.

5. 5. The process according to claim 1, wherein the concentration of sulfinic acid in the batch is at least 1 g / l.

6. 6. The method of any one of claims 1 to 5, wherein the sulfinic acid is 2-aminoethanesulfinic acid (hypotaurine) and the sulfonic acid formed is 2-aminoethanesulfonic acid (taurine).

7. The process according to any one of claims 1 to 6, wherein the sulfonic acid is isolated from the reaction batch.

8. 8. The method of claim 6 or 7, wherein the hypotaurine is hypotaurine from fermentation production.

9. 9. The method of claim 8, wherein the hypotaurine is derived from bacterial production, i.e., produced using a bacterial production strain.

10. 10. The method of claim 9, wherein the bacterial production strain is a strain of the E. coli species.

11. 11. The method of claim 9 or 10, wherein the bacterial production strain is a strain with a deregulated cysteine ​​biosynthetic pathway.

12. 12. The method of any one of claims 9 to 11, wherein the bacterial production strain is one of the E. coli strains W3110xpCys-CDOrn-CSADhs or W3110-ppsA-MHIxpCys-CDOrn-CSADhs.

13. 4. The method of claim 1, wherein the sulfinic acid is cysteine ​​sulfinic acid and the sulfonic acid formed is cysteic acid.

14. The compound of formula H used in this reaction ２ N-CH(R)-CH ２ -SO ２ H and sulfinic acids of formula H ２ N-CH(R)-CH ２ -SO ３ The formula H for the total molar concentration of sulfonic acid H ２ N-CH(R)-CH ２ -SO ３ 14. The process according to any one of claims 1 to 3 and 13, wherein the molar yield of sulfonic acid of H is at least 60%, and the group R is selected from the group consisting of hydrogen and organic groups, linear groups, branched groups, cyclic groups, saturated groups, unsaturated groups, aromatic groups and heteroaromatic groups, with or without substituents, said substituents being -CN, -NCO, -COOH, -halogen, -(meth)acryloyl, -epoxy, -SH or -OH.

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