METHOD FOR THE PRODUCTION OF TAURINE

Description

[0001] The invention relates to a process for the production of taurine from O-acetyl-L-serine (OAS) by biotransformation, wherein in a first process step (biotransformation 1) L-cysteic acid is produced from O-acetyl-L-serine (OAS) using an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of a salt of sulfurous acid and then in a second process step (biotransformation 2) L-cysteic acid is decarboxylated to taurine, wherein in the first process step the biotransformation is carried out under active pH control, the OAS concentration in the mixture is at least 10 g / L and the OAS sulfhydrylase is CysM. Taurine is provided by this biotransformation.

[0002] Taurine (2-aminoethanesulfonic acid, CAS number 107-35-7) is an aminosulfonic acid that occurs naturally as a breakdown product of the amino acids cysteine ​​and methionine. Taurine is a component of energy drinks and is also used in pet food, e.g., for cats or in fish farming (Salze and Davis (2015) Aquaculture 437: 215-229). However, taurine is also believed to have health-promoting effects (Ripps and Shen (2012), Molecular Vision 18: 2673-2686).

[0003] In nature, taurine occurs almost exclusively in the animal kingdom, with only a few examples of its occurrence in bacteria, algae, or plants. There are various biosynthetic pathways to taurine (for an overview, see, for example, the KEGG Pathway Database: "Taurine and hypotaurine metabolism"), including starting from L-cysteine. The most important synthesis steps leading from L-cysteine ​​to taurine are shown in equations (1) to (5). L − Cystein + O 2 − > L − Cysteinsulfinsäure L − Cysteinsulfinsäure + 1 2 O 2 − > L − Cysteinsäure L − Cysteinsulfinsäure − > Hypotaurin + CO 2 Hypotaurin + 1 2 O 2 − > Taurin L − Cysteinsäure − > Taurin + CO 2 (1) In a first step, L-cysteine ​​is oxidized by the enzyme cysteine ​​dioxygenase (CDO, EC 1.13.11.20) to L-cysteine ​​sulfinic acid (3-sulfinoalanine, CAS number 207121-48-0). (2) Cysteine ​​sulfinate oxidase (so far a rather hypothetical enzymatic step) further oxidizes L-cysteine ​​sulfinic acid to L-cysteine ​​acid ((R)-2-amino-3-sulfopropionic acid, CAS number 23537-25-9). (3) Cysteine ​​sulfinic acid decarboxylase (CSAD, EC 4.1.1.29) decarboxylates L-cysteine ​​sulfinic acid to hypotaurine (2-aminoethane sulfinic acid, CAS number 300-84-5). (4) It is not yet fully understood whether hypotaurine is oxidized to taurine. (5) In a step similar to (3), L-cysteic acid is decarboxylated to taurine by suitable CSAD enzymes.

[0004] For commercial use, taurine is currently produced chemically. A well-known example is a process developed by Changshu Yudong Chemical Factory that starts with ethylene and converts taurine via ethyleneimine. With the trend driven by consumer demand, away from chemically produced ingredients and toward sustainably produced ones, biotechnological processes for the production of taurine are increasingly being investigated. The current state of the art is based on metabolic engineering approaches, in which suitable biosynthesis genes are heterologously expressed in a production strain and produce taurine or its biosynthetic precursor, hypotaurine.

[0005] Honjoh et al. (2010), Amino Acids 38: 173-1183 describe a genetically modified yeast strain that carries the CDO and CSAD genes from carp ( Cyprinus carpio) heterologously expressed. When L-cysteine ​​was added to the culture of the genetically modified strain, the production of hypotaurine was observed as the main product, along with a smaller amount of taurine. Hypotaurine could be oxidized to taurine by treatment with H 2 O 2. Although S. cerevisiae is capable of producing cysteine ​​from its own metabolism, the external addition of L-cysteine ​​to the culture medium was necessary to produce hypotaurine and taurine.

[0006] Tevatia et al. (2019), Algal Research 40: 101491, also use heterologously expressed CDO and CSAD genes from carp to produce taurine in the algae Chlamydomonas reinhardtii The intracellular taurine yield was 0.14 mg taurine / g dry biomass.

[0007] Joo et al. (2018), J. Agric. Food Chem. 66: 13454 - 13463, describe a genetically modified strain of the bacterium Corynebacterium glutamicumwith a production capacity of 0.5 g / L taurine in shake flask culture. Taurine was evidently accumulated intracellularly and not secreted into the culture medium. For taurine synthesis, the genes for L-cysteine ​​acid synthase, cysteine ​​dioxygenase, and L-cysteine ​​sulfinic acid decarboxylase were heterologously expressed in the strain. Furthermore, a repressor gene for methionine and cysteine ​​biosynthesis was inactivated, which simultaneously resulted in improved sulfur uptake. In addition to the low yield, the fact that taurine is produced intracellularly is considered a fundamental disadvantage with regard to product isolation. This requires the laborious disruption of the cells to release the taurine for further processing.

[0008] WO17213142A1 (Ajinomoto) describes a taurine-producing strain obtained by heterologous expression of a cysteine ​​dioxygenase and an L-cysteine ​​sulfinic acid decarboxylase in a strain capable of cysteine ​​production. The main product was hypotaurine with a maximum yield of 450 µM, which could only be converted to taurine by subsequent alkaline treatment, and even then, only in low yield.

[0009] Ono et al. (Free Radical Biology and Medicine 106, pp. 69-79, 2017) revealed that the OAS sulfhydrylases CysM and CysK from Salmonella entericum LT2 analogous to the enzymes from E. coli (see Maier, Nature Biotechnology 21, pp. 422-427, 2003, Table 1) are able to form cysteine ​​from the substrates OAS and sulfide in high yields of over 70%. Ono further reveals that CysM and CysK are Salmonella entericumLT2 can also form the cysteine ​​derivative L-cysteine ​​acid (cysteine ​​sulfonate, salt of cysteic acid) from OAS in the presence of Na 2 SO 3 under otherwise identical reaction conditions in small amounts, ie in a molar yield of less than 0.2%.

[0010] US9267148B2, US2012 / 0222148A1, US2018 / 0028474A1, US2019 / 0085339, WO2017 / 176277A1, and WO2019 / 094051 (Plant Sensory Systems) describe taurine-producing strains and organisms that, in various forms, comprise the heterologous expression of a cysteine ​​dioxygenase and an L-cysteine ​​sulfinic acid decarboxylase. The applications primarily target the production of taurine in plants, but no statements regarding yields are made.

[0011] US20190062757A1 (KnipBio) describes heterologous production strains for the production of taurine or its precursors.

[0012] The state of the art thus reveals various metabolic engineering approaches for producing hypotaurine or taurine in heterologous production systems. While these metabolic engineering approaches yield product yields, they are too low for industrial use.

[0013] The state of the art further provides processes for the production of non-proteinogenic (unnatural) amino acids, e.g., by direct fermentation of microorganisms (EP 1 191 106 B1, Wacker) or biotransformation of OAS catalyzed by O-acetyl-L-serine sulfhydrylase (OAS sulfhydrylase) (EP 1 247 869 B1, Wacker). These processes are based on an OAS sulfhydrylase catalyzing the reaction of OAS with a nucleophile to form a non-proteinogenic amino acid according to the general equation (6): OAS + Nucleophil − > unnat . Aminosäure + Acetat

[0014] In EP 1 247 869 B1 (Wacker), a variety of different nucleophiles were investigated for their suitability as nucleophiles for the reaction with OAS catalyzed by an OAS sulfhydrylase, including selenides, selenol, azides, cyanides, azoles, and isoxazolinones. In addition, sulfur compounds from the group of thiosulfates and thiols of the general formula HSR were also used, where the radical R was a monovalent substituted or unsubstituted alkyl, alkoxy, aryl, or heteroaryl radical.

[0015] These processes are not suitable for the production of L-cysteine ​​acid and / or taurine.

[0016] The object of the present invention was to provide a biotechnological process for the production of taurine by biotransformations, bypassing a heterologous production strain produced by metabolic engineering.

[0017] The problem is solved by a process for the production of taurine from O-acetyl-L-serine (OAS) by means of biotransformation, wherein in a first process step (biotransformation 1) L-cysteic acid is produced from O-acetyl-L-serine (OAS) with an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of a salt of sulfurous acid and then in a second process step (biotransformation 2) L-cysteic acid is decarboxylated to taurine, wherein in the first process step the biotransformation is carried out under active pH control, the OAS concentration in the mixture is at least 10 g / L and the OAS sulfhydrylase is CysM.

[0018] As disclosed in the accompanying application EP 4 441 239 A1 and the examples of the present invention, it was surprisingly found that salts of sulfurous acid (hereinafter referred to as sulfites or SO 3 2-<) are suitable as nucleophiles in reaction (6) and, in extension of EP 1 247 869 B1 (Wacker), enable the synthesis of the non-proteinogenic amino acid L-cysteic acid in a reaction according to equation (7) (biotransformation 1). OAS + SO 3 2 − − > L − Cysteinsäure + Acetat

[0019] The L-cysteic acid produced in biotransformation 1 could also unexpectedly be decarboxylated to taurine without further processing according to equation (5) by a recombinantly produced CSAD enzyme (biotransformation 2).

[0020] The advantage of the process according to the invention for the production of taurine is that it is a sustainable and technically feasible, economical biotransformation process for the production of taurine. Environmentally hazardous chemicals can be dispensed with. No raw materials of fossil origin are consumed, and no toxic chemical waste and / or waste gases are generated. The production process of the present invention is therefore environmentally friendly and sustainable. Furthermore, the process requires neither extreme reaction conditions nor special equipment and is therefore easily implemented technically. Such processes are in increasing demand.

[0021] Taurine from the process according to the invention can either be used directly without further processing steps or can be enriched using known methods.

[0022] In the context of the present invention, manufacturing processes are distinguished as follows: 1. Chemical processes 2. Biotechnological processes: a) by metabolic engineering Metabolic Engineering (also “Pathwaydesign.” In contrast to biotransformation, biotransformation is a method of biotechnology in which the metabolic pathways of an organism are modified by optimizing or modifying genetic and regulatory processes. By supplementing the genome with enzyme genes, new or modified enzymes can be introduced into an organism, or genes of endogenous enzymes can be expressed in an increased or reduced form, thereby establishing new metabolic pathways in an organism or strengthening or weakening existing metabolic pathways. Metabolic Engineering is that the organism produces a metabolite either newly or a cellular metabolite with increased yield. Metabolic Engineering-Verfahrenno starting materials specific to the metabolite, such as an enzyme substrate, such as OAS in the present invention, are used, but only a nutrient medium, also referred to as a culture medium, which is required for the growth of the organism in question and is composed of a C source (e.g. glucose), an N source (e.g. an ammonium salt or a complex amino acid mixture such as peptone or yeast extract) and other salts required for growth. Such nutrient media are known to the person skilled in the art from microbiological practice. b) by biotransformation Biotransformation is defined as the conversion of one or more reactants into a product under enzymatic catalysis, wherein the enzyme substrate is added to a reaction mixture with the enzyme. In the reaction mixture, the added enzyme substrate, such as OAS or L-cysteic acid in the present invention, is enzymatically converted.In the present invention, this is done for OAS by a CysM enzyme from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of a salt of sulfurous acid, according to equation (7), and for L-cysteic acid by an enzyme selected from the class of cysteine ​​sulfinic acid decarboxylases (CSAD, EC 4.1.1.29), according to equation (5). The reactant(s) can originate from chemical or biotechnological production. The OAS used in the process according to the invention can originate, for example, from chemical synthesis or from biotechnological production by fermentation of a production strain. The L-cysteic acid used in the process can originate, for example, from chemical synthesis or from biotechnological production by biotransformation of OAS. The enzyme(s) used for the enzymatic catalysis originate either from biotechnological production by culturing production strains, e.g.by fermentation, or biological material containing the enzyme(s) is used (e.g., plants, fungi, algae, animal organs). The biomass from the cultivation of the production strain or the biological material can be used directly, or the enzyme can be isolated from it, depending on the requirements of the biotransformation. The enzymes CysM and CSADcc used in the process according to the invention originate from biotechnological production by fermentation of a production strain.

[0023] In the context of the present invention, a reaction mixture is defined as a mixture of reactant (starting material), enzyme and optionally other reactants, in which the reactant is converted into a product.

[0024] The yield of the reaction within the meaning of the invention is defined as the amount of reactant used that is converted to product under reaction conditions. The yield can be expressed as an absolute amount (g or mmol), as a volume yield in absolute, volume-related amount of product (mM or g / L), or as a relative yield of product as a percentage of the reactant used (taking into account the molecular weights of the reactant and the product), also referred to as a percentage yield. Fermentation is a process step for the production (cultivation) of cell cultures on an industrial scale, in which a preferably microbial production strain is induced to grow under defined conditions of culture medium, temperature, pH, oxygen supply, and medium mixing.The aim of fermentation is, depending on the configuration (genetic makeup) of the production strain, the production of a protein / enzyme or a metabolite, in each case with the highest possible yield for further use. The components of the process according to the invention, OAS, OAS sulfhydrylase CysM and cysteine ​​sulfinic acid decarboxylase, can be produced by fermentation. The end product of the fermentation is a fermenter broth consisting of the biomass of the cells of the production strain (fermenter cells) and the fermentation medium freed of biomass (fermentation supernatant), which was formed during the fermentation from the culture medium and the metabolites secreted by the fermenter cells. The target products of the fermentation can be found in the fermenter cells or in the fermentation medium. For example, OAS is found in the fermentation medium, while the enzymes OAS sulfhydrylase CysM and CSADcc are found in the. Find fermenter cells.

[0025] The open reading frame (ORF, synonymous with cds, coding sequence) is the region of DNA or RNA that begins with a start codon and ends with a stop codon and encodes the amino acid sequence of a protein. The ORF is also called the coding region or structural gene.

[0026] A gene is the DNA segment that contains all the basic information needed to produce biologically active RNA. A gene contains the DNA segment from which a single-stranded RNA copy is produced through transcription, as well as the expression signals involved in regulating this copying process. Expression signals include, for example, at least a promoter, a transcription start site, a translation start site, and a ribosome binding site (RBS). Further expression signals can include a terminator and one or more operators.

[0027] An mRNA, also called messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for building a protein. With an mRNA, the blueprint for a specific protein is available in a cell. The mRNA molecule carries the message necessary for protein construction from the genetic information (DNA) to the protein-building ribosomes. In a cell, it is formed as a transcript of a section of DNA belonging to a gene. The genetic information stored in the DNA is not altered in the process.

[0028] Genes of eukaryotic organisms are predominantly so-called mosaic genes and, in contrast to prokaryotic genes, also contain non-coding sections, so-called introns. intr agenic regi ounces ). Coding sequences, so-called exons (English ex pressed regi ounces ) are DNA segments of a eukaryotic gene that, after transcription into RNA, are translated by the ribosomes into the amino acid sequence of a protein. The introns are spliced ​​from the primary transcript after transcription of DNA into RNA. The protein-coding RNA freed of introns is called messenger RNA (mRNA), also known as "mature" mRNA. This undergoes further modifications such as capping and polyadenylation. The coding region of the mature mRNA is then translated into the protein sequence. If a eukaryotic gene with an exon / intron structure is to be expressed in prokaryotic organisms, it is necessary to retranslate the protein sequence or the coding region of the mature mRNA into intron-free DNA, since the processing of the exon / intron structure does not occur in prokaryotes. If, within the scope of this invention, from the protein sequence or from the gene sequences of the mRNSThis is precisely the process of backtranslation. It is preferable that the backtranslation of the protein sequence or mRNA sequence into DNA sequence be accompanied by sequence optimization, i.e., adaptation to the codon usage of the corresponding prokaryote (codon optimization).

[0029] A gene construct is a DNA molecule in which a gene is linked to other genetic elements (e.g., promoter, terminator, selection marker, origin of replication). A gene construct within the scope of the invention is a circular DNA molecule and is referred to as a plasmid, vector, or expression vector. The genetic elements of the gene construct cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the gene.

[0030] The abbreviation WT (Wt) stands for wild type. A wild-type gene is the form of a gene that has arisen naturally through evolution and is present in the wild-type genome. The DNA sequence of Wt genes is publicly available in databases such as NCBI. Biotransformation 1:

[0031] A prerequisite for the process according to the invention for producing taurine is the availability of OAS. Chemical processes for producing OAS are conceivable, e.g., by acetylation of L-serine, which is expensive due to the high price of L-serine, or the production of the racemate O-acetyl-D / L-serine, which can be used directly, or OAS is previously obtained from the racemate, e.g., by racemate resolution. During direct acetylation, N-acetyl-L-serine (NAS) can be formed as a by-product, e.g., by non-selective acetylation at the hydroxy or amino group of L-serine or the known rearrangement of OAS to NAS at neutral to alkaline pH values ​​(Tai et al. (1995), Biochemistry 34: 12311-12322), which reduces yields or requires the prior introduction of a protecting group at the amino group of L-serine. For this reason, direct acetylation of L-serine is not practical for an economical process.

[0032] The biotechnological production of OAS is known. This involves the use of organisms that exhibit deregulated cysteine ​​metabolism and therefore provide high levels of OAS. A fermentative process for the production of OAS is disclosed in EP 1 233 067 B1 (Wacker) and described in Example 1 of the present invention.

[0033] Preference is given to the biotechnological production of OAS, particularly preferably using the Escherichia coli (E. coli) strain W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1), deposited under the Budapest Treaty at the DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 13495.

[0034] An advantage of the present invention is that an OAS-containing fermenter broth, such as that obtained, for example, from a fermentation conducted according to EP 1 233 067, can be used as a fermentation supernatant after separation of the particulate biomass, such as by centrifugation, directly in the process according to the invention as an OAS source without further processing, purification, or isolation steps such as extraction, adsorption, ion exchange chromatography, precipitation, or crystallization. This procedure is particularly economical and avoids the isolation of an unstable compound.

[0035] The skilled person can use isotope analysis to determine whether a substance, such as OAS or L-cysteic acid, that they intend to use in the process originates from chemical or fermentative production. An isotope analysis method suitable for distinguishing between the two is described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527 and is based on determining the isotope ratios for, for example, C or N, which vary depending on whether a product originates from chemical (petroleum-based) or fermentative (plant-based raw materials) production.

[0036] OAS sulfhydrylases have been isolated from various plants and microorganisms. E. coliFor example, two OAS sulfhydrylase enzymes exist, designated CysK and CysM. The corresponding genes are also known and are designated cysK and cysM, respectively. CysM OAS sulfhydrylases within the meaning of the present invention are characterized by their ability to catalyze the synthesis of the proteinogenic amino acid L-cysteine ​​from OAS according to equation (8), with sulfide serving as the nucleophile in this case. OAS + S 2 − − > L − Cystein + Acetat

[0037] Although both enzymes have a very similar reaction mechanism and are involved in the biosynthesis of L-cysteine, CysM, in contrast to CysK, has a variable substrate spectrum with respect to the nucleophile that can react with OAS according to equation (6). For example, CysM, unlike CysK, is known to be able to catalyze the reaction of OAS with thiosulfate to form S-sulfocysteine ​​(CAS number 1637-71-4). This reaction plays an important role in the growth of bacteria with thiosulfate as the sole sulfur source. Furthermore, the use of CysM for the production of non-proteinogenic amino acids is known from EP 1 247 869 B1 (Wacker).

[0038] Preferably, the method is characterized in that the OAS sulfhydrylase CysM is a bacterial enzyme, particularly preferably CysM of the strain E. coli is.

[0039] The process is preferably characterized in that the OAS sulfhydrylase CysM originates from fermentative production, particularly preferably from the fermentation of a E. coli strain and particularly preferably from the fermentation of the strain E. coli DH5α / pFL145.

[0040] A fermentative biotechnological production of CysM with the strain E. coli DH5α / pFL145 is disclosed in Example 2. The production strain consists of a host strain, as in this case E. coli DH5α and a gene construct suitable for the expression of OAS sulfhydrylase, preferably the gene construct pFL145. The host strain and gene construct, as well as the preparation of the production strain, are described in EP 1 247 869 B1. The production strain is deposited under the Budapest Treaty at the DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Brauschweig) under the number DSM 14088.

[0041] The fermenter broth obtained by fermentation of the production strain consists of the fermenter cells containing the OAS sulfhydrylase and the fermentation medium (fermentation supernatant). In the process according to the invention, either the non-further processed fermenter broth can be used or a suspension of the fermenter cells (resuspension of the fermenter cells), e.g., in a buffer, after their isolation from the fermenter broth, e.g., by centrifugation or filtration. The production of CysM-containing, resuspended fermenter cells of the strain E . coli DH5α / pFL145 is described in Example 2.

[0042] It is also conceivable that the OAS sulfhydrylase is used in the form of a cell homogenate after mechanical disruption of the fermenter cells or in the form of chemically permeabilized cells (e.g., with chloroform), or as a cell extract after separation of particulate components from the cell homogenate, or as a chromatographically purified enzyme, for example. The use of the OAS sulfhydrylase is preferred as fermenter broth that has not been further processed, as a cell suspension of resuspended fermenter cells, or as a cell homogenate after mechanical disruption of resuspended fermenter cells, or in the form of chemically permeabilized cells (e.g., with chloroform). Particular preference is given to using the OAS sulfhydrylase in the form of resuspended fermenter cells or as a cell homogenate. The use of resuspended fermenter cells of the OAS sulfhydrylase production strain is particularly preferred.

[0043] In the process according to the invention, OAS reacts with sulfite under catalysis of an OAS sulfhydrylase CysM according to equation (7) to form L-cysteic acid.

[0044] Sulfurous acid forms a multitude of chemical species coexisting in reversible equilibria, whose respective suitability as nucleophiles in the biotransformation according to the invention was unpredictable. Thus, it is known that sulfurous acid (H 2 SO 3 ) is the aqueous solution of gaseous SO 2 and, as a dibasic acid, exists in different equilibria, depending on the pH of the aqueous solution, whose species also have different suitability as nucleophiles. The following equilibria (9) to (13) are known: SO 2 gasförgmig < − > SO 2 gelöst SO 2 gelöst + H 2 O < − > H 2 SO 3 H 2 SO 3 < − > HSO 3 − + H + HSO 3 − < − > SO 3 2 − + H + 2 HSO 3 − < − > S 2 O 5 2 − + H 2 O

[0045] Sulfurous acid and its salts are used as preservatives in the food industry because of their antimicrobial properties. This means that sulfurous acid and its salts can kill microorganisms by inactivating the enzymes necessary for the microorganism's viability.

[0046] Therefore, the skilled person would expect that the CysM enzyme would also be inactivated when using sulphurous acid or its salts and that L-cysteic acid would not be accessible by the process disclosed in EP 1 247 869 B1.

[0047] For the reasons mentioned above, it was surprising to the person skilled in the art that L-cysteic acid can be produced as a starting compound for a biotechnological process for the production of taurine using sulfite and OAS in a biotransformation.

[0048] In principle, all conceivable salts of sulfurous acid are suitable for the reaction. The process is preferably characterized in that Na 2 SO 3 , K 2 SO 3 , (NH 4 ) 2 SO 3 , NaHSO 3 (or its anhydride Na 2 S 2 O 5 ) or KHSO 3 is used as the salt of a sulfurous acid. Particular preference is given to using Na 2 SO 3 , NaHSO 3 (or its anhydride Na 2 S 2 O 5 ) or (NH 4 ) 2 SO 3 as the salt of a sulfurous acid, and especially preferably Na 2 SO 3 or NaHSO 3 (or its anhydride Na 2 S 2 O 5 ).

[0049] It is conceivable to use gaseous sulfur dioxide, the anhydride of sulfurous acid, which can be introduced into the reaction mixture, where it hydrates to the sulfurous acid H 2 SO 3 and, depending on the pH, is in equilibrium with the deprotonated forms HSO 3 -< and SO 3 2-<.

[0050] The process is preferably characterized in that the concentration of the sulfurous acid salt is at least equimolar to OAS. Particularly preferably, the sulfurous acid salt is present in at least a 1.5-fold molar excess, particularly preferably in at least a five-fold molar excess, relative to OAS.

[0051] In a particularly preferred embodiment, the process for producing taurine is characterized in that both the OAS sulfhydrylase CysM and OAS are produced by fermentation.

[0052] OAS, the starting material in the biotransformation process according to the invention, isomerizes to N-acetyl-L-serine (NAS) at pH values ​​above approximately 7 and is then no longer suitable for reaction with sulfite to form L-cysteic acid. The mechanism of the reaction was investigated in Tai et al. (1995), Biochemistry 34: 12311-12322 and is based on an intramolecular, nucleophilic attack of the deprotonated amino group at the carbonyl carbon of the acyl residue. This reaction is suppressed with decreasing pH, so that the compound is stable, for example, at pH 4.0.

[0053] The biotransformation process according to the invention is thus preferably characterized in that the reaction is carried out under pH conditions which minimize the isomerization of OAS to NAS.

[0054] The reaction temperature of biotransformation 1 is preferably chosen between 5°C and 70°C. A reaction temperature between 10°C and 60°C is preferred, particularly preferably between 15°C and 50°C, and especially preferably between 20°C and 40°C.

[0055] The OAS concentration in the mixture is at least 10 g / L and preferably at least 40 g / L.

[0056] In the biotransformation of OAS, the molar yield of L-cysteic acid based on the molar amount of OAS used is preferably at least 60%, particularly preferably at least 70% and especially preferably at least 80%.

[0057] In a further preferred embodiment of biotransformation process 1, the substrate OAS is added from a receiver in a so-called feed process to the reaction mixture comprising OAS sulfhydrylase CysM and sulfite (Example 5). To avoid the isomerization of OAS to NAS, the OAS-containing receiver is preferably set to a pH of ≤6.5, more preferably to a pH of ≤6.0, and especially preferably to a pH of ≤5.5. At the same time, the pH in the reaction mixture is adjusted to favor the reaction to L-cysteic acid, preferably pH ≤7.5, more preferably pH ≤7.0, and especially preferably pH ≤6.5.

[0058] According to equation (7), acetic acid is released in stoichiometric amounts during the reaction of OAS to L-cysteic acid, which can lead to a reduction in the pH value in the mixture during the reaction. Since a pH that is too low impairs the activity of OAS sulfhydrylase, an excessive drop in the pH value should preferably be prevented. This can be achieved passively by adding a suitable highly concentrated buffer to the mixture or, according to the invention, actively by means of a measuring and control unit. According to the invention, active pH control is achieved by a measuring and control unit, as disclosed in Example 5, which, if the pH value deviates from the target value, resets the desired pH value by adding an alkali or acid (so-called pH-stat method).

[0059] Biotransformation 1 can be operated discontinuously or continuously. In discontinuous operation (batch operation), all reactants are added to the mixture during the course of the reaction, and the mixture is processed after completion of the reaction. In continuous operation, OAS, CysM enzyme, and a salt of sulfurous acid are continuously added during the reaction, and a solution containing the product L-cysteic acid is simultaneously withdrawn from the mixture. A steady state is established in which the reactants are added such that they can react to form the product L-cysteic acid during the residence time in the reaction vessel. A process for the continuous production of unnatural amino acids is disclosed, for example, in EP 1 247 869 B1 (Wacker). Biotransformation 1 is preferably a discontinuous process. Biotransformation 2:

[0060] The process for producing taurine involves the production of L-cysteic acid in biotransformation 1 followed by the decarboxylation of L-cysteic acid to taurine in biotransformation 2.

[0061] The decarboxylation of L-cysteic acid to taurine can be carried out chemically or, according to the invention, enzymatically catalyzed, in a biotransformation. Thermal decarboxylation at high temperatures under metal catalysis is known, although not considered sustainable. However, this method suffers from the fact that it is energy-intensive and produces a high proportion of byproducts.

[0062] The process is preferably characterized in that an enzyme from the class of L-cysteine ​​sulfinic acid decarboxylases (CSAD, EC 4.1.1.29), aspartate 1-decarboxylases (EC 4.1.1.11) or glutamate decarboxylases (EC 4.1.1.15) is used for the decarboxylation of L-cysteine ​​acid to taurine.

[0063] For the enzymatically catalyzed decarboxylation of L-cysteine ​​acid to taurine according to the invention, enzymes from the class of L-cysteine ​​sulfinic acid decarboxylases (CSAD, EC 4.1.1.29) are particularly preferred. CSAD enzymes are known to decarboxylate L-cysteine ​​sulfinic acid to hypotaurine according to equation (3). To varying degrees, these enzymes are also capable of decarboxylating L-cysteine ​​acid as a substrate to taurine according to equation (5). As disclosed in Examples 7 to 11, for example, the CSADcc enzyme from Cyprinus carpio (carp) capable of decarboxylating L-cysteine ​​acid to taurine.

[0064] Enzymes of class EC 4.1.1.29 (CSAD) are found mainly in metazoans (multicellular animals), including mammals. Enzymes with CSAD activity can also be found in unicellular organisms, such as algae from the genus Synechoccocus,also in bacteria or fungi. The process according to the invention for producing taurine is preferably characterized in that CSAD enzymes of class EC 4.1.1.29 from mammals, selected from humans ( Homo sapiens ) , Beef ( Taurus boss ) , Rat (Rattus norvegicus) or mouse (Mus musculus) as well as from fish such as carp ( Cyprinus carpio ), particularly preferably enzymes from humans ( Homo sapiens ) , the rat ( Rattus norvegicus ) or the carp ( Cyprinus carpio ) .

[0065] Particularly preferred is the CSAD enzyme from carp (CSADcc, Cyprinus carpio ) . In the examples used is a CSADcc enzyme derived from the protein sequence of the Wt-CSAD enzyme from Cyprinus carpio,with a DNA sequence of the cds as disclosed in SEQ ID NO: 1, nt 31 - 1530 and designated CSADcc-cds, encoding a protein with the amino acid sequence in SEQ ID NO: 2 and designated CSADcc. The DNA sequence underlying the Wt-CSADcc amino acid sequence is accessible in the NCBI (National Center for Biotechnology Information) database under Genbank Sequence ID: AB220585.1 (cds: nt 82 - 1584). From the corresponding Wt-CSADcc amino acid sequence, a E. coli A codon-optimized CSADcc-cds DNA sequence was derived (SEQ ID NO: 1, nt 31-1530), which encodes the identical amino acid sequence. Publicly available software programs are available for codon optimization, such as the Eurofins Genomics GENEius software used in Example 6. The DNA from SEQ ID NO: 1 was synthetically produced in a known manner, as offered by service providers such as Eurofins Genomics.

[0066] Particularly preferably, the L-cysteine ​​sulfinic acid decarboxylase is SEQ ID NO: 2 or a sequence homologous to this sequence.

[0067] Homologous sequences are understood to mean that the DNA or amino acid sequences are at least 80%, preferably at least 90% and particularly preferably at least 95% identical, wherein each change in the homologous sequence is selected from insertion, addition, deletion and substitution of one or more nucleotides or amino acids.

[0068] The degree of DNA identity is determined by the program "nucleotide blast", found on the page http: / / blast.ncbi.nlm.nih.gov / , which is based on the blastn algorithm. The preset parameters were used as algorithm parameters for aligning two or more nucleotide sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding preset scoring parameters are: Match / Mismatch Scores = 1,-2; Gap Costs = Linear.

[0069] For the comparison of protein sequences, the program "protein blast" is used, on the page http: / / blast.ncbi.nlm.nih.gov / ,This program uses the blastp algorithm. The preset parameters were used as algorithm parameters for aligning two or more protein sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. The preset scoring parameters are: Matrix = BLOSUM62; Gap Costs = Existence: 11; Extension: 1; Compositional adjustments = Conditional compositional score matrix adjustment.

[0070] Preferably, the process is characterized in that the L-cysteine ​​sulfinic acid decarboxylase originates from fermentative production. A corresponding recombinant production of the CSADcc enzyme in a E. coliProduction strain is disclosed in Example 6. The CSADcc-cds is cloned in a known manner into an expression vector, e.g. the vector pKKj, and the gene construct pCSADcc-pKKj is produced ( Figs. 1 To produce a production strain, the gene construct pCSADcc-pKKj is also introduced into a E. coli Host strain, e.g. the strain E. coli JM105, transformed and the resulting production strain E. coli JM105 x pCSADcc-pKKj is also used in a known manner to produce the CSADcc enzyme. The CSADcc enzyme can be produced for laboratory purposes on a shake flask scale or by fermentation (Example 6).

[0071] CSAD enzymes contain pyridoxal phosphate (PLP, CAS No. 54-47-7) as a cofactor. Supplementing the culture medium or biotransformation approaches for converting L-cysteic acid to taurine with PLP therefore offers an opportunity for process improvement. Since PLP belongs to the B6 vitamin family, supplementation with other members of the vitamin B6 family, such as pyridoxine (CAS No. 65-23-6), pyridoxal (CAS No. 66-72-8), or pyridoxamine (CAS No. 85-87-0), is also suitable for process improvement. The biotransformation process for converting L-cysteic acid to taurine is preferably characterized in that it is carried out in the presence of ≥20 mg / L, more preferably ≥10 mg / L, and especially preferably ≥4 mg / L of PLP.

[0072] The CSAD enzyme, preferably CSADcc, obtained by culture in a shake flask or by fermentation can be used in the process according to the invention either as a fermenter broth that has not been further processed or as a cell suspension after re-isolation of the cells, e.g., by centrifugation and resuspension of the fermenter cells, e.g., in a buffer (resuspension of the fermenter cells). Furthermore, the CSAD enzyme, preferably CSADcc, can be used in the form of a cell homogenate after mechanical disruption of the resuspended fermenter cells or in the form of chemically permeabilized cells (e.g., with chloroform), or as a cell extract after separation of particulate components from the cell homogenate, or as a chromatographically purified enzyme, e.g., as a cell extract.

[0073] The CSAD enzyme (CSADcc) is preferably used as unprocessed fermenter broth, as resuspended fermenter cells, or as a cell homogenate after mechanical disruption of the resuspended fermenter cells. The CSAD enzyme is particularly preferred as resuspended fermenter cells or as a cell homogenate after mechanical disruption of the resuspended fermenter cells. The CSAD enzyme is particularly preferred in the form of resuspended fermenter cells.

[0074] The biotransformation of L-cysteic acid to taurine by the CSAD enzyme, preferably CSADcc, is preferably carried out under pH and temperature conditions that enable efficient decarboxylation of L-cysteic acid to taurine. The preferred pH range at which biotransformation 2 is carried out is between pH 5.0 and 9.0, more preferably between pH 6.0 and 8.5, and especially preferably between pH 6.5 and 8.0.

[0075] Biotransformation 2 is carried out at a temperature preferably <70°C, preferably <60°C, particularly preferably <50°C and especially preferably <40°C.

[0076] The molar yield of taurine from the biotransformation of L-cysteic acid in biotransformation 2 is preferably at least 60%, more preferably at least 80% and especially preferably at least 90%.

[0077] The biotransformation for the production of taurine from L-cysteic acid can be carried out discontinuously or continuously. In discontinuous operation (batch operation), all reactants are added to the reaction mixture during the reaction, and the mixture is processed after completion of the reaction. In continuous operation, the CSAD enzyme is introduced as the stationary phase, e.g., in a membrane reactor or immobilized on a support, and the substrate L-cysteic acid is added as the mobile phase. The contact time of the mobile phase with the stationary phase is adjusted so that the substrate L-cysteic acid can react to form the product taurine. Discontinuous operation (batch operation) is preferred.

[0078] The preferred process for the production of taurine from OAS, comprising biotransformation 1 and biotransformation 2, is characterized by comprising the following steps: a) OAS is produced by fermentation, b) the enzymes from the class of OAS sulfhydrylases (EC 4.2.99.8) CysM and the class of cysteine ​​sulfinic acid decarboxylases (EC 4.1.1.29), such as CSADcc, are produced by fermentation, c) OAS and sulfurous acid, or a salt of sulfurous acid, react under enzymatic catalysis of the OAS sulfhydrylase from point b to form L-cysteic acid and d) L-cysteic acid from point c is decarboxylated by the CSAD enzyme from point b to form taurine.

[0079] Particularly preferred is the process comprising steps a, b, c and d, characterized in that in biotransformation 1, OAS is reacted with Na 2 SO 3 or the salt of its anhydride Na 2 S 2 O 5 in a CysM-catalyzed reaction to form L-cysteic acid, and in biotransformation 2, the L-cysteic acid from biotransformation 1 is decarboxylated to taurine in a CSAD-catalyzed biotransformation according to equation (5). These process steps are disclosed in Examples 7 to 10. OAS from biotransformation 1 can be produced synthetically or by culturing a production strain, as described in Example 1 of the present invention.

[0080] Production of OAS by cultivation of a production strain. The CysM enzyme of biotransformation 1 can originate from the fermentation of a production strain, as described in Example 2. The CSAD enzyme used in biotransformation 2 can originate from the cultivation of a production strain, preferably the CSADcc enzyme from the cultivation of the strain E. coli JM105 x pCSADcc-pKKj, as described in Example 6. L-cysteic acid from Biotransformation 1 can be used in Biotransformation 2 without further processing, as described in Examples 8 to 10, or after prior processing. Various methods are known to the person skilled in the art, such as filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization. The use of L-cysteic acid from Biotransformation 1 without further processing in Biotransformation 2 is preferred.

[0081] The process for producing taurine combines, in a simple and efficient manner, the enzymatic production of L-cysteic acid from OAS and a salt of sulfurous acid, also referred to as sulfite, according to equation (7) (biotransformation 1) with the enzymatic decarboxylation of L-cysteic acid to taurine, according to equation (5) (biotransformation 2). This means that the present invention comprises a two-step biotransformation process combining biotransformation 1 with biotransformation 2 for producing taurine from OAS.

[0082] Preferably, the process steps (biotransformation 1 and biotransformation 2) for producing taurine are carried out sequentially, i.e., one after the other. While the process comprises steps ad in the preferred embodiment already described, in an alternative preferred embodiment it is characterized in that all process steps take place in one reaction batch. When all process steps take place in one reaction batch, it is also referred to as a one-pot process or one-pot reaction. This has the advantage that all reactants for producing taurine are already present in the reaction batch or can simply be added. Carrying out the process in one reaction batch is particularly economically advantageous.

[0083] Example 11 of the present invention discloses that the process steps can be carried out in a single batch (one-pot process), whereby OAS is reacted with a sulfite (salt of sulfurous acid) in the presence of the enzymes CysM and CSADcc. The first reaction produces L-cysteic acid according to equation (7), which is decarboxylated in situ by CSADcc to taurine according to equation (5).

[0084] The product distribution of L-cysteic acid and taurine from the reaction of OAS in the one-pot process is determined by the activity of CysM relative to CSADcc. With sufficient dosage of CSADcc, the L-cysteic acid formed from OAS can be quantitatively converted to taurine. A process in which the OAS used is converted to L-cysteic acid and taurine is preferred. The total molar yield of L-cysteic acid and taurine, based on the molar amount of OAS used, is preferably more than 60%, particularly preferably more than 70%, and especially preferably more than 80%. Based on the molar amount of OAS used, the molar yield of taurine is preferably more than 25%, particularly preferably more than 50%, and especially preferably more than 80%.

[0085] With regard to a one-pot process, it is conceivable that the expression of the genes for OAS sulfhydrylase and L-cysteine ​​sulfinic acid decarboxylase can occur together in one strain and that the cells from the culture of this strain are reacted with OAS in a biotransformation in the presence of a sulfite, with taurine being formed as the end product.

[0086] It is also conceivable that, in the sense of a metabolic engineering approach, the expression of the genes for an OAS sulfhydrylase and an L-cysteine ​​sulfinic acid decarboxylase takes place in the OAS production strain and taurine is produced by cultivating such a production strain in the presence of a sulfite (salt of sulfurous acid).

[0087] The figure shows the plasmid used in the examples. Figs. 1 : pCSADcc-pKKj Abbreviations used in the figure:

[0088] AmpR: Gene that confers resistance to ampicillin (ß-lactamase) Ori: Origin of replication Ptac: tac promoter EcoRI: Site for the restriction enzyme EcoRI HindIII: Site for the restriction enzyme HindIII CSADcc: CSAD (cysteine ​​sulfinic acid decarboxylase) C. carpio CDs

[0089] The invention is further illustrated by the following examples: Example 1: Production of OAS

[0090] The strain disclosed in EP 1 233 067 B1 (Wacker) was used E. coliW3110 / pACYC-cysEX-GAPDH-ORF306, deposited under the Budapest Treaty with the DSMZ Deutsche Sammlung für Mikroorganismen und Zellkulturen GmbH (Braunschweig) under the number DSM 13495. OAS was produced by fermentation as described in EP 1 233 067 B1. At the end of fermentation, a pH of 4.5 was adjusted with 21% (v / v) phosphoric acid to stabilize OAS. The cells were separated by centrifugation for 10 min at 4000 rpm (Heraeus Megafuge 1.0 R). The OAS content in the fermentation supernatant, determined by HPLC, was 15.3 g / L.

[0091] HPLC analysis of OAS, L-cysteic acid, and taurine: For the quantitative determination of the compounds analyzed in the examples, an HPLC method calibrated for OAS, L-cysteic acid, and taurine was used. All reference substances used for calibration were commercially available (Sigma-Aldrich). An Agilent HPLC instrument, Model 1260 Infinity II, was used. It was equipped with a pre-column derivatization with o-phthalaldehyde (OPA derivatization) from the same manufacturer, a process known from the analysis of amino acids. The HPLC instrument was equipped with a fluorescence detector for the detection of the OPA-derivatized products OAS, L-cysteic acid, and taurine. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. Furthermore, an Accucore™< aQ column from Thermo Scientific™<, length 100 mm, inner diameter 4.6 mm, particle size 2.6 µm, heated to 40°C in a column oven was used.Mobile phase A: 25 mM Na phosphate, pH 6.0. Mobile phase B: methanol. The separation was performed in gradient mode: 0–25 min, 10% mobile phase B to 60% mobile phase B, followed by 2 min of 60% mobile phase B to 100% mobile phase B, followed by another 2 min of 100% mobile phase B, at a flow rate of 0.5 ml / min. Retention time of L-cysteic acid: 3.2 min. Retention time of taurine: 14.8 min. Retention time of OAS: 17.0 min. Example 2: Production of the enzyme CysM

[0092] The strain disclosed in EP 1 247 869 B1 (Wacker) was used E. coli DH5α / pFL145, deposited under the Budapest Treaty at the DSMZ German Collection of Microorganisms and Cell Cultures GmbH (Brauschweig) under the number DSM 14088. CysM enzyme was produced by both shake flask cultivation and fermentation. A) Cultivation in shake flask: From the strain E. coliA preculture of DH5α / pFL145 was established in LBamp medium (10 g / L tryptone (GIBCO™), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 100 mg / L ampicillin (Sigma-Aldrich)) and grown overnight at 37°C and 120 rpm. A 25 ml preculture was used as the inoculum for a main culture of 250 ml LBamp medium (1 L baffled Erlenmeyer flask). The main culture was shaken at 30°C and 110 rpm. After 4 h, a cell density OD 600 of 1.0 / ml was reached (OD 600 : photometric determination of the cell density / ml cell suspension by determining the absorbance at 600 nm; Genesys™< 10S UV-Vis spectrophotometer from Thermo Scientific™< ). The inducer tetracycline (Sigma-Aldrich, 3 mg / L final concentration) was then added, and cultivation continued for another 20 h at 30°C and 110 rpm. After completion of cultivation, the cell density OD 600 was 3 / ml. B) Fermentative production of CysM with the strain E. coliDH5α / pFL145 is disclosed in EP 1 247 869 B1. The cells from the fermentation were separated by centrifugation at 4000 rpm for 10 min (Heraeus Megafuge 1.0 R) and suspended in KPi6.5 buffer (0.1 M potassium phosphate, pH 6.5) so that the cell density was OD 600 90 / ml.

[0093] Cells from shake flask culture or fermentation were isolated for further use by centrifugation (10 min at 15,000 rpm, Sorvall RC5C centrifuge equipped with an SS34 rotor). For further use to prepare a cell homogenate, as described below, the cell pellet was resuspended in KPi6.5 buffer as a cell suspension. To prepare the cell suspension, sufficient KPi6.5 buffer was used to achieve a cell density of OD 600 of 30 / ml: for example, 50 ml of cells from shake flask culture with an OD 600 of 3 / ml were centrifuged and resuspended in 5 ml of KPi6.5 buffer (10-fold concentration), or 1 ml of cells from fermentation with an OD 600 of 90 / ml were resuspended in 3 ml of KPi6.5 buffer (3-fold dilution).

[0094] In this way, the cells of the strain isolated and resuspended from the fermenter broth E. coliDH5α / pFL145, which were subsequently used as OAS sulfhydrylase CysM in the process according to the invention.

[0095] The FastPrep-24™<5G cell homogenizer from MP Biomedicals was used to prepare a cell homogenate. 1 ml of cell suspension in KPi6.5 buffer with a cell density of OD 600 30 / ml was disrupted in 1.5 ml tubes pre-filled by the manufacturer with glass beads ("Lysing Matrix B") (3 x 20 sec at a shaking frequency of 6000 rpm with a 30 sec break between each interval). The resulting cell homogenate was used directly as OAS sulfhydrylase (CysM enzyme) in the method according to the invention or used to prepare a cell extract.

[0096] To prepare a cell extract, the resulting cell homogenate was centrifuged (10 min 15000 rpm, Sorvall centrifuge RC5C, equipped with an SS34 rotor) and the supernatant was labeled cell extract and used as OAS sulfhydrylase (CysM enzyme) in the method according to the invention or further used to determine the CysM enzyme activity.

[0097] The protein content of the cell extract was determined using a Qubit 3.0 fluorometer from Thermo Fisher Scientific using the Qubit® Protein Assay Kit according to the manufacturer's instructions. The protein content of the cell extract from the shake flask culture was 5.3 mg / ml. The protein content of the cell extract from the fermentation was 4.0 mg / ml.

[0098] CysM enzyme activity was determined as described in EP 1 247 869 B1 (Wacker). For this purpose, OAS (Sigma-Aldrich) was used in the presence of Na 2 S and cell extract from the cultured strain E. coliDH5α / pFL145 was incubated at 37°C. The assay mixture (0.4 ml final volume) in KPi6.5 buffer contained 10 mM OAS (added from a 200 mM stock solution in 500 mM sodium succinate buffer pH 5.5), 10 mM sodium sulfide (Na 2 S), and 5 µl of cell extract containing CysM. The cysteine ​​produced in the CysM reaction was determined with ninhydrin (Sigma-Aldrich) according to Gaitonde (1967), Biochem. J. 104: 627-633. The CysM enzyme activity in the cell extract from the culture of the strain E. coliThe DH5α / pFL145 concentration in the shake flask was 57.1 U / ml. Since the cells from the shake flask culture (OD 600 of 3 / ml) were concentrated 10-fold to prepare the cell extract, the enzyme activity in the cells from the shake flask culture was 5.7 U / ml. The CysM enzyme activity in the cell extract after fermentation of the E. coli DH5α / pFL145 strain was 58.1 U / ml. Since the cells from the fermentation (OD 600 of 90 / ml) were diluted to an OD 600 of 30 / ml to prepare the cell extract, the enzyme activity in the concentrated (OD 600 of 90 / ml) cell suspension of the fermenter cells was 174.4 U / ml.

[0099] The specific CysM enzyme activity of the cell extract from the culture of the strain E. coli DH5α / pFL145 in the shake flask was 10.8 U / mg protein. The specific CysM enzyme activity of the cell extract after fermentation of the strain E. coliDH5α / pFL145 was 14.5 U / mg. Assuming that CysM activity was completely released from the cells during cell extract preparation, the CysM enzyme activity determined in the cell extracts was equated with the enzyme activity contained in CysM cell suspensions in the following examples.

[0100] 1 U / ml CysM enzyme activity is defined as the production of 1 µmol cysteine / min from OAS and Na 2 S under test conditions in 1 ml of cell extract (volume activity). The specific CysM enzyme activity in U / mg protein is obtained by dividing the volume activity of the cell extract (U / ml) by the protein concentration of the cell extract (mg / ml) and is defined as the CysM enzyme activity in U per 1 mg of protein in the cell extract. Example 3: Production of L-cysteic acid from commercially available OAS and Na 2 SO 3 using CysM produced in shake flask culture

[0101] Two approaches were carried out in parallel: Step 1:8.25 ml of NaPi6.5 buffer (50 mM Na phosphate, pH 6.5) were placed in a 100 ml Erlenmeyer flask, and 1 ml of a 0.2 M solution of Na 2 SO 3 in NaPi6.5 buffer, 0.4 ml of CysM cell extract from the shake flask culture (from Example 2A) with an activity of 57.1 U / ml (2.3 U / ml final concentration in the mixture), and 350 µl of a 0.2 M solution of OAS x HCl (Sigma-Aldrich) in 0.5 M Na succinate, pH 5.5, were added successively. The mixture volume was 10 ml. Step 2: The mixture (comparison mixture without Na 2 SO 3 ) had the same composition as mixture 1. Instead of the Na 2 SO 3 solution, mixture 2 contained 1 ml of NaPi6.5 buffer.

[0102] Both batches were incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 1 h and 3 h, 1 ml of each batch was incubated for 5 min at 80°C to stop the reaction, centrifuged, and the supernatant analyzed by HPLC. The amount of L-cysteic acid detected by HPLC is shown in Table 1. Table 1: Amount of L-cysteic acid detected by HPLC as a function of reaction time using commercially available OAS, Na 2 SO 3 and a CysM-containing cell extract. Time [h] Answer 1 with Na 2 SO 3 L-cysteine ​​acid [mg / L] Ansatz 2 without Na 2 SO 3 L-Cysteinic acid [mg / L] 0 0,0 0,0 1 78,0 0,0 3 95,8 0,0 Example 4: Production of L-cysteic acid from OAS-containing fermentation supernatant and Na 2 SO 3 using CysM produced in shake flask culture

[0103] In a 100 ml Erlenmeyer flask, 1 ml of cell culture supernatant from the fermentation of the strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 with an OAS content of 15.3 g / L (from Example 1) was added, and 6 ml of NaPi6.5 buffer, 1 ml of a 1 M solution of Na2SO3 in NaPi6.5 buffer, and 2 ml of CysM cell suspension from the shake flask culture (from Example 2A, cell density OD600 30 / ml; 57.1 U / ml CysM enzyme activity) were added successively. The reaction volume was 10 ml. The CysM enzyme activity in the reaction was 11.4 U / ml. The reaction was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 2 h, 1 ml of the reaction mixture was incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed by HPLC for OAS and L-cysteic acid content. The time course of the reaction is summarized in Table 2. Table 2: Amount of L-cysteic acid and OAS detected by HPLC using an OAS-containing cell culture supernatant, Na 2 SO 3 and a CysM-containing cell suspension. Time [h] OAS [mg / L] L-cysteine ​​acid [mg / L] 0 1530,0 0,0 2 0,0 1473,3 Example 5: Preparative production of L-cysteic acid by biotransformation of OAS at constant pH

[0104] A double-walled 0.5 L thermostattable glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and heated to 37°C. 50 ml of CysM-containing cell suspension in KPi6.5 buffer (OD 600 90 / ml, 8720 U CysM enzyme activity) from the fermentation of strain DH5α / pFL145 (from Example 2B) and 6.6 ml of a 400 g / L solution of Na2S2O5 (13.9 mmol, molecular weight 190.1 g / mol) in KPi6.5 buffer were added. In dissolved form, this corresponded to 27.8 mmol of NaHSO3 (1.78-fold molar excess over the 15.6 mmol of OAS added later). The mixture was stirred with a magnetic stirrer. The mixture was further equipped with a pH electrode (Mettler Toledo) connected to a pH control unit (Titrator TitroLine alpha, Schott) operated in pH-stat mode according to the manufacturer's instructions.Under pH-stat conditions, the pH in the reaction vessel was kept constant at the set pH 6.5 throughout the reaction run by adding 2 M NaOH from a burette connected to the control unit. 150 ml of OAS-containing cell culture supernatant (OAS content 15.3 g / L, 2.3 g; 15.64 mmol) from the fermentation of strain . E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) was added to the mixture from a receiver via a pump (Watson Marlow peristaltic pump 101U / R) at a flow rate of 0.35 ml / min.

[0105] The reaction time was 19 h. Since the reaction was carried out in an open reaction vessel, the reaction volume was 185 ml due to evaporation after the reaction. At 0.5 h, 3 h, and 19 h after the start of the reaction, 1 ml aliquots of the reaction were taken, and the L-cysteic acid content was analyzed by HPLC. The time course of the formation of L-cysteic acid is summarized in Table 3. After 19 h of reaction, the L-cysteic acid content in the reaction was 12,970 mg / L (76.65 mM), which, with a reaction volume of 185 ml, corresponded to an absolute molar yield of 14.18 mmol of L-cysteic acid. Based on the amount of OAS used (15.64 mmol), this corresponded to a yield of 90.1%. Table 3: Amount of L-cysteine ​​acid detected by HPLC as a function of reaction time using an OAS-containing fermentation supernatant, NaHSO 3 and a cell suspension of CysM-containing fermenter cells Time [h] L-cysteine ​​acid [mg / L] L-cysteine ​​acid [mM] 0,5 758,0 4,47 3 4244,0 25,08 19 12970,0 76,65 Example 6: Recombinant production of CSADcc from Cyprinus carpio (carp) in E. coli Vector pCSADcc-pKKj:

[0106] The cDNA gene (cDNA: complementary DNA, isolated from mRNA by reverse transcription) of cysteine ​​sulfinic acid decarboxylase (CSAD) from Cyprinus carpio (carp) was isolated by Honjoh et al. (2010), Amino Acids 38: 1173-1183 and the DNA sequence was disclosed in the NCBI (National Center for Biotechnology Information) database under Genbank Sequence ID: AB220585.1 (cds: nt 82 - 1584). From the corresponding amino acid sequence, a gene suitable for expression in E. coliA codon-optimized DNA sequence was derived (publicly available Eurofins Genomics GENEius software) and synthetically produced (Eurofins Genomics). The synthetically produced DNA had the sequence disclosed in SEQ ID NO: 1 and contained the cds of the gene, hereinafter referred to as CSADcc-cds (SEQ ID NO: 1, nt 31-1530), encoding a protein with the amino acid sequence disclosed in SEQ ID NO: 2 and referred to as CSADcc. For cloning purposes, the synthetically produced DNA contained an EcoRI site at the 5' end (SEQ ID NO: 1, nt 25 to 30) and a HindIII site at the 3' end (SEQ ID NO: 1, nt 1532 to 1537).

[0107] For the production of the vector pCSADcc-pKKj ( Fig. 1), the synthetically produced DNA was cleaved with EcoRI and HindIII and cloned in a known manner as an EcoRI / HindIII fragment into the EcoRI and HindIII-cleaved vector pKKj. The expression vector pKKj, disclosed in EP 2 670 837 A1 (Wacker), is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database under accession number M77749.1. Approximately 1.7 kb was removed from the 4.6 kb plasmid (bp 262 - 1947 of the DNA sequence disclosed in M77749.1), resulting in the 2.9 kb expression vector pKKj. Production strain E. coli JM105 x pCSADcc-pKKj:

[0108] For the expression of CSADcc-cds in E. coli The vector pCSADcc-pKKj was introduced into the strain E. coli K12 JM105 transformed. The strain E. coliK12 JM105 is commercially available under strain number DSM 3949 from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH. Clones of the transformation were selected on LBamp plates containing 10 g / l tryptone (GIBCO™), 5 g / l yeast extract (BD Biosciences), 5 g / l NaCl, 15 g / l agar, and 100 mg / L ampicillin (Sigma-Aldrich). One clone was selected for shake flask cultivation and fermentation. The CSADcc-producing strain was designated E. coli JM105 x pCSADcc-pKKj. Expression of CSADcc-cds was carried out in E . coli JM105 x pCSADcc-pKKj in a known manner under the control of the IPTG-inducible tac promoter functionally linked to the CSADcc-cds (IPTG: isopropyl-β-thiogalactoside, Sigma-Aldrich). Cultivation in shake flasks:

[0109] From the tribe E. coli JM105 x pCSADcc-pKKj was precultured in LBamp medium (grown at 37°C and 120 rpm overnight, Infors chest shaker).

[0110] Two ml of preculture was used as inoculum for a main culture of 100 ml of SM3 medium (1 L Erlenmeyer flask) supplemented with 15 g / L glucose, 5 mg / L pyridoxal phosphate (PLP, Sigma-Aldrich), and 100 mg / L ampicillin. The main culture was shaken in a chest shaker (Infors) at 30°C and 140 rpm. After 4 h of incubation, a cell density (OD 600 ) of 2.0 was reached. Then, the inducer IPTG (Sigma-Aldrich, 0.4 mM final concentration) was added, and culture was continued for another 20 h in a chest shaker (Infors) at 30°C and 140 rpm.

[0111] Composition of the SM3 medium: 12 g / LK 2 HPO 4 , 3 g / L KH 2 PO 4 , 5 g / L (NH 4 ) 2 SO 4 , 0.3 g / L MgSO 4 x 7 H 2 O, 0.015 g / L CaCl 2 x 2 H 2 O, 0.002 g / L FeSO 4 x 7 H 2 O, 1 g / L Na 3 citrate x 2 H 2 O, 0.1 g / L NaCl; 5 g / L peptone (oxoid); 2.5 g / L yeast extract (BD Biosciences); 0.005 g / L vitamin B1; 1 ml / L trace element solution. Composition of the trace element solution: 0.15 g / L Na 2 MoO 4 x 2 H 2 O, 2.5 g / LH 3 BO 3 , 0.7 g / L CoCl 2 x 6 H 2 O, 0.25 g / L CuSO 4 x 5 H 2 O, 1.6 g / L MnCl 2 x 4 H 2 O, 0.3 g / L ZnSO 4 x 7H2O

[0112] The cells from the shake flask culture were isolated by centrifugation. To prepare a cell suspension, the cell pellet from 50 ml of shake flask culture was resuspended in 2 ml of 50 mM Na phosphate, pH 7.0 (NaPi 7.0 buffer). The cell suspension was used either directly for biotransformation experiments or to prepare a cell homogenate.

[0113] The FastPrep-24™ 5G cell homogenizer from MP Biomedicals was used to prepare a cell homogenate. Two 1 ml cell suspensions were lysed in 1.5 ml tubes pre-filled by the manufacturer with glass beads ("Lysing Matrix B") (3 x 20 sec at a shaking frequency of 6000 rpm with a 30 sec break between each interval). The resulting cell homogenate (2 ml volume) was used without further processing for the biotransformation of L-cysteic acid to taurine. Cultivation by fermentation:

[0114] The production strain used for fermentation was E . coli JM105 x pCSADcc-pKKj. Fermentation was carried out in a Biostat B fermenter (2 L working volume) from Sartorius BBI Systems GmbH. Shake flask preculture:

[0115] 100 ml of LBamp medium in a 1 L Erlenmeyer flask was inoculated from an agar plate with the strain JM105 x pCSADcc-pKKj and incubated on an incubation shaker (Infors) for 7 - 8 h at 30°C and a speed of 120 rpm until a cell density OD 600 of 2 / ml - 4 / ml was reached. Pre-fermenter:

[0116] 1.5 L of FM2 medium, supplemented with 40 g / L glucose and 100 mg / L ampicillin, were inoculated with 21.3 ml of the shake flask preculture. The fermentation conditions were: temperature 30°C; pH constant 7.0 (automatic correction with 25% NH 4 OH and 6.8 NH 3 PO 4 ); foam control by automatic dosing of 4% v / v Struktol J673 in H 2 O (Schill & Seilacher); stirrer speed 450 - 1300 rpm; aeration with compressed air sterilized through a sterile filter constant 1.7 vvm (vvm: input of compressed air into the fermentation mixture expressed in liters of compressed air per liter of fermentation volume per minute); pO 2 ≥ 50%. The oxygen partial pressure pO 2 was regulated by the stirring speed. After a fermentation time of 16 h, a cell density OD 600 of 45 / ml was reached. Production fermenter:

[0117] 1.35 L of FM2 medium, pH 7.0, supplemented with 20 g / L glucose, 0.36 g / L pyridoxine (vitamin B6, Sigma-Aldrich), and 100 mg / L ampicillin, were inoculated with 150 ml of pre-fermenter culture. The fermentation conditions were: temperature 30°C; pH constant 7.0 (automatic correction with 25% NH 4 OH and 6.8% NH 3 PO 4 ); foam control by automatic addition of 4% v / v Struktol J673 in H 2 O (Schill & Seilacher); stirrer speed 450–1300 rpm; aeration constant 1.7 vvm; pO 2 ≥ 50%. The oxygen partial pressure pO 2 was regulated by the stirring speed. The fermentation time was 30 h.

[0118] FM2 medium: (NH 4 ) 2 SO 4 , 5 g / L; NaCl, 0.50 g / L; FeSO 4 x 7 H 2 O, 0.075 g / L; Na 3 citrate, 1 g / L, MgSO 4 x 7 H 2 O, 0.30 g / L, CaCl 2 x 2 H 2 O, 0.015 g / L, KH 2 PO 4 , 1.50 g / L, vitamin B1 (Sigma-Aldrich), 0.005 g / L; peptone (Oxoid), 5.00 g / L; yeast extract (Oxoid), 2.50 g / L; trace element solution, 10 ml / L (equivalent to that used for shake flask culture).

[0119] The pH in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH 4 OH solution. During fermentation, the pH was maintained at 7.0 by automatic correction with 25% NH 4 OH or 6.8% NH 3 PO 4 . For inoculation, 150 ml of pre-fermenter culture was pumped into the fermenter vessel. The initial volume was thus 1.5 L. The cultures were initially stirred at 350 rpm and aerated at an aeration rate of 1.7 vvm. Under these starting conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0120] The target value for O2 saturation (pO2) during fermentation was set to 50%. Once O2 saturation fell below the target value, a regulation cascade was initiated to return O2 saturation to the target value. The stirring speed was continuously increased (up to a maximum of 1,300 rpm).

[0121] Fermentation was carried out at a temperature of 30°C. Once the glucose content in the fermenter had dropped from an initial 20 g / L to approximately 5 g / L, a 60% (w / w) glucose solution was continuously added. The feed rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / L. Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0122] After the cell density in the fermenter reached an OD 600 of 50 / ml (8 h fermentation time), expression of the CSADcc gene was initiated by a single addition of the inducer IPTG (0.2 mM final concentration). Fermentation was stopped 22 h after induction, corresponding to a total fermentation time of 30 h. The cell density OD 600 was 164 / ml at this time point. 1 L of the fermenter broth was centrifuged (10 min at 15,000 rpm, Sorvall Centrifuge RC5C equipped with an SS34 rotor), the fermentation supernatant was discarded, the cells were resuspended in 1 L NaPi7.0 buffer, and stored in 50 ml aliquots at -20°C for further use. Example 7: Production of taurine from commercially available L-cysteine ​​acid by biotransformation

[0123] In a 100 ml Erlenmeyer flask, 12 mg of L-cysteic acid x H2O (Sigma-Aldrich) were weighed and dissolved in 9.7 ml of NaPi 7.0 buffer. The reaction was started by adding 0.3 ml of cell homogenate from the shake flask culture of JM105 x pCSADcc-pKKj (Example 6). The reaction volume was 10 ml. The molar concentration of L-cysteic acid x H2O was 6.41 mM (molecular weight of L-cysteic acid x H2O: 187.2 g / mol). The reaction was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 3 h, 1 ml of the reaction was incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed by HPLC. The L-cysteic acid used was completely consumed. The amount of taurine formed was 789.4 mg / L, corresponding to a molar concentration of 6.31 mM (molecular weight of taurine: 125.1 g / mol). Accordingly, the molar yield of taurine formed from 6.41 mM L-cysteic acid x H 2 O was 98.4%. Example 8: Production of taurine from commercially available OAS by biotransformation Reaction 1:Production of L-cysteine ​​acid from OAS:

[0124] 6.6 ml of KPi6.5 buffer were placed in a 100 ml Erlenmeyer flask, and 0.4 ml of a 0.2 M stock solution of OAS x HCl (Sigma-Aldrich) dissolved in 0.5 M Na succinate, pH 5.5, 1 ml of 1 M Na 2 SO 3 in KPi6.5 buffer, and 2 ml of cell suspension of cysM cells from shake flask culture (from Example 2A; CysM enzyme activity 57.1 U / ml) were added in rapid succession. The reaction volume was 10 ml. The dosage of CysM enzyme in the reaction mixture was 11.4 U / ml. The molar concentration of OAS x HCl was 8.00 mM (1.47 g / L; OAS x HCl molecular weight: 183.6 g / mol). The mixture was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 3 h, 1 ml of the mixture was incubated at 80°C for 5 min, centrifuged, and the supernatant analyzed by HPLC. The OAS used was completely consumed. The amount of L-cysteic acid formed was 1350.4 mg / L, corresponding to a molar concentration of 7.98 mM (molecular weight of L-cysteic acid: 169.2 g / mol).The molar yield of L-cysteic acid formed from 8.00 mM OAS x HCl was 99.7%. Reaction 2: Production of taurine from L-cysteine ​​acid synthesized in reaction 1:

[0125] In a 100 ml Erlenmeyer flask, 9 ml of the mixture from reaction 1 were placed, the pH was adjusted to 7.0 with 1 M KOH, and 1 ml of cell homogenate from the shake flask culture of the strain JM105 x pCSADcc-pKKj was added (Example 6). The mixture volume was 10 ml. Starting from an L-cysteic acid content of 1350.4 mg / L from reaction 1, the L-cysteic acid content at the beginning of reaction 2 was 1215.4 mg / L (7.18 mM at a molecular weight of 169.2 for L-cysteic acid). The mixture was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 3 h, 1 ml of the mixture was incubated for 5 min at 80°C, centrifuged, and the supernatant was analyzed by HPLC. The applied L-cysteic acid was completely consumed. The taurine content was 825.7 mg / L (6.60 mM with a taurine molecular weight of 125.1 g / mol). The molar yield of taurine formed from 7.18 mM L-cysteic acid was 91.9%. Example 9: Production of taurine by CSADcc-catalyzed conversion of L-cysteic acid from the reaction of fermentatively produced OAS with NaHSO 3 and fermentatively produced CysM enzyme

[0126] In a 100 ml Erlenmeyer flask, 7 ml of NaPi7.0 buffer containing 5 mg / L PLP, 1 ml of the mixture from Example 5 with an L-cysteic acid content of 12970 mg / L, and 2 ml of CSADcc cells in NaPi7.0 buffer (Example 6) were mixed. The mixture volume was 10 ml. The L-cysteic acid content in the mixture was 1297 mg / L, corresponding to a molar concentration of 7.67 mM (molecular weight of L-cysteic acid: 169.2 g / mol). The mixture was incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 4 h, 1 ml of the mixture was incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed by HPLC. The L-cysteic acid used was completely consumed. The amount of taurine formed was 925.3 mg / L, corresponding to a molar concentration of 7.40 mM (molecular weight of taurine: 125.1 g / mol). The molar yield of taurine formed from 7.67 mM L-cysteic acid was 96.4%. Example 10: Preparative production of taurine Biotransformation 1:

[0127] A double-walled 0.5 L thermostattable glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and heated to 37°C. 50 ml of CysM-containing cell suspension in KPi6.5 buffer (OD 600 90 / ml, 8720 U CysM enzyme activity) from the fermentation of strain DH5α / pFL145 (from Example 2) and 20 ml of a 400 g / L solution of Na2S2O5 (42.1 mmol, molecular weight 190.1 g / mol) in KPi6.5 buffer were added. In dissolved form, this corresponded to 84.2 mmol of NaHSO3 (1.63-fold molar excess over the 51.7 mmol of OAS added later). The mixture was stirred with a magnetic stirrer. The mixture was further equipped with a pH electrode (Mettler Toledo) connected to a pH control unit (Titrator TitroLine alpha, Schott) operated in pH-stat mode according to the manufacturer's instructions.Under pH-stat conditions, the pH in the reaction vessel was kept constant at the set pH 6.5 throughout the reaction by adding 2 M NaOH from a burette connected to the control unit. 400 ml of OAS-containing fermentation supernatant (OAS content 19.1 g / L, 7.6 g; 51.7 mmol) from the fermentation of strain . E. coliW3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1) was added to the reaction mixture from a receiver via a pump (Watson Marlow peristaltic pump 101U / R) at a flow rate of 0.2 ml / min. The total reaction time was 46 h. The reaction volume was 500 ml after completion of the reaction. At 1 h, 3 h, 20 h, 28 h, and 46 h after the start of the reaction, 1 ml aliquots of the mixture were taken, incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed for L-cysteic acid content by HPLC. The time course of L-cysteic acid formation is summarized in Table 4. After a reaction time of 46 hours, the L-cysteic acid content in the mixture was 15,370 mg / L (90.8 mM), which corresponded to an absolute molar yield of 45.4 mmol of L-cysteic acid at a reaction volume of 500 ml. Based on the amount of OAS used (51.7 mmol), this corresponds to a yield of 87.8%. Table 4: Amount of L-cysteine ​​acid detected by HPLC as a function of reaction time using an OAS-containing fermentation supernatant, NaHSO 3 and a cell suspension of CysM-containing fermenter cells Time [h] L-cysteine ​​acid [mg / L] L-cysteine ​​acid [mM] 1 709,0 4,2 3 1427,0 8,4 20 9814,0 58,0 28 10560,0 62,4 46 15370,0 90,8 Biotransformation 2:

[0128] A double-walled 0.3 L thermostatic glass vessel (Diehm) was connected to a thermostat (Lauda) via a hose connection and heated to 37°C. 100 ml of L-cysteic acid-containing Biotransformation 1 was adjusted to pH 7.0 with 2.5 M NaOH. Further, 1 ml of 1 M DTE (dithioerythrol, Sigma-Aldrich) dissolved in H2O, 1 ml of 500 mg / L PLP (4 mg / L final concentration) dissolved in NaPi7.0 buffer, and 20 ml of CSADcc-containing fermenter cells resuspended in NaPi7.0 buffer were added. The mixture was stirred with a magnetic stirrer. The reaction volume was 122 ml. At the beginning of the reaction and then after 2 h, 4 h, 6 h, and 24 h, 1 ml aliquots of the reaction were taken, incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed for L-cysteic acid and taurine content by HPLC. The results are summarized in Table 5. Based on the molar amount of L-cysteic acid used in the reaction of 74.4 mM, a taurine yield (8.8 g / L, 70.2 mM) of 94.3% was achieved. Table 5: Amount of L-cysteine ​​acid and taurine detected by HPLC as a function of reaction time using an L-cysteine-containing fermentation supernatant from Biotransformation 1 and a cell suspension of CSADcc-containing fermenter cells Time [h] L-cysteine ​​acid [mg / L] L-cysteine ​​acid [MM] Taurine [mg / L] Taurine [mM] 0 12592,5 74,4 0,0 0, 0 2 11821,1 69,88 1792,2 14,3 4 9106,7 53,83 2737,1 21,9 6 5159,6 30,5 4023,9 32,2 24 321,2 1,9 8783,6 70,2 Example 11: Production of taurine from OAS in a "one-pot reaction"

[0129] The reaction mixture was composed of 3 ml KPi6.5 buffer, 2 ml OAS-containing fermentation supernatant from the fermentation of the strain E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1), 1 ml of 1 M Na 2 SO 3 in KPi6.5 buffer, 2 ml of a cell suspension of CysM-containing cells from the fermentation of strain DH5α / pFL145 (Example 2B), and 2 ml of a cell suspension of CSADcc-containing cells from the shake flask culture of strain JM105 x pCSADcc-pKKj (Example 6). The reaction volume was 10 ml. The OAS concentration in the reaction mixture was 3.1 g / L (20.80 mM). The Na 2 SO 3 concentration in the reaction mixture was 100 mM. The CysM enzyme activity in the reaction mixture was 34.9 U / ml.

[0130] The reaction was carried out at pH 6.5. The mixture was incubated in a chest shaker (Infors) at 37°C and 140 rpm. 24 h after the start of the reaction, 1 ml of the mixture was incubated for 5 min at 80°C, centrifuged, and the supernatant analyzed by HPLC. The L-cysteic acid content was 1.3 g / L (7.68 mM, with a molecular weight of 169.2 g / mol for L-cysteic acid). The taurine content was 721 mg / L (5.76 mM, with a molecular weight of 125.1 g / mol for taurine). The molar yield of 7.68 mM L-cysteic acid formed from 20.80 mM OAS was 36.9%. The molar yield of 5.76 mM taurine formed from 20.80 mM OAS was 27.7%. The overall molar yield of L-cysteic acid and taurine formed from OAS was 64.6%.

Claims

1. Process for producing taurine from O-acetyl-L-serine (OAS) by means of biotransformation, wherein i) in a 1st process step (biotransformation 1), L-cysteic acid is produced from O-acetyl-L-serine (OAS) using an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of a salt of sulfurous acid, wherein the biotransformation is carried out under active pH control, and then ii) in a 2nd process step (biotransformation 2), L-cysteic acid is decarboxylated to taurine, wherein the OAS concentration in the batch is at least 10 g / L and wherein the OAS sulfhydrylase is CysM.

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

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

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

5. Process according to one or more of Claims 1 to 4, characterized in that the concentration of the salt of sulfurous acid is at least in equimolar concentration to OAS.

6. Process according to one or more of Claims 1 to 5, characterized in that L-cysteic acid is decarboxylated to taurine using an enzyme from the class of L-cysteine sulfinic acid decarboxylases (EC 4.1.1.29), aspartate 1-decarboxylases (EC 4.1.1.11) or glutamate decarboxylases (EC 4.1.1.15).

7. Process according to Claim 6, characterized in that the L-cysteine sulfinic acid decarboxylase is SEQ ID NO: 2 or a sequence homologous to this sequence.

8. Process according to one or both of Claims 6 and 7, characterized in that the L-cysteine sulfinic acid decarboxylase stems from fermentative production.

9. Process according to one or more of Claims 1 to 8, characterized in that a) OAS is produced by fermentation, b) the enzymes from the class of OAS sulfhydrylases (EC 4.2.99.8) and the class of cysteine sulfinic acid decarboxylases (EC 4.1.1.29) are produced by fermentation, c) OAS and a salt of sulfurous acid react to form L-cysteic acid under enzymatic catalysis by the OAS sulfhydrylase from point b, and d) L-cysteic acid from point c is decarboxylated to taurine by the CSAD enzyme from point b.

10. Process according to Claim 9, characterized in that all the process steps take place in one reaction batch.

11. Process according to one or more of Claims 1 to 10, characterized in that the molar yield of taurine from the biotransformation of L-cysteic acid in biotransformation 2 is preferably at least 60%.