Taurine production method

Abstract

The present invention is a method for producing taurine from O-acetyl-L-serine (OAS) by biotransformation. In a first process step (biotransformation 1), L-cysteic acid is produced from O-acetyl-L-serine (OAS) in the presence of sulfite using an enzyme selected from the group of OAS sulfhydrylases (EC 4.2.99.8), and then in a second process step (biotransformation 2), L-cysteic acid is decarboxylated to taurine. This type of biotransformation results in taurine.

Description

[Technical Field]

[0001] The present invention relates to a method for producing taurine from O-acetyl-L-serine (OAS) by biotransformation, comprising the steps of: in a first process step (biotransformation 1), generating L-cysteic acid from O-acetyl-L-serine (OAS) in the presence of sulfite using an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8); and then, in a second process step (biotransformation 2), decarboxylating L-cysteic acid to taurine, which results from 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 an ingredient in energy drinks and is also used in pet food for cats and farmed fish (Salze and Davis (2015) Aquaculture 437: 215-229). However, taurine is also thought to have health-promoting properties (Ripps and Shen (2012), Molecular Vision 18: 2673-2686).

[0003] In nature, taurine is found almost exclusively in the animal kingdom, with only a few examples in bacteria, algae, or plants. The biosynthetic pathways to taurine are diverse (for an overview, see, for example, "Taurine and hypotaurine metabolism" in the KEGG Pathway Database), and begin primarily with L-cysteine. The most important steps leading to taurine from L-cysteine ​​are shown in Equations (1) through (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

[0004] (1) In the 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 (previously a rather hypothetical enzymatic step) further oxidizes L-cysteine ​​sulfinate to L-cysteic acid ((R)-2-amino-3-sulfopropionic acid, CAS number 23537-25-9). (3) L-cysteine ​​sulfinic acid is decarboxylated to hypotaurine (2-aminoethanesulfinic acid, CAS number 300-84-5) by cysteine ​​sulfinic acid decarboxylase (CSAD, EC 4.1.1.29). (4) Hypotaurine is oxidized to taurine, a process that has not been adequately explained until now. (5) In a similar process to (3), L-cysteic acid is decarboxylated to taurine by the appropriate CSAD enzyme.

[0005] Currently, commercially available taurine is produced chemically. One known method, for example, that of the Changshu Yudong Chemical Factory, starts with ethylene and then ethyleneimine to produce taurine. Due to the consumer-driven trend away from chemically produced raw materials and toward sustainably produced raw materials, biotechnology methods for taurine production are increasingly being researched. Conventional techniques rely on metabolic engineering approaches, in which appropriate biosynthetic genes are heterologously expressed in production strains to produce taurine or its biosynthetic precursor, hypotaurine.

[0006] Honjoh et al. (2010), Amino Acids 38: 173-1183, described a genetically engineered yeast strain heterologously expressing the carp (Cyprinus carpio) CDO and CSAD genes. When L-cysteine ​​was added to the growth of the genetically engineered strain, hypotaurine was produced as the major product, with a relatively small proportion of taurine also being produced. Treatment with H2O2 oxidized hypotaurine to taurine. Although S. cerevisiae itself can produce cysteine ​​from its own metabolism, exogenous L-cysteine ​​had to be added to the growth medium to produce hypotaurine and taurine.

[0007] Tevatia et al. (2019), Algal Research 40:101491, also produced taurine in the algae Chlamydomonas reinhardtii using heterologously expressed CDO and CSAD genes from carp. The intracellular taurine yield was 0.14 mg taurine per gram of dry biomass.

[0008] Joo et al. (2018), J. Agric. Food Chem. 66:13454-13463, described a genetically engineered strain of the bacterium Corynebacterium glutamicum that produced 0.5 g / L of taurine when grown in shake flasks. It was clear that taurine accumulated intracellularly and was not secreted into the growth medium. Taurine synthesis was achieved by heterologously expressing the genes for L-cysteine ​​acid synthase, cysteine ​​dioxygenase, and L-cysteine ​​sulfinic acid decarboxylase in the strain. Furthermore, repressor genes related to methionine and cysteine ​​biosynthesis were inactivated, simultaneously improving sulfur absorption. In addition to the low yield, the fact that taurine is produced intracellularly is considered a fundamental drawback from the perspective of product isolation. Complex cell disruption is required to release taurine for further processing.

[0009] WO17213142A1 (Ajinomoto) describes a taurine-producing strain obtained by heterologous expression of cysteine ​​dioxygenase and L-cysteine ​​sulfinic acid decarboxylase in a strain capable of producing cysteine. The main product is hypotaurine with a maximum yield of 450 μM, which can then be converted to taurine only in low yield by alkaline treatment.

[0010] US9267148B2, US2012 / 0222148A1, US2018 / 0028474A1, US2019 / 0085339, WO2017 / 176277A1, and WO2019 / 094051 (Plant Sensory Systems) describe taurine-producing strains and organisms that variously involve heterologous expression of cysteine ​​dioxygenase and L-cysteine ​​sulfinic acid decarboxylase. The primary focus of these applications is taurine production in plants, but nothing is said about yield.

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

[0012] The prior art discloses various metabolic engineering approaches to produce hypotaurine or taurine in heterologous production systems. Although production yields from metabolic engineering approaches have been disclosed, the yields are too low for industrial use.

[0013] The prior art also provides methods for producing non-proteinogenic (unnatural) amino acids, for example, by direct microbial fermentation (EP 1191106 B1, Wacker) or biotransformation of OAS catalyzed by O-acetyl-L-serine sulfhydrylase (OAS sulfhydrylase) (EP 1247869 B1, Wacker). These methods catalyze the reaction of OAS with a nucleophile to form a non-proteinogenic amino acid according to the general formula (6). (6) OAS + nucleophile -> unsaturated amino acid + acetic acid

[0014] In EP 1247869 B1 (Wacker), a number of different nucleophiles are tested for their suitability as nucleophiles for reactions with OAS catalyzed by OAS sulfhydrylase, including selenides, selenols, azides, cyanides, azoles, and isoxazolinones. In addition, sulfur compounds from the group of thiosulfates and thiols of the general formula HSR are also used, where the radical R is a monovalent substituted or unsubstituted alkyl, alkoxy, aryl, or heteroaryl radical.

[0015] These methods are not suitable for the production of L-cysteic acid and / or taurine.

[0016] The object of the present invention is to provide a biotechnological method for producing taurine by biotransformation, bypassing heterologous production strains generated by metabolic engineering.

[0017] This object is achieved by a method for producing taurine from O-acetyl-L-serine (OAS) by biotransformation, which comprises, in a first process step (biotransformation 1), generating L-cysteic acid from O-acetyl-L-serine (OAS) in the presence of sulfite using an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8), followed by decarboxylation of L-cysteic acid to taurine in a second process step (biotransformation 2).

[0018] As disclosed in the accompanying application Co12102 and in the examples of the present invention, it has been surprisingly found that sulfites (hereinafter referred to as sulfites or SO3 2- ) was found to be suitable as a nucleophile in reaction (6), enabling the previously unknown synthesis of the non-proteinogenic amino acid L-cysteic acid (biotransformation 1) in reaction (7), as an extension of EP1247869B1 (Wacker). (7) OAS+SO3 2- -> L-cysteic acid + acetic acid

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

[0020] The advantage of the taurine production method of the present invention is that it is a sustainable, technically feasible, and economically viable biotransformation method for producing taurine. No environmentally harmful chemicals are required. No fossil raw materials are consumed, and no toxic chemical waste and / or waste gases are produced. Therefore, the production method of the present invention is environmentally friendly and sustainable. Furthermore, the method of the present invention does not require extreme reaction conditions or special equipment, and is therefore technically easy to implement. There is a growing demand for such a method.

[0021] The taurine from the method of the present invention can be used directly without further processing steps or can be concentrated by known methods.

[0022] In the context of the present invention, a distinction is made between the following manufacturing methods: 1.Chemical method 2. Biotechnology methods a) Metabolic engineering Metabolic engineering (also called "pathway design"), in contrast to biotransformation, is a biotechnology method for altering the metabolic pathways of organisms by optimizing or modifying genes and regulatory processes. Novel or modified enzymes can be introduced into organisms by supplementing the genome with the enzyme's genes, or endogenous enzyme genes can be expressed at enhanced or attenuated levels, thereby establishing new metabolic pathways in the organism or enhancing or attenuating existing metabolic pathways. The goal of metabolic engineering is to produce new metabolites or endogenous metabolites in organisms at higher yields. Metabolic engineering processes do not use starting materials specific to metabolites, such as enzyme substrates (e.g., OAS in the present invention). Instead, they use only a nutrient medium, also known as a growth medium, required for the growth of the target organism and consisting of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts or complex amino acid mixtures such as peptone or yeast extract), and other salts necessary for growth. Such nutrient media are known to those skilled in the art from microbiological practice. b) Biotransformation Biotransformation is defined as the enzymatic conversion of one or more reactants into products, where an enzyme substrate is added to a reaction batch along with the enzyme. In the reaction batch, the added enzyme substrate, such as OAS or L-cysteic acid in the present invention, is enzymatically converted. In the present invention, this is achieved by an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8) in the presence of sulfite according to formula (7) for OAS, and by an enzyme selected from the class of cysteine ​​sulfinic acid decarboxylases (CSAD, EC 4.1.1.29) according to formula (5) for L-cysteic acid. The reactants can be derived from chemical or biotechnological production. The OAS used in the methods of the present invention can be derived, for example, from chemical synthesis or biotechnological production by fermentation of a producing strain. The L-cysteic acid used in the methods of the present invention can be obtained, for example, from chemical synthesis or biotechnological production by biotransformation of OAS. The enzymes used in the enzymatic catalysis can be obtained from biotechnological production, for example, by growing a production strain by fermentation, or biological materials containing the enzymes (e.g., plants, fungi, algae, animal organs) can be used. The biomass or biological materials from growing a production strain can be used as is, or the enzymes can be isolated therefrom depending on the requirements of the biotransformation. The enzymes CysM and CSADcc used in the method of the present invention can be obtained from biotechnological production by fermentation of a production strain.

[0023] In the context of the present invention, a reaction batch is defined as a mixture of reactants (starting materials), enzymes, and optionally other reactants, in which the reactants are converted into products.

[0024] The yield of a reaction in the sense of the present invention is defined as the amount of reactant used that is converted into product under the reaction conditions. The yield can be expressed as an absolute amount (g or mmol), a volumetric yield expressed as the absolute amount of product per unit volume (mM or g / L), or a relative yield, also called percent yield, expressed as the proportion of reactants used (taking into account the molecular weights of the reactants and products).

[0025] Fermentation is a process step for the production (cultivation) of cell cultures on an industrial scale, preferably by growing a microbial production strain under defined conditions of medium, temperature, pH, oxygen supply, and medium mixture. Depending on the genetic makeup of the production strain, the goal of fermentation is to produce proteins / enzymes or metabolic products, in each case in the highest possible yield for further use. The OAS, OAS sulfhydrylase, and cysteine ​​sulfinic acid decarboxylase components of the method of the present invention can be produced by fermentation. The end product of fermentation is a fermentation broth consisting of the cellular biomass of the production strain (fermentation cells) and a fermentation medium (fermentation supernatant) removed from the biomass and formed from the growth medium and metabolic products secreted by the fermentation cells during fermentation. The target product of fermentation can be present in the fermentation cells or the fermentation medium. For example, OAS is contained in the fermentation medium, while the enzymes OAS sulfhydrylase and CSADcc are contained in the fermentation cells.

[0026] An open reading frame (ORF, synonymous with cds or coding sequence) is a region of DNA or RNA that begins with a start codon, ends with a stop codon, and codes for the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0027] A gene is a DNA section that contains all the basic information for producing biologically active RNA. A gene includes both the DNA section that is transcribed to produce a single-stranded RNA copy and the expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, a transcription start site, a translation start site, and a ribosome binding site (RBS). Terminators and one or more operators are additional possible expression signals.

[0028] 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 specific proteins within cells. mRNA molecules transmit the messages needed for protein synthesis from the genetic information (DNA) to the ribosomes responsible for protein synthesis. Within cells, mRNA molecules are formed as transcripts of the segments of DNA that correspond to genes. The genetic information stored in DNA remains unchanged during this process.

[0029] Eukaryotic genes are primarily mosaic genes, and unlike prokaryotic genes, they also contain non-coding regions called introns (intragenic regions). Coding sequences, called exons (expressed regions), are portions of DNA in eukaryotic genes that are transcribed into RNA and then translated by ribosomes into the amino acid sequence of a protein. After DNA is transcribed into RNA, introns are cleaved from the primary transcript. The protein-coding RNA from which the introns have been removed is called messenger RNA (mRNA) or "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. When eukaryotic genes containing exon / intron structures are expressed in prokaryotes, the protein sequence or coding region of the mature mRNA must be reverse-translated into intron-free DNA, since prokaryotes do not process the exon / intron structure. In the context of this invention, when referring to a gene sequence derived from a protein sequence or a gene sequence derived from an mRNA, it is this reverse translation process that is meant. Preferably, the protein or mRNA sequence is reverse-translated into a DNA sequence and at the same time the sequence is optimised, ie adapted to the codon usage of the corresponding prokaryote (codon optimisation).

[0030] A gene construct refers to a DNA molecule in which a gene is linked to other genetic elements (such as a promoter, terminator, selection marker, origin of replication, etc.). A gene construct in the context of the present invention is a circular DNA molecule, also called a plasmid, vector, or expression vector. The genetic elements of a gene construct cause its extrachromosomal inheritance during cell growth, producing the protein encoded by the gene.

[0031] The abbreviation WT (Wt) stands for wild-type. A wild-type gene refers to the form of a gene that arises naturally through evolution and is present in the wild-type genome. The DNA sequence of the Wt gene is publicly available in databases such as NCBI.

[0032] Biotransformation 1: The method for producing taurine of the present invention requires the availability of OAS. Chemical methods for producing OAS include, for example, acetylation of L-serine, which is costly due to the high price of L-serine, or the generation of racemic O-acetyl-D / L-serine, which can be used directly, or OAS can be obtained in advance from the racemate, for example, by dissolution. In the case of direct acetylation, N-acetyl-L-serine (NAS) may be formed as a by-product, for example, by nonselective acetylation of the hydroxyl or amino groups of L-serine, or by the known rearrangement of OAS to NAS at neutral to alkaline pH values ​​(Tai et al. (1995), Biochemistry 34: 12311-12322), but these methods either result in reduced yields or require prior introduction of a protecting group on the amino group of L-serine. Therefore, direct acetylation of L-serine is not practical for an economically viable process.

[0033] Biotechnological production of OAS is known. This involves the use of organisms in which cysteine ​​metabolism is deregulated, thereby providing high levels of OAS. A fermentation process for producing OAS is disclosed in EP 1233067 B1 (WACKER) and described in Example 1 of the present invention.

[0034] The production of OAS using Escherichia coli is preferred, and it is particularly preferred to use Escherichia coli, which has been deposited under the number DSM 13495 at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) in accordance with the Budapest Treaty.

[0035] An advantage of the present invention is that the OAS-containing fermentation broth, e.g., obtained from a fermentation carried out according to EP 1233067, can be used directly as a source of OAS in the process of the present invention as a fermentation supernatant after removing the particulate biomass, e.g., by centrifugation, without further workup, purification, or isolation steps, including extraction, adsorption, ion exchange chromatography, precipitation, or crystallization. This procedure is particularly economical and avoids the isolation of unstable compounds.

[0036] Those skilled in the art can use isotope analysis to determine whether a substance, such as OAS or L-cysteic acid, desired for use in the methods of the present invention is derived from chemical or fermentative production. Distinguishing isotope analysis methods are described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527, and are based on measuring, for example, carbon and nitrogen isotope ratios, which differ depending on whether the product is derived from chemical (petroleum-based) or fermentative production (from plant-based raw materials).

[0037] OAS sulfhydrylases have been isolated from various plants and microorganisms. For example, Escherichia coli has two OAS sulfhydrylase enzymes, CysK and CysM. The related genes are also known and are called cysK and cysM, respectively.

[0038] OAS sulfhydrylases in the sense 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), where the nucleophile used is sulfide. Thus, both CysM-related enzymes and CysK-related enzymes are OAS sulfhydrylases in the sense of the present invention. (8) OAS+S 2- -> L-cysteine ​​+ acetic acid

[0039] Both enzymes have very similar reaction mechanisms and are involved in the biosynthesis of L-cysteine. However, unlike CysK, CysM has a more flexible substrate spectrum with respect to the nucleophiles that can react with OAS according to equation (6). For example, unlike CysK, CysM 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 that use thiosulfate as their sole sulfur source. Furthermore, EP 1247869 B1 (Wacker) discloses the use of CysM for the production of non-proteinogenic amino acids.

[0040] Preferably, the method according to the invention is characterized in that the OAS sulfhydrylase is a bacterial enzyme, particularly preferably CysM, particularly preferably CysM from an Escherichia coli strain.

[0041] The CysM-containing OAS sulfhydrylase is characterized in that it is produced by fermentation, particularly preferably by fermentation of an Escherichia coli strain, particularly preferably by fermentation of the Escherichia coli strain DH5α / pFL145.

[0042] Example 2 discloses a procedure for the fermentative biotechnological production of CysM using the E. coli DH5α / pFL145 strain. The production strain consists in this case of a host strain, such as E. coli DH5α, and a suitable genetic construct for expressing OAS sulfhydrylase, preferably the genetic construct pFL145. The host strain and genetic construct, as well as the production of the production strain, are described in EP 1247869 B1. The production strain has been deposited under the number DSM 14088 at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) in accordance with the Budapest Treaty.

[0043] The fermentation broth obtained by fermentation of the production strain consists of fermentation cells containing OAS sulfhydrylase and a fermentation medium (fermentation supernatant). In the method of the present invention, the fermentation broth can be used directly without further treatment, or a suspension of fermenter cells in, for example, a buffer solution (resuspension fermenter cells) can be used after separation from the fermentation broth, for example, by centrifugation or filtration. Example 2 describes the production of resuspended CysM-containing fermentation cells of the E. coli DH5α / pFL145 strain.

[0044] Furthermore, it is contemplated that OAS sulfhydrylase may be used in the form of a cell homogenate after mechanical disruption of fermentation cells, or in the form of chemically permeabilized cells (e.g., with chloroform), or as a cell extract after removal of particulate components from the cell homogenate, or as an enzyme purified by chromatography or the like. OAS sulfhydrylase is preferably used as a fermentation broth without further treatment, 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). It is particularly preferred to use OAS sulfhydrylase in the form of resuspended fermentation cells or as a cell homogenate. It is particularly preferred to use resuspended fermentation cells of an OAS sulfhydrylase-producing strain.

[0045] In the method of the present invention, OAS reacts with sulfite under the catalysis of OAS sulfhydrylase to form L-cysteic acid according to equation (7).

[0046] Sulfurous acid forms multiple species that exist simultaneously in reversible equilibrium, and the suitability of each species as a nucleophile in the biotransformation of the present invention was unpredictable. Sulfurous acid (HSO) is an aqueous solution of gaseous SO, and as a dibasic acid, it exists in different equilibria depending on the pH of the aqueous solution, and the species at these equilibria are known to have different suitabilities as nucleophiles. The following equilibria (9) to (13) are known: (9) SO2 (gas) <-> SO2 (dissolved) (10)SO2(dissolved)+H2O <-> H2SO3 (11) H2SO3<-> HSO3 - +H + (12)HSO3 - <->SO3 2- +H + (13)2HSO3 - <-> S2O5 2- +H2O

[0047] Sulfite and its salts have antibacterial properties and are therefore used as preservatives in the food industry. This means that sulfite and its salts may kill microorganisms, which is due to the inactivation of enzymes necessary for the survival of microorganisms. Therefore, those skilled in the art would expect that the use of sulfite or its salts would also inactivate the CysM enzyme, making it impossible to produce L-cysteic acid using the method disclosed in EP1247869B1.

[0048] It was surprising to those skilled in the art that sulfite and OAS could be used in biotransformations to produce L-cysteic acid as a starting compound for biotechnological processes to produce taurine.

[0049] In principle, all possible sulfite salts are suitable for this reaction. Preferably, the method of the present invention is characterized in that the sulfite salt used is Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its anhydride Na2SO5) or KHSO3. Particularly preferably, the sulfite salt used is Na2SO3, NaHSO3 (or its anhydride Na2SO5) or (NH4)2SO3, particularly preferably Na2SO3 or NaHSO3 (or its anhydride Na2SO5).

[0050] It is possible to use gaseous sulfur dioxide, the anhydride of sulfurous acid, which, when introduced into the reaction batch, hydrates to sulfurous acid H2SO3 and, depending on the pH, to the deprotonated form HSO3 - and SO3 2- and reaches equilibrium.

[0051] Preferably, the method of the invention is characterized in that the concentration of sulfite is at least equimolar with respect to OAS, particularly preferably at least a 1.5-fold molar excess of sulfite, and especially preferably at least a 5-fold molar excess.

[0052] In a particularly preferred embodiment, the method for producing taurine is characterized in that both the OAS sulfhydrylase and the OAS are produced by fermentation.

[0053] OAS, as a reactant in the biotransformation process of the present invention, isomerizes to N-acetyl-L-serine (NAS) from a pH of about 7, which is then unsuitable for reaction with sulfite to form L-cysteic acid. The reaction mechanism was studied by Tai et al. (1995), Biochemistry 34: 12311-12322, and involves an intramolecular nucleophilic attack by a deprotonated amino group on the carbonyl carbon of the acyl radical. This reaction is inhibited with decreasing pH, making the compound stable at, for example, pH 4.0.

[0054] Therefore, the biotransformation process according to the present invention is preferably characterized in that the reaction is carried out under pH conditions that minimize the isomerization of OAS to NAS.

[0055] The reaction temperature for biotransformation 1 is preferably selected between 5°C and 70°C. The reaction temperature is preferably between 10°C and 60°C, particularly preferably between 15°C and 50°C, and particularly preferably between 20°C and 40°C.

[0056] The OAS concentration in the batch is preferably at least 1 g / L, particularly preferably at least 10 g / L, particularly preferably at least 40 g / L.

[0057] 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%, particularly preferably at least 80%.

[0058] In a further preferred embodiment of biotransformation process 1, the substrate OAS is metered from a reservoir into a reaction batch consisting of OAS sulfhydrylase and sulfite in a so-called feed process (Example 5). Isomerization of OAS to NAS in the OAS-containing reservoir is preferably avoided by setting the pH at ≦6.5, particularly preferably at ≦6.0, and particularly preferably at ≦5.5. At the same time, the pH in the reaction batch is adjusted to promote the reaction forming L-cysteic acid, preferably at ≦7.5, particularly preferably at ≦7.0, and particularly preferably at ≦6.5.

[0059] According to equation (7), the reaction of OAS to form L-cysteic acid releases a stoichiometric amount of acetic acid, resulting in a decrease in the pH of the batch as the reaction proceeds. Because an excessively low pH affects the activity of OAS sulfhydrylase, preventing an excessively large decrease in pH is a priority. This can be achieved passively by using an appropriate high-concentration buffer in the batch or actively by a measurement and control unit.

[0060] Particularly preferred is active pH control by a measurement and control unit, as disclosed in Example 5, which restores the desired pH by metered addition of alkaline solution or acid if the pH deviates from the target value (the so-called pH-stat method).

[0061] Biotransformation 1 can be carried out in a discontinuous or continuous manner. In a discontinuous (batch) operation, all reactants are added to the batch during the reaction, and the batch is processed after the reaction is complete. In a continuous operation, OAS, CysM enzyme, and sulfite are constantly metered into the reaction, and a solution containing the product L-cysteic acid is simultaneously removed from the batch. A steady state is established in which the reactants are metered in so that they can completely react to form the product L-cysteic acid during the residence time in the reaction vessel. A continuous method for producing unnatural amino acids is disclosed, for example, in EP1247869B1 (Wacker). Biotransformation 1 is preferably a discontinuous process.

[0062] Biotransformation 2: The method for producing taurine of the present invention comprises biotransformation 1 to produce L-cysteic acid, followed by biotransformation 2 to decarboxylate L-cysteic acid to produce taurine.

[0063] Decarboxylation of cysteic acid to taurine can be carried out chemically or by enzymatic catalysis in biotransformations. Thermal decarboxylation at high temperatures using metal catalysts is known, but is considered unsustainable and has the disadvantages of being energy intensive and producing a high proportion of by-products.

[0064] Preferably, the method of the present invention is characterized by decarboxylating L-cysteic acid to taurine using an enzyme from the class of L-cysteine ​​sulfinic acid decarboxylase (CSAD, EC 4.1.1.29), aspartate 1-decarboxylase (EC 4.1.1.11), or glutamic acid decarboxylase (EC 4.1.1.15).

[0065] For the enzymatic decarboxylation of L-cysteic acid to taurine, enzymes of the L-cysteine ​​sulfinic acid decarboxylase (CSAD, EC 4.1.1.29) class are particularly preferred. CSAD enzymes are known to decarboxylate L-cysteine ​​sulfinic acid to hypotaurine according to equation (3). These enzymes can also decarboxylate L-cysteic acid as a substrate to taurine, to varying degrees, according to equation (5). For example, the CSADcc enzyme from Cyprinus carpio (carp) is suitable for decarboxylating L-cysteic acid to taurine, as disclosed in Examples 7-11.

[0066] Enzymes of the class EC 4.1.1.29 (CSAD) are found primarily in metazoans (multicellular animals), including mammals. Enzymes with CSAD activity are also found in unicellular organisms, such as algae of the genus Synechococus, as well as bacteria and fungi. Preferably, the method for producing taurine of the present invention uses a CSAD enzyme of the class EC 4.1.1.29 from a mammal selected from humans (Homo sapiens), cattle (Bos taurus), rats (Rattus norvegicus), or mice (Mus musculus), and fish such as carp (Cyprinus carpio), particularly preferably from humans (Homo sapiens), rats (Rattus norvegicus), or carp (Cyprinus carpio).

[0067] The CSAD enzyme from carp (CSADcc, Cyprinus carpio) is particularly preferred. In the examples, a CSADcc enzyme derived from the protein sequence of the wild-type CSAD enzyme from Cyprinus carpio, designated CSADcc, is used, which is disclosed in nt 31-1530 of SEQ ID NO: 1 and has the DNA sequence of the cds designated CSADcc cds, and encodes a protein having the amino acid sequence of SEQ ID NO: 2.

[0068] The DNA sequence underlying the Wt CSADcc amino acid sequence is available in the NCBI (National Center for Biotechnology Information) database under GenBank sequence ID: AB220585.1 (cds: nt 82-1584). Derived from the corresponding Wt CSADcc amino acid sequence is the CSADcc cds DNA sequence (nt 31-1530 of SEQ ID NO: 1) that has been codon-optimized for expression in E. coli and encodes the same 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 of SEQ ID NO: 1 is synthesized using known methods provided by commercial suppliers, such as Eurofins Genomics.

[0069] Particularly preferably, the L-cysteine ​​sulfinic acid decarboxylase is SEQ ID NO: 2 or a sequence homologous to this sequence.

[0070] Homologous sequences are understood to mean that the DNA or amino acid sequences are at least 80%, preferably at least 90%, particularly preferably at least 95% identical, and that each change in the homologous sequence is selected from insertions, additions, deletions and substitutions of one or more nucleotides or amino acids.

[0071] The degree of DNA identity is determined by the "nucleotide blast" program, which is based on the blastn algorithm at http: / / blast.ncbi.nlm.nih.gov / . The algorithm parameters used to align two or more nucleotide sequences are default parameters. The default 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 default scoring parameters are: Match / Mismatch Scores=1, -2; Gap Costs=Linear.

[0072] Protein sequences are compared using the "protein blast" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences are the default parameters. The default 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 default scoring parameters are: Matrix=BLOSUM62; Gap Costs=Existence:11 Extension: 1; Compositional adjustments=Conditional compositional score template adjustment.

[0073] Preferably, the method of the present invention is characterized in that the L-cysteine ​​sulfinic acid decarboxylase is derived from fermentation production. Recombinant production of the corresponding CSADcc enzyme in an E. coli production strain is disclosed in Example 6. For this purpose, the CSADcc cds are cloned into an expression vector, for example, the vector pKKj, by known methods, resulting in the gene construct pCSADcc-pKKj (Figure 1). The production strain is produced by transforming the gene construct pCSADcc-pKKj into an E. coli host strain, for example, the E. coli JM105 strain, by known methods, and using the resulting production strain E. coli JM105 x pCSADcc-pKKj to produce the CSADcc enzyme, also by known methods. The CSADcc enzyme can be produced on a shake flask scale for laboratory purposes or by fermentation (Example 6).

[0074] The CSAD enzyme contains pyridoxal phosphate (PLP, CAS No. 54-47-7) as a cofactor. Therefore, supplementing the growth medium or biotransformation batch for converting L-cysteic acid to taurine with PLP is one way to achieve process improvement. Because PLP belongs to the vitamin B6 family, supplementing 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 a suitable alternative for process improvement. Preferably, the biotransformation process for converting L-cysteic acid to taurine is carried out in the presence of PLP at a concentration of ≥ 20 mg / L, particularly preferably ≥ 10 mg / L, and particularly preferably ≥ 4 mg / L.

[0075] In the method of the present invention, the CSAD enzyme, preferably CSADcc, obtained by growth or fermentation in shake flasks can be used as a fermentation broth without further processing, or as a cell suspension after reisolation of the cells, for example by centrifugation and resuspension of the fermented cells in a buffer solution (resuspended fermented cells). Furthermore, the CSAD enzyme, preferably CSADcc, can be used in the form of a cell homogenate after mechanical disruption of the resuspended fermented cells, or in the form of chemically permeabilized cells (e.g., with chloroform), or as a cell extract after removal of particulate components from the cell homogenate, or as an enzyme purified by chromatography, etc.

[0076] The CSAD enzyme (CSADcc) is preferably used as a fermentation broth without further treatment, as resuspended fermented cells, or as a cell homogenate after mechanical disruption of resuspended fermented cells. Particularly preferably, the CSAD enzyme is used as a resuspended fermented cell or as a cell homogenate after mechanical disruption of resuspended fermented cells. Particularly preferably, the CSAD enzyme is used in the form of resuspended fermented cells.

[0077] The biotransformation of L-cysteic acid to taurine by a CSAD enzyme, preferably CSADcc, is preferably carried out under pH and temperature conditions that allow efficient decarboxylation of L-cysteic acid to taurine.

[0078] The preferred pH range in which Biotransformation 2 is carried out is pH 5.0 to 9.0, particularly preferably pH 6.0 to 8.5, and particularly preferably pH 6.5 to 8.0.

[0079] Biotransformation 2 is preferably carried out at temperatures below 70°C, preferably below 60°C, particularly preferably below 50°C, particularly preferably below 40°C.

[0080] The molar yield of taurine from the biotransformation of cysteic acid is preferably at least 60%, particularly preferably at least 80%, particularly preferably at least 90%.

[0081] The biotransformation of cysteic acid to produce taurine can be carried out in a discontinuous or continuous manner. In a discontinuous (batch) operation, all reactants are added to a batch during the reaction, and the batch is processed after the reaction is complete. In a continuous operation, the CSAD enzyme is introduced as a stationary phase, for example, in a membrane reactor or immobilized on a support, and the substrate L-cysteic acid is metered in as a mobile phase. The contact time between the mobile phase and the stationary phase is set so that the substrate L-cysteic acid can be completely reacted to form the product taurine. A discontinuous (batch) operation is preferred.

[0082] A preferred process for producing taurine from OAS, including biotransformation 1 and biotransformation 2, is characterized in that it comprises the following steps: a) OAS is produced by fermentation, b) enzymes of the class of OAS sulfhydrylases (EC 4.2.99.8), such as 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 sulfite react under enzymatic catalysis by OAS sulfhydrylase in point b to form L-cysteic acid; d) L-cysteic acid at point c is decarboxylated to taurine by the CSAD enzyme at point b.

[0083] Particularly preferred is a method comprising steps a, b, c, and d, characterized in that in biotransformation 1, OAS is reacted with NaSO or its anhydrous salt, NaSO, in a CysM-catalyzed reaction to form L-cysteic acid, and in biotransformation 2, L-cysteic acid from biotransformation 1 is decarboxylated to taurine by CSAD-catalyzed biotransformation according to formula (5). These method steps are disclosed in Examples 7 to 10. To this end, OAS for biotransformation 1 can be produced synthetically or by growing a production strain, as described in Example 1 of the present invention. Producing OAS by growing a production strain is preferred. The CysM enzyme for biotransformation 1 can be obtained from fermentation of a production strain, as described in Example 2. The CSAD enzyme used in biotransformation 2 can be obtained from growing a production strain, preferably the CSADcc enzyme from growing the E. coli JM105×pCSADcc-pKKj strain, as described in Example 6. L-cysteic acid from biotransformation 1 can be used in biotransformation 2 without further workup or after prior workup, as described in Examples 8-10. Those skilled in the art are familiar with various methods for this purpose, such as filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization, etc. It is preferred to use L-cysteic acid from biotransformation 1 in biotransformation 2 without further workup.

[0084] The method for producing taurine simply and efficiently combines a previously undescribed method for enzymatically producing L-cysteic acid from OAS and sulfite (also called sulfurous acid) according to Equation (7) (Biotransformation 1) with a method for enzymatically decarboxylating L-cysteic acid to taurine according to Equation (5) (Biotransformation 2). That is, the present invention is a two-step biotransformation method combining Biotransformation 1 and Biotransformation 2 to produce taurine from OAS.

[0085] Preferably, the process steps (biotransformation 1 and biotransformation 2) for the production of taurine proceed continuously, i.e., one after the other. If the process comprises steps a to d of the preferred embodiment already described, an alternative preferred embodiment is characterized in that all of the process steps are carried out in one reaction batch. If all of the process steps are carried out in one reaction batch, the process is also called a one-pot process or one-pot reaction. This has the advantage that all reactants for the production of taurine are already present in the reaction batch or can be easily metered in. Carrying out the process in one reaction batch is particularly important in terms of economic feasibility.

[0086] For example, according to the disclosure of Example 11 of the present invention, the method steps can be carried out in one batch (one-pot process) in which OAS reacts with sulfite (a salt of sulfurous acid) in the presence of the enzymes CysM and CSADcc. L-cysteic acid is produced in the first reaction according to equation (7) and is decarboxylated to taurine "in situ" by CSADcc according to equation (5).

[0087] The product distribution of L-cysteic acid and taurine from the reaction of OAS in a one-pot process is determined by the activity of CysM relative to CSADcc. By adding a sufficient amount of CSADcc, the L-cysteic acid formed from OAS can be quantitatively converted to taurine. A method 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 60% or more, particularly preferably 70% or more, and particularly preferably 80% or more. The molar yield of taurine based on the molar amount of OAS used is preferably 25% or more, particularly preferably 50% or more, and particularly preferably 80% or more.

[0088] For a one-pot process, it is conceivable to co-express the genes for OAS sulfhydrylase and L-cysteine ​​sulfinic acid decarboxylase in one strain and then react cells from the growth of that strain with OAS in the presence of sulfite for biotransformation to produce taurine as the end product.

[0089] In one variation of the present invention, in the context of a metabolic engineering approach, genes for OAS sulfhydrylase and L-cysteine ​​sulfinic acid decarboxylase are expressed in OAS-producing strains, and taurine is produced by growth of such strains in the presence of sulfite (a salt of sulfurous acid).

[0090] The figure shows the plasmids used in the examples. [Brief explanation of the drawings]

[0091] [Figure 1] pCSADcc-pKKj

[0092] Abbreviations used in the drawings: AmpR: a gene that confers ampicillin (β-lactamase) resistance Ori: origin of replication Ptac:tac promoter ECoRI: Restriction enzyme ECoRI cleavage site HindIII: Restriction enzyme HindIII cleavage site CSADcc: CSAD (cysteine ​​sulfinic acid decarboxylase) C.carpio cds [Example]

[0093] The present invention is further illustrated by, but not limited to, the following examples.

[0094] Example 1: Preparation of OAS The E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 strain, described in EP 1233067 B1 (WACKER) and deposited under the Budapest Treaty at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 13495, was used. OAS was produced by fermentation as described in EP 1233067 B1. At the end of the fermentation, the OAS was stabilized by setting the pH to 4.5 with 21% (v / v) phosphoric acid. The cells were removed by centrifugation (Heraeus Megafuge 1.0 R) at 4000 rpm for 10 min. The OAS content in the fermentation supernatant was 15.3 g / L, as determined by HPLC.

[0095] HPLC analysis of cysteic acid and taurine: The compounds analyzed in the examples were quantified using an HPLC method calibrated for OAS, L-cysteic acid, and taurine, respectively. All reference materials used for calibration were commercially available (Sigma-Aldrich). As known for the analysis of amino acids, an Agilent 1260 Infinity II HPLC system equipped with a unit from the same manufacturer for precolumn derivatization with o-phthaldialdehyde (OPA derivatization) was used. To detect the OPA derivatization products, OAS, L-cysteic acid, and taurine, the HPLC system was equipped with a fluorescence detector. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. A Thermo Scientific Accucore aQ Scientific column, 100 mm long, 4.6 mm internal diameter, and 2.6 μm particle size, was used, thermally equilibrated at 40°C in the column oven.

[0096] Eluent A: 25 mM sodium phosphate, pH 6.0. Eluent B: methanol. The separation was performed in gradient mode, going from 10% Eluent B to 60% Eluent B over 0-25 min, then from 60% Eluent B to 100% Eluent B over 2 min, then to 100% Eluent B for another 2 min 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.

[0097] Example 2: Production of the enzyme CysM The Escherichia coli DH5α / pFL145 strain was used, as described in EP 1247869 B1 (Wacker) and deposited in accordance with the Budapest Treaty at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig) under the number DSM 14088. CysM enzyme was produced both by growth in shake flasks and by fermentation. A) Growth in shake flasks: A preculture of E. coli DH5α / pFL145 strain was prepared in LBamp medium (10 g / l tryptone (GIBCO™), 5 g / l yeast extract (BD Biosciences), 5 g / l NaCl, 100 mg / L ampicillin (Sigma-Aldrich)) (grown overnight at 37°C and 120 rpm). 25 ml of the preculture was used as inoculum for a main culture of 250 ml of LBamp medium (1 L baffled Erlenmeyer flask). The main culture was shaken at 30°C and 110 rpm. After 4 hours, the cell density OD 600 reached 1.0 / ml (OD 600 The cell density per ml of cell suspension was measured photometrically by measuring absorbance at 600 nm (using a Thermo Scientific™ Genesys™ 10S UV-Vis spectrophotometer). The inducer tetracycline (Sigma-Aldrich, final concentration 3 mg / L) was then added, and the culture was continued for another 20 hours at 30°C and 110 rpm. The cell density at the end of the culture was OD 600 was 3 / ml. B) Fermentative production of CysM using E. coli DH5α / pFL145 strain is disclosed in EP 1247869 B1. Cells from the fermentation were removed by centrifugation (Heraeus Megafuge 1.0 R) at 4000 rpm for 10 min, suspended in KPi6.5 buffer (0.1 M K phosphate, pH 6.5), and analyzed at a cell density (OD ). 600 The concentration was adjusted to 90 / ml.

[0098] Cells grown or fermented in shake flasks were separated by centrifugation (15,000 rpm for 10 minutes in a Sorvall RC5C centrifuge with an SS34 rotor) for further use. The cell pellet was resuspended in KPi6.5 buffer as a cell suspension for further use in the production of cell homogenates, as described below. The cell suspension was prepared at a cell density of OD 600 Prepare a sample using a sufficient amount of KPi6.5 buffer to achieve an OD of 30 / ml. For example, 60050 ml of cells from a shake flask culture with an OD of 3 / ml were centrifuged and resuspended in 5 ml of KPi6.5 buffer (10x concentration) or 600 1 ml of cells from a fermentation with a % ATP content of 90 / ml was resuspended in 3 ml of KPi6.5 buffer (3-fold dilution).

[0099] This resulted in the production of cells of the E. coli strain DH5α / pFL145, which were separated from the fermentation broth, resuspended, and used as the OAS sulfhydrylase CysM in the method of the present invention.

[0100] To prepare the cell homogenate, a FastPrep-24™ 5G cell homogenizer from MP Biomedicals was used. Cell density OD 600 One ml of cell suspension in KPi6.5 buffer at 30 / ml was disrupted in a manufacturer-prepared 1.5 ml tube containing glass beads ("Lysing Matrix B") (3 x 20 sec at a shaking frequency of 6000 rpm with a 30 sec pause between each interval). The resulting cell homogenate was used directly as OAS sulfhydrylase (CysM enzyme) in the method of the present invention or for the preparation of cell extracts.

[0101] To prepare the cell extract, the resulting cell homogenate was centrifuged (15,000 rpm for 10 minutes in a Sorvall RC5C centrifuge with an SS34 rotor), and the supernatant was referred to as the cell extract and was used as the OAS sulfhydrylase (CysM enzyme) in the method of the present invention or further used to measure CysM enzyme activity.

[0102] The protein content of the cell extracts was measured using the Qubit® Protein Assay Kit on a Thermo Fisher Scientific Qubit 3.0 Fluorometer. 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.

[0103] CysM enzyme activity was measured as described in EP 1247869B1 (Wacker). For this purpose, OAS (Sigma-Aldrich) was incubated at 37°C in the presence of NaS and cell extract from growth of E. coli DH5α / pFL145. Assays in KPi6.5 buffer (final volume 0.4 ml) contained 10 mM OAS (added from a 200 mM stock solution in 500 mM sodium succinate buffer, pH 5.5), 10 mM sodium sulfide (NaS), and 5 μl of CysM-containing cell extract. Cysteine ​​produced in the CysM reaction was measured using ninhydrin (Sigma-Aldrich) according to the method of Gaitonde (1967), Biochem. J. 104: 627-633. The CysM enzyme activity in the cell extract of E. coli DH5α / pFL145 strain cultured in a shake flask was 57.1 U / ml. 600 The enzyme activity of the shake flask cultured cells was 5.7 U / ml because the OD of the fermented cells (OD 3 / ml) was concentrated 10-fold for the preparation of the cell extract. The CysM enzyme activity in the cell extract after fermentation of E. coli DH5α / pFL145 was 58.1 U / ml. 600 90 / ml) is the OD for the preparation of cell extracts 600 The OD was diluted to 30 / ml, which is the concentration of fermented cells. 600 The enzyme activity in the cell suspension was 174.4 U / ml.

[0104] The specific CysM enzyme activity of the cell extract of E. coli DH5α / pFL145 strain was 10.8 U / mg of protein. The enzyme activity of the cell extract after fermentation of the specific CysM E. coli DH5α / pFL145 strain was 14.5 U / mg. Assuming that CysM activity was completely released from the cells during preparation of the cell extract, the CysM enzyme activity measured in the cell extract was equivalent to the enzyme activity present in the CysM cell suspension in the following examples.

[0105] 1 U / ml CysM enzyme activity means that 1 μmol of cysteine ​​is produced per minute from OAS and Na2S in 1 ml of cell extract under assay conditions (volume activity). Specific CysM enzyme activity in U per mg of 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 based on 1 mg of protein in the cell extract.

[0106] Example 3: Commercial OAS and Na using CysM produced in shake flask culture 2 SO 3 Production of L-cysteic acid from Two batches were run in parallel. Batch 1: A 100 ml Erlenmeyer flask was charged with 8.25 ml of NaPi6.5 buffer (50 mM sodium phosphate, pH 6.5), and the following were added sequentially: 1 ml of 0.2 M NaSO in NaPi6.5 buffer, 0.4 ml of CysM cell extract from a shake flask culture (Example 2A) with an activity of 57.1 U / ml (final concentration in the batch: 2.3 U / ml), and 350 μl of 0.2 M OAS×HCl (Sigma-Aldrich) in 0.5 M sodium succinate, pH 5.5. The batch volume was 10 ml. Batch 2: Batch 1 (a control batch without Na2SO3) had the same composition as batch 1. Batch 2 contained 1 ml of NaPi6.5 buffer instead of Na2SO3 solution.

[0107] Both batches were incubated in a chest shaker (Infors) at 37°C and 140 rpm. After 1 and 3 hours, 1 ml of each batch was incubated at 80°C for 5 minutes to stop the reaction, centrifuged, and the supernatant was analyzed by HPLC. The amount of L-cysteic acid detected by HPLC is shown in Table 1.

[0108] [Table 1]

[0109] Example 4: OAS-containing culture supernatant and NaCl from fermentations using CysM produced in shake flask cultures 2 SO 3 Production of L-cysteic acid from A 100 ml Erlenmeyer flask was charged with 1 ml of cell culture supernatant from the fermentation of E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 strain with an OAS content of 15.3 g / L (from Example 1), followed by 6 ml of NaPi6.5 buffer, 1 ml of a 1 M NaSO solution in NaPi6.5 buffer, and 2 ml of CysM cell suspension from shake flask growth (from Example 2A, cell density OD ). 600 The batch volume was 10 ml. The CysM enzyme activity in the batch was 11.4 U / ml. The batch was incubated at 37°C and 140 rpm in a chest shaker (Infors). After 2 hours, 1 ml of the batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC to determine the OAS and L-cysteic acid content. The progress of the reaction over time is summarized in Table 2.

[0110] [Table 2]

[0111] Example 5: Preproduction of L-cysteic acid by biotransformation of OAS at constant pH A 0.5 L thermostated double-walled glass vessel (Diehm) was connected via a hose connection to a thermostat (Lauda) and the temperature was adjusted to 37°C.

[0112] First, 50 ml (OD ) of a CysM-containing cell suspension in KPi6.5 buffer from a fermentation of strain DH5α / pFL145 (from Example 2B) was cultured. 600The reaction mixture was charged with 6.6 ml of a 400 g / L solution of NaSO (13.9 mmol, molecular weight 190.1 g / mol) in KPi6.5 buffer (90 / ml, CysM enzyme activity 8720 U). In the dissolved state, this corresponded to 27.8 mmol of NaHSO (a 1.78-fold molar excess over the 15.6 mmol of OAS that was subsequently metered in). The batch was stirred with a magnetic stirrer. The batch was also equipped with a pH electrode (Mettler Toledo), which was connected to a pH control unit (TitroLine α titrator, Schott) and operated in pH-stat mode according to the manufacturer's instructions. Under pH-stat conditions, the pH in the reaction vessel was kept constant at a set pH of 6.5 throughout the reaction by metering 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 E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 strain (Example 1) was metered into the batch from a reservoir via a pump (Watson Marlow 101U / R peristaltic pump) at a flow rate of 0.35 ml / min.

[0113] The reaction time was 19 hours. Because the batch was run in an open reaction vessel, the batch volume at the end of the reaction was 185 ml due to evaporation. At 0.5, 3, and 19 hours after the start of the reaction, 1 ml aliquots were removed from the batch and analyzed for L-cysteic acid content by HPLC. The time course of L-cysteic acid production is summarized in Table 3. After 19 hours of reaction time, the L-cysteic acid content in the batch was 12,970 mg / L (76.65 mM), corresponding to an absolute molar yield of 14.18 mmol of L-cysteic acid for a batch volume of 185 ml. Based on the amount of OAS used (15.64 mmol), this corresponds to a 90.1% yield.

[0114] [Table 3]

[0115] Example 6: Recombinant production of CSADcc from Caprinus carpio (carp) in Escherichia coli Vector pCSADcc-pKKj: The cDNA gene (cDNA: complementary DNA, isolated from mRNA by reverse transcription) for cysteine ​​sulfinic acid decarboxylase (CSAD) from common carp (Cyprinus carpio) was isolated by Honjoh et al. (2010), Amino Acids 38: 1173-1183, and the DNA sequence is published in the NCBI (National Center for Biotechnology Information) database as GenBank sequence ID: AB220585.1 (cds: nt 82-1584). The corresponding amino acid sequence was used to derive a codon-optimized DNA sequence for expression in E. coli (published Eurofins Genomics GENEius software) and synthetically generated (Eurofins Genomics). The synthesized DNA had the sequence disclosed in SEQ ID NO: 1 and contained the cds of the gene (hereafter referred to as CSADcc). cds (SEQ ID NO: 1, nt 31-1530) has the amino acid sequence disclosed in SEQ ID NO: 2 and encodes a protein called CSADcc. For cloning purposes, the synthesized DNA had an ECoRI cleavage site (SEQ ID NO: 1, nt 25-30) at the 5' end and a HindIII cleavage site (SEQ ID NO: 1, nt 1532-1537) at the 3' end.

[0116] The vector pCSADcc-pKKjcds (Figure 1), suitable for recombinant expression of CSADcc, was generated by digesting the synthetic DNA with ECoRI and HindIII and cloning it as an ECoRI / HindIII fragment into the ECoRI and HindIII-digested vector pKKj using known methods. The expression vector pKKj, disclosed in EP 2670837 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 (bp 262 to 1947 of the DNA sequence disclosed in M77749.1) was removed from the 4.6 kb plasmid to obtain the 2.9 kb expression vector pKKj.

[0117] E. coli JM105xpCSADcc-pKKj production strain: The CSADcccds were expressed in E. coli by transforming the vector pCSADcc-pKKj into the E. coli K12JM105 strain by known methods. The E. coli K12JM105 strain is commercially available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM3949.

[0118] Clones from the transformation were selected on LB amp 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). Clones were selected for growth and fermentation in shake flasks. The CSADcc-producing strain was designated E. coli JM105×pCSADcc-pKKj. The CSADcc cds were expressed in E. coli JM105×pCSADcc-pKKj by known methods under the control of an IPTG-inducible tac promoter (IPTG: isopropyl β-thiogalactoside, Sigma-Aldrich), operably linked to the CSADcc cds.

[0119] Growth in shake flasks: A preculture of E. coli JM105×pCSADcc-pKKj strain was prepared in LBamp medium (incubated overnight at 37°C and 120 rpm in an Infors chest shaker).

[0120] Two ml of the preculture was used as inoculum for the 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 at 30°C and 140 rpm in a chest shaker (Infors). After 4 h of incubation, the cell density OD 600 The pH reached 2.0. The inducer IPTG (Sigma-Aldrich, final concentration 0.4 mM) was then added and growth continued for a further 20 h at 30°C and 140 rpm on a chest shaker (Infors).

[0121] SM3 medium contains 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 0.002 g / L FeSO4 x 7H2O, 1 g / L Na3 Citrate x 2H2O, 0.1 g / L NaCl, 5 g / L peptone (Oxoid), 2.5 g / L yeast extract (BD Biosciences), 0.005 g / L vitamin B1, and 1 ml / L trace element solution.

[0122] 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.

[0123] Cells from shake flask cultures were separated by centrifugation. A cell suspension was prepared by resuspending the cell pellet from 50 ml of shake flask culture in 2 ml of 50 mm sodium phosphate, pH 7.0 (NaPi 7.0 buffer). The cell suspension was used directly in biotransformation studies or for the preparation of cell homogenates.

[0124] To prepare cell homogenates, an MP Biomedicals FastPrep-24™ 5G cell homogenizer was used. Two 1 ml aliquots of cell suspension were disrupted in a manufacturer-prepared 1.5 ml tube containing glass beads ("Lysing Matrix B") (3 x 20 s at a shaking frequency of 6000 rpm, with a 30 s pause between each interval).

[0125] The resulting cell homogenate (2 ml volume) was used for the biotransformation of L-cysteic acid to taurine without further treatment.

[0126] Fermentative growth: The production strain E. coli JM105xpCSADcc-pKKj was used for the fermentation, which was carried out in a Biostat B fermentor (working volume 2 L) from Sartorius BBI Systems GmbH.

[0127] Shake flask preculture: 100 ml of LBamp medium in a 1-L Erlenmeyer flask was inoculated from the agar medium containing the JM105×pCSADcc-pKKj strain and cultured in an incubation shaker (Infors) at 30°C and 120 rpm for 7 to 8 hours to reach a cell density of OD 600 The culture was continued until the number of cells reached 2-4 / ml.

[0128] Fermenter: 1.5 L of FM2 medium supplemented with 40 g / L glucose and 100 mg / L ampicillin was inoculated with 21.3 ml of the shake flask preculture. Fermentation conditions were: temperature 30°C, constant pH 7.0 (automatically corrected with 25% NH4OH and 6.8 N H3PO4), foam control by automated metered addition of 4% v / v Struktol J673 (Schill & Seilacher) in water, stirrer speed 450–1300 rpm, constant aeration with sterile-filtered compressed air at 1.7 vvm (vvm: the amount of compressed air introduced into the fermentation batch expressed as liters of compressed air per liter of fermentation volume per minute), and pO2 ≥ 50%. The oxygen partial pressure pO2 was regulated by the agitation speed. After 16 h of fermentation, the cell density OD 600 reached 45 / ml.

[0129] Production fermenter: 150 ml of pre-fermentor culture was inoculated into 1.35 L of FM2 medium at pH 7.0 supplemented with 20 g / L glucose, 0.36 g / L pyridoxine (vitamin B6, Sigma-Aldrich), and 100 mg / L ampicillin. Fermentation conditions were: temperature 30°C, constant pH 7.0 (automatically corrected with 25% NH4OH and 6.8 N H3PO4), foam control by automated metered addition of 4% v / v Struktol J673 (Schill & Seilacher) in water, stirrer speed 450–1300 rpm, constant aeration 1.7 vvm, and pO2 ≥ 50%. Oxygen partial pressure (pO2) was controlled by the stirrer speed. Fermentation time was 30 h.

[0130] FM2 medium: 5 g / L (NH4)2SO4, 0.50 g / L NaCl, 0.075 g / L FeSO4 x 7H2O, 1 g / L Na3 citrate, 0.30 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 1.50 g / L KH2PO4, 0.005 g / L vitamin B1 (Sigma-Aldrich), 5.00 g / L peptone (Oxoid), 2.50 g / L yeast extract (Oxoid), 0 ml / L trace element solution (equivalent to that used for shake flask culture).

[0131] First, the pH in the fermenter was adjusted to 7.0 by injecting a 25% NH4OH solution. During the fermentation, the pH was maintained at 7.0 by automatic correction with 25% NH4OH or 6.8N H3PO4. For inoculation, 150 ml of preculture was injected into the fermenter vessel. Thus, the initial volume was 1.5 L. The culture was 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.

[0132] The target value for O2 saturation (pO2) during the fermentation was set at 50%. After the O2 saturation fell below the target value, a control cascade was initiated to bring the O2 saturation back to the target value. In connection with this, the agitation speed was continuously increased (up to a maximum of 1300 rpm).

[0133] Fermentation was carried out at a temperature of 30°C. When the glucose content in the fermenter decreased from an initial 20 g / L to approximately 5 g / L, a 60% (w / w) glucose solution was continuously metered in. The feed rate was adjusted so that the glucose concentration in the fermenter did not subsequently exceed 2 g / L. Glucose was measured using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0134] OD 600 When the cell density (OD) reached 50 / ml (8 hours of fermentation), the inducer IPTG (final concentration 0.2 mM) was added once to initiate CSADcc gene expression. Fermentation was stopped 22 hours after induction, i.e., after a total fermentation time of 30 hours. At this point, the cell density (OD) 600 One liter of fermentation broth was centrifuged (15,000 rpm for 10 minutes in a Sorvall RC5C centrifuge equipped with an SS34 rotor), the fermentation supernatant was discarded, and the cells were resuspended in 1 liter of NaPi7.0 buffer and stored in 50 ml aliquots at −20°C for further use.

[0135] Example 7: Production of taurine by biotransformation of commercially available L-cysteic acid 12 mg of L-cysteic acid x HO (Sigma-Aldrich) was weighed into a 100 ml Erlenmeyer flask and dissolved in 9.7 ml of NaPi 7.0 buffer. The reaction was initiated by the addition of 0.3 ml of cell homogenate from shake flask growth of JM105 x pCSADcc-pKKj (Example 6). The batch volume was 10 ml. The molarity of L-cysteic acid x HO was 6.41 mM (molecular weight of L-cysteic acid x HO: 187.2 g / mol). The batch was incubated at 37 °C and 140 rpm on a chest shaker (Infors). After 3 hours, 1 ml of the batch was incubated at 80 °C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The L-cysteic acid used was completely consumed. The amount of taurine produced was 789.4 mg / L, corresponding to a molar content of 6.31 mM (molecular weight of taurine: 125.1 g / mol). Therefore, the molar yield of taurine produced from 6.41 mM L-cysteic acid × HO was 98.4%.

[0136] Example 8: Production of taurine by biotransformation from commercially available OAS Response 1: Production of L-cysteic acid from OAS: A 100 ml Erlenmeyer flask was first charged with 6.6 ml of KPi6.5 buffer, followed by the addition of 0.4 ml of a 0.2 M OASxHCl (Sigma-Aldrich) stock solution dissolved in 0.5 M sodium succinate (pH 5.5), 1 ml of 1 M NaSO in KPi6.5 buffer, and 2 ml of a cell suspension of cysM cells from shake flask growth (CysM enzyme activity 57.1 U / ml, from Example 2A). The batch volume was 10 ml. The measured amount of CysM enzyme in the batch was 11.4 U / ml. The molarity of OASxHCl was 8.00 mM (1.47 g / L, molecular weight of OASxHCl: 183.6 g / mol). The batch was incubated at 37 °C and 140 rpm on a chest shaker (Infors). After 3 hours, 1 ml of the batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The OAS used was completely consumed. The amount of L-cysteic acid produced was 1350.4 mg / L, corresponding to a molar content of 7.98 mM (molecular weight of L-cysteic acid: 169.2 g / mol). The molar yield of L-cysteic acid produced from 8.00 mM OAS × HCl was 99.7%.

[0137] Reaction 2: Production of taurine from L-cysteic acid synthesized in reaction 1: A 9 ml batch of reaction 1 was placed in a 100 ml Erlenmeyer flask, the pH was adjusted to 7.0 with 1 M KOH, and 1 ml of cell homogenate from shake flask growth of JM105×pCSADcc-pKKj strain (Example 6) was added. The batch volume was 10 ml. The L-cysteic acid content from reaction 1 was 1350.4 mg / L, and the L-cysteic acid content at the start of reaction 2 was 1215.4 mg / L (7.18 mM, with a molecular weight of 169.2 for L-cysteic acid). The batch was incubated at 37°C and 140 rpm on a chest shaker (Infors). After 3 hours, 1 ml of the batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The L-cysteic acid used was completely consumed. The taurine content was 825.7 mg / L (6.60 mM with a molecular weight of 125.1 g / mol). The molar yield of taurine produced from 7.18 mM L-cysteic acid was 91.9%.

[0138] Example 9: Fermentative production of OAS and NaHSO 3 Taurine production by CSADcc-catalyzed conversion of L-cysteic acid from the reaction with fermentatively produced CysM enzyme In a 100 ml Erlenmeyer flask, 7 ml of NaPi7.0 buffer containing 5 mg / L LPLP, 1 ml of the batch from Example 5 with an L-cysteic acid content of 12,970 mg / L, and 2 ml of CSADcc cells (Example 6) in NaPi7.0 buffer were mixed. The batch volume was 10 ml. The L-cysteic acid content in the batch was 1,297 mg / L, corresponding to a molar content of 7.67 mM (molecular weight of L-cysteic acid: 169.2 g / mol). The batch was incubated at 37°C and 140 rpm on a chest shaker (Infors). After 4 hours, 1 ml of the batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The L-cysteic acid used was completely consumed. The amount of taurine produced was 925.3 mg / L, corresponding to a molar content of 7.40 mM (molecular weight of taurine: 125.1 g / mol). The molar yield of taurine produced from 7.67 mM L-cysteic acid was 96.4%.

[0139] Example 10: Preproduction of taurine Biotransformation 1: A 0.5 L thermostated double-walled glass vessel (Diehm) was connected via a hose connection to a thermostat (Lauda) and the temperature was adjusted to 37°C.

[0140] First, 50 ml (OD ) of CysM-containing cell suspension in KPi6.5 buffer from the fermentation of strain DH5α / pFL145 (Example 2) was cultured. 600The reaction mixture was charged with 20 ml of a 400 g / L solution of NaSO (42.1 mmol, molecular weight 190.1 g / mol) in KPi6.5 buffer (90 U / ml, CysM enzyme activity 8720 U). In the dissolved state, this corresponded to 84.2 mmol of NaHSO (a 1.63-fold molar excess over the 51.7 mmol of OAS metered in later). The batch was stirred with a magnetic stirrer. The batch was also equipped with a pH electrode (Mettler Toledo), which was connected to a pH control unit (TitroLine α titrator, Schott) and operated in pH-stat mode according to the manufacturer's instructions. Under pH-stat conditions, the pH in the reaction vessel was kept constant at a set pH of 6.5 throughout the reaction by metering 2 M NaOH from a burette connected to the control unit. Four hundred ml of OAS-containing fermentation supernatant (OAS content: 19.1 g / L, 7.6 g, 51.7 mmol) from the fermentation of E. coli W3110 / pACYC-cysEX-GAPDH-ORF306 strain (Example 1) was metered into the batch from a reservoir via a pump (Watson Marlow 101U / R peristaltic pump) at a flow rate of 0.2 ml / min. The total reaction time was 46 h. The batch volume at the end of the reaction was 500 ml. After 1, 3, 20, 28, and 46 h from the start of the reaction, 1 ml aliquots were removed from the batch in each case, incubated at 80 °C for 5 min, centrifuged, and the supernatant was analyzed by HPLC to determine the L-cysteic acid content. The formation of L-cysteic acid over time is summarized in Table 4. After 46 hours of reaction time, the L-cysteic acid content in the batch was 15,370 mg / L (90.8 mM), which corresponds to an absolute molar yield of 45.4 mmol of L-cysteic acid for a batch volume of 500 ml. Based on the amount of OAS used, 51.7 mmol, this corresponds to a yield of 87.8%.

[0141] [Table 4]

[0142] Biotransformation 2: A 0.3 L thermostated double-walled glass vessel (Diehm) was connected via a hose connection to a thermostat (Lauda) and the temperature was adjusted to 37°C.

[0143] 100 ml of L-cysteic acid-containing biotransformation 1 was adjusted to pH 7.0 with 2.5 M NaOH. Additionally, 1 ml of 1 M DTE (dithioerythritol, Sigma-Aldrich) dissolved in HO, 1 ml of 500 mg / L LPLP (final concentration 4 mg / L) dissolved in NaPi 7.0 buffer, and 20 ml of CSADcc-containing fermented cells resuspended in NaPi 7.0 buffer were added. The batch was stirred magnetically. The batch volume was 122 ml. At the start of the reaction and after 2, 4, 6, and 24 hours, 1 ml aliquots were removed from the batch in each case, incubated at 80 °C for 5 min, centrifuged, and the supernatant analyzed for L-cysteine ​​and taurine content by HPLC. The results are summarized in Table 5. Based on the molar amount of L-cysteic acid used in the batch of 74.4 mM, the taurine yield (8.8 g / L, 70.2 mM) was 94.3%.

[0144] [Table 5]

[0145] Example 11: Production of taurine from OAS by a "one-pot reaction" The reaction batch consisted of 3 ml of KPi6.5 buffer, 2 ml of OAS-containing fermentation supernatant from the fermentation of E. coli strain W3110 / pACYC-cysEX-GAPDH-ORF306 (Example 1), 1 ml of 1 M NaSO 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 shake flask growth of strain JM105xpCSADcc-pKKj (Example 6). The batch volume was 10 ml. The OAS concentration in the batch was 3.1 g / L (20.80 mM). The NaSO concentration in the batch was 100 mM. The CysM enzyme activity in the batch was 34.9 U / ml.

[0146] The reaction was carried out at pH 6.5. The batch was incubated at 37°C and 140 rpm on a chest shaker (Infors). 24 h after the start of the reaction, 1 ml of the batch was incubated at 80°C for 5 min, centrifuged, and the supernatant was 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 produced from 20.80 mM OAS was 36.9%. The molar yield of 5.76 mM taurine produced from 20.80 mM OAS was 27.7%. Overall, the molar yield of L-cysteic acid and taurine produced from OAS was 64.6%.

Claims

1. A method for producing taurine from O-acetyl-L-serine (OAS) by biotransformation, comprising: i) in a first step (biotransformation 1), L-cysteic acid is produced from O-acetyl-L-serine (OAS) in the presence of sulfite using an enzyme selected from the class of OAS sulfhydrylases (EC 4.2.99.8), said biotransformation being carried out under active pH control, and then ii) in a second step (biotransformation 2), L-cysteic acid is decarboxylated to taurine; the OAS concentration in the batch is at least 10 g / L; The OAS sulfhydrylase is CysM; The method.

2. 2. The method of claim 1, wherein the OAS sulfhydrylase is a bacterial enzyme.

3. 3. The method of claim 1, wherein the OAS sulfhydrylase is CysM derived from an Escherichia coli strain.

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

5. 5. The method of claim 1, wherein the concentration of the sulfite is at least equimolar to the OAS.

6. 6. The method according to any one of claims 1 to 5, wherein L-cysteic acid is decarboxylated to taurine using an enzyme from the class of L-cysteine ​​sulfinic acid decarboxylase (EC 4.1.1.29), aspartate 1-decarboxylase (EC 4.1.1.11) or glutamate decarboxylase (EC 4.1.1.15).

7. 7. The method of claim 6, wherein the L-cysteine ​​sulfinic acid decarboxylase has a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:

2.

8. The method according to claim 6 or 7, wherein the L-cysteine ​​sulfinic acid decarboxylase is derived from fermentation production.

9. a) OAS is produced by fermentation; b) enzymes of the class of OAS sulfhydrylases (EC 4.2.99.8) and of the class of cysteine ​​sulfinic acid decarboxylases (EC 4.1.1.29) are produced by fermentation; c) OAS and sulfite react under enzymatic catalysis by OAS sulfhydrylase of point b to form L-cysteic acid; d) L-cysteic acid at point c is decarboxylated to taurine by the CSAD enzyme at point b; The method according to any one of claims 1 to 8.

10. 10. The method according to claim 9, wherein all process steps are carried out in one reaction batch.

11. 11. The method according to any one of claims 1 to 10, wherein the molar yield of taurine from the biotransformation of L-cysteic acid in biotransformation 2 is at least 60%.

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