Method for isolating taurine from fermentation medium containing taurine and hypotaurine after microbial fermentation

Abstract

The present invention relates to a method for isolating taurine from a fermentation broth after microbial fermentation of a production strain, comprising the following isolation steps in the given order: (i) isolating a fermentation supernatant by separating biomass from the fermentation broth, (ii) precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1 (e.g., ethanol), (iii) isolating the taurine precipitate and dissolving it in solvent 2 (water), (iv) crystallizing the dissolved taurine, and (v) isolating and optionally drying the crystallized taurine, whereby the isolated taurine has a purity of >80% (w / w). Thus, a simple method for isolating fermentatively prepared, highly pure taurine is provided.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating taurine from fermenter broth after microbial fermentation of a production strain, comprising the following isolation steps in the order shown: (i) isolating a fermentation supernatant by removing biomass from the fermenter broth, (ii) precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1, (iii) isolating the taurine precipitate and dissolving it in solvent 2, (iv) crystallizing the dissolved taurine, and (v) isolating and optionally drying the crystallized taurine, whereby the isolated taurine is obtained with a purity of >80% (w / w). This provides a simple method for isolating fermentatively produced taurine with high purity. [Background technology]

[0002] Taurine (2-aminoethanesulfonic acid, CAS number 107-35-7) is an aminosulfonic acid that occurs in nature as a breakdown product of the amino acids cysteine ​​and methionine. Taurine is economically important and is a component of energy drinks and is also used, for example, in cat or fish feed. However, taurine is also said to have health-promoting effects.

[0003] Commercially available taurine is currently produced chemically. One known method, for example, that of the Changshu Yudong Chemical Factory, starts with ethylene and produces taurine via ethyleneimine. As consumers move away from chemically produced ingredients, biotechnological methods for producing taurine are increasingly being researched.

[0004] In nature, taurine occurs almost exclusively in the animal kingdom, with only a few examples occurring in plants, algae, or bacteria. There are various biosynthetic pathways to taurine (see, for example, the KEGG Pathway Database: "Taurine and hypotaurine metabolism"), particularly those starting from L-cysteine. The most important steps leading to taurine from L-cysteine ​​are shown in Equations (1) to (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

[0005] (1) In the first step, L-cysteine ​​is oxidized to L-cysteine ​​sulfinic acid (3-sulfinoalanine, CAS number 207121-48-0) by the enzyme cysteine ​​dioxygenase (CDO, EC 1.13.11.20).

[0006] (3) and (5): Cysteine ​​sulfinate decarboxylase (CSAD, EC 4.1.1.29) can decarboxylate L-cysteine ​​sulfinic acid to hypotaurine (2-aminoethanesulfinic acid, CAS number 300-84-5) and, in a similar reaction, decarboxylate L-cysteic acid to taurine.

[0007] (2) and (4): The oxidation of cysteine ​​sulfinic acid to cysteic acid and the oxidation of hypotaurine to taurine are not yet clearly understood.

[0008] An alternative metabolic engineering pathway to taurine is possible using the enzyme cysteate synthase (CS, EC 2.5.1.76), known from methanogenic archaea, where L-cysteic acid is formed in a side reaction of coenzyme M biosynthesis according to equation (6).

[0009] (6) O-phospho-L-serine + SO 2- →L-cysteic acid + HPO4 2-

[0010] The L-cysteic acid formed in this reaction can then be decarboxylated by the CSAD enzyme according to equation (5). O-phospho-L-serine (OPS) is used as a biosynthetic precursor of L-serine in L-cysteine ​​metabolism, for example, by Escherichia coli (see, for example, KEGG Pathway Database: "Cysteine ​​and methionine metabolism").

[0011] A drawback of known biotechnological methods for producing taurine is that a product mixture consisting of hypotaurine and taurine is formed, with hypotaurine generally being the major product. Furthermore, the yields of known biotechnological methods for producing taurine and hypotaurine have so far been very low, and there are no known methods for isolating the products from the fermentation process.

[0012] Honjoh et al. (2010) Amino Acids 38:173-1183 described a genetically engineered yeast strain heterologously expressing the CDO and CSAD genes from common carp (Cyprinus carpio). When L-cysteine ​​was added to the growth of the genetically engineered strain, hypotaurine was observed as the major product, with a relatively low proportion of taurine. Although S. cerevisiae itself can produce cysteine ​​from its own metabolism, hypotaurine production required the external addition of L-cysteine ​​to the growth medium. Analytical measurements of intracellularly formed taurine were performed with and without HO treatment (Honjoh et al., Table 1). Although hypotaurine analysis was listed in the methods section of Honjoh et al., analytical quantification of hypotaurine was not performed. However, the significantly higher taurine levels after HO treatment suggested that the majority of the intracellularly formed product was in the form of hypotaurine. No method for isolating taurine from yeast cells has been described.

[0013] WO 17 / 213142 (Ajinomoto) describes a taurine-producing strain obtained by heterologous expression of cysteine ​​dioxygenase and L-cysteine ​​sulfinic acid decarboxylase in a strain originally producing cysteine. The main product was hypotaurine with a maximum yield of 450 μM, which could be converted to taurine in low yield by subsequent chemical treatment with alkali. Common methods from the prior art for isolating amino acids, such as cation exchange chromatography, membrane filtration, and crystallization, were mentioned, but no information was provided regarding the suitability of the method for isolating taurine, the yield, and the purity achieved for the taurine product.

[0014] US Patent Application Publication No. 2019 / 0062757 (KnipBio) describes a heterologous production strain for producing taurine or its precursors, with the maximum yield achieved being only 419 ng / ml hypotaurine. Common methods from the prior art for isolating taurine, such as centrifugation, filtration, or ion exchange chromatography, were mentioned, but no purification method was disclosed, and it was not demonstrated whether the method was suitable for isolating taurine. Furthermore, no information was provided regarding the purity achieved for the taurine product.

[0015] Ma et al., Food Anal. Methods (2018) 11:415-425, described a laboratory process for isolating taurine from bovine liver. Liver tissue was first disrupted by ultrasound to prepare a protein-free extract, from which taurine was isolated after activated carbon treatment and subsequent crystallization by cation exchange chromatography.

[0016] Lin et al., Ind. Eng. Chem. Res. (2013) 52:13449-13458, describes the crystallization of taurine from chemical production.

[0017] The prior art discloses methods for isolating taurine from chemical production or biological materials, including animal organs such as bovine liver, rather than from microbial fermentation. Recombinant microorganisms with heterologous metabolic pathways leading to taurine and, in particular, hypotaurine, are known, such as Escherichia coli, baker's yeast, or Corynebacterium glutamicum. The disclosed yields of both hypotaurine and taurine are consistently low. No efficient method is known for the preparative conversion of hypotaurine to taurine as a step in the taurine workup process. Only a general method for taurine isolation from amino acid workup has been described. There are no known methods for isolating fermentatively produced taurine. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] International Publication No. 2017 / 213142 [Patent Document 2] US Patent Application Publication No. 2019 / 0062757 [Non-patent literature]

[0019] [Non-Patent Document 1] Honjoh et al. (2010), Amino Acids 38:173-1183 [Non-patent document 2] Ma et al.,Food Anal.Methods(2018)11:415-425 [Non-patent document 3] Lin et al.,Ind.Eng.Chem.Res.(2013)52:13449-13458 Summary of the Invention

[0020] It is an object of the present invention to provide a simple method for isolating fermentatively produced taurine in high purity and high yield while avoiding chromatographic steps.

[0021] The object of the present invention is achieved by a method for isolating taurine from a fermenter broth after microbial fermentation of a production strain, the method comprising the following isolation steps in the given order: i. isolating the fermentation supernatant by removing the biomass from the fermenter broth; ii. Precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1; iii. isolating the taurine precipitate and dissolving it in solvent 2; iv. Crystallizing the dissolved taurine; v. Isolating and optionally drying the crystallized taurine, wherein the isolated taurine has a purity of >80% (w / w).

[0022] A major advantage of the present invention is that it does not require a chromatographic step and therefore provides a simple method for isolating fermentatively produced taurine in high purity.

[0023] Chromatography is used to separate compounds, but the distribution of individual components differs between the stationary and mobile phases. In this way, relatively pure substances can be obtained. The prior art method for isolating taurine by Ma (2018, supra) also includes a chromatography step. Surprisingly, this method does not use a chromatography step, yet can still isolate taurine with a purity of over 80% (w / w). Therefore, the method for isolating taurine according to the present invention reduces the number of work steps and materials, making it more economically viable overall.

[0024] A process in the context of the present invention is defined as a multi-step sequence of working steps, in which the product from a batch is separated in a predetermined order of successive processing steps, also called work-up or DSP (downstream processing). The number of processing steps is variable and depends on the desired purity of the product. The present invention for isolating taurine from a fermentation batch is one process.

[0025] Fermentation is a process for the production (cultivation) of cell cultures on an industrial scale, in which a microbial production strain is preferably grown under defined conditions of culture medium (growth medium), temperature, pH, oxygen supply, and medium mixing. Optionally, to optimize the formation of biomass and target products, medium components such as glucose (carbon source) or a complex amino acid mixture such as yeast extract (nitrogen source) are continuously supplied as a feed in so-called fed-batch fermentation. Depending on the genetic makeup of the production strain, the goal of fermentation is to produce the highest possible yield of proteins / enzymes or metabolic products for further use. Taurine and hypotaurine products can be produced by fermentation. The end product of fermentation is a fermenter broth consisting of the biomass of cells of the production strain (fermentor cells) and a biomass-free fermentation medium (fermentation supernatant) formed over the course of fermentation from the growth medium and metabolites secreted by the fermentor cells. In contrast to the growth medium, which is defined by its chemical composition, the composition of the fermentation medium is not clearly defined because metabolic product formation is unpredictable. In the present invention, taurine and hypotaurine products are produced by fermentation. The products of the fermentation can be present in the fermenter cells or in the fermentation supernatant.

[0026] Yield in the context of the present invention is defined as the amount of product obtained in a work-up step or treatment. Yield can be expressed as an absolute amount of product (mmol or g), as a volumetric yield (concentration) as an absolute amount of product per unit volume (mM or g / L), or as a relative yield of the amount of product obtained in a work-up step or treatment as a percentage of the amount of product used in the work-up step or treatment, also called percent yield.

[0027] In the case of a chemical reaction rather than a work-up treatment, the yield of the reaction can also be expressed as the absolute amount of product (mmol or g), as a volumetric yield (concentration) as the absolute amount of product per unit volume (mM or g / L), or as a relative yield of product as a percentage of the reactants used (taking into account the molecular weights of the reactants and product), also called percent yield. In the context of the present invention, hypotaurine in the fermentation supernatant is oxidized to taurine by HO, although taurine may also already be present in the fermentation supernatant together with hypotaurine. The molar yield of taurine from the oxidation of hypotaurine- and taurine-containing fermentation supernatant is defined as the total molar amount of taurine after oxidation relative to the sum of the molar amounts of hypotaurine and taurine present in the fermentation supernatant, expressed as a percentage.

[0028] The amount of product detected analytically in a workup fraction, e.g., by HPLC, is defined as product content or purity. If the fraction is solid, the purity is expressed as g product / g solid, or this value is expressed as a percentage. For example, if the product content is 0.8 g in 1 g solid, this is called 80% content or purity. The fraction may also be a solution. In this case, the purity is expressed as g product / g liquid fraction.

[0029] A solvent is a substance capable of diluting or dissolving a gas, liquid, or solid without chemical reactions occurring between the solute and the solvent during the process. In the context of the present invention, water-miscible organic solvents are used. Non-limiting examples of such solvents are organic solvents, such as ethanol, methanol, 1-propanol, 2-propanol (isopropanol), and acetone. A water-miscible solvent is defined as a solvent that produces only one liquid phase at any mixing ratio with water, whereas a water-immiscible solvent has limited solubility in water depending on its chemical properties (especially nonpolarity) and undergoes phase separation into an aqueous phase and a solvent phase when the concentration in the mixture exceeds the concentration characteristic of the solvent in question. Examples of water-immiscible solvents are hydrocarbons such as n-pentane, n-hexane, or n-heptane, and esters such as ethyl acetate or butyl acetate.

[0030] An open reading frame (ORF, synonymous with cds or coding sequence) is a region of DNA or RNA that begins with a start codon and ends with a stop codon and codes for the amino acid sequence of a protein. ORFs are also called coding regions or structural genes.

[0031] A gene, cistron, or expression unit refers to a segment of DNA that contains all the basic information for producing biologically active RNA. A gene comprises a segment of DNA that is transcribed to produce a single-stranded RNA copy, and expression signals involved in regulating this copying process. Expression signals include at least one promoter, transcription initiation, translation initiation, and ribosome binding site. Terminators and one or more operators are additional possible expression signals.

[0032] Alleles define the states of a gene that can be converted into one another by mutation, i.e., by changes in the nucleotide sequence of DNA. A gene that occurs naturally in a microorganism is called a wild-type allele, and any variant derived from it is called a mutant allele of the gene.

[0033] A promoter is a nucleotide sequence upstream of the 5' end of a cDNA fragment that allows for gene expression. In the direction of synthesis, the promoter precedes the RNA coding region. A promoter mediates the initiation of gene transcription by RNA polymerase and contains regions that interact specifically with DNA-binding proteins called transcription factors.

[0034] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for protein synthesis. mRNA provides building instructions for specific proteins within a cell. mRNA molecules transmit the messages needed for protein synthesis from the genetic information (DNA) to the ribosomes responsible for protein synthesis. In cells, they are formed as transcripts of segments of DNA corresponding to genes. The genetic information stored in DNA remains unchanged during this process.

[0035] Eukaryotic genes are primarily known as mosaic genes and, unlike prokaryotic genes, also contain non-coding regions known as introns (intragenic regions). Coding sequences, known as 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 transcription of DNA into RNA, introns are spliced ​​out of the primary transcript. The protein-coding RNA from which the introns have been removed is called messenger RNA (mRNA) or "mature" mRNA. 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, because processing of the exon / intron structure does not occur in prokaryotes. Whenever the terms "expressed gene sequence derived from a protein sequence" or "gene sequence derived from an mRNA" are used in connection with the present invention, this reverse translation process is precisely what is meant. Sequence optimization, ie adaptation to the codon usage of the corresponding prokaryote (codon optimization), is preferably carried out simultaneously with the back-translation of the protein or mRNA sequence into a DNA sequence.

[0036] A homologous gene or sequence is to be understood as meaning that the DNA sequences or DNA portions of said genes are at least 80% identical, preferably at least 90% identical, particularly preferably at least 95% identical.

[0037] The degree of DNA identity was determined by the "nucleotide blast" program, which can be found at http: / / blast.ncbi.nlm.nih.gov / and is based on the blastn algorithm. The algorithm parameters used to align two or more nucleotide sequences were default parameters. The default general parameters were: 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 were: Match / Mismatch Scores = 1, -2; Gap Costs = Linear.

[0038] Homologous protein sequences are to be understood as meaning that the protein sequences or protein parts of these proteins are at least 80% identical, preferably at least 90% identical, particularly preferably at least 95% identical.

[0039] Protein sequences were 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 were 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 matrix adjustment.

[0040] A gene construct refers to a DNA molecule in which at least one cds is linked to other genetic elements (e.g., promoter, ribosome binding site (RBS), terminator, selection marker, origin of replication). 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 and result in the production of the protein encoded by the gene.

[0041] An operon is defined as a high-level expression unit in which multiple genes are transcribed under the control of a single promoter, but each is translated from its own ribosome binding site. Operons are often found in the genomes of bacterial microorganisms, but can also be specifically created by cloning (artificial operons). In the expression vector of the present invention (Example 1), the expression unit a) serA317, CDOrn and CSADcc (expression vector pCys-CDOrn-CSADcc, Figure 2); b) serA317, CSma and CSADcc (expression vector pCys-CSma-CSADcc, Figure 3); c) serA317, CSma, CSADcc, and CDOrn (expression vector pCys-CSma-CSADcc-CDOrn, Figure 4) Each of these forms an artificial operon, and their expression is under the control of the serA promoter.

[0042] The abbreviation WT (Wt) refers to the wild-type form of a gene. 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.

[0043] In contrast to biotransformation, metabolic engineering (also called "pathway design") is a biotechnological method in which an organism's metabolic pathways are modified by optimizing or modifying genes and / or regulatory processes. New or modified enzymes can be introduced into an organism by supplementing the genome with the enzyme's gene, or the expression level of an endogenous enzyme's gene can be increased or decreased, thereby establishing new metabolic pathways in the organism or enhancing or weakening existing metabolic pathways. The goal of metabolic engineering is for an organism to produce either a new metabolite or a cell-resident metabolite with high yield. The result of metabolic engineering is a producing strain. Metabolic engineering methods do not use metabolite-specific starting materials, e.g., enzyme substrates such as L-cysteine ​​as a starting compound for producing hypotaurine. Instead, they use only a nutrient medium, also called a growth medium, necessary for the growth of the organism in question and composed 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. In the present invention, metabolically engineered strains for fermentative production of hypotaurine and taurine are used.

[0044] In the context of the present invention, a microorganism produced by metabolic engineering and capable of producing hypotaurine and / or taurine by fermentation is called a producer strain. Particularly preferably, the producer strain contains at least one gene construct containing genes of a biosynthetic pathway that results in the production of hypotaurine, taurine, or a mixture of hypotaurine and taurine after expression in the producer strain. A microorganism strain that does not contain a gene construct is also called a host strain. Thus, a producer strain is the product of a host strain plus a gene construct.

[0045] The microbial production strain for producing hypotaurine and / or taurine is preferably 1) metabolic pathways leading to hypotaurine and / or taurine; and 2) Deregulated cysteine ​​biosynthetic pathway The present invention is characterized by comprising:

[0046] 1) The metabolic pathway leading to hypotaurine and / or taurine is preferably: i) the reaction of L-cysteine ​​to form hypotaurine according to equations (1) and (3), i.e. the production strain contains at least a cds encoding the enzyme CDO and a cds encoding the enzyme CSAD, and / or ii) the reaction of OPS to form taurine according to equations (6) and (5), i.e. the production strain contains at least a cds encoding the enzyme CS and a cds encoding the enzyme CSAD; and / or iii) In the combination of i) and ii), the reaction to form a mixture of hypotaurine and taurine is included, i.e., the production strain contains at least cds encoding the enzymes CDO, cds encoding the enzymes CSAD, and cds encoding the enzymes CS. It is characterized by:

[0047] Particularly preferably, the producing strain is characterized in that it contains at least a cds encoding the enzyme CDO and a cds encoding the enzyme CSAD, and thus catalyzes the reaction of L-cysteine ​​to form hypotaurine according to equations (1) and (3).

[0048] The genes of the production strain that constitute the hypotaurine and / or taurine metabolic pathway may be derived from the host strain (homologous expression), or may be foreign genes produced synthetically (which allows, inter alia, the adaptation of cds by so-called codon optimization for expression in the host strain) or isolated from other strains (heterologous expression), or a combination of homologous and heterologous expression may be performed.

[0049] Heterologous expression of genes of the hypotaurine and / or taurine constitutive metabolic pathway in the production strain is preferred, with heterologous expression of synthetically engineered genes being particularly preferred, and heterologous expression of codon-optimized synthetically engineered genes of the hypotaurine and / or taurine constitutive metabolic pathway in the production strain being especially preferred.

[0050] 2) Because metabolic pathways i) to iii) to hypotaurine and / or taurine corresponding to Equations (1) and (3) and Equations (6) and (5) proceed from the metabolite OPS and L-cysteine ​​from the L-cysteine ​​biosynthetic pathway (see, for example, the KEGG pathway database: "Cysteine ​​and methionine metabolism"), a production strain containing a deregulated cysteine ​​biosynthetic pathway and capable of cysteine ​​production is preferred. As described, for example, in Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73:48-54, cysteine ​​production in wild-type microorganisms is tightly regulated, which means that microbial strains with a regulated cysteine ​​biosynthetic pathway cannot produce commercially viable amounts of hypotaurine and / or taurine, as demonstrated in the prior art with low yields (e.g., production of up to 2 μM cysteine ​​in WO 17 / 213142 A1, Table 5). Thus, although wild-type microorganisms contain a cysteine ​​biosynthetic pathway, it is highly regulated by product inhibition (so-called feedback inhibition) of key biosynthetic enzymes, and only minimal, if any, amounts of cysteine ​​are formed during growth of these microorganisms (see WO 17 / 213142 A1, Table 5, maximum cysteine ​​production of 2 μM). Therefore, the term "cysteine-producing ability" encompasses only microorganisms with a deregulated cysteine ​​biosynthetic pathway. Therefore, preferred are producer strains with a deregulated cysteine ​​biosynthetic pathway and capable of producing hypotaurine and taurine. Such strains are disclosed in Example 2 of the present invention.

[0051] Microorganisms containing a deregulated cysteine ​​biosynthetic pathway and thus capable of producing cysteine ​​are characterized by containing at least one of the following modifications:

[0052] a) The microbial strain is characterized by a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) in which feedback inhibition by L-serine is reduced by at least two-fold compared to the corresponding wild-type enzyme, and SerA enzyme activity can be determined photometrically, e.g., by oxidation of NADH dependent on the SerA substrate 3-phosphohydroxypyruvate, as described, e.g., by McKitrick and Pizer, J. Bacteriol. (1980) 141:235-245.

[0053] In particularly preferred mutants of 3-phosphoglycerate dehydrogenase (serA), feedback inhibition by L-serine is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment by at least 50-fold compared to the corresponding wild-type enzyme.

[0054] Examples of serA mutants that encode SerA enzymes with reduced feedback inhibition are known from the prior art and are preferred embodiments in the context of the present invention. - serA317: a feedback-resistant SerA mutant used in the vector pCys of the present invention (Fig. 1). This is a C-terminal deletion mutant of SerA containing the N-terminal 317 amino acids of the full-length 410 amino acid SerA WT protein (Bell et al., Eur. J. Biochem., 2002, 269:4176-4184; referred to herein as "NSD:317"; see also Example 1). -serA G349E: mutation of glycine to glutamic acid at position 349 of the WT SerA enzyme. -serA G349D: mutation of glycine to aspartic acid at position 349 of the WT SerA enzyme. -serA T372X: A mutation of the threonine at position 372 of the WT SerA enzyme to X. X can be any naturally occurring amino acid except threonine. -serA mutation: 25% of the C-terminal amino acids are altered compared to WT, and the alterations i) within the 50 C-terminal amino acids of the WT protein, and ii) the modification comprises a C-terminal deletion, and iii) The modification comprises an insertion into the WT sequence.

[0055] and / or b) The microbial strain comprises a cds encoding a serine O-acetyltransferase (CysE) whose feedback inhibition by cysteine ​​is reduced by at least two-fold compared to the corresponding wild-type enzyme (e.g., as described in Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611), and CysE enzyme activity can be determined, for example, photometrically, by the consumption of the CysE substrate acetyl-CoA resulting from the reaction with L-serine to form O-acetyl-L-serine (e.g., as described in Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611). In particularly preferred mutants of serine O-acetyltransferase (cysE), feedback inhibition by cysteine ​​is reduced by at least five-fold, particularly preferably at least ten-fold, and in a more preferred embodiment at least 50-fold compared to the corresponding wild-type enzyme.

[0056] Examples of cysE mutants that encode CysE enzymes with reduced feedback inhibition, and preferred embodiments in the context of the present invention, are as follows: -cysE mutation: a mutation in the sequence region from position 97 to position 273. -cysE deletion: deletion in the sequence region from position 248 to position 259.

[0057] Specific mutations that are preferred embodiments in the context of the present invention are as follows: -cysEII:G238S, mutation of glycine at position 238 to serine. -cysEIII:G165D, mutation of glycine at position 165 to aspartic acid. -cysEIV: A237V, an alanine to valine mutation at position 237, and G238S, a glycine to serine mutation at position 238 (double mutation). -cysEV: A237V, alanine to valine mutation at position 237, G238S, glycine to serine mutation at position 238, and M256I, methionine to isoleucine mutation at position 256 (triple mutation). -cysEVI:G238S, a glycine to serine mutation at position 238, and Met256I, a methionine to isoleucine mutation at position 256 (double mutation). -cysEVII:A237V, mutation of alanine at position 237 to valine. -cysEVIII:Met256I, a methionine to isoleucine mutation at position 256, and A237V, an alanine to valine mutation at position 237 (double mutation). -cysEX:T167A, mutation of threonine to alanine at position 167 (used in vector pCys, Example 1). -cysEXI:T167A, a threonine to alanine mutation at position 167, and G245S, a glycine to serine mutation at position 245 (double mutation). -cysEXII: K97Q, a lysine to glutamine mutation at position 97, and G238S, a glycine to serine mutation at position 238, and F267L, a phenylalanine to leucine mutation at position 267 (triple mutation). -cysEXIII: V164A, a mutation of valine at position 164 to alanine, and F267L, a mutation of phenylalanine at position 267 to leucine (double mutation). -cysEXIV:T167A, mutation of threonine at position 167 to alanine, and M256Stop, mutation of methionine at position 256 to Stop (double mutation). -cysEXVI:D250G, mutation of aspartic acid at position 250 to glycine. -cysEXVII: G165D, a glycine to aspartic acid mutation at position 165, and T167A, a threonine to alanine mutation at position 167 (double mutation). -cysEXXIII: T167A, a threonine to alanine mutation at position 167, and A237V, an alanine to valine mutation at position 237, and G238S, a glycine to serine mutation at position 238 (triple mutation). -cysE M256X: mutation of methionine at position 256 of the WT CysE enzyme to X. X can be any natural amino acid except methionine. -cysE Arabidopsis thaliana (plant): A. thaliana contains three cysE genes, two of which are feedback resistant to cysteine. -cysE M256I: mutation of methionine to isoleucine at position 256 of the WT CysE enzyme.

[0058] and / or c) The microbial strain comprises cds encoding a cysteine ​​exporter (efflux protein) such that the cells of the producing strain are distinguished in that cysteine ​​export from the cells of the producing strain is increased by at least two-fold compared to the corresponding wild-type cells, and cysteine ​​export can be determined by photometric measurement of the extracellular cysteine ​​content (including cysteine, cystine and the adduct (R)-2-methylthiazolidine-2,4-dicarboxylic acid formed from cysteine ​​and pyruvate) according to Gaitonde, Biochem. J. (1967) 104:627-633, as described, for example, in U.S. Pat. No. 5,972,663.

[0059] The additional expression of the cysteine ​​exporter preferably increases the export of cysteine ​​from the cell by at least 5-fold, particularly preferably by at least 10-fold, especially preferably by at least 20-fold compared to wild-type cells.

[0060] The cysteine ​​exporter-encoding cds are preferably one or more sequences selected from the group consisting of ydeD, yfiK, cydDC, bcr and emrAB of Escherichia coli (E. coli), or a DNA sequence at least 80%, particularly preferably at least 90%, particularly preferably at least 95% identical thereto.

[0061] The modification involving a deregulated cysteine ​​biosynthetic pathway can be located on the gene construct, in the genome of the production strain, or both on the gene construct and in the genome of the production strain. Preferably, the modification involving a deregulated cysteine ​​biosynthetic pathway is located on the gene construct.

[0062] Suitable microorganisms / host strains for generating the production strain are any microorganisms suitable for recombinant DNA technology and for the fermentative production of recombinant proteins. Suitable microbial strains include bacterial strains selected from the Corynebacteriaceae or Enterobacteriaceae families, yeasts (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica), or fungi (e.g., Aspergillus niger). Preferred microbial strains are bacterial strains selected from Corynebacterium glutamicum, Pantoea ananatis, or Escherichia coli. Particularly preferred is a method for isolating taurine from a fermenter broth after microbial fermentation of a production strain, characterized in that the microbial production strain is a strain of the Escherichia coli species. Particularly preferred is the Escherichia coli (E. coli) K12 W3110 strain commercially available from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM 5911.

[0063] A preferred genetic construct that, after transformation into a host strain, results in a production strain with a deregulated cysteine ​​biosynthetic pathway, capable of cysteine ​​production, is the vector pCys, disclosed in WO2021 / 259491 (Wacker) and described in Example 1 (Figure 1).

[0064] The gene constructs for the hypotaurine and / or taurine biosynthetic pathway and the deregulated cysteine ​​biosynthesis can be present extrachromosomally within the microbial strain as autonomously replicating vectors or can be integrated into the genome of the microbial strain. Preferably, the autonomously replicating vectors for the hypotaurine and / or taurine biosynthetic pathway and the gene constructs for the deregulated cysteine ​​biosynthesis are combined on a single vector or separated into multiple vectors. A gene construct that includes both the hypotaurine and / or taurine biosynthetic pathway and the deregulated cysteine ​​biosynthetic pathway combined on a single autonomously replicating vector is particularly preferred. The gene constructs pCys-CDOrn-CSADcc (FIG. 2), pCys-CSma-CSADcc (FIG. 3), and pCys-CSma-CSADcc-CDOrn (FIG. 4) disclosed in Example 1 are particularly preferred.

[0065] A parent vector for generating a genetic construct according to the present invention is, for example, the vector pCys, which is disclosed in WO 2021 / 259491 (Wacker) and described in Example 1 of the present invention (FIG. 1). As further described in Example 1, pCys was extended with polynucleotides encoding cysteine ​​dioxygenase (CDO), L-cysteine ​​sulfinic acid decarboxylase (CSAD), and / or cysteine ​​synthase (CS). These genetic constructs are therefore distinct in that they encompass the hypotaurine and / or taurine biosynthetic pathway as well as a deregulated cysteine ​​biosynthetic pathway.

[0066] The gene and protein sequences of CDO enzymes are available, for example, in the NCBI database using the search term "cysteine ​​dioxygenase." Any CDS encoding proteins with CDO activity can be used, including CDO enzymes selected from Rattus norvegicus (rat), Homo sapiens (human), Cyprinus carpio (carp), Bos taurus (cow), Capra hircus (goat), Gallus gallus (chicken), Synechococcus (algae), and homologous amino acid sequences. CDSs derived from Rattus norvegicus (rn) or homologous CDSs encoding CDO enzymes are particularly preferred. Particularly preferably, the CDOrn cds has the sequence specified in SEQ ID NO: 1 (nt 1 to nt 603) and encodes the CDOrn enzyme having SEQ ID NO: 2. Thus, in a preferred embodiment, the method is characterized in that the production strain comprises cds encoding the protein having SEQ ID NO: 2.

[0067] The CDO enzyme activity assay can be performed as follows.

[0068] i) Producing the enzyme to be tested: Enzymes produced by growth in shake flasks or by fermentation can be used in reactions as follows. as an aliquot from the culture broth without further workup, or as an aliquot of cell suspension after re-isolation of the cells from the culture broth, for example by centrifugation, or - in the form of aliquots of cell homogenate a) after mechanical disruption of the cell suspension, or b) in the form of chemically permeabilized cells (e.g., with chloroform) or as a cell extract after removal of particulate matter from a cell homogenate, or - for example as an enzyme purified by chromatography.

[0069] The total protein concentration obtained in each case can be determined, for example, using a Qubit 3.0 Fluorometer commercially available from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions.

[0070] ii) Measuring CDO enzyme activity L-cysteine ​​(final concentration 10 mM) is initially added to a solution buffered to pH 7 with potassium phosphate, and the reaction is initiated by adding the enzyme from i). The assay volume is 10 ml. The temperature at which the assay is performed is 30°C. The amount of enzyme from i) used depends on its purity. If culture broth, a cell suspension of reisolated cells, a cell homogenate, or a cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of purified enzyme, at least 10 μg of the purified enzyme fraction is used. 1 hour, 2 hours, and 4 hours after the start of the reaction, 1 ml of assay is removed in each case, centrifuged for 10 minutes, and the content of L-cysteine ​​sulfinic acid is determined by calibrated HPLC (see Example 3). The standards used for calibration are commercially available (Sigma-Aldrich).

[0071] The detection limit for the HPLC method is 1 mg / L of L-cysteine ​​sulfinic acid. If less than 1 mg / L of L-cysteine ​​sulfinic acid is formed under the above assay conditions, the assay does not contain active CDO.

[0072] Gene and protein sequences for CSAD enzymes are available, for example, in the NCBI database using the search term "cysteine ​​sulfinic acid decarboxylase." Any cds encoding proteins with CSAD activity can be used, including CSAD enzymes selected from Cyprinus carpio (carp), Rattus norvegicus (rat), Homo sapiens (human), Bos taurus (cow), Capra hircus (goat), Gallus gallus (chicken), Escherichia coli, Synechococcus (algae), and homologous amino acid sequences. Cyprinus carpio (cc)-derived cds or homologous cds encoding CSAD enzymes are particularly preferred. Particularly preferably, the CSADcc cds have the sequence specified in SEQ ID NO: 3 (nt 1 to nt 1503) and encode the CSADcc enzyme having SEQ ID NO: 4. Thus, in a preferred embodiment, the method is characterized in that the production strain comprises cds encoding the protein having SEQ ID NO: 4.

[0073] The CSAD enzyme activity assay can be performed as follows.

[0074] i) Producing the enzyme to be tested: Enzymes produced by growth in shake flasks or by fermentation can be used in reactions as follows. as an aliquot from the culture broth without further workup, or as an aliquot of cell suspension after re-isolation of the cells from the culture broth, for example by centrifugation, or - in the form of aliquots of cell homogenate a) after mechanical disruption of the cell suspension, or b) in the form of chemically permeabilized cells (e.g., with chloroform) or as a cell extract after removal of particulate matter from a cell homogenate, or - for example as an enzyme purified by chromatography.

[0075] The total protein concentration obtained in each case can be determined, for example, using a Qubit 3.0 Fluorometer commercially available from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions.

[0076] ii) Measuring CSAD enzyme activity A solution buffered to pH 7 with potassium phosphate is first charged with L-cysteine ​​sulfinic acid (final concentration 10 mM), and the reaction is initiated by adding the enzyme from i). The assay volume is 10 ml. The assay is performed at 30°C. The amount of enzyme from i) used depends on its purity. If culture broth, a cell suspension of reisolated cells, a cell homogenate, or a cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of purified enzyme, at least 10 μg of the purified enzyme fraction is used. 1 ml of assay is removed in each case 1 hour, 2 hours, and 4 hours after the start of the reaction, centrifuged for 10 minutes, and the hypotaurine content is determined by calibrated HPLC (see Example 3). The standards used for calibration are commercially available (Sigma-Aldrich).

[0077] The detection limit for the HPLC method is 1 mg / L hypotaurine. If less than 1 mg / L hypotaurine is formed under the above assay conditions, the assay does not contain active CSAD.

[0078] Gene and protein sequences for CS enzymes are available, for example, in the NCBI database using the search term "cysteate synthase." Any cds encoding proteins with CS activity can be used, including CS enzymes selected from Methanosarcina acetivorans, Methanosarcina barkeri, Methanocella paludicola, Methanoculleus marisnigri, Methanolinea mesophila, and homologous amino acid sequences. CS-encoding cds selected from Methanosarcina acetivorans (ma), Methanosarcina barkeri, or homologous sequences are particularly preferred. Particularly preferably, the CSma cds have the sequence specified in SEQ ID NO: 5 and encode the CSma enzyme having the amino acid sequence of SEQ ID NO: 6. Thus, in a preferred embodiment, the method is characterized in that the production strain comprises cds encoding the protein having SEQ ID NO: 6.

[0079] The CS enzyme activity assay can be performed as follows.

[0080] i) Producing the enzyme to be tested: Enzymes produced by growth in shake flasks or by fermentation can be used in reactions as follows. as an aliquot from the culture broth without further workup, or as an aliquot of cell suspension after re-isolation of the cells from the culture broth, for example by centrifugation, or - in the form of aliquots of cell homogenate a) after mechanical disruption of the cell suspension, or b) in the form of chemically permeabilized cells (e.g., with chloroform) or as a cell extract after removal of particulate matter from a cell homogenate, or - for example as an enzyme purified by chromatography.

[0081] The total protein concentration obtained in each case can be determined, for example, using a Qubit 3.0 Fluorometer commercially available from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions.

[0082] ii) Measuring CS enzyme activity ii) A solution buffered to pH 7 with potassium phosphate and containing 100 mM KCl is initially charged with NaSO (final concentration 20 mM) and O-phospho-L-serine (final concentration 7 mM), and the reaction is initiated by the addition of the enzyme from i). The assay volume is 10 ml. The assay is performed at 30°C. The amount of enzyme from i) used depends on its purity. If culture broth, a cell suspension of reisolated cells, a cell homogenate, or a cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of purified enzyme, at least 10 μg of the purified enzyme fraction is used. 1 ml of assay aliquots are removed in each case 1 hour, 2 hours, and 4 hours after the start of the reaction, centrifuged for 10 minutes, and the L-cysteic acid content is determined by calibrated HPLC (see Example 3). The standards used for calibration are commercially available (Sigma-Aldrich).

[0083] The detection limit of the HPLC method is 1 mg / L of L-cysteic acid. If less than 1 mg / L of L-cysteic acid is formed under the above assay conditions, the assay does not contain active CS.

[0084] The gene construct is prepared according to prior art techniques, preferably using recombinant DNA techniques, as described in Example 1 and well known to those skilled in the art. For this purpose, the cds of the CDO gene, preferably CDOrn, the cds of the CSAD gene, preferably CSADcc, and the cds of the CS gene, preferably CSma, are cloned in the desired order into a suitable vector, preferably the vector pCys described in Example 1. The polynucleotides encoding CDO, CSAD, and CS are preferably cloned in the form of an artificial operon, each with its own ribosome binding site (RBS) but without its own promoter, after the serA317 gene present in pCys, so that their expression is under the control of the serA317 promoter.

[0085] Examples of preferred genetic constructs are the vectors pCys-CDOrn-CSADcc (hypotaurine production, Figure 2), pCys-CSma-CSADcc (taurine production, Figure 3), and pCys-CSma-CSADcc-CDOrn (hypotaurine + taurine production, Figure 4), which are described in the Examples. Each genetic construct also contains deregulated genetic elements of the cysteine ​​biosynthetic pathway, namely feedback-resistant alleles of the cysE and serA genes, and the efflux gene ydeD.

[0086] pCys-CDOrn-CSADcc (Figure 2) further contains expression units of the CDOrn gene (CDO Rattus norvegicus) and the CSADcc gene (CSAD Cyprinus carpio) in the form of an operon linked to the serA317 gene to produce hypotaurine according to equations (1) and (3).

[0087] pCys-CSma-CSADcc (Figure 3) further contains expression units of the CSma gene (CS Methanosarcina acetivorans) and the CSADcc gene (CSAD Cyprinus carpio) in the form of an operon linked to the serA317 gene to produce taurine according to equations (6) and (5).

[0088] pCys-CSma-CSADcc-CDOrn (Figure 4) further contains expression units of the CSma gene (CS Methanosarcina acetivorans), the CSADcc gene (CSAD Cyprinus carpio), and the CDOrn gene (CDO Rattus norvegicus) in the form of an operon linked to the serA317 gene to produce a mixture of hypotaurine and taurine according to equations (1) and (3) and equations (6) and (5), respectively.

[0089] Any other order of the CDO and CSAD configuration, the CS and CSAD configuration, and the CS, CSAD and CDO configuration in the above genetic constructs is also possible.

[0090] Furthermore, the CDO, CS, and CSAD genes can be cloned into the pCys vector as independent expression units, each with its own promoter and, optionally, terminator. It is also conceivable to clone the CDO, CS, and CSAD genes as separate expression units into separate vectors independent of pCys, or to integrate them into the genome of a microorganism such as Escherichia coli (E. coli) as a host organism. Combinations of expression units present in vectors and expression units integrated into the genome are also conceivable.

[0091] The hypotaurine and / or taurine-producing strain preferably comprises a microbial strain, particularly preferably the Escherichia coli (E. coli) K12 W3110 strain, and a genetic construct according to the present invention, particularly preferably a genetic construct selected from the genetic constructs pCys-CDOrn-CSADcc, pCys-CSma-CSADcc, or pCys-CSma-CSADcc-CDOrn. Transformation of the genetic construct into a microbial strain, preferably Escherichia coli (E. coli) W3110, in a known manner results in a producing strain. Example 2 discloses the generation of production strains E. coli W3110×pCys-CDOrn-CSADcc for the production of hypotaurine, E. coli W3110×pCys-CSma-CSADcc for the production of taurine, and E. coli W3110×pCys-CSma-CSADcc-CDOrn for the production of a mixture of hypotaurine and taurine.

[0092] Production of hypotaurine, taurine, or a mixture of hypotaurine and taurine using the described production strains can be carried out by growth in shake flasks as described in Example 3, or by production-scale fermentation, preferably fed-batch fermentation of the production strains, as described in Example 4. Growth in shake flasks or fermentative growth results in a shake culture broth or fermenter broth containing hypotaurine or taurine, or a mixture of hypotaurine and taurine, and the taurine / hypotaurine content in the cell culture supernatant (fermentation supernatant) after fermentation is preferably at least 10 g / L.

[0093] After microbial fermentation of the production strain and optionally lowering the pH and / or increasing the temperature and possible incubation of the fermenter broth (see below), the fermentation supernatant is isolated by removing the biomass from the fermenter broth (step i). The prior art provides various methods for biomass removal. Preferred methods for biomass removal are centrifugation (sedimentation) and filtration, of which filtration is particularly preferred. The two methods can be carried out batchwise or continuously, with continuous treatments being preferred, of which continuous centrifugation (decanter, separator) and cross-flow filtration (tangential flow filtration) are particularly preferred. Cross-flow filtration is particularly preferred. In cross-flow filtration, the pore size of the filtration module is selected so that the biomass and other macromolecules remain (retained) in the retentate, while hypotaurine and taurine are recovered in the filtrate. The removed filtrate reduces the volume of the retentate, which still contains a high proportion of unfiltered product. To increase the yield, water may be added back to the retentate, followed by further filtration (so-called diafiltration).

[0094] Depending on the pore size of the filtration module, a distinction is made between microfiltration (pore size >100 nm), ultrafiltration (pore size 2 nm to 100 nm) and nanofiltration (pore size up to 2 nm). Microfiltration and ultrafiltration are preferred, with microfiltration being particularly preferred. Microfiltration followed by ultrafiltration is also conceivable.

[0095] In practice, in order to reduce the consumption of solvent 1, step i) is preferably followed by a volume reduction (see below) before solvent 1 is added first.

[0096] After removing the biomass and optionally isolating the fermentation supernatant by volume reduction (see below), taurine is precipitated from the fermentation supernatant by adding water-miscible solvent 1 (step ii). Taurine precipitation is based on selecting a solvent 1 in which taurine is not soluble or only slightly soluble. Adding solvent 1 to the taurine-containing fermentation supernatant results in a set mixing ratio of solvent 1 to the aqueous fermentation supernatant. As the mixing ratio increases (i.e., as the proportion of solvent 1 in the mixture increases), the solubility of taurine decreases until taurine no longer dissolves in the mixture and precipitates, and an equilibrium is established between taurine in precipitated form and taurine still in solution. This proportion of solvent 1 in the mixture depends on the chemical properties of solvent 1 and is preferably at least 40% v / v, particularly preferably at least 60% v / v, and particularly preferably at least 80% v / v.

[0097] The solubility of compounds is known to depend on temperature and may decrease at low temperatures. This means that cooling the mixture can shift the equilibrium between precipitated and dissolved taurine toward precipitated taurine, thereby increasing the yield of step II. Preferably, the mixture of solvent 1 and the taurine-containing aqueous fermentation supernatant is cooled to 8°C, particularly preferably to 0°C, and particularly preferably to -20°C, as long as the mixture does not freeze.

[0098] The definition given above for the term "solvent" is applicable to solvent 1. Preferably, the method for isolating taurine is characterized in that the water-miscible solvent 1 is a solvent selected from the group consisting of methanol, ethanol, 1-propanol or 2-propanol (isopropanol) and mixtures thereof with HO, particularly preferably a solvent selected from the group consisting of ethanol, isopropanol and mixtures thereof with HO, particularly preferably ethanol.

[0099] The taurine precipitate is then isolated from the mixture and dissolved in solvent 2 (step iii).

[0100] Isolation of the taurine precipitate, i.e., removal of the taurine precipitate from the mixture, can be carried out by centrifugation, sedimentation or filtration, preferably by filtration.

[0101] Preferably, the taurine precipitate is first suspended in solvent E (extraction solvent), which is insoluble in the taurine unlike the impurities, at least once, particularly preferably twice, particularly preferably three times, and then removed from the suspension again as described above. The procedure of suspending a compound such as taurine in solvent E and removing it from the suspension again is called extraction. This extraction step of suspension and removal is used to remove (deplete) the impurities present in the precipitate along with taurine, and can essentially be repeated as many times as necessary. The definition given above for the term "solvent" is also applicable to solvent E, and the solvent E used for extraction is preferably solvent 1, although solvents other than solvent 1 are also contemplated.

[0102] For the removal of impurities by extraction, material properties of different solubility are utilized, for example, the fact that taurine is insoluble or poorly soluble in pure water-miscible alcohols such as methanol, ethanol, n-propanol, and isopropanol. However, other water-miscible organic solvents, such as acetone, are also suitable as solvent E for the extraction of impurities. Any mixture of water-miscible organic solvents can also be used for the extraction of impurities. Water-miscible alcohols such as methanol, ethanol, n-propanol, and isopropanol are preferred, with ethanol and isopropanol being particularly preferred, and ethanol being particularly preferred. Ethanol can be used as pure ethanol or denatured ethanol.

[0103] The volume of water-immiscible alcohol, preferably ethanol, used for extraction can be freely selected, preferably at least the same volume as the volume of the fermenter broth used to isolate taurine, particularly preferably at least 5 times that volume, and particularly preferably at least 10 times that volume. The volume used for extraction can be used completely in one extraction or divided into multiple batches for repeated extractions. In a preferred embodiment, ethanol in a mixture with water having an ethanol content of 100% (v / v) to 30% (v / v) ethanol, particularly preferably 100% (v / v) to 50% (v / v) ethanol, and particularly preferably 100% (v / v) to 80% (v / v) ethanol can be used for extraction. In a further preferred embodiment of the present invention, the extractant, preferably ethanol, is recovered from the extraction filtrate (e.g., by distillation) to increase the economic viability of the method and to increase the sustainability of the method by minimizing chemical consumption.

[0104] The solubility of taurine in an ethanol / HO mixture depends on the ethanol content and the incubation temperature. At room temperature, taurine is insoluble in commercially available 99% (v / v) ethanol and insoluble in 80% (v / v) ethanol. Low solubility is observed at room temperature in 60% (v / v) ethanol, and it increases significantly in 50% (v / v) and 30% (v / v) ethanol, but can be significantly reduced by cooling to 0°C. Ethanol is preferably metered into the fermentation supernatant from step i), the volume of which is particularly preferably reduced as described below, resulting in an ethanol concentration of at least 40% (v / v), particularly preferably at least 60% (v / v), and particularly preferably at least 80% (v / v). When the ethanol content is specified as a percentage, it is meant as a volume percentage. 80% ethanol means 80 ml of ethanol + 20 ml of fermentation supernatant in a total volume of 100 ml. Depending on the ethanol content, the temperature may be reduced as above to further reduce taurine solubility.

[0105] To further remove impurities, the taurine-containing precipitate may be extracted multiple times with an ethanol / water mixture, preferably containing at least 40% (v / v) ethanol, particularly preferably at least 60% (v / v) ethanol, and particularly preferably at least 80% (v / v) ethanol.

[0106] The isolated taurine precipitate is dissolved in solvent 2. The definition given above for the term "solvent" is applicable to solvent 2. Solvent 2 is used as a mixture with water, and the proportion of water in the mixture is preferably 100% v / v (pure water) to 50% v / v, particularly preferably 100% v / v to 75% v / v, and particularly preferably 100% v / v to 90% v / v.

[0107] In a preferred embodiment, the method for isolating taurine is characterized in that solvent 2 is water.

[0108] In step iv, the taurine precipitate is isolated and optionally subjected to extraction to remove impurities, followed by crystallization of the taurine precipitate dissolved in solvent 2.

[0109] Preferably, the taurine concentration after dissolving the taurine precipitate in solvent 2 is at least 100 g / L, particularly preferably at least 200 g / L, and particularly preferably at least 300 g / L. Taurine at these concentrations is dissolved by heating to a temperature below the boiling point of solvent 2, preferably up to 70° C., particularly preferably up to 80° C., and particularly preferably up to 90° C. Taurine crystallizes upon cooling to room temperature, or, to further increase the yield, upon cooling to a temperature at which solvent 2 does not yet freeze, preferably 0° C.

[0110] This crystallization step of dissolving and cooling can be repeated as many times as necessary to achieve higher purity of taurine. Preferably, the crystallization step is carried out at least once, particularly preferably at least twice, and particularly preferably at least three times.

[0111] Furthermore, it is preferable to optimize the crystallization by adding a water-miscible solvent 3, for example, to increase the yield of crystallized taurine and reduce the taurine content in the mother liquor.

[0112] When a compound is crystallized from a solution, a distribution equilibrium is established between the crystallized product that has precipitated from the solution and the compound that is still in solution. The term "mother liquor" refers to the soluble portion of the crystallization without the precipitated crystallized product. The definition given above for the term "solvent" is applicable to solvent 3. Water-miscible solvents 3 for optimizing crystallization are preferably methanol, ethanol, n-propanol and isopropanol, particularly preferably ethanol and isopropanol, and particularly preferably ethanol. The proportion of solvent 3 in a mixture with water is preferably at most 30% v / v, particularly preferably at most 20% v / v, and particularly preferably at most 10% v / v.

[0113] In step v, the crystallized taurine is isolated and optionally dried. The crystallized taurine is preferably isolated by sedimentation, centrifugation, or filtration. The dissolved taurine still present in the supernatant is not necessarily lost, but is preferably recycled to one of the preceding workup steps i to iii, particularly preferably to workup step ii.

[0114] Crystallized taurine can be preferably dried to constant weight by drying at high temperature with or without vacuum, for example, in a vacuum dryer.The drying temperature of taurine is preferably at least 30°C, particularly preferably at least 50°C, particularly preferably at least 70°C.Drying is preferably carried out without vacuum, particularly preferably under a vacuum of ≦300 mbar, particularly preferably under a vacuum of ≦200 mbar.In a further preferred embodiment, crystallized taurine can also be dried by freeze-drying or spray-drying, and in the case of spray-drying, crystallized taurine first needs to be redissolved, preferably in H2O.

[0115] For further use, the obtained taurine is preferably ground and can be sieved to obtain a final product with the desired particle characteristics.

[0116] The taurine isolated by the method according to the invention has a purity of more than 80% (w / w), preferably more than 90%, particularly preferably more than 95%.

[0117] To determine the purity and estimate the impurities still present, the taurine content in the crystallized product is determined, which is referred to as content determination. For this purpose, a product solution is preferably prepared by weighing a small amount of the crystallized product and dissolving it in HO, for example, as a 20 g / L solution. The taurine content of the product solution is then determined, preferably by calibrated HPLC. The taurine content in the crystallized product is expressed as the percentage ratio of the taurine concentration (g / L) determined by HPLC to the concentration (g / L) of the product solution. For example, if the concentration of the product solution is 20 g / L and the taurine content determined by HPLC is 19 g / L, the taurine content in the crystallized product is 95%. The taurine content determined in this manner is equivalent to the purity of the taurine product.

[0118] To further increase the purity, additional purification steps are conceivable, such as treatment with activated carbon or ion exchange chromatography, as known from the prior art.

[0119] The method according to the invention therefore allows for the isolation of highly pure taurine from hypotaurine- and / or taurine-containing fermenter broth by simple and cost-effective processing steps for applications in the food, feed and pharmaceutical sectors.

[0120] At the beginning of the method, the content of hypotaurine and taurine from the shaken culture broth or fermenter broth may be quantified. For example, this can be done by measuring the cell density OD 600This is done by taking a 1 ml aliquot of shake culture broth or fermenter broth where the NA / ml is at least 1.0 / ml, incubating it at 80°C for 5 minutes, then removing all solid components, e.g., by centrifugation for 5 minutes at maximum speed in a benchtop centrifuge, and quantifying the supernatant by HPLC calibrated for hypotaurine and taurine, e.g., as described for hypotaurine and taurine in Example 3.

[0121] In a preferred embodiment, the method for isolating taurine is characterized in that the added molar content of hypotaurine, taurine or a mixture thereof in the fermenter broth is at least 91.6 mM (corresponding to a pure hypotaurine content of 10 g / L (molecular weight 109.2 g / mol) or a pure taurine content of 11.5 g / L (molecular weight 125.2 g / mol)), particularly preferably at least 183.2 mM (corresponding to a pure hypotaurine content of 20 g / L or a pure taurine content of 23 g / L), and particularly preferably at least 274.7 mM (corresponding to a pure hypotaurine content of 30 g / L or a pure taurine content of 34.4 g / L). The added molar content of hypotaurine and / or taurine means that the fermenter broth with the preferred added molar content can contain hypotaurine and taurine in any ratio.

[0122] Step i serves to remove biomass and other polymeric components (e.g., polysaccharides, proteins, nucleic acids) from the fermenter broth that may arise as by-products of fermentation or through partial cell disruption in the final stages of fermentation. Thus, biomass removal is often complicated by the small particle size of microbial cells and the high viscosity of the fermenter broth due to the polymeric by-products of fermentation. One means to improve polymer removability is to denature the polymers by lowering the pH of the fermenter broth and / or incubating the fermenter broth at elevated temperatures.

[0123] Preferably, the method for isolating taurine is characterized in that the removal of biomass (step i) is preceded by adjusting the pH of the fermenter broth to below 6.0, particularly preferably below 5.0, and particularly preferably below 4.0 by adding an acid. This step involves lowering the pH, which does not change during the remainder of the method according to the invention. Optionally, incubation, i.e., allowing to occur, can be carried out at the adjusted pH value, preferably for at least 5 minutes, particularly preferably for at least 10 minutes, particularly preferably for at least 20 minutes, and particularly preferably for at least 30 minutes. Suitable acids for adjusting the pH are all known acids, preferably mineral acids, such as particularly preferred sulfuric acid, hydrochloric acid, and phosphoric acid, particularly preferred sulfuric acid. Particularly preferably, the method for isolating taurine is characterized in that adjusting the pH of the fermenter broth and optionally incubating are carried out as a continuous process.

[0124] In a further preferred embodiment, the method for isolating taurine is characterized in that the temperature of the fermenter broth is increased to at least 50°C, particularly preferably at least 60°C, particularly preferably at least 70°C, and the fermenter broth is incubated at this temperature for at least 5 minutes, preferably at least 10 minutes, particularly preferably at least 20 minutes, particularly preferably at least 30 minutes, i.e., the selected temperature acts on the fermenter broth under these conditions. Preferably, the method for isolating taurine is characterized in that the increasing the temperature of the fermenter broth and the incubation are carried out in a continuous process.

[0125] In a preferred embodiment, the method for isolating taurine is characterized in that, prior to the removal of biomass (step i), at least one measure selected from the group consisting of: (1) adjusting the pH of the fermenter broth to a pH of 6.0 or less by adding an acid; and (2) increasing the temperature of the fermenter broth to at least 50° C. and incubating under these conditions for at least 5 minutes.

[0126] It is particularly preferred to simultaneously lower the pH of the fermenter broth and increase the temperature, with particular preference being given to a combination of pH≦5.0 and temperature≧50° C., and even more particularly preferred to a combination of pH≦4.0 and temperature≧70° C. Both the lowering of the pH of the fermenter broth and the increase in temperature, which are associated with the denaturation of the macromolecules, provide a means for improving the removability of the macromolecules from the fermenter broth. The incubation time (working time) of the fermenter broth at low pH and high temperature is preferably at least 5 minutes, particularly preferably at least 20 minutes, and especially preferably at least 30 minutes.

[0127] Particularly preferably, the method for isolating taurine is characterized in that at least one of the selected steps is carried out continuously, the term "step" referring to (1) adjusting the pH of the fermenter broth to a pH of 6.0 or less by adding an acid, and / or (2) increasing the temperature of the fermenter broth to at least 50° C. and incubating for at least 5 minutes.

[0128] It is particularly preferred to both adjust the pH of the fermentor broth and to increase the temperature of the fermentor broth followed by incubation for at least 5 minutes, and to perform both steps together sequentially.

[0129] The incubation time is particularly preferably at least 10 minutes, particularly preferably at least 20 minutes, and most particularly preferably at least 30 minutes.

[0130] The continuous treatment is preferably carried out as follows.

[0131] - The fermenter broth is pumped into a reactor that has been preheated to the desired temperature.

[0132] The reactor is preferably equipped with a metering section for acid and an active pH control. The desired pH of the fermenter broth is preferably set by metering in acid. The active pH control can be achieved by a measurement and control unit which readjusts the desired pH by metering in acid if the pH deviates from the target value due to the metering in of the fermenter broth (so-called pH-stat method).

[0133] After a predetermined residence time for incubation at high temperature (temperature treatment) and / or low pH, the fermenter broth is pumped from the reactor to a reservoir for further work-up steps, which may or may not be temperature controlled.

[0134] The rate at which the fermenter broth is pumped into the reactor determines the residence time in the reactor and therefore the incubation time.

[0135] The advantage of continuous processing is greater economic viability, since there is no need to bring the entire fermenter broth to the desired temperature and / or pH simultaneously, which would mean high energy and time consumption, especially in the industrial sector.

[0136] In a preferred embodiment, the method for isolating taurine is characterized in that an oxidizing agent is added and incubated with the oxidizing agent before the addition of solvent 1 (step ii). The addition of an oxidizing agent and incubation with the oxidizing agent, also called oxidation treatment, results in the oxidation of hypotaurine to taurine. The oxidation treatment means adding an oxidizing agent, preferably at room temperature or optionally under cooling, followed by incubation for preferably at least 5 minutes, particularly preferably at least 0.5 hours, particularly preferably at least 2 hours, and particularly preferably at least 4 hours.

[0137] The oxidation treatment can be carried out directly in the fermenter broth (see, e.g., Example 6, Batch 1B) or in the fermentation supernatant after biomass removal (see, e.g., Example 6, Batch 1A). Thus, the oxidation treatment can be carried out before or after step i (biomass removal). When carrying out the oxidation treatment of the fermentation supernatant, the previously clear fermentation supernatant becomes cloudy after biomass removal, which allows the removal of further impurities as a precipitate, preferably by filtration or sedimentation (centrifugation). When the oxidation treatment is carried out directly in the fermenter broth, the resulting precipitate can be removed together with the biomass in one step, which saves one removal step and improves the economic viability of the method.

[0138] Preferably, the direct oxidation treatment of the fermentor broth for oxidation of hypotaurine to taurine is followed by removal of the biomass along with the precipitate formed by the oxidation treatment, as described in Example 6, Batch 1B.

[0139] The oxidizing agent used may be, for example, air, oxygen, ozone, hydrogen peroxide, organic peroxide, or a mixture thereof. Oxidizing agents that produce no or no problematic by-products are preferred. Therefore, preferred oxidizing agents are air, oxygen (O), ozone (O), and hydrogen peroxide (H0), and particularly preferred are ozone and hydrogen peroxide.

[0140] Particularly preferably, the method for isolating taurine from a fermentation batch after microbial fermentation is characterized in that the oxidizing agent is H2O2. Preferably, the oxidizing agent is not NaOH, which, due to its chemical properties, is a base (alkaline solution) and is not known as an oxidizing agent.

[0141] Hydrogen peroxide HO is preferably added in its commercially available form as a 30% (w / w) or 50% (w / w) aqueous solution with stirring. The HO reaction is exothermic, meaning that temperature control is necessary. Temperature control can be achieved by external cooling or by a suitable metering profile for adding HO. Preferably, temperatures do not exceed 70°C, particularly preferably 50°C, and particularly preferably 40°C. The amount of HO used depends on the hypotaurine content of the fermentation supernatant. According to equation (7), one molecule of HO is consumed for oxidation per molecule of hypotaurine.

[0142] (7) Hypotaurine + H2O2 → Taurine + H2O The molar ratio of H2O2 used, based on the hypotaurine content, is preferably at least 1:1, particularly preferably at least 1.2:1, particularly preferably at least 1.5:1.

[0143] In the case of oxidation with H2O2, the molar yield of taurine, based on the molar amount of hypotaurine used, is preferably at least 70%, particularly preferably at least 80%, particularly preferably at least 90%.

[0144] The method according to the invention is therefore characterized in that the incubation with the oxidizing agent can be before or after the removal of the biomass from the fermenter broth (step i).

[0145] For example, as disclosed in the example for the E. coli W3110×pCys-CSma-CSADcc strain, if the fermentation batch contains only taurine, there is no need to incubate with an oxidizing agent.

[0146] In a further preferred embodiment, the method for isolating taurine from fermenter broth after microbial fermentation is characterized in that the incubation with the oxidizing agent is performed continuously. In a preferred configuration of continuous processing, the fermenter broth before biomass removal or the fermentation supernatant after biomass removal is pumped into a reactor for oxidation. The reactor may have active cooling to facilitate temperature control of the reaction. The reactor further comprises a metering section for the oxidizing agent and active temperature control. Active temperature control is achieved by a measurement and control unit that controls the metering of the oxidizing agent and the fermenter broth or fermentation supernatant so that the temperature due to the exothermic oxidation of hypotaurine to taurine does not exceed a target value, preferably 70°C or less, particularly preferably 50°C or less, and particularly preferably 40°C or less. The rate at which the fermenter broth or fermentation supernatant and the oxidizing agent are metered into the reactor determines the residence time and therefore the oxidation treatment time. The residence time / oxidation treatment time is selected so that hypotaurine is quantitatively oxidized to taurine at a specific temperature target value. This depends on the hypotaurine content and can be empirically determined in advance. The residence time in the reactor is preferably at least 5 minutes, particularly preferably at least 20 minutes, particularly preferably at least 40 minutes.

[0147] An advantage of continuous processing is greater economic viability, since the entire fermenter broth does not need to be subjected to oxidation treatment simultaneously, which means less material is needed for corrosion-resistant reactors due to the corrosive nature of the oxidizing agent. Furthermore, reaction safety is a positive aspect of continuous operation, since the exothermic reaction of hypotaurine oxidation to taurine is easier to control in a relatively small (cooled) reactor than in a large fermentation batch.

[0148] After incubation with the oxidizing agent, the fermenter broth or fermentation supernatant is pumped to a reservoir for further work-up steps.

[0149] In a preferred embodiment, the method for isolating taurine is characterized in that the volume of the fermentation supernatant is reduced before the addition of solvent 1 (step ii). If the incubation with the oxidizing agent and the volume reduction are to be carried out before step ii, the order of the treatment steps is preferably such that, starting from the fermenter broth, it is first incubated with the oxidizing agent, followed by the removal of the biomass (step i), then the volume reduction, and then step ii. If the biomass is removed first (step i), this is followed by the incubation with the oxidizing agent, optionally followed by the removal of a precipitate formed during oxidation, then the volume reduction, and then step ii.

[0150] For volume reduction or concentration of the fermentation supernatant, preferably a part of the aqueous medium or the entire aqueous medium is removed, particularly preferably a part thereof. Various methods for volume reduction are available, including evaporation with or without application of a vacuum (distillation of H2O), evaporation of H2O on a thin film evaporator, reverse osmosis, electrodialysis, and nanofiltration. Removal of H2O by freeze-drying is also possible. Evaporation of the taurine-containing fermentation supernatant with or without application of a vacuum, evaporation of H2O on a thin film evaporator, and reverse osmosis are preferred. Evaporation of the taurine-containing fermentation supernatant with or without application of a vacuum and reverse osmosis are particularly preferred. Evaporation of the taurine-containing fermentation supernatant with application of a vacuum is particularly preferred, as disclosed in Example 7 of the present invention.

[0151] The H2O can be evaporated, for example, by heating the fermentation supernatant to preferably at least 60°C, particularly preferably at least 70°C, particularly preferably at least 80°C. At the same time, a vacuum of preferably less than 300 mbar, particularly preferably less than 200 mbar, particularly preferably less than 150 mbar is applied.

[0152] The extent of the volume reduction depends on how the taurine-containing fermentation supernatant is further used. The HO may be evaporated to complete dryness or to a predetermined volume. It is preferred to reduce the volume to 50% or less of the initial volume of the fermenter broth used for taurine isolation, particularly preferably 30% or less, particularly preferably 20% or less of the initial volume of the fermenter broth used for taurine isolation.

[0153] As disclosed in Example 8 of the present invention, the evaporated concentrated taurine-containing batch from Example 7 was mixed with ethanol in an amount such that the final ethanol concentration in the resulting mixture was 80% (v / v). Under these conditions, taurine precipitated from the mixture, which could be further enhanced by further cooling the mixture to a temperature below 0°C. Taurine was isolated from this mixture by filtration. Further removal of impurities from the precipitated taurine fraction was achieved by resuspension with 80% (v / v) ethanol, followed by filtration without dissolving the taurine.

[0154] As already discussed above, the sequence of resuspension and filtration of the precipitated taurine fraction is called extraction and can be repeated as desired. The volume of 80% (v / v) ethanol used can be freely selected, preferably at least the same volume as the initial volume of the fermenter broth used to isolate taurine, particularly preferably at least 5 times that volume, and particularly preferably at least 10 times that volume. The ethanol / water mixture is not limited to an ethanol content of 80% (v / v). In a further preferred embodiment, ethanol in a mixture with water of 100% (v / v) to 30% (v / v) ethanol, particularly preferably 100% (v / v) to 50% (v / v) ethanol, and particularly preferably 100% (v / v) to 80% (v / v) ethanol can be used repeatedly for extraction.

[0155] In a further preferred embodiment of the present invention, the extractant, preferably ethanol, is recovered from the extraction filtrate (e.g., by distillation) to increase the economic viability of the process and to increase the sustainability of the process by minimizing chemical consumption. In addition to ethanol, all other water-miscible organic solvents mentioned above are also suitable for extraction in the manner described for ethanol.

[0156] A preferred method for isolating taurine from the fermenter broth after microbial fermentation of the production strain comprises the following isolation steps: - adjusting the pH to below 5, raising the temperature of the fermenter broth to above 50°C, and incubating the fermenter broth at the low pH and high temperature for at least 5 minutes in a continuous process as described above; - carrying out an oxidation treatment of the fermentor broth with H2O2 in a continuous process as described above at 70°C or less for at least 5 minutes, wherein the molar ratio of H2O2:hypotaurine is at least 1:1; - isolating the fermentation supernatant by removing the biomass from the fermenter broth by filtration (microfiltration combined with diafiltration) in a continuous process as described above; - reducing the volume of the fermentation supernatant to less than 50% of the initial volume of the fermenter broth used by evaporation at a temperature of at least 60°C and a vacuum of less than 300 mbar; - precipitating taurine from the volume-reduced fermentation supernatant by adding ethanol to obtain an ethanol / water mixture containing 80% ethanol (v / v), followed by cooling to below 0°C; - isolating the taurine precipitate by filtration or sedimentation; - performing at least one extraction of the taurine precipitate with an ethanol / water mixture containing at least 60% (v / v) ethanol, and cooling the extraction mixture consisting of the taurine precipitate and the ethanol / water mixture to below 0°C; - isolating the taurine precipitate and dissolving it in HO at a concentration of at least 200 g / L at a temperature of at least 50°C; - crystallizing taurine at or below room temperature; isolating the crystallized taurine by filtration; - drying the crystallized taurine at at least 50°C and a vacuum of ≦300 mbar.

[0157] Preferably, the method for isolating taurine from a fermenter broth after microbial fermentation of a production strain is characterized in that the molar yield of taurine achieved is at least 50%, particularly preferably at least 60%, and particularly preferably at least 70%, based on the molar amount of starting product used, which is present in the fermentation supernatant and consists of at least one compound selected from the group consisting of hypotaurine and taurine. The yield refers to the amount of isolated taurine with a purity of >80% after crystallization, based on the amount of taurine used that was present in the fermentation supernatant after step i and optional pre-oxidation of hypotaurine, but before possible volume reduction of the fermentation supernatant.

[0158] The recovery rate corresponds to the sum of the taurine isolated after crystallization and the taurine still present in the crystallization mother liquor and is preferably at least 80%, particularly preferably at least 90%, particularly preferably at least 95%, based on the amount of taurine used that was present in the fermentation supernatant after step i and, optionally, after pre-oxidation of hypotaurine.

[0159] The figure shows the plasmids used in the examples. [Brief explanation of the drawings]

[0160] [Figure 1] Figure 1: pCys. [Figure 2] Figure 2: pCys-CDOrn-CSADcc. [Figure 3] Figure 3: pCys-CSma-CSADcc. [Figure 4] Figure 4: pCys-CSma-CSADcc-CDOrn.

[0161] Abbreviations used in the diagram: TetR: a gene that confers tetracycline resistance P15A ORI: Origin of replication serA317:serA (3-phosphoglycerate dehydrogenase gene encoding amino acids 1 to 317) cds cysE X:cysE (serine O-acetyltransferase gene, feedback resistant) cds ORF306: ydeD (cysteine ​​excretion gene) cds ScaI: Restriction enzyme ScaI cleavage site PpuMI: Cleavage site of the restriction enzyme PpuMI CDOrn:CDO (cysteine ​​dioxygenase) R. norvegicus cds CSADcc: CSAD (cysteine ​​sulfinic acid decarboxylase) Cyprinus carpio (C. carpio) cds CSma:CS (cysteate synthase) Methanosarcina acetivorans (M. acetivorans) cds RBS: ribosome binding site [Example]

[0162] The following examples serve to further elucidate the present invention.

[0163] [Example 1]: Preparation of DNA constructs All DNA constructs were derived from the vector pCys (Figure 1) disclosed in WO 2021 / 259491 (Wacker).

[0164] pCys: pCys is disclosed in WO 2021 / 259491 (Wacker) and is a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306. Plasmid pACYC184-cysEX-GAPDH-ORF306 contains not only an origin of replication and a tetracycline resistance gene (parent vector pACYC184), but also a cysEX allele (synonymous with the cyseE allele) encoding a serine O-acetyltransferase with reduced cysteine ​​feedback inhibition, and the efflux gene ydeD (ORF306), whose expression is controlled by a constitutive GAPDH promoter.

[0165] Additionally, pCys contains the serA317 gene fragment, which is cloned after the ydeD (ORF306) efflux gene and encodes the N-terminal 317 amino acids of the SerA protein (full length: 410 amino acids). The Escherichia coli (E. coli) serA gene is listed in the GenBank gene database under gene ID 945258. serA317 is described in Bell et al., Eur. J. Biochem. (2002) 269:4176-4184, where it is designated "NSD:317" and encodes a serine feedback-resistant mutant of 3-phosphoglycerate dehydrogenase. Expression of serA317 is controlled by the serA promoter. The serA promoter is described in Rex et al. (1991), J. Bacteriol. 173:5944-5953, Figure 2.

[0166] For cloning, the plasmid DNA of the vector pCys was cleaved with ScaI and PpuMI, and the 6.1 kb vector fragment (hereafter referred to as pCys-ScaI / PpuMI) was isolated by preparative agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

[0167] The amino acid sequence of the cysteine ​​dioxygenase from Rattus norvegicus derived from the mRNA of the CDOrn-rrnB gene is disclosed in the NCBI (National Center for Biotechnology Information) database under sequence ID: AAH70509.1. Using this amino acid sequence, a codon-optimized DNA sequence for expression in Escherichia coli (E. coli) was derived (using the publicly available Eurofins Genomics GENEius software), which is referred to as CDOrn cds. The DNA sequence of CDOrn cds is disclosed in SEQ ID NO: 1, nt 1 to 603, and encodes a protein with the amino acid sequence from SEQ ID NO: 2. The 3' end of CDOrn cds was ligated to the DNA sequence of the E. coli rrnB terminator (SEQ ID NO: 1, nt 604 to 936). The DNA sequence of the rrnB terminator is disclosed in Orosz et al. (1991), Eur. J. Biochem. 201:653-659. CDO-rrnB DNA consisting of the CDOrn cds and the rrnB terminator was synthetically produced (Eurofins Genomics).

[0168] CSADcc-rrnB: The cDNA gene (cDNA: complementary DNA isolated by reverse transcription from mRNA) for cysteine ​​sulfinic acid decarboxylase (CSAD) from Cyprinus carpio (carp) was isolated by Honjoh et al. (2010), Amino Acids 38:1173-183. The DNA sequence is published in the NCBI (National Center for Biotechnology Information) database under GenBank sequence ID: AB220585.1 (cds: nt 82-1584). Using the amino acid sequence, a codon-optimized DNA sequence for expression in Escherichia coli (E. coli) was derived (using the publicly available Eurofins Genomics GENEius software), designated CSADcc cds. The CSADcc cds DNA sequence is published in SEQ ID NO:3, nt 1-1503, and encodes a protein with the amino acid sequence from SEQ ID NO:4. The 3' end of the CSADcc cds was ligated to the DNA sequence of the Escherichia coli (E. coli) rrnB terminator (SEQ ID NO: 3, nt 1504-1834). The DNA sequence of the rrnB terminator is disclosed in Orosz et al. (1991) (see above). CSADcc-rrnB DNA, consisting of the CSADcc cds and the rrnB terminator, was synthetically constructed (Eurofins Genomics).

[0169] CSma: Cysteine ​​synthase (CSma) from Methanosarcina acetivorans was used. The amino acid sequence of the CSma enzyme is available in the NCBI database under accession ID WP_048066469. The amino acid sequence was used to derive a codon-optimized DNA sequence for expression in Escherichia coli (E. coli) (using the publicly available Eurofins Genomics GENEius software). The CSma DNA sequence is disclosed in SEQ ID NO: 5 and encodes a protein with the amino acid sequence from SEQ ID NO: 6. CSma DNA was synthetically produced (Eurofins Genomics).

[0170] Primers used: cdorn-1f (SEQ ID NO: 7) Cdorn-3r (SEQ ID NO: 8) cdorn-9f (SEQ ID NO: 9) glf-2r (SEQ ID NO: 10) csadcc-1f (SEQ ID NO: 11) csadcc-10f (SEQ ID NO: 12) csadcc-3r (SEQ ID NO: 13) csma-1f (SEQ ID NO: 14) csma-2r (SEQ ID NO: 15)

[0171] PCR product: Table 1 summarizes the names and sizes of the PCR products used for cloning, as well as the synthetic DNA (i.e., template DNA) and primers used. PCR products PCR1 to PCR6 were generated from each template DNA by PCR (Phusion™ High-Fidelity DNA polymerase, Thermo Scientific™) using appropriate primers and isolated by agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

[0172] [Table 1]

[0173] Vector construction: The vectors were generated using the NEBuilder® Cloning Kit (NEB New England Biolabs) according to the manufacturer's instructions. As summarized in Table 2, the 6.1 kb pCys-ScaI / PpuMI vector fragment was ligated with the corresponding PCR product in each case, following the manufacturer's instructions regarding the amount of each DNA fragment used in the ligation mixture. The ligation mixture was then transformed into Escherichia coli (E. coli) NEB® 10-beta (NEB New England Biolabs) according to the manufacturer's instructions. Clones from the transformation were selected on LBtet plates, which contained 10 g / L tryptone (GIBCO™), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 15 g / L agar, and 15 mg / L tetracycline (Sigma-Aldrich). Plasmid DNA was isolated from the transformed clones (Qiagen Kit) and correct clones were selected in each case to generate the vectors pCDOrn-CSADcc (Fig. 2), pCSma-CSADcc (Fig. 3), and pCSma-CSADcc-CDOrn (Fig. 4), listed in Table 2.

[0174] [Table 2]

[0175] [Example 2]: Construction of a production strain The parent strain (host strain) for generating the production strains was the microbial strain Escherichia coli K12 W3110 (commercially available from DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under strain number DSM 5911). E. coli W3110 was in each case transformed in a known manner with the vectors pCys-CDOrn-CSADcc, pCys-CSma-CSADcc, and pCys-CSma-CSADcc-CDOrn, and the transformants were selected on LBtet plates. One transformant in each case was selected as the production strain. The producing strains were designated E. coli W3110×pCys-CDOrn-CSADcc (production of hypotaurine), E. coli W3110×pCys-CSma-CSADcc (production of taurine), and E. coli W3110×pCys-CSma-CSADcc-CDOrn (production of a mixture of hypotaurine and taurine).

[0176] Example 3: Preparation of hypotaurine and taurine in shake flasks Production strains: The production strains were Escherichia coli (E. coli) W3110×pCys-CDOrn-CSADcc (production of hypotaurine), Escherichia coli (E. coli) W3110×pCys-CSma-CSADcc (production of taurine), and Escherichia coli (E. coli) W3110×pCys-CSma-CSADcc-CDOrn (production of a mixture of hypotaurine and taurine).

[0177] Preculture: A preculture of the production strain was prepared in LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L tetracycline) (20 ml LBtet medium in a 100 ml Erlenmeyer flask, grown overnight at 37°C and 120 rpm).

[0178] Main culture: For each production strain, 0.5 ml of preculture was transferred to a 300 ml Erlenmeyer flask (with baffles) containing 30 ml of SM1 medium supplemented with 15 g / L glucose, 2 g / L NaSO 5H O, 0.1 g / L L-isoleucine, 0.1 g / L D,L-methionine, 0.1 g / L L-threonine, 5 mg / L vitamin B1, and 15 mg / L tetracycline.

[0179] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L NH4 sulfate, 0.3 g / L MgSO4 x 7H2O, 0.015 g / L CaCl2 x 2H2O, 0.002 g / L FeSO4 x 7H2O, 1 g / L Nacitrate x 2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0180] Composition of trace element solution: 0.15 g / L Na2MoO4x2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2x6H2O, 0.25 g / L CuSO4x5H2O, 1.6 g / L MnCl2x4H2O, 0.3 g / L ZnSO4x7H2O.

[0181] The main culture was incubated for 24 hours in an incubator shaker (Infors) at 30°C and 140 rpm. After 24 hours, a 1 ml sample was taken and the cell density OD 600 The optical density of the main culture was measured photometrically at 600 nm (mg / ml) using a Thermo Scientific™ Genesys™ 10S UV / Visible spectrophotometer, and the hypotaurine and taurine contents were determined by HPLC. The taurine and hypotaurine yields are summarized in Table 3.

[0182] [Table 3]

[0183] Sample preparation for the determination of hypotaurine and taurine by HPLC: One ml of culture broth from shake flask growth or fermenter broth from fermentation was incubated at 80°C for 5 minutes and then centrifuged at 13,000 rpm for 5 minutes (Heraeus™ Fresco™ 21 centrifuge). Cell culture supernatant from shake flask growth was analyzed directly by HPLC for hypotaurine and taurine content. Cell culture supernatant from fermentation was diluted 10-fold with HO and analyzed by HPLC for hypotaurine and taurine content.

[0184] HPLC analysis of L-cysteic acid, L-cysteine ​​sulfinic acid, hypotaurine, and taurine: For quantitative measurement of the compounds quantitatively analyzed in the Examples, as well as L-cysteic acid and L-cysteine ​​sulfinic acid, HPLC methods calibrated for each of the four analytes described above were used. Reference materials used for calibration were commercially available (Sigma-Aldrich). As known from the analysis of amino acids, an Agilent 1260 Infinity II HPLC system equipped with a unit from the same manufacturer was used for precolumn derivatization with o-phthaldialdehyde (OPA derivatization). For detection of the OPA-derivatized products of hypotaurine 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 column (100 mm length, 4.6 mm internal diameter, 2.6 μm particle size) was also used, thermally equilibrated at 40°C in the column oven.

[0185] Eluent A: 25 mM Na phosphate, pH 6.0. Eluent B: methanol. Separation was performed in gradient mode, from 10% Eluent B to 60% Eluent B over 0-25 min, followed by 60% Eluent B to 100% Eluent B over 2 min, followed by 100% Eluent B at a flow rate of 0.5 ml / min for another 2 min. Retention time of L-cysteic acid: 3.2 min. Retention time of L-cysteine ​​sulfinic acid: 4.1 min. Retention time of taurine: 14.8 min. Retention time of hypotaurine: 15.7 min.

[0186] [Example 4]: Production of hypotaurine and taurine by fermentation Preculture 1: Twenty ml of LBtet medium was inoculated into a 100 ml Erlenmeyer flask with each of the production strains: E. coli W3110×pCys-CDOrn-CSADcc (production of hypotaurine), E. coli W3110×pCys-CSma-CSADcc (production of taurine), or E. coli W3110×pCys-CSma-CSADcc-CDOrn (production of a mixture of hypotaurine and taurine), and the mixture was incubated on a shaker (150 rpm, 30°C) for 7 hours.

[0187] Preculture 2: Thereafter, each preculture 1 was completely transferred to 100 ml of SM1 medium supplemented with 5 g / L glucose, 5 mg / L vitamin B1, and 15 mg / L tetracycline (see Example 3 for the composition of SM1 medium).

[0188] The cultures were each shaken in Erlenmeyer flasks (1 L volume) at 30°C and 150 rpm for 17 hours (Infors incubator shaker). After this incubation, the cell density OD 600 / ml was 3-5.

[0189] Main culture: Fermentations were carried out in Biostat B fermenters (2 l working volume) from Sartorius BBI Systems GmbH.

[0190] The fermentation medium (900 ml) consisted of 15 g / L glucose, 10 g / L tryptone (Difco), 5 g / L yeast extract (Difco), 2.4 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.25 g / L NaCl, 0.6 g / L MgSO4 x 7H2O, 0.03 g / L CaCl2 x 2H2O, 0.15 g / L FeSO4 x 7H2O, and 1 g / L NaCitrate x 2H2O. and 1 ml of trace element solution (see Example 3), 0.9 g / L L-isoleucine (Sigma-Aldrich), 0.6 g / L D,L-methionine (Sigma-Aldrich), 0.018 g / L vitamin B1 (Sigma-Aldrich), 0.09 g / L pyridoxine × HCl (vitamin B6, Sigma-Aldrich), and 15 mg / L tetracycline.

[0191] The pH in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH4OH solution. During the fermentation, the pH was maintained at a value of 7.0 by automatic correction with 25% NH4OH.

[0192] Foam control was achieved by automated metered addition of 4% v / v Struktol J673 in H2O (Schill & Seilacher).

[0193] For inoculation, 100 ml of preculture 2 was pumped into the fermenter. The initial volume was therefore approximately 1 L. The culture was initially stirred at 400 rpm and aerated with sterilized compressed air through a sterile filter at an aeration rate of 2 vvm (vvm: introduction of compressed air into the fermentation batch, specified in liters of compressed air per liter of fermentation volume per minute). Under these starting conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0194] The target value for O2 saturation during fermentation was set at 30%. After O2 saturation fell below the target value, a regulating cascade was initiated to bring O2 saturation back to the target value. This involved first continuously increasing the gas supply (up to 5 vvm) and then continuously increasing the agitation speed (up to 1500 rpm).

[0195] The fermentation was carried out at a temperature of 30° C. After 2 hours of fermentation time, a sulfur source in the form of a sterile 60% (w / v) stock solution of sodium thiosulfate×5H 2 O was fed at a rate of 1.5 ml / hour.

[0196] When the glucose content in the fermenter decreased from the initial 15 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously metered in. The feed rate was then adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L. Glucose was measured using a YSI (Yellow Springs, OH, USA) glucose analyzer.

[0197] The fermentation time was 65 hours. Following this, samples of the fermenter broth were taken and the contents of hypotaurine and taurine in the culture supernatant were determined by HPLC. The results are summarized in Table 4.

[0198] [Table 4]

[0199] [Example 5]: Biomass removal As described in the Examples below, the workup of the fermenter broth from Example 4 is shown as follows: Batch 1: Fermentation of W3110×pCys-CDOrn-CSADcc Batch 2: Fermentation of W3110×pCys-CSma-CSADcc Batch 3: Fermentation of W3110×pCys-CSma-CSADcc-CDOrn

[0200] Batch 1: 720 ml of fermenter broth of the W3110×pCys-CDOrn-CSADcc strain from Example 4 was titrated to pH 3.0 with 4 M H2SO4 and then incubated at 80°C for 30 minutes. After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes) and the fermentation supernatant was removed for further use. 630 ml of fermentation supernatant was obtained. The hypotaurine content was 56.3 g / L, corresponding to 35.5 g or 324.8 mmol of hypotaurine.

[0201] Batch 2: 1000 ml of fermenter broth of the W3110×pCys-CSma-CSADcc strain from Example 4 was titrated to pH 3.0 with 25 ml of 4 M HSO and then incubated in an autoclave (H+P Varioklav 135S) at 121°C for 20 minutes. After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes) and the fermentation supernatant was removed for further use. 870 ml of fermentation supernatant was obtained. The taurine content was 13.0 g / L, corresponding to 11.3 g (90.3 mmol) of taurine used for further workup.

[0202] Batch 3: 880 ml of fermenter broth of strain W3110xpCys-CSma-CSADcc-CDOrn from Example 4 was titrated to pH 3.0 with 4 M H2SO4 and then incubated at 80°C for 30 minutes. After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes) and the fermentation supernatant was removed for further use. 830 ml of fermentation supernatant was obtained. An aliquot of the fermentation supernatant was used to determine the hypotaurine and taurine contents by HPLC. The hypotaurine content was 8.3 g / L, corresponding to 6.9 g or 63.1 mmol of hypotaurine. The taurine content was 14.4 g / L, corresponding to 11.95 g or 95.4 mmol of taurine.

[0203] [Example 6]: Oxidation with H2O2 Batch 1A: 630 ml of fermentation supernatant of the W3110×pCys-CDOrn-CSADcc strain from Example 5 was initially placed in a beaker on a magnetic stirrer, and 55 ml of 30% (w / w) HO (Sigma-Aldrich) in a dropping funnel was added dropwise with stirring for 1 hour, ensuring that the batch temperature did not exceed 60°C. After the HO addition, the batch was stirred for another hour. Following this, the batch was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes). 1 ml of the centrifugation supernatant (660 ml volume) was used to determine the hypotaurine and taurine content by HPLC. Hypotaurine was no longer detectable. The taurine content was 62.7 g / L, corresponding to 41.4 g of taurine in the 660 ml centrifugation supernatant used for further workup.

[0204] Batch 1B: 860 ml of fermenter broth from the fermentation of the W3110×pCys-CDOrn-CSADcc strain as in Example 4, having a hypotaurine content of 55.2 g / L (0.5 mol hypotaurine), was titrated to pH 3.0 with 4 M H2SO4 and then incubated at 80°C for 30 min. After cooling, the fermenter broth was first placed in a beaker on a magnetic stirrer, and 70 ml of 30% (w / w) H2O2 (Sigma-Aldrich), corresponding to 0.62 mol H2O2 in a dropping funnel (a molar ratio of H2O2:hypotaurine of 1.22:1), was added dropwise with stirring for 1 h, ensuring that the batch temperature did not exceed 60°C. After the metered addition of H2O2, the batch was stirred for an additional 1 h. A 1 ml aliquot of the batch was centrifuged (Heraeus™ Fresco™ 21 centrifuge) and the centrifugation supernatant was used to determine the hypotaurine and taurine content by HPLC. Hypotaurine was no longer detectable. The taurine content was 58.2 g / L.

[0205] Batch 3: 830 ml of fermentation supernatant of the W3110xpCys-CSma-CSADcc-CDOrn strain from Example 5 was initially placed in a beaker on a magnetic stirrer, and 12 ml of 30% (w / w) HO (Sigma-Aldrich) in a dropping funnel was added dropwise with stirring for 1 hour, ensuring that the batch temperature did not exceed 60°C. After the HO addition, the batch was stirred for another hour. Following this, the batch was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes). 1 ml of the centrifugation supernatant (840 ml volume) was used to determine the hypotaurine and taurine contents by HPLC. The taurine content was 21.7 g / L, corresponding to 18.2 g of taurine in the 840 ml centrifugation supernatant used for further workup.

[0206] [Example 7]: Concentration of fermentation supernatant Batch 1: The centrifugation supernatant of the W3110×pCys-CDOrn-CSADcc strain after H 2 O 2 incubation from Example 6, Batch 1A was concentrated to a volume of 130 ml in a rotary evaporator (Buchi Rotavapor R-205, bath temperature 80° C., vacuum 140 mbar).

[0207] Batch 2: The centrifugation supernatant after biomass removal from Example 5 was concentrated to a volume of 70 ml in a rotary evaporator (Buchi Rotavapor R-205, bath temperature 80° C., vacuum 140 mbar).

[0208] Batch 3: The centrifugation supernatant of the W3110×pCys-CSma-CSADcc-CDOrn strain after H 2 O 2 treatment from Example 6 was concentrated to a volume of 86 ml in a rotary evaporator (Buchi Rotavapor R-205, bath temperature 80° C., vacuum 140 mbar).

[0209] [Example 8]: Ethanol extraction Batch 1: A 130 ml volume of the evaporated concentrated batch of W3110×pCys-CDOrn-CSADcc strain from Example 7 was mixed with 530 ml of ethanol at room temperature to a final ethanol concentration of 80% (v / v) in the mixture. The batch was stirred for 30 minutes (magnetic stirrer), cooled to 0°C in an ice bath (overnight), and then centrifuged in a centrifuge precooled to 4°C (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes). The supernatant was removed, and the precipitate was stirred with 720 ml of 80% (v / v) ethanol at room temperature for 30 minutes, followed by centrifugation at 6300 rpm for 10 minutes. The supernatant was removed, and the precipitate was used further.

[0210] Batch 2: A 70 ml volume of the evaporated concentrated batch from Example 7 was mixed with 280 ml of ethanol at room temperature to a final ethanol concentration of 80% (v / v) in the mixture. The batch was stirred (magnetically) for 30 minutes, incubated overnight at 0°C in an ice bath, and then centrifuged in a centrifuge precooled to 4°C (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 6300 rpm for 10 minutes). The supernatant was removed, and the precipitate was stirred with 1000 ml of 80% (v / v) ethanol at room temperature for 30 minutes, followed by centrifugation at 6300 rpm for 10 minutes. The supernatant was separated, and the precipitate was used further.

[0211] Batch 3: A volume of 86 ml of the evaporated batch of strain W3110xpCys-CSma-CSADcc-CDOrn from Example 7 was mixed with 342 ml of ethanol at room temperature to a final ethanol concentration of 80% (v / v) in the mixture. The batch was stirred (magnetically) for 30 minutes, cooled to 0°C in an ice bath, and then filtered through a folded cellulose filter (Fisherbrand™ QL115; diameter 185 mm, pore size 2-3 μm). The filter cake was washed with 880 ml of 80% (v / v) ethanol. The filtrate was discarded. The undried filter cake was used further.

[0212] [Example 9]: Crystallization Batch 1: The precipitate from Example 8 was dissolved in 200 ml of HO by heating in a 90°C water bath, then slowly cooled to RT (overnight) with the water bath turned off and gentle stirring (200 rpm), then cooled to 0°C in an ice bath, and subsequently filtered through a folded filter (Fisherbrand™ QL115; diameter 185 mm, pore size 2-3 μm). The filtrate (mother liquor) was removed, and an aliquot was used to determine the taurine content by HPLC. The taurine content was 53.8 g / L (10.8 g in 200 ml of filtrate). The crystallized material was washed with 100 ml of ethanol and dried at 37°C. The dried crystallized material was weighed. The weighed amount of crystallized material was 28.5 g. 21.3 mg of the crystallized material was weighed and dissolved in 1 ml of HO (concentration 21.3 g / L), and the taurine content was measured by HPLC. The taurine content was 20.7 g / L, corresponding to 97.2% of the taurine content of the crystallized product. Therefore, the taurine yield in 28.5 g of crystallized product was 27.7 g. The percent taurine yield was 66.9% based on the amount of 41.4 g of taurine used from Example 6, Batch 1A. Including the 10.8 g taurine content of the reusable mother liquor, the amount of taurine recovered was 38.5 g (92.9% of the amount of 41.4 g of taurine used in Example 6, Batch 1A).

[0213] Batch 2: The precipitate from Example 8 was dissolved in 40 ml of HO by heating in a 90°C water bath, then slowly cooled to RT (overnight) with the water bath turned off and gentle stirring (200 rpm), then cooled to 0°C in an ice bath, and subsequently filtered through a folded filter (Fisherbrand™ QL115; diameter 185 mm, pore size 2-3 μm). The filtrate (mother liquor) was removed, and an aliquot was used to determine the taurine content by HPLC. The taurine content was 67.1 g / L (2.3 g in 35 ml of filtrate). The crystallized material was washed with 50 ml of ethanol and dried at 37°C. The dried crystallized material was weighed. The weighed amount of crystallized material was 8.4 g. 20.0 mg of the crystallized material was weighed and dissolved in 1 ml of HO (concentration 20 g / L), and the taurine content was measured by HPLC. The taurine content was 19.2 g / L, corresponding to 96.0% of the taurine content of the crystallized product. Therefore, the taurine yield in 8.4 g of crystallized product was 8.1 g. The taurine yield percentage based on the amount of 11.3 g of taurine used from Example 5 was 71.7%. Including the taurine content of 2.3 g of the reusable mother liquor, the amount of taurine recovered was 10.4 g (92.0% of the amount of 11.3 g of taurine used).

[0214] Batch 3: The filter cake of strain W3110xpCys-CSma-CSADcc-CDOrn from Example 8 was dissolved in 50 ml of HO by heating in a water bath at 90 °C, then slowly cooled to RT (overnight) with the water bath turned off and gentle stirring (200 rpm), then cooled to 0 °C in an ice bath, and subsequently filtered through a folded filter (Fisherbrand™ QL 115; diameter 185 mm, pore size 2-3 μm). A volume of 80 ml of filtrate (mother liquor) was removed, and an aliquot was used to determine the taurine content by HPLC. The taurine content was 83.0 g / L (6.6 g in 80 ml of filtrate). The crystallized product was washed with 100 ml of ethanol and dried at 37 °C. The dried crystallized product was weighed. The weighed amount of crystallized product was 10.1 g. 20 mg of the crystallized material was weighed and dissolved in 1 ml of HO, and the taurine content was measured by HPLC. The taurine content was 19.54 g / L, corresponding to a taurine content of 97.7% in the crystallized material. Therefore, the taurine yield in 10.1 g of crystallized material was 9.9 g. The taurine yield percentage based on the amount of 18.2 g of taurine used from Example 6 was 54.2%. Including the taurine content of 6.6 g of the reusable mother liquor, the amount of taurine recovered was 16.5 g (90.7% of the amount of 18.2 g of taurine used from Batch 3 of Example 6).

Claims

1. 1. A method for isolating taurine from a fermenter broth after microbial fermentation of a production strain, comprising the following isolation steps: i. isolating a fermentation supernatant by removing biomass from the fermenter broth; ii. Precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1; iii. Isolating the taurine precipitate and dissolving it in solvent 2; iv. Crystallizing the dissolved taurine; v. isolating and optionally drying the crystallized taurine, wherein the isolated taurine is obtained with a purity of >80% (w / w); A method comprising:

2. 2. The method according to claim 1, characterized in that the microbial production strain is a strain of the species Escherichia coli.

3. 3. The method according to claim 1 or 2, characterized in that an oxidizing agent is added and incubated with said oxidizing agent before the addition of solvent 1 (step ii).

4. The oxidizing agent is H ２ O ２ The method according to claim 3, wherein

5. 5. The method according to claim 3 or 4, wherein the incubation with the oxidizing agent is carried out in a continuous process.

6. 6. The method according to claim 1, wherein the fermenter broth has a combined molar content of hypotaurine and taurine in the fermentation supernatant of at least 91.6 mM.

7. 7. The method according to one or more of claims 1 to 6, characterized in that prior to the removal of biomass (step i), at least one measure selected from the group consisting of: (1) adjusting the pH of the fermentor broth to a pH of 6.0 or less; and (2) increasing the temperature of the fermentor broth to at least 50°C and incubating for at least 5 minutes.

8. 8. The method of claim 7, wherein at least one of the selected measures is performed continuously.

9. Water-miscible solvent 1 is ethanol, isopropanol and their H ２ 9. The method according to claim 1, wherein the solvent is selected from the group consisting of mixtures of methyl methyl ketone with methyl ketone and methyl ketone.

10. 10. The method according to one or more of claims 1 to 9, characterized in that the volume of the fermentation supernatant is reduced before the addition of solvent 1 (step ii).

11. 11. The method according to one or more of claims 1 to 10, characterized in that the solvent 2 is water.

12. 12. The method according to claim 1, wherein the molar yield of taurine achieved is at least 50%, based on the molar amount of starting product used, present in the fermentation supernatant and consisting of at least one compound selected from the group consisting of hypotaurine and taurine.

13. The method according to one or more of claims 1 to 12, characterized in that the production strain contains a cds encoding the protein having SEQ ID NO:

2.

14. The method according to one or more of claims 1 to 13, characterized in that the production strain contains a cds encoding the protein having SEQ ID NO:

4.

15. The method according to one or more of claims 1 to 14, characterized in that the production strain contains a cds encoding the protein having SEQ ID NO:6.

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