WO2024125795A1

WO2024125795A1 - Method for isolating taurine from taurine- and hypotaurine-containing fermentation media after microbial fermentation

Info

Publication number: WO2024125795A1
Application number: PCT/EP2022/086041
Authority: WO
Inventors: Rupert Pfaller
Original assignee: Wacker Chemie Ag
Priority date: 2022-12-15
Filing date: 2022-12-15
Publication date: 2024-06-20

Abstract

The invention relates to a method for isolating taurine from a fermentation broth following the microbial fermentation of a production strain, having the following isolation steps in the given order: (i) isolating the fermentation supernatant by separating the biomass from the fermentation broth, (ii) precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1 (e.g. ethanol), (iii) isolating and dissolving the taurine precipitate in a solvent 2 (water), (iv) crystallizing the dissolved taurine, and (v) isolating and optionally drying the crystallized taurine, wherein the isolated taurine is obtained with a degree of purity of >80% (w/w). In this manner, a simple method is provided for isolating fermentatively prepared taurine with a high degree of purity.

Description

Method for the isolation of taurine from taurine and hypotaurine-containing fermentation media of microbial fermentation

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

Taurine (2-aminoethanesulfonic acid, CAS number 107-35-7) is an aminosulfonic acid that occurs naturally as a degradation product of the amino acids cysteine and methionine. Taurine is economically important, is a component of energy drinks and is also used in animal feed, e.g. for cats or in fish farming. However, taurine is also said to have health-promoting effects.

Taurine is currently produced chemically for commercial use. One well-known process is the Changshu Yudong Chemical Factory, which starts with ethylene and leads to taurine via ethyleneimine. With the trend away from chemically produced ingredients, driven by consumer demands, biotechnological processes for producing taurine are increasingly being investigated. In nature, taurine occurs almost exclusively in the animal kingdom, with only a few examples of occurrence in plants, algae or bacteria. There are various biosynthetic pathways to taurine (see eg the KEGG Pathway database: "Taurine and hypotaurine metabolism"), including starting from L-cysteine. The most important synthesis steps that lead from L-cysteine to taurine are shown in equations (1) to (5).

(1) L-cysteine + O2 -> L-cysteine sulfinic acid

(2) L-cysteine sulfinic acid +

O2 -> L-cysteine acid

(3) L-cysteine sulfinic acid -> hypotaurine + CO2

(4) Hypotaurine

-> Taurine

(5) L-cysteine acid -> taurine + CO2

(1) : In a first step, L-cysteine is converted by the enzyme cysteine dioxygenase (CDO, EC 1.13.11.20) to L-

Cysteine sulfinic acid (3-sulfinoalanine, CAS number 207121-48-0) oxidized.

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

(2) and (4): The oxidation of cysteine sulfinic acid to cysteic acid and the oxidation of hypotaurine to taurine have not yet been clearly clarified.

An alternative route of metabolic engineering to taurine is possible using the enzyme cysteate synthase (CS, EC 2.5.1.76) known from methanogenic archaebacteria, where L-cysteic acid is formed in a partial reaction of coenzyme M biosynthesis according to equation (6) :

(6) O-phospho-L-serine + SO3 ² “ -> L-cysteic acid + HPO4 ² “ The L-cysteine acid produced in this reaction can then be decarboxylated by a CSAD enzyme according to equation (5). O-phospho-L-serine (OPS) serves as a biosynthetic precursor of L-serine in the L-cysteine metabolism of, for example, Escherichia coli (see, for example, the KEGG Pathway database: "Cysteine and methionine metabolism").

A disadvantage of the known biotechnological processes for producing taurine is that product mixtures of hypotaurine and taurine are produced and hypotaurine is often the main product. In addition, the yields of known biotechnological processes for producing taurine and hypotaurine are very low and no process for isolating the product from fermentative processes is known.

Honjoh et al. (2010) , Amino Acids 38: 173-1183 describe a genetically modified yeast strain that heterologously expresses the CDO and CSAD genes from carp (Cyprinus carpio). When L-cysteine was added to the culture of the genetically modified strain, the production of hypotaurine was observed as the main product, along with a smaller amount of taurine. Although S. cerevisiae is capable of producing cysteine from its own metabolism, the external addition of L-cysteine to the culture medium was necessary to produce hypotaurine. Intracellularly formed taurine was determined analytically, with and without H ₂ O2 treatment (Table 1 in Honjoh et al.). From the significantly higher taurine values after H ₂ O2 treatment, it was concluded that a large part of the intracellularly formed product was present as hypotaurine, although hypotaurine was not quantified analytically, even though hypotaurine analysis was described in the methods section of Honjoh et al. No method for isolating taurine from yeast cells was described. WO 17/213142 A1 (Ajinomoto) describes a taurine-producing strain that was obtained by heterologous expression of a cysteine dioxygenase and an L-cysteine sulfinic acid decarboxylase in an originally cysteine-producing strain. The main product was hypotaurine with a maximum yield of 450 pM, which could be converted to taurine by subsequent chemical-alkaline treatment and in low yield. General methods from the state of the art for isolating amino acids, such as cation exchange chromatography, membrane filtration and crystallization, were mentioned, but no information was given on the suitability of the methods for isolating taurine, on yields and on the purity of a taurine product that can be achieved.

US 2019/0062757 Al (KnipBio) describes heterologous production strains for the production of taurine or its precursor substances, whereby maximum yields of only 419 ng/ml hypotaurine were achieved. General state-of-the-art methods for isolating taurine were given, such as centrifugation, filtration or ion exchange chromatography, but without disclosing a purification process and without proving whether these methods are suitable for isolating taurine. In addition, no information was given on the purity of a taurine product that could be achieved.

Ma et al., Food Anal. Methods (2018) 11:415-425 describe a laboratory procedure for isolating taurine from bovine liver. The liver tissue is first disrupted by ultrasound and a protein-free extract is prepared from which taurine was isolated by cation exchange chromatography and subsequent crystallization after treatment with activated carbon.

Lin et al., Ind. Eng. Chem. Res. (2013) 52: 13449-13458 describe the crystallization of taurine from chemical preparation. The state of the art discloses methods for isolating taurine from chemical production or from biological material, including animal organs such as beef liver, but not from the fermentation of microorganisms. Recombinant microorganisms with heterologous metabolic pathways that lead to taurine and especially hypotaurine are known, such as Escherichia coli, baker's yeast or Coryn ebacteri um glutamicum. The yields disclosed for both hypotaurine and taurine are consistently low. An efficient method for the preparative conversion of hypotaurine to taurine as a step in a taurine processing process is not known. Only general methods from the processing of amino acids are given for isolating taurine. A method for isolating taurine produced by fermentation is not known.

The object of the present invention was to provide a simple process for isolating taurine produced by fermentation in high purity and with high yields while avoiding chromatographic process steps.

The object was achieved by a method for isolating taurine from a fermenter broth after microbial fermentation of a production strain, which comprises the following isolation steps in the order given: i. isolating the fermentation supernatant by separating the biomass from the fermenter broth, ii. precipitating taurine from the fermentation supernatant by adding a water-miscible solvent 1, iii. isolating and dissolving the taurine precipitate in a solvent 2, iv. crystallizing the dissolved taurine, v. isolating and optionally drying the crystallized taurine, whereby the isolated taurine is obtained in a purity of >80% (w/w).

A significant advantage of the invention is that no chromatographic process steps are necessary, so that a simple process for isolating fermentatively produced taurine in high purity is provided.

Chromatographic processes are used to separate a mixture of substances by differently distributing its individual components between a stationary and a mobile phase. In this way, relatively pure substances can be obtained. The state-of-the-art method for isolating taurine by Ma (2018, see above) also includes a chromatographic step.

Surprisingly, the present method does not require any chromatographic process steps and is nevertheless able to isolate taurine with a purity of >80% (w/w). The method according to the invention for isolating taurine thus saves work steps and material and is overall more economical.

A process in the sense of the invention is defined as a multi-stage sequence of work steps in which a product is isolated from a batch in a predefined order of successive process steps, also referred to as a processing process or DSP (downstream processing). The number of process steps is variable and depends on the desired degree of purity of the product. The present invention for isolating taurine from fermentation batches is a process.

Fermentation is a process step for the production (cultivation) of cell cultures on an industrial scale, in which a preferably microbial production strain is grown under defined conditions of culture medium (cultivation medium), Temperature, pH, oxygen supply and medium mixing are used to make the culture medium grow. If necessary, the formation of biomass and target product is optimized by continuously adding media components such as glucose (C source) or a complex amino acid mixture such as yeast extract (N source) as feed in a so-called fed-batch fermentation. The aim of the fermentation is, depending on the configuration (genetic makeup) of the production strain, to produce a protein/enzyme or a metabolic product, in each case with the highest possible yield for further use. The products taurine and hypotaurine can be produced by fermentation. The end product of the fermentation is a fermenter broth consisting of the biomass of the cells of the production strain (fermenter cells) and the fermentation medium freed from the biomass (fermentation supernatant) which was formed in the course of the fermentation from the culture medium and the metabolic products secreted by the fermenter cells. In contrast to the culture medium which is defined by its chemical composition, the composition of the fermentation medium is not clearly defined due to the unpredictable formation of metabolic products. In the present invention, the products taurine and hypotaurine are produced by fermentation. The products of the fermentation can be found in the fermenter cells or in the fermentation supernatant.

Yield in the sense of the invention is defined as the amount of product which is obtained in a processing step or processing process. The yield can be stated in absolute amount of product (mmol or g), as volume yield (concentration) based on the volume of absolute amount of product (mM or g/L) or as relative yield of the product obtained in a processing step or processing process.

Amount of product obtained in the processing step or processing process in percent of the amount of product used, also referred to as percentage yield.

If it is not a work-up process but a chemical reaction, the yield of the reaction can also be given as the absolute amount of product (mmol or g), as the volume yield (concentration) of the absolute amount of product related to the volume (mM or g/L) or as the relative yield of product as a percentage of the reactant used (taking into account the molecular weights of the reactant and the product), also referred to as the percentage yield. In the context of the invention, hypotaurine in fermentation supernatants is oxidized to taurine by H2O2, whereby the fermentation supernatants can already contain taurine in addition to hypotaurine. The molar yield of taurine from the oxidation of hypotaurine and taurine-containing fermentation supernatants is then defined as the total molar amount of taurine after the oxidation in relation to the sum of the molar amounts of hypotaurine and taurine contained in the fermentation supernatant, expressed as a percentage.

The content or purity of the product is defined as the amount of product in a fraction of the processing process, as determined analytically, e.g. by HPLC. If the fraction is a solid, the purity is given as g product/g solid, or this value is expressed as a percentage. If, for example, the product content is 0.8 g in 1 g solid, this is referred to as a content or purity of 80%. The fraction can also be a solution. The purity is then expressed in g product/g liquid fraction.

A solvent (also called solvent or solvents) is a substance that can dissolve or dilute gases, liquids or solids without causing chemical reactions between the dissolved substance and the solvent. In this invention, water-miscible organic solvents are used. Examples of such solvents, but not limited to these, are organic solvents such as ethanol, methanol, 1-propanol, 2-propanol (isopropanol), and acetone. Water-miscible solvents are defined as solvents which, in any mixing ratio with water, only produce a liquid phase, in contrast to water-immiscible solvents which, depending on their chemical properties (especially apolarity), are only partially soluble in water and, above a concentration in a mixture that is characteristic of the solvent in question, lead to phase separation into the water phase and the solvent phase. 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.

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

A gene, cistron or expression unit is the DNA section that contains all the basic information for producing a biologically active RNA. A gene contains the DNA section from which a single-stranded RNA copy is produced by transcription and the expression signals that are involved in regulating this copying process. The expression signals include at least one promoter, one transcription start, one translation start and one ribosome binding site. Other possible expression signals are a terminator and one or more operators. Alleles are defined as the states of a gene that can be converted into one another by mutation, i.e. by changes in the nucleotide sequence of the DNA. The gene that occurs naturally in a microorganism is called the wild-type allele and the variants derived from it are called mutated alleles of the gene.

A promoter is a nucleotide sequence upstream of the 5' end of the cds that enables the expression of a gene. The promoter is located in the direction of synthesis before the RNA coding region. The promoter contains regions of specific interaction with DNA-binding proteins that mediate the start of transcription of the gene by the RNA polymerase and are referred to as transcription factors.

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

Genes of eukaryotic organisms are predominantly so-called mosaic genes and, in contrast to prokaryotic genes, also contain non-coding sections, so-called introns (intragenic regions). Coding sequences, so-called exons (expressed regions), are DNA sections of a eukaryotic gene that are translated into the amino acid sequence of a protein by the ribosomes after transcription into RNA. The introns are spliced from the primary transcript after transcription of the DNA into RNA. The introns are freed from the introns. Protein-coding RNA is referred to as messenger RNA (mRNA), also known as "mature" mRNA. This is subjected to further modifications such as capping and polyadenylation. The coding region of the mature mRNA is then translated into the protein sequence. If a eukaryotic gene with an exon/intron structure is to be expressed in prokaryotic organisms, it is necessary to translate the protein sequence or the coding region of the mature mRNA back into intron-free DNA, since in prokaryotes the processing of the exon/intron structure does not take place. When, in the context of this invention, reference is made to gene sequences derived from the protein sequence or from the mRNA, this is precisely the process of back translation that is meant. It is preferred that at the same time as the protein sequence or the mRNA sequence is translated back into DNA sequence, a sequence optimization, i.e. adaptation to the codon usage of the corresponding prokaryote (codon optimization) takes place.

Homologous genes or homologous sequences mean that the DNA sequences of these genes or DNA segments are at least 80%, preferably at least 90% and particularly preferably at least 95% identical.

The degree of DNA identity is determined by the program "nucleotide blast", found at http://blast. ncbi . nlm. nih . gov/ , which is based on the blastn algorithm. The preset parameters were used as algorithm parameters for an alignment of two or more nucleotide sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding preset scoring parameters are: Match/Mismatch Scores = 1,-2; Gap Costs = Linear. Homologous protein sequences mean that the protein sequences of these proteins or protein segments are at least 80%, preferably at least 90% and particularly preferably at least 95% identical.

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

A gene construct is 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, replication origin). A gene construct within the scope of the invention is a circular DNA molecule and is referred to as a plasmid, vector or expression vector. The genetic elements of the gene construct cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the gene.

An operon is defined as a higher-level expression unit in which several genes are transcribed under the control of only one promoter, but are each translated by its own ribosome binding site. Operons are often found in the genomes of bacterial microorganisms, but can also be specifically produced by cloning. (artificial operons). In the expression vectors of the present invention (Example 1), the expression units a) serA317, CDOrn and CSADcc (expression vector pCys-CDOrn-CSADcc, Fig. 2), b) serA317, CSma and CSADcc (expression vector pCys-CSma-CSADcc, Fig. 3), c) serA317, CSma, CSADcc and CDOrn (expression vector pCys-CSma-CSADcc-CDOrn, Fig. 4) each form artificial operons, with expression taking place under the control of the serA promoter.

The abbreviation WT (Wt) means wild type. A wild type gene is the form of the gene that has arisen naturally through evolution and is present in the wild type genome. The DNA sequence of Wt genes is publicly available in databases such as NCBI.

In contrast to biotransformation, metabolism engineering (also called "pathway design") is a biotechnology method in which the metabolic pathways of an organism are modified by optimizing or changing genetic and/or regulatory processes. By supplementing the genome with enzyme genes, new or modified enzymes can be introduced into an organism, or genes of endogenous enzymes can be expressed in a stronger or weaker manner, thereby establishing new metabolic pathways in an organism or strengthening or weakening existing metabolic pathways. The aim of metabolism engineering is for the organism to produce either a new metabolic product or a cell-specific metabolic product with increased yield. The result of metabolism engineering is a production strain. In a metabolism engineering process, no starting materials specific to the metabolic product are used, such as an enzyme substrate, such as L-cysteine as a starting compound for the production of hypotaurine. but merely a nutrient medium, also referred to as a culture medium, which is required for the growth of the organism in question and is composed of a C source (e.g. glucose), an N source (e.g. an ammonium salt or a complex amino acid mixture such as peptone or yeast extract) and other salts required for growth. Such nutrient media are known to the person skilled in the art from microbiological practice. In the present invention, production strains for the fermentative production of hypotaurine and taurine are used, which were produced by metabolic engineering.

In the context of the present invention, a microorganism produced by metabolic engineering, with which hypotaurine and/or taurine can be produced by fermentation, is referred to as a production strain. The production strain particularly preferably comprises at least one gene construct, comprising genes of a biosynthesis pathway which, after expression in the production strain, lead to the production of hypotaurine, taurine or a mixture of hypotaurine and taurine. The microorganism strain without a gene construct is also referred to as a host strain. The production strain is thus the product of the host strain plus the gene construct.

A microbial production strain for the production of hypotaurine and/or taurine is preferably characterized in that it

1) a metabolic pathway leading to hypotaurine and/or taurine and

2) involves a deregulated cysteine biosynthesis pathway.

1) The metabolic pathway leading to hypotaurine and/or taurine is preferably characterized in that it comprises i) the reaction of L-cysteine to hypotaurine according to equations (1) and (3), ie the production strain contains at least one cds encoding the enzyme CDO and one cds encoding the enzyme CSAD and/or ii) according to equations (6) and (5) comprises the reaction of OPS to taurine, i.e. the production strain contains at least one cds encoding the enzyme CS and one cds encoding the enzyme CSAD and/or iii) in a combination of i) and ii) comprises the reaction to a mixture of hypotaurine and taurine, i.e. the production strain contains at least one cds encoding the enzyme CDO, one cds encoding the enzyme CSAD and one cds encoding the enzyme CS.

Particularly preferably, the production strain is characterized in that it contains at least one cds encoding the enzyme CDO and one cds encoding the enzyme CSAD and thus catalyzes the reaction of L-cysteine to hypotaurine according to equations (1) and (3).

The genes constituting the hypotaurine and/or taurine metabolic pathway in the production strain can originate from the host strain (homologous expression) or they can be foreign genes (heterologous expression), either synthetically produced (including the adaptation of the cds to the expression in the host strain by so-called codon optimization) or isolated from other organisms, or it can be a combination of homologous and heterologous expression.

Preference is given to the heterologous expression of the genes of the metabolic pathway constituting hypotaurine and/or taurine in the production strain, particularly preference is given to the heterologous expression of synthetically produced genes and particularly preference is given to the heterologous expression of codon-optimized, synthetically produced genes of the hypotaurine and/or Taurine constitutive metabolic pathway in

Product ion strain.

2) Since the metabolic pathways i) to iii) to hypotaurine and/or taurine according to equations (1) and (3) or (6) and (5) originate from the metabolites OPS and L-cysteine from the L-cysteine biosynthesis pathway (see e.g. the KEGG Pathway Database: “Cysteine and methionine metabolism”), a production strain is preferred which comprises a deregulated cysteine biosynthesis pathway and is suitable for cysteine production. As described e.g. by Wada and Takagi, Appl. Microbiol. Biotechnol. (2006) 73: 48-54, cysteine production in a wild-type microorganism is strictly regulated, so that a microorganism strain with a regulated cysteine biosynthesis pathway, as documented in the prior art by the low yields (e.g. a max. cysteine production of 2 pM in WO 17/213142 A1, Tab.

5) is not suitable for the production of economically viable amounts of hypotaurine and/or taurine. Wild-type microorganisms therefore contain a cysteine biosynthesis pathway, but this is so strongly regulated by product inhibition of central enzymes of the biosynthesis (so-called feedback inhibition) that no or only minimal amounts of cysteine are formed when these microorganisms are cultivated (see 2 pM max. cysteine production in WO 17/213142 A1, Table 5). The term "suitable for cysteine production" therefore only includes microorganisms with a deregulated cysteine biosynthesis pathway. A production strain with a deregulated cysteine biosynthesis pathway suitable for hypotaurine and taurine production is therefore preferred. Such strains are disclosed in Example 2 of the present invention.

A microorganism comprising a deregulated cysteine biosynthesis pathway and thus suitable for cysteine production is characterized by containing at least one of the following changes: a) The microorganism strain is characterized by a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with a feedback inhibition by L-serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme, whereby the SerA enzyme activity can be determined, for example, photometrically by the oxidation of NADH dependent on the SerA substrate 3-phosphohydroxypyruvate, as described, for example, by McKitrick and Pizer, J. Bacteriol. (1980) 141: 235 - 245.

Particularly preferred variants of 3-phosphoglycerate dehydrogenase (serA) have a feedback inhibition by L-serine that is reduced by at least a factor of 5, particularly preferably by at least a factor of 10 and, in a further preferred embodiment, by at least a factor of 50 compared to the corresponding wild-type enzyme.

Examples of serA variants encoding SerA enzymes with reduced feedback inhibition are known from the prior art and represent preferred embodiments within the scope of this invention:

- serA317 : feedback-resistant SerA mutant as used in the present invention in the vector pCys (Fig. 1). It is a C-terminal deletion mutant of SerA comprising the N-terminal 317 amino acids of the SerA WT protein with a total length of 410 amino acids (Bell et al., Eur. J. Biochem., 2002, 269: 4176-4184, referred to therein as "NSD:317"; see also Example 1).

- serA G349E: Mutation of glycine at position 349 of the WT SerA enzyme to glutamic acid.

- serA G349D: Mutation of glycine at position 349 of the WT SerA enzyme to aspartic acid.

- serA T372X: Mutation of threonine at position 372 of the WT SerA enzyme to X, where X can be any natural amino acid except threonine.

- serA mutated: serA gene in which 25% of the C-terminal amino acids are changed compared to WT, whereby the change i) is located in the 50 C-terminal amino acids of the WT protein and ii) the change comprises a C-terminal deletion and iii) the change comprises an insertion into the WT sequence. and/or b) The microorganism strain contains a cds encoding a serine O-acetyl transferase (CysE) which, compared to the corresponding wild-type enzyme, has a feedback inhibition by cysteine that is reduced by at least a factor of two (as described, for example, in Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611), where the CysE enzyme activity can be determined, for example, photometrically by the consumption of the CysE substrate acetyl-CoA as a result of the reaction with L-serine to form O-acetyl-L-serine, as described, for example, by Nakamori et al., Appl. Env. Microbiol. (1998) 64: 1607-1611. Particularly preferred variants of serine-O-acetyl-transferase (cysE) have a feedback inhibition by cysteine that is reduced by at least a factor of 5, particularly preferably by at least a factor of 10 and, in a further preferred embodiment, by at least a factor of 50 compared to the corresponding wild-type enzyme.

Examples of cysE variants encoding CysE enzymes with reduced feedback inhibition and preferred embodiments within the scope of this invention are:

- cysE -mutated: Mutation in the sequence region position 97 to 273 inclusive.

- cysE-deleted: Deletion in the sequence region position 248 to 259 inclusive.

Preferred embodiments of specific mutations within the scope of this invention are:

- cysEII: G238S, mutation of glycine at position 238 to serine.

- cysEIII: G165D Mutation of glycine at position 165 to aspartic acid. - cysEIV: A237V, mutation of alanine at position 237 to valine and G238S, mutation of glycine at position 238 to serine (double mutant).

- cysEV: A237V, mutation of alanine at position 237 to valine and G238S, mutation of glycine at position 238 to serine and M256I, mutation of methionine at position 256 to isoleucine (triple mutant).

- cysEVI : G238S, mutation of glycine at position 238 to serine and Met256I, mutation of methionine at position 256 to isoleucine (double mutant).

- cysEVII: A237V, mutation of alanine at position 237 to valine.

- cysEVIII: Met256I, mutation of methionine at position 256 to isoleucine and A237V, mutation of alanine at position 237 to valine (double mutant).

- cysEX: T167A, mutation of threonine at position 167 to alanine (used in the vector pCys, example 1).

- cysEXI : T167A, mutation of threonine at position 167 to alanine and G245S, mutation of glycine at position 245 to serine (double mutant).

- cysEXII: K97Q, mutation of lysine at position 97 to glutamine and G238S, mutation of glycine at position 238 to serine and F267L, mutation of phenylalanine at position 267 to leucine (triple mutant).

- cysEXIII: V164A, mutation of valine at position 164 to alanine and F267L, mutation of phenylalanine at position 267 to leucine (double mutant).

- cysEXIV: T167A, mutation of threonine at position 167 to alanine and M256Stop, mutation of methionine at position 256 to stop (double mutant).

- cysEXVI : D250G, mutation of aspartic acid at position 250 to glycine.

- cysEXVII: G165D, mutation of glycine at position 165 to aspartic acid and T167A, mutation of threonine at position 167 to alanine (double mutant). - cysEXXIII : T167A, mutation of threonine at position 167 to alanine and A237V, mutation of alanine at position 237 to valine and G238S, mutation of glycine at position 238 to serine (triple mutant) .

- cysE M256X : Mutation of methionine at position 256 of the WT CysE enzyme to X, where 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 at position 256 of the WT CysE enzyme to I soleucine. and/or c) the microorganism strain has a cds encoding a cysteine exporter (Ef fluxprotein), so that the cell of the production strain is characterized by a cysteine export from the cell that is increased by at least a factor of two compared to the corresponding wild-type cell, where the cysteine export can be determined by photometric measurement of the extracellular cysteine content according to Gaitonde, Biochem. J. (1967) 104: 627 - 633 (comprising cysteine, cystine and the adduct 2-methyl-thiazolidine-2,4-(R)-dicarboxylic acid formed from cysteine and pyruvate), such as e.g. described in US 5 , 972 , 663 B .

The additional expression of a cysteine exporter preferably leads to an increase in cysteine export from the cell by at least a factor of 5, particularly preferably by at least a factor of 10, especially preferably by at least a factor of 20, compared to a wild-type cell.

The cds encoding the cysteine exporter is preferably one or more sequences selected from the group consisting of ydeD, yfiK, cydDC, bcr and emrAB from E. coli or a DNA sequence that is at least 80%, particularly preferably at least 90% and especially preferably at least 95% identical thereto. The changes comprising a deregulated cysteine biosynthesis 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. It is preferred that the changes comprising a deregulated cysteine biosynthesis pathway are located on the gene construct.

Any microorganism that is accessible to recombinant DNA techniques and that is suitable for the fermentative production of recombinant proteins is suitable as a microorganism/host strain for producing a production strain. Suitable microorganism strains include bacterial strains selected from the Corynebacteriaceae family or the Enterobacteriaceae family, yeasts (e.g. Saccharomyces cerevisiae, Yarrowia lipolytica) or fungi (e.g. Aspergillus niger). Preferred microorganism strains are bacterial strains selected from Corynebacterium glutamicum, Pantoea ananatis or E. coli. The method for isolating taurine from a fermenter broth after microbial fermentation of a production strain is particularly preferably characterized in that the microbial production strain is a strain of the species Escherichia coli. Particularly preferred is the strain E. coli K12 W3110, commercially available under the strain number DSM 5911 from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH.

A preferred gene construct which, when transformed into a host strain, leads to a production strain with a deregulated cysteine biosynthesis pathway, which is thus suitable for cysteine production, is the vector pCys disclosed in WO 2021/259491 (Wacker) and described in Example 1 (Fig. 1) •

Gene constructs for a hypotaurine and/or taurine

Biosynthesis pathway and for deregulated cysteine biosynthesis can either be present extrachromosomally as autonomously replicating vectors in the microorganism strain or integrated into its genome. Autonomously replicating vectors for gene constructs of a hypotaurine and/or taurine biosynthesis pathway and a deregulated cysteine biosynthesis are preferred, either combined on one vector or divided into several vectors. A gene construct is particularly preferred which comprises both a hypotaurine and/or taurine biosynthesis pathway and a deregulated cysteine biosynthesis pathway combined on an autonomously replicating vector. 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.

The starting vector for producing gene constructs according to the invention is, for example, the vector pCys 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 expanded to include polynucleotides encoding cysteine dioxygenase (CDO), L-cysteine sulfinic acid decarboxylase (CSAD) and/or cysteate synthase (CS). These gene constructs are thus characterized by the fact that they comprise a hypotaurine and/or taurine biosynthesis pathway and a deregulated cysteine biosynthesis pathway.

Gene and protein sequences of CDO enzymes are accessible, for example, in the NCBI database under the search term "cysteine dioxygenase". All cds that code for proteins with CDO activity, including CDO enzymes selected from Rattus norvegicus (rat), Homo sapiens (human), Cyprinus carpio (carp), Bos taurus (cattle), Capra hircus (goat), Gallus gallus (rooster), Synechococcus (algae) and homologous amino acid sequences can be used. Particularly preferred is the cds from Rattus norvegicus (rn), encoding the CDO enzyme or a cds homologous to it. Particularly preferred is the CDOrn-cds that has the sequence given in SEQ ID NO: 1 (nt 1 to nt 603). Sequence encoding a CDOrn enzyme having SEQ ID NO: 2. In a preferred embodiment, the method is therefore characterized in that the production strain comprises a CDs encoding a protein having SEQ ID NO: 2.

The CDO enzyme activity test can be carried out as follows: i) Preparation of the enzyme to be tested:

A culture obtained by cultivation in a shake flask or in a

Enzyme produced by fermentation can be used in the reaction as follows: as an aliquot from the culture broth that has not been further processed or as an aliquot of the cell suspension after re-isolation of the cells from the culture broth, e.g. by centrifugation or in the form of an aliquot of the cell homogenate a) after mechanical disruption of the cell suspension or b) in the form of chemically permeabilized cells (e.g. by chloroform) or as a cell extract after separation of particulate components from the cell homogenate or as an enzyme that has been purified e.g. by chromatography.

The total protein concentration obtained in each case can be determined, for example, with a commercially available Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions. ii) Determination of CDO enzyme activity

L-cysteine (10 mM final concentration) is placed in a solution buffered to pH 7 with potassium phosphate and the reaction is started by adding the enzyme from i). The test volume is 10 ml. The temperature at which the test is carried out is 30°C. The amount of enzyme from i) used depends on the degree of purification. If culture broth, cell suspension of the reisolated cells, cell homogenate or cell extract are used, at least 0.1 mg of the enzyme fractions prepared in i) are used. In the case of purified enzyme, at least 10 pg of the purified enzyme fraction are used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the test mixture is taken, centrifuged for 10 min and the content of L-cysteine sulfinic acid is determined by calibrated HPLC (see Example 3), whereby the reference substance used for calibration is commercially available (Sigma-Aldrich).

The detection limit of the HPLC method is 1 mg/L L-cysteine sulfinic acid. If less than 1 mg/L L-cysteine sulfinic acid is formed under the test conditions mentioned, then the mixture does not contain any active CDO.

Gene and protein sequences of CSAD enzymes are accessible, for example, in the NCBI database under the search term "cysteine sulfinic acid decarboxylase". All CDs that code for proteins with CSAD activity, including CSAD enzymes selected from Cyprinus carpio (carp), Rattus norvegicus (rat), Homo sapiens (human), Bos taurus (cattle), Capra hircus (goat), Gallus gallus (rooster), E. coli, Synechococcus (algae) and homologous amino acid sequences can be used. Particularly preferred is the CDS from Cyprinus carpio (cc), coding for the CSAD enzyme or a CDS homologous to it. Particularly preferred is the CSADcc-CDS having the sequence given in SEQ ID NO: 3 (nt 1 to nt 1503), coding for a CSADcc enzyme with SEQ ID NO: 4. In a preferred embodiment, the method is therefore characterized in that the production strain comprises a cds encoding a protein with SEQ ID No. 4.

The CSAD enzyme activity test can be performed as follows: i) Preparation of the enzyme to be tested:

An enzyme produced by cultivation in a shake flask or in a fermentation can be used in the reaction as follows: as an aliquot from the culture broth that has not been further processed or as an aliquot of the cell suspension after re-isolation of the cells from the culture broth, e.g. by centrifugation or in the form of an aliquot of the cell homogenate a) after mechanical disruption of the cell suspension or b) in the form of chemically permeabilized cells (e.g. by chloroform) or as a cell extract after separation of particulate components from the cell homogenate or as e.g. chromatographically purified enzyme. The total protein concentration obtained in each case can be determined, for example, with a commercially available Qubit 3.0 fluorometer from Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions. ii) Determination of CSAD enzyme activity

L-cysteine sulfinic acid (10 mM final concentration) is placed in a solution buffered to pH 7 with potassium phosphate and the reaction is started by adding the enzyme from i). The test volume is 10 ml. The temperature at which the test is carried out is 30°C. The amount of enzyme from i) used depends on the degree of purification. If culture broth, cell suspension of the reisolated cells, cell homogenate or cell extract are used, at least 0.1 mg of the enzyme fractions prepared in i) are used. In the case of purified enzyme, at least 10 pg of the purified enzyme fraction are used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the test mixture is taken and centrifuged for 10 min. and the content of hypotaurine is determined by calibrated HPLC (see Example 3), whereby the reference substance used for calibration is commercially available (Sigma-Aldrich).

The detection limit of the HPLC method is 1 mg/L hypotaurine. If less than 1 mg/L hypotaurine is formed under the test conditions mentioned, then the mixture does not contain active CSAD.

Gene and protein sequences of CS enzymes are e.g. accessible in the NCBI database under the search term "cysteate synthase". All cds that code for proteins with CS activity, including CS enzymes selected from Methanosarcina acetivorans, Methanosarcina barker!, Methanocella pal udi cola, Methanoculll eus mari snigri, Methanolinea mesophila and amino acid sequences homologous thereto can be used. The CS-coding cds is particularly preferably selected from Methanosarcina aceti vorans (ma), Methanosarcina barker! or a sequence homologous thereto. The CSma-cds particularly preferably has the sequence given in SEQ ID NO: 5, coding for a CSma enzyme with the amino acid sequence with SEQ ID NO: 6. In a preferred embodiment, the method is therefore characterized in that the production strain comprises a cds, coding for a protein having SEQ ID No. 6 .

The CS enzyme activity test can be carried out as follows: i) Preparation of the enzyme to be tested:

An enzyme produced by cultivation in a shake flask or in a fermentation can be used in the reaction as follows : as an aliquot from the not further processed

Culture broth or as an aliquot of the cell suspension after re-isolation of the cells from the culture broth, e.g. by centrifugation or in the form of an aliquot of the cell homogenate a) after mechanical disruption of the cell suspension or b) in the form of chemically permeabilized cells (e.g. by chloroform) or as a cell extract after separation of particulate components from the cell homogenate or as e.g. chromatographically purified enzyme.

The total protein concentration obtained in each case can be determined, for example, using a commercially available Qubit 3.0 fluorometer from the company . Thermo Fisher Scientific using the "Qubit® Protein Assay Kit" according to the manufacturer's instructions. ii) Determination of CS enzyme activity Na2SO3 (20 mM final concentration) and O-phospho-L-serine (7 mM final concentration) are placed in a solution buffered with potassium phosphate to pH 7 and containing 100 mM KCl and the reaction is started by adding the enzyme from i). The test volume is 10 ml. The temperature at which the test is carried out is 30°C. The amount of enzyme from i) used depends on the degree of purification. If culture broth, cell suspension of the re-isolated cells, cell homogenate or cell extract are used, at least 0.1 mg of the enzyme fractions prepared in i) are used. In the case of purified enzyme, at least 10 pg of the purified enzyme fraction are used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the test mixture is taken and centrifuged for 10 min. and the content of L-cysteic acid is determined by calibrated HPLC (see Example 3), whereby the reference substance used for calibration is commercially available (Sigma-Aldrich). The detection limit of the HPLC method is 1 mg/L L-cysteic acid. If less than 1 mg/L L-cysteic acid is formed under the test conditions mentioned, then the mixture does not contain any active CS.

The gene constructs are produced in accordance with the state of the art, preferably using recombinant DNA techniques, as described in Example 1 and as are familiar to the person skilled in the art. For this purpose, the cds of a CDO gene, preferably CDOrn, the cds of a CSAD gene, preferably CSADcc, or the cds of a CS gene, preferably CSma, are cloned in the desired order into a suitable vector, preferably the vector pCys described in Example 1. The polynucleotides coding for CDO, CSAD and CS are cloned behind the serA317 gene contained in pCys, preferably in the form of an artificial operon, each with its own ribosome binding site (RBS) but without its own promoter, so that their expression occurs under the control of the serA317 promoter.

Examples of preferred gene constructs are the vectors pCys-CDOrn-CSAdcc (hypotaurine production, Fig. 2), pCys-CSma-CSADcc (taurine production, Fig. 3) and pCys-CSma-CSADcc-CDOrn (hypotaurine + taurine production Fig. 4) described in the examples. The gene constructs each contain genetic elements of a deregulated cysteine biosynthesis pathway, namely feedback-resistant alleles of the cysE and serA genes as well as the efflux gene ydeD. pCys-CDOrn-CSADcc (Fig. 2) also contains, in operon form linked to the serA317 gene, expression units for the CDOrn (CDO Rattus norvegicus) and the CSADcc gene (CSAD Cyprinus carpio) for the production of hypotaurine according to equations (1) and (3). pCys-CSma-CSADcc (Fig. 3) also contains, in operon form linked to the serA317 gene, expression units for the CSma (CS Methanosarci na acetivorans) and the CSADcc Gene (CSAD Cyprinus carpio) for the production of taurine according to equations (6) and (5) . pCys-CSma-CSADcc-CDOrn (Fig. 4) also contains, linked in operon form to the serA317 gene, expression units for the CSma (CS Methanosarcina acetivorans), the CSADcc (CSAD Cyprinus carpio) and the CDOrn gene (CDO Rattus norvegicus) for the production of a mixture of hypotaurine and taurine according to equations (1) and (3) , or (6) and (5) .

The configuration of CDO and CSAD, CS and CSAD, or CS, CSAD and CDO in the above-mentioned gene constructs in any other order is also possible.

Furthermore, it is possible to clone the CDO, CS and CSAD genes, each with its own promoter and possibly terminator, as independent expression units into the vector pCys. It is also conceivable to clone the CDO, CS and CSAD genes as separate expression units independent of pCys into a separate vector, or to integrate them into the genome of a microorganism as a host organism such as E. coli. The combination of vector-based and genome-integrated expression units is also conceivable.

A production strain suitable for hypotaurine and/or taurine production preferably comprises a microorganism strain, particularly preferably the microorganism strain E. coli K12 W3110 and a gene construct according to the invention, particularly preferably selected from the gene constructs pCys-CDOrn-CSADcc, pCys- CSma-CSADcc or pCys-CSma-CSADcc-CDOrn. The production strains are obtained by transforming the gene constructs in a known manner into a microorganism strain, preferably E. coli W3110. Example 2 shows the production of the production strains E. coli W3110 x pCys-CDOrn-CSADcc for the production of hypotaurine, E. coli W3110 x pCys-CSma-CSADcc for the production of taurine and E. coli W3110 x pCys-CSmaCSADcc- CDOrn for the production of a mixture of hypotaurine and taurine disclosed.

The production of hypotaurine, taurine or a mixture of hypotaurine and taurine using the described production strains can be carried out by cultivation in a shake flask, as described in example 3, or on a production scale by fermentation, preferably fed-batch fermentation of the production strains, as described in example 4. Cultivation in a shake flask or by fermentation produces a shake culture or fermenter broth containing hypotaurine or taurine, or a mixture of hypotaurine and taurine, wherein the taurine or hypotaurine content in the cell culture supernatant after fermentation (fermentation supernatant) is preferably at least 10 g/L.

After microbial fermentation of the production strain and, if appropriate, a reduction in pH and/or increase in temperature and possible incubation of the fermenter broth (see below), the fermentation supernatant is isolated by separating the biomass from the fermenter broth (step i). The state of the art offers various processes for separating biomass. Preferred processes for separating biomass are centrifugation (sedimentation) and filtration, particularly preferred among these is filtration. Both processes can be operated batchwise or continuously, with continuous processes being preferred, particularly preferred among these are continuous centrifugation (decanters, separators) and cross-flow filtration (tangential flow filtration, cross-flow filtration). Cross-flow filtration is particularly preferred. In cross-flow filtration, the pore size of the filtration modules is selected so that biomass and other macromolecules remain in the retentate (are retained) and hypotaurine and taurine are found in the filtrate. Through the separated filtrate the volume of the retentate, which still contains a high proportion of non-titrated product, is reduced. To increase the yield, the retentate can be refilled with water and further filtered (so-called diafiltration).

Depending on the pore size of the filtration modules, a distinction is made between microfiltration (pore size > 100 nm), ultrafiltration (pore size between 2 nm and 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.

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

After isolating the fermentation supernatant by separating off the biomass and, if necessary, reducing the volume of the fermentation supernatant (see below), taurine is precipitated from the fermentation supernatant by adding a water-miscible solvent 1 (step ii). The basis for the precipitation of taurine is that solvent 1 is chosen such that taurine is not or only slightly soluble in it. By adding solvent 1 to the taurine-containing fermentation supernatant, a mixing ratio of solvent 1 to aqueous fermentation supernatant is set. As the mixing ratio increases (i.e. increasing proportion of solvent 1 in the mixture), the taurine solubility decreases until taurine is no longer soluble in the mixture and precipitates, whereby an equilibrium is established between precipitated taurine 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 especially preferably at least 80% v/v. The solubility of a compound is known to be dependent on temperature and can be reduced at low temperatures. This means that by cooling the mixture, the equilibrium of precipitated to dissolved taurine can be shifted towards the precipitated taurine, which increases the yield of step ii. Preferably, the mixture of solvent 1 and aqueous, taurine-containing fermentation supernatant is cooled to 8 °C, particularly preferably to 0 °C and, if the mixture does not freeze, particularly preferably to -20 °C.

The definition of the term solvent given above applies to solvent 1. The process for isolating taurine is preferably 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 H ₂ 0, particularly preferably a solvent selected from the group consisting of ethanol, isopropanol and mixtures thereof with H ₂ 0 and especially preferably ethanol.

The taurine precipitate is then isolated from the mixture and dissolved in a solvent 2 (step iii). The isolation of the taurine precipitate, i.e. separation of the taurine precipitate from the mixture, can be carried out by centrifugation, sedimentation or filtration, preferably by filtration.

Preferably, the taurine precipitate is first suspended at least once, particularly preferably twice and especially preferably three times in a solvent E (extraction solvent) in which it is insoluble in contrast to the impurities and is then separated again from the suspension as described above. The process of suspending a compound such as taurine in a solvent E and separating it again from the suspension is called extraction This extraction step from suspension and separation serves to remove (deplete) impurities present in the precipitate in addition to taurine and can be repeated as often as required. The definition of solvent given above also applies to solvent E, whereby the solvent E used for extraction is preferably solvent 1, but a solvent other than solvent 1 is also conceivable.

To separate impurities by extraction, the material property of different solubilities is used, e.g. that taurine is not or only slightly soluble in pure, water-miscible alcohols such as methanol, ethanol, n-propanol and isopropanol. However, other water-miscible organic solvents such as solvent E are also suitable for extracting impurities. Any mixture of water-miscible organic solvents can be used to extract impurities. Preference is given to water-miscible alcohols such as methanol, ethanol, n-propanol and isopropanol, with ethanol and isopropanol being particularly preferred and ethanol being especially preferred. Ethanol can be used as pure ethanol or as denatured ethanol.

The volume of water-miscible alcohol, preferably ethanol, used for extraction can be chosen arbitrarily and is preferably at least the same volume, particularly preferably at least five times the volume and especially preferably at least 10 times the volume of the fermenter broth used to isolate taurine. The volume used for extraction can be used entirely in one extraction or divided into several batches for repeated extraction. In a preferred embodiment, ethanol can be used in a mixture with water with an ethanol content of 100% (v/v) up to 30% (v/v) ethanol, particularly preferably 100% (v/v) up to 50% (v/v) ethanol and especially preferably from 100% (v/v) up to 80% (v/v) ethanol can be used for extraction. In a further preferred embodiment of the invention, the extraction agent, preferably ethanol, is recovered from the extraction filtrate (e.g. by distillation) in order to increase the economic efficiency and the sustainability of the process by minimizing the consumption of chemicals.

The solubility of taurine in ethanol/H ₂ 0 mixtures depends on the ethanol content and the incubation temperature. At room temperature, taurine is not soluble in commercially available 99% (v/v) ethanol, nor in 80% (v/v) ethanol. A low solubility is observed in 60% (v/v) ethanol at room temperature, which increases significantly in 50% (v/v) and 30% (v/v) ethanol, but can be greatly reduced by cooling to 0 °C. It is preferred to dose ethanol into the fermentation supernatant from step i), the volume of which has particularly preferably been reduced as described below, so that the ethanol concentration is at least 40% (v/v), particularly preferably at least 60% (v/v) and especially preferably at least 80% (v/v). When the percentage of ethanol content is stated, it is a volume percentage. 80% ethanol then means 80 ml ethanol + 20 ml fermentation supernatant in 100 ml total volume. Depending on the ethanol content, the temperature can be lowered as described above to further reduce taurine solubility.

The taurine-containing precipitate can be extracted several times with ethanol/water mixtures for further separation of impurities, wherein the ethanol/water mixture preferably contains at least 40% (v/v) ethanol, particularly preferably at least 60% (v/v) ethanol and especially preferably at least 80% (v/v) ethanol.

The isolated taurine precipitate is dissolved in solvent 2. For solvent 2, the above-mentioned Solvent as defined. Solvent 2 is used as a mixture with water, the proportion of water in the mixture preferably being 100% v/v (pure water) to 50% v/v, particularly preferably 100% v/v to 75% v/v and especially preferably 100% v/v to 90% v/v.

In a preferred embodiment, the process for isolating taurine is characterized in that the solvent 2 is water.

In step iv, the isolated taurine precipitate, optionally freed from impurities by extraction and then dissolved in solvent 2, is crystallized. Preferably, the taurine concentration after dissolving the taurine precipitate in solvent 2 is at least 100 g/L, more preferably at least 200 g/L, and especially preferably at least 300 g/L. Taurine in these concentrations is dissolved by heating to temperatures below the boiling point of solvent 2, preferably up to 70 °C, more preferably up to 80 °C, and especially preferably up to 90 °C. Taurine crystallizes on cooling to room temperature and, to further increase the yield, to a temperature at which solvent 2 does not yet freeze, preferably to 0 °C.

This crystallization step of dissolving and cooling can be repeated as often as desired in order to achieve a higher taurine purity. The crystallization step is preferably carried out at least once, particularly preferably at least twice and especially preferably at least three times. It is also preferred that the crystallization is optimized by adding a water-miscible solvent 3 in order, for example, to increase the yield of crystallized taurine and to reduce the taurine content in the mother liquor. When a compound is crystallized from a solution, a distribution equilibrium is established between the crystals precipitated from the solution and the compound still in solution. The term Mother liquor is the soluble portion of a crystallization without the precipitated portion of the crystals. The definition given above for the term solvent applies to solvent 3. Preferred water-miscible solvents 3 for optimizing crystallization are methanol, ethanol, n-propanol and isopropanol, particularly preferably ethanol and isopropanol and especially preferably ethanol. The proportion of solvent 3 in the mixture with water is preferably a maximum of 30% v/v, particularly preferably a maximum of 20% v/v and especially preferably a maximum of 10% v/v.

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 contained in the supernatant is not necessarily lost, but is preferably returned to one of the preceding processing steps i to iii, with processing step ii being particularly preferred.

The crystallized taurine can be dried to constant weight, preferably by drying at elevated temperature, with and without vacuum, e.g. in a vacuum dryer. The temperature for drying taurine is preferably at least 30 ° C, more preferably at least 50 ° C and especially preferably at least 70 ° C. Drying is preferably carried out without a vacuum, more preferably at a vacuum of <300 mbar and especially preferably at a vacuum of <200 mbar. In a further preferred embodiment, the crystallized taurine can also be dried by freeze-drying or spray-drying, whereby in the case of spray-drying the crystallized taurine first has to be redissolved, preferably in H ₂ O. For further use, the taurine obtained can preferably be ground and sieved to obtain a final product with the desired particle properties.

The taurine isolated according to the process of the invention has a purity of over 80% (w/w), preferably over 90% and particularly preferably over 95%. To determine the purity and estimate any impurities that may still be present, the taurine content in the crystals is determined, referred to as content determination. For this purpose, a product solution is preferably prepared by weighing out a small amount of the crystals and dissolving it in _H2O , e.g. as a 20 g/L solution. The taurine content of the product solution is then preferably determined by calibrated HPLC. The taurine content in the crystals is determined by the ratio, expressed as a percentage, of the taurine concentration in g/L determined by HPLC to the concentration of the product solution, also in g/L. If, for example, 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 crystals is 95%. The taurine content determined in this way is synonymous with the purity of the taurine product.

To further increase the purity, additional purification steps are conceivable, such as the state-of-the-art clay exchange chromatography or treatment with activated carbon.

The process according to the invention thus enables the isolation of taurine from hypotaurine- and/or taurine-containing fermentation broths by simple and cost-effective process steps in high purity for applications in the food, feed and pharmaceutical sectors. At the beginning of the process, the content of hypotaurine and taurine can be quantified from the shake culture or fermenter broth. For example, an aliquot of 1 ml is taken from the shake culture or fermenter broth with a cell density ODgoo/ml of at least 1.0/ml, incubated for 5 minutes at 80°C, then all solid components are separated, for example by centrifuging for 5 minutes at maximum speed in a table centrifuge and the supernatant is quantified by HPLC calibrated for hypotaurine and taurine, as described for example in Example 3 for hypotaurine and taurine.

In a preferred embodiment, the process for isolating taurine is characterized in that the fermenter broth has an additive molar content of hypotaurine, taurine or a mixture thereof of 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 in the fermentation supernatant. Additive molar content of hypotaurine and/or taurine means that the fermenter broth with preferred additive molar content can contain hypotaurine and taurine in any ratio.

Step i serves to separate biomass and other macromolecular components from the fermenter broth (e.g. polysaccharides, proteins, nucleic acids) that can arise as by-products of fermentation or through partial rupture of cells in the final phase of fermentation. The separation of biomass is therefore often hampered by the small particle size of microbial cells and by a high viscosity of the fermenter broth due to macromolecular by-products of fermentation. One measure to improve the separability of macromolecules is their denaturation by lowering the pH of the fermentation broth and/or incubating the fermentation broth at an elevated temperature.

The process for isolating taurine is preferably characterized in that, before separating the biomass (step i), the pH of the fermenter broth is adjusted to a pH of at most 6.0, particularly preferably to a pH of at most 5.0 and especially preferably to a pH of at most 4.0 by adding an acid. This step comprises lowering the pH, which is no longer changed in the further course of the process according to the invention. If necessary, incubation, i.e. allowing to act, can take place at the adjusted pH for preferably at least 5 minutes, particularly preferably at least 10 minutes, especially preferably at least 20 minutes and especially preferably at least 30 minutes. All known acids are suitable as acids for adjusting the pH, preferably mineral acids, including particularly preferably sulfuric acid, hydrochloric acid and phosphoric acid and especially preferably sulfuric acid. Particularly preferably, the process for isolating taurine is characterized in that the adjustment of the pH of the fermenter broth and optionally incubation are carried out in a continuous process.

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 to at least 60 ° C and especially preferably to at least 70 ° C and the fermenter broth is incubated at this temperature for at least 5 min, preferably at least 10 min, particularly preferably at least 20 min and especially preferably at least 30 min, i.e. the selected temperature under these conditions is Fermenter broth. It is preferred that the process for isolating taurine is characterized in that the temperature increase of the fermenter broth and incubation takes place in a continuous process.

In a preferred embodiment, the process for isolating taurine is characterized in that before separating the biomass (step i), at least one measure selected from the group consisting of (1) adjusting the pH of the fermenter broth by adding an acid to a pH of at most 6.0 and (2) increasing the temperature of the fermenter broth to at least 50°C and incubating under these conditions for at least 5 min is carried out.

Particularly preferred is the simultaneous reduction of the pH and increase of the temperature of the fermenter broth, including particularly preferred a combination of pH <5.0 and a temperature of >50°C and furthermore especially preferred a combination of pH <4.0 and a temperature of >70°C. Both the reduction of the pH and the increase of the temperature of the fermenter broth combined with a denaturation of macromolecules represent measures for improving the separability of macromolecules from the fermenter broth. The duration of the incubation (exposure time) of the fermenter broth at reduced pH and increased temperature is preferably at least 5 min, particularly preferably at least 20 min and especially preferably at least 30 min.

The process for isolating taurine is particularly preferably characterized in that at least one of the selected measures is carried out in continuous form. The term measure refers to (1) adjusting the pH of the fermenter broth by adding an acid to a pH of at most 6.0 and/or (2) Increase the temperature of the fermenter broth to at least 50 ° C and incubate for at least 5 min .

It is particularly preferred that both an adjustment of the pH value of the fermenter broth and an increase in the temperature of the fermenter broth are carried out, followed by an incubation for at least 5 minutes and that both processes are carried out together in a continuous manner.

The incubation time is particularly preferably at least 10 minutes, especially preferably at least 20 minutes and moreover especially preferably at least 30 minutes.

A continuous process is preferably carried out as follows:

- The fermentation broth is pumped into a reactor preheated to the desired temperature.

- The reactor is preferably equipped with a dosing section for the acid and an active pH control. The desired pH value of the fermenter broth is preferably set by dosing in the acid. The active pH control can be carried out by a measuring and control unit which, if the pH value deviates from the target value as a result of the dosing in of the fermenter broth, readjusts the desired pH value by dosing in the acid (so-called pH-stat method).

- After the predetermined residence time for incubation at elevated temperature (temperature treatment) and/or reduced pH, the fermentation broth is pumped from the reactor into a receiver for the further processing steps. The receiver can be tempered or not tempered.

- The speed at which the fermentation broth is pumped into the reactor determines the residence time in the reactor and thus the duration of incubation.

The advantage of a continuous process is the higher economic efficiency, since not the entire fermentation broth has to be heated to the desired temperature and/or The pH value must be brought to the desired level, which would require a high amount of time and energy, especially on a large scale.

In a preferred embodiment, the process for isolating taurine is characterized in that an oxidizing agent is added and incubated with it before adding the solvent 1 (step ii). The addition of an oxidizing agent and incubation with it is also referred to as oxidative treatment and causes the oxidation of hypotaurine to taurine. The oxidative treatment means that an oxidizing agent is added, preferably at room temperature or optionally with cooling, and then incubated for preferably at least 5 minutes, particularly preferably at least 0.5 hours, especially preferably at least 2 hours and especially preferably at least 4 hours. The oxidative treatment can be carried out directly in the fermenter broth (see, for example, Example 6, batch 1B) or after separation of the biomass in the fermentation supernatant (see, for example, Example 6, batch 1A). The oxidative treatment can thus be carried out before or after step i (separation of the biomass). If the fermentation supernatant is treated oxidatively, the fermentation supernatant, which was previously clear after the biomass has been separated, becomes cloudy, which enables further impurities to be separated as a precipitate, preferably by filtration or by sedimentation (centrifugation). If the oxidative treatment is carried out directly in the fermenter broth, the resulting precipitate can be separated in one step with the biomass, which saves a separation step and improves the economic efficiency of the process.

Preferred is the direct oxidative treatment of the fermenter broth to oxidize hypotaurine to taurine, followed by separation of the biomass together with the precipitate formed by the oxidative treatment, as described in Example 6, Approach 1B. The oxidizing agent used can be, for example, air, oxygen, ozone, hydrogen peroxide, organic peroxides or a mixture thereof. Preference is given to oxidizing agents which produce no or unproblematic by-products. Preferred oxidizing agents are therefore air, oxygen (0 ₂ ), ozone (0 ₃ ) and hydrogen peroxide (H ₂ O ₂ ), with ozone and hydrogen peroxide being particularly preferred.

The method for isolating taurine from a fermentation batch after microbial fermentation is particularly preferably characterized in that the oxidizing agent is H ₂ O ₂ . It is preferred that the oxidizing agent is not NaOH. In terms of its chemical properties, NaOH is a base (lye) and is not known as an oxidizing agent.

Hydrogen peroxide H ₂ O ₂ is preferably added in commercially available form, as a 30% (w/w) or 50% (w/w) aqueous solution with stirring. The reaction of H ₂ O ₂ is exothermic, so temperature control is necessary. Temperature control can be achieved by external cooling or appropriate dosing profiles for the H ₂ O ₂ addition. A temperature of preferably 70°C, particularly preferably 50°C and especially preferably 40°C is not exceeded. The amount of H ₂ O ₂ used is based on the hypotaurine content of the fermentation supernatant. According to equation (7), one molecule of H ₂ O ₂ is consumed for each molecule of hypotaurine for the oxidation.

(7) Hypotaurine + H ₂ O ₂ -> Taurine + H ₂ O

The molar ratio of H ₂ O ₂ used relative to the hypotaurine content is preferably at least 1:1, particularly preferably at least 1.2:1 and especially preferably at least 1.5:1. The molar yield of taurine, based on the molar amount of hypotaurine used, is preferably at least 70%, particularly preferably at least 80% and especially preferably at least 90% when oxidized by H2O2.

The process according to the invention is thus characterized in that the incubation with an oxidizing agent can be carried out before or after the separation of the biomass from the fermenter broth (step i).

If the fermentation mixture contains only taurine, as disclosed in the examples for the strain E. coli W3110 x pCys-CSma-CSADcc, then incubation with an oxidizing agent is not necessary.

In a further preferred embodiment, the method for isolating taurine from a fermenter broth after microbial fermentation is characterized in that the incubation with the oxidizing agent takes place in a continuous process. A continuous process preferably involves pumping the fermenter broth before separation of the biomass or the fermentation supernatant after separation of the biomass into a reactor for oxidative treatment. The reactor can have active cooling, which makes it easier to control the temperature of the reaction. The reactor is also equipped with a metering section for the oxidizing agent and an active temperature control. The active temperature control is carried out by a measuring and control unit which regulates the metering of the oxidizing agent and the fermenter broth or the fermentation supernatant in such a way that the temperature as a result of the exothermic oxidation of hypotaurine to taurine does not exceed the target value of preferably at most 70 °C, particularly preferably at most 50 °C and especially preferably at most 40 °C. The speed at which fermenter broth, or fermentation supernatant and oxidizing agent are added to the reactor determines the residence time and thus the Duration of the oxidative treatment. The residence time/duration of the oxidative treatment is selected so that hypotaurine is quantitatively oxidized to taurine at a given target temperature. It depends on the hypotaurine content and is determined empirically beforehand. The residence time in the reactor is preferably at least 5 minutes, particularly preferably at least 20 minutes and especially preferably at least 40 minutes.

The advantage of the continuous process is the higher economic efficiency, since the entire fermentation broth does not have to be subjected to the oxidative treatment at the same time, which requires less material for a corrosion-resistant reactor due to the corrosive nature of the oxidizing agent. In addition, aspects of reaction safety speak in favor of continuous operation, since the exothermic reaction of the oxidation of hypotaurine to taurine is easier to control in a (cooled) reactor of smaller volume than in the large volume of the fermentation batch.

After incubation with the oxidizing agent, the fermentation broth or fermentation supernatant is pumped into a receiver for further processing steps.

In a preferred embodiment, the process for isolating taurine is characterized in that the volume of the fermentation supernatant is reduced before the addition of the solvent 1 (step ii). If incubation with an oxidizing agent and the volume reduction take place before step ii, the order of the process steps is preferably such that, starting from the fermenter broth, first the incubation with an oxidizing agent takes place, then the separation of the biomass (step i), then the volume reduction and then step ii. In the event that the biomass is separated first (step i), this is followed by incubation with an oxidizing agent, then the possible subsequent separation of the precipitate formed during the oxidation, then the volume reduction and then step ii. To reduce the volume or concentrate the fermentation supernatant, the aqueous medium is preferably partially or completely removed, particularly preferably partially. Various methods for reducing the volume are available, including evaporation with and without applying a vacuum (distillation of H ₂ O), evaporation of H ₂ O via a thin-film evaporator, reverse osmosis, electrodialysis and nanofiltration. Separation of H ₂ O by freeze-drying is also conceivable. Preference is given to evaporating the taurine-containing fermentation supernatant with and without applying a vacuum, evaporation of H ₂ O via a thin-film evaporator and reverse osmosis. Particularly preferred is evaporating the taurine-containing fermentation supernatant with and without applying a vacuum and reverse osmosis. Particularly preferred is the evaporation of the taurine-containing fermentation supernatant by applying a vacuum, as disclosed in Example 7 of the present invention.

The evaporation of H ₂ 0 can be carried out, for example, by heating the fermentation supernatant preferably to at least 60 ° C, particularly preferably to at least 70 ° C and especially preferably to at least 80 ° C. A vacuum of preferably less than 300 mbar, particularly preferably less than 200 mbar and especially preferably less than 150 mbar is applied.

The degree of volume reduction depends on the further use of the taurine-containing fermentation supernatant. The evaporation of H ₂ O can be carried out until it is completely dry or to a predetermined volume. The reduction of the volume to 50% or less of the initial volume of the fermenter broth used for taurine isolation is preferred, particularly preferably to 30% or less and especially preferably to 20% or less of the Initial volume of the taurine isolation

Fermenter broth .

As disclosed in Example 8 of the present invention, a taurine-containing evaporated batch from Example 7 was added with sufficient ethanol 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 of 0 °C or less. Taurine was isolated from this mixture by filtration. The precipitated taurine fraction was further freed from impurities by resuspension with 80% (v/v) ethanol followed by filtration, during which the taurine did not go into solution.

As already explained above, the sequence of resuspension of the precipitated taurine fraction and filtration is referred to as extraction and can be repeated as desired. The volume of 80% (v/v) ethanol used can be chosen as desired and is preferably at least the same volume, particularly preferably at least five times the volume and especially preferably at least 10 times the volume of the initial volume of the fermenter broth used for taurine isolation. The ethanol/water mixture is not fixed to an ethanol content of 80% (v/v). In a further preferred embodiment, ethanol in a mixture with water of 100% (v/v) up to 30% (v/v) ethanol, particularly preferably 100% (v/v) up to 50% (v/v) ethanol and especially preferably 100% (v/v) up to 80% (v/v) ethanol can be used for repeated extraction.

In a further preferred embodiment of the invention, the extraction agent, preferably ethanol, is recovered from the extraction filtrate (e.g. by distillation) in order to increase the economic efficiency and the sustainability of the process by minimizing the use of chemicals. In addition to ethanol, all other mentioned water-miscible organic solvent in the manner described for ethanol for extraction.

A preferred method for isolating taurine from a fermenter broth after microbial fermentation of a production strain comprises the following isolation steps:

Adjusting the pH to <5 and increasing the temperature of the fermenter broth to >50 °C and incubating the fermenter broth at reduced pH and elevated temperature for at least 5 min in a continuous process as described above; oxidatively treating the fermenter broth with H2O2 for at least 5 min at a maximum of 70 °C in a continuous process as described above, wherein the molar ratio of H2O2:hypotaurine is at least 1:1;

I sol uting the fermentation supernatant by separating the biomass from the fermenter broth by filtration (microfiltration combined with diafiltration) in a continuous process as described above;

Reduction of the volume of the fermentation supernatant to 50% or less 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; precipitation of taurine from the volume-reduced fermentation supernatant by addition of ethanol to give an ethanol/water mixture with a proportion of 80% (v/v) ethanol and cooling to 0 °C or less;

I solation of the taurine precipitate by filtration or sedimentation; extraction of the taurine precipitate at least once with an ethanol/water mixture, wherein the ethanol/water mixture contains at least 60% (v/v) ethanol and the extraction mixture of taurine precipitate and ethanol/water mixture is cooled to 0 °C or less; I Isolating the taurine precipitate and dissolving it in H ₂ 0 in a concentration of at least 200 g/L at a temperature of at least 50 ° C;

Crystallisation of taurine at room temperature or lower; isolation of the crystallised taurine by filtration; drying of the crystallised taurine at at least 50 °C and a vacuum of <300 mbar.

Preferably, the process 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 especially preferably at least 70% based on the molar amount of starting product used and contained in the fermentation supernatant, consisting of at least one of the compounds selected from the group consisting of hypotaurine and taurine. The yield refers to the amount of isolated taurine with a purity >80% after crystallization based on the amount of taurine used, which was contained in the fermentation supernatant after step i and after any prior oxidation of hypotaurine, but before a possible volume reduction of the fermentation supernatant.

The recovery, equivalent to the sum of isolated taurine after crystallization and the taurine still contained in the mother layer of the crystallization, is preferably at least 80%, particularly preferably at least 90% and especially preferably at least 95% based on the amount of taurine used which was contained in the fermentation supernatant after step i and optionally after prior oxidation of hypotaurine. The figures show the plasmids used in the examples.

Fig . 1 : pCys .

Fig. 2: pCys-CDOrn-CSADcc .

Fig. 3: pCys-CSma-CSADcc .

Fig. 4: pCys-CSma-CSADcc-CDOrn .

Abbreviations used in the figures:

TetR: gene that confers resistance to tetracycline.

P15A ORI : Origin of replication. serA317: serA (3-phosphoglycerate dehydrogenase gene, encodes amino acids 1 to 317) cds cysE X: cysE (serine-O-acetyltransferase gene, feedback resistant) cds

ORF306: ydeD (cysteine efflux gene) cds

Seal: cleavage site for the restriction enzyme Seal

PpuMI : cleavage site for the restriction enzyme PpuMI

CDOrn: CDO (cysteine dioxygenase) R. norvegicus cds

CSADcc: CSAD (cysteine sulfinic acid decarboxylase) C. carpio cds

CSma: CS (cysteate synthase) M. acetivorans cds

RBS : Ribosome binding site

The following examples serve to further illustrate the invention.

Production of DNA constructs

All DNA constructs are derived from the vector pCys (Fig. 1) , disclosed in WO 2021/259491 (Wacker) . pCys : pCys is disclosed in WO 2021/259491 (Wacker) and is a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306. The plasmid pACYC184-cysEX-GAPDH-ORF306 contains, in addition to the replication origin and a tetracycline resistance gene (starting vector pACYC184), the cysEX allele (synonym cysE allele) , which encodes a serine-O-acetyl transferase with reduced feedback inhibition by cysteine, as well as the efflux gene ydeD (ORF306) , the expression of which is controlled by the constitutive GAPDH promoter. pCys also contains, cloned behind the ydeD (ORF306) efflux gene, the serA317 gene fragment coding for the N-terminal 317 amino acids of the SerA protein (total length 410 amino acids). The E. coli serA gene is disclosed in the "GenBank" gene database with the gene ID 945258. serA317 is disclosed in Bell et al., Eur. J. Biochem. (2002) 269: 4176- 4184, referred to therein as "NSD:317" and encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. The expression of serA317 is controlled by the serA promoter . The serA promoter is described in Rex et al. (1991) , J. Bacteriol. 173: 5944-5953, Fig. 2.

For cloning purposes, plasmid DNA of the vector pCys was cut with Seal and PpuMI and the 6.1 kb vector fragment, hereinafter referred to as pCys-Scal/PpuMI, was isolated after preparative agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

CDOrn-rrnB: The amino acid sequence of the cysteine dioxygenase from Rattus norvegicus, derived from the mRNA of the gene, is disclosed in the NCBI (National Center for Biotechnology Information) database under the sequence ID: AAH70509.1. From the amino acid sequence, a gene for expression in E. coli was codon-optimized DNA sequence (publicly available Eurofins Genomics GENEius software), designated CDOrn-cds . The DNA sequence of CDOrn-cds is disclosed in SEQ ID NO: 1, nt 1 to 603, encoding a protein having the amino acid sequence of SEQ ID NO: 2. To the 3' end of CDOrn-cds was linked 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. The CDO-rrnB DNA, consisting of the CDOrn-cds and the rrnB terminator, was produced synthetically (Eurofins Genomics).

CSADcc-rrnB: The cDNA gene (cDNA: complementary DNA isolated from mRNA by reverse transcription) of cysteine sulfinic acid decarboxylase (CSAD) from Cyprlnus carpio (common carp) was isolated by Honjoh et al. (2010) , Amino Acids 38: 1173-183 and the DNA sequence was disclosed in the NCBI (National Center for Biotechnology Information) database under Genbank Sequence ID: AB220585.1 (cds: nt 82 - 1584). From the amino acid sequence, a DNA sequence codon-optimized for expression in E. coli (publicly available Eurofins Genomics GENEius software) was derived, designated CSADcc-cds. The CSADcc-cds DNA sequence is disclosed in SEQ ID NO: 3, nt 1 to 1503, encoding a protein having the amino acid sequence of SEQ ID NO: 4. The DNA sequence of the E. coli rrnB terminator (SEQ ID NO: 3, nt 1504 to 1834) was linked to the 3' end of the CSADcc-cds. The DNA sequence of the rrnB terminator is disclosed in Orosz et al. (1991), see above. The CSADcc-rrnB DNA, consisting of the CSADcc-cds and the rrnB terminator, was produced synthetically (Eurofins Genomics).

CSma: Cysteate synthase from Methanosarci na acetivorans (CSma) was used. The amino acid sequence of the CSma enzyme is available in the NCBI database under the accession ID WP_048066469 . From the amino acid sequence, a Expression in E. coli was derived from codon-optimized DNA sequence (publicly available Eurofins Genomics GENEius software).

The CSma DNA sequence is disclosed in SEQ ID NO: 5, encoding a protein having the amino acid sequence of SEQ ID NO: 6.

The CSma DNA was produced synthetically (Eurofins Genomics).

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

PCR products:

Table 1 summarizes the name of the PCR products used for cloning, their size, and the synthetic DNA (so-called template DNA) and primers used. The PCR products PCRI to PCR6 were produced from the respective template DNA by PCR ("Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) with the appropriate primers and isolated by agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen).

Table 1: PCR products for the production of gene constructs

Production of vectors:

The NEBuilder® cloning kit (NEB New England Biolabs) was used to produce the vectors according to the manufacturer's instructions. As summarized in Table 2, the 6.1 kb pCys-Scal/PpuMI vector fragment was ligated with the corresponding PCR products, following the manufacturer's instructions regarding the amounts of the individual DNA fragments used in the ligation mixture. The ligation mixture was then transformed into E. coli NEB® 10-beta (NEB New England Biolabs) according to the manufacturer's instructions. Clones from the transformation were selected on LBtet plates. LBtet plates 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 clones of the transformations (Qiagen kit) and one correct clone was selected in each case. The vectors pCDOrn-CSADcc (Fig. 2), pCSma-CSADcc (Fig. 3) and pCSma-CSADcc-CDOrn (Fig. 4) listed in Table 2 were produced.

Table 2: Overview of the DNA fragments used to produce gene constructs

Example 2: Production of product strains The starting strain (host strain) for the production of production strains was the microorganism strain Escherichia coli K12 W3110 (available for purchase under the strain number DSM 5911 from the DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). E. coli W3110 was transformed in a known manner using the vectors pCys-CDOrn-CSADcc, pCys-CSma-CSADcc and pCys-CSma-CSADcc-CDOrn and transformants were selected on LBtet plates. One transformant was selected as the production strain in each case. The production strains were named E. coli W3110 x pCys-CDOrn-CSADcc (production of hypotaurine), E. coli W3110 x pCys-CSma-CSADcc (production of taurine) and E. coli W3110 x pCys-CSma-CSADcc-CDOrn (production of a mixture of hypotaurine and taurine).

Example 3 : Production of hypotaurine and taurine in shake flask

Production strains: Production strains were E. coli W3110 x pCys-CDOrn-CSADcc (production of hypotaurine), E. coli W3110 x pCys-CSma-CSADcc (production of taurine) and E. coli W3110 x pCys-CSma-CSADcc-CDOrn (production of a mixture of hypotaurine and taurine). Pre-culture: A pre-culture was prepared from the production strains 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 100 ml Erlenmeyer flask, cultured at 37 °C and 120 rpm overnight).

Main culture: For each production strain, 0.5 ml of the preculture was transferred into a 300 ml Erlenmeyer flask (with baffle) with 30 ml SMl medium, further containing 15 g/L glucose, 2 g/L Na2S203 x 5 _H2O , 0.1 g/L L-isoleucine, 0.1 g/L L-methionine, 0.1 g/L L-threonine, 5 mg/L vitamin B1 and 15 mg/L tetracycline.

Composition of SM1 medium: 12 g/L K2HPO4, 3 g/L KH2PO4, 5 g/L NH ₄ sulfate, 0.3 g/L MgSO ₄ x 7 H ₂ O, 0.015 g/L CaCl ₂ x 2 H ₂ O, 0.002 g/L FeSO ₄ x 7 H ₂ O, 1 g/L Na ₃ citrate x 2 H ₂ O, 0.1 g/L NaCl;

1 ml/L trace element solution.

Composition of the trace element solution: 0.15 g/L Na2MoC>4 x 2 H ₂ 0, 2.5 g/L H3BO3, 0.7 g/L CoCl ₂ x 6 H ₂ 0, 0.25 g/L CuSO ₄ x 5 H ₂ O, 1.6 g/L MnCl ₂ x 4 H ₂ 0, 0.3 g/L ZnSO ₄ x 7 H ₂ 0.

The main cultures were incubated for 24 hours at 30°C and 140 rpm in a chest shaker (Infors). After 24 hours, 1 ml samples were taken and the cell density OD ₆ oo/ml (optical density of the main culture, measured photometrically at 600 nm) was measured with a Genesys™ 10S UV-Vis spectrophotometer from Thermo-Scientific™, and the hypotaurine and taurine content was determined by HPLC. The yields of taurine and hypotaurine are summarized in Table 3.

Tab. 3: Hypotaurine and taurine production of

Production strains in shake flask

Sample preparation for quantification of hypotaurine and taurine by HPLC:

1 ml of culture broth from the shake flask culture or fermenter broth from the fermentation was incubated for 5 min at 80°C and then centrifuged for 5 min at 13000 rpm (Heraeus™ Fresco™ 21 centrifuge). Cell culture supernatants from the shake flask culture were directly analyzed by HPLC for the content of hypotaurine and taurine. Cell culture supernatants from the fermentation were diluted 10-fold in H ₂ O and analyzed by HPLC for the content of hypotaurine and taurine.

HPLC analysis of L-cysteic acid, L-cysteine sulfinic acid, hypotaurine and taurine: For the quantitative determination of the compounds quantitatively analyzed in the examples as well as for L-cysteic acid and L-cysteine sulfinic acid, an HPLC method calibrated for each of the four analytes mentioned was used. The reference substances used for calibration were commercially available (Sigma-Aldrich). An HPLC instrument from Agilent, model 1260 Infinity II, was used, equipped with a pre-column derivatization with o-phthaldialdehyde (OPA derivatization) from the same manufacturer, which is familiar from the analysis of amino acids. To detect the OPA-derivatized products of hypotaurine and taurine, the HPLC instrument was equipped with a fluorescence detector. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. An Accucore™ aQ column from Agilent was also used. Thermo Scientific™, length 100 mM, inner diameter 4.6 mm, particle size 2.6 pm, tempered to 40°C in the column oven. Mobile phase A: 25 mM Na phosphate, pH 6.0. Mobile phase B: methanol. The separation was carried out in gradient mode: 0 - 25 min, 10% mobile phase B to 60% mobile phase B, followed by 2 min 60% mobile phase B to 100% mobile phase B, followed by another 2 min 100% mobile phase B, at a flow rate of 0.5 ml/min.

Retention time of L-cysteine 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.

Example 4 : Production of hypotaurine and taurine by fermentation

Pre-culture 1 :

20 ml of LBtet medium were inoculated in a 100 ml Erlenmeyer flask with the respective production strain E. coli W3110 x pCys-CDOrn- CSADcc (production of hypotaurine), E. coli W3110 x pCys-CSma- CSADcc (production of taurine) or E. coli W3110 x pCys-CSma- CSADcc-CDOrn (production of a mixture of hypotaurine and taurine) and incubated for 7 h on a shaker (150 rpm, 30°C).

Pre-culture 2 :

Subsequently, the respective precultures 1 were each completely transferred into 100 ml of SMl medium supplemented with 5 g/L glucose, 5 mg/L vitamin B1 and 15 mg/L tetracycline (composition of SMl medium see Example 3).

The cultures were each shaken in an Erlenmeyer flask (1 L volume) at 30°C for 17 h at 150 rpm (Infors chest shaker). After this incubation, the cell density ODgoo/ml was between 3 and 5.

Main culture:

The fermentations were carried out in Biostat B fermenters (2 1 working volume) from Sartorius BBI Systems GmbH. The fermentation medium (900 ml) contained 15 g/L glucose, 10 g/L tryptone (Difco), 5 g/L yeast extract (Difco), 2.4 g/L (NH ₄ ) ₂ SO ₄ , 5 g/L KH ₂ PO ₄ , 0.25 g/L NaCl, 0.6 g/L MgSO ₄ x 7 H ₂ O, 0.03 g/L CaCl ₂ x 2 H ₂ O, 0.15 g/L FeSO ₄ x 7 H ₂ O, 1 g/L Na ₃ citrate x 2 H ₂ O and 1 ml trace element solution (see example 3), 0.9 g/L L-isoleucine (Sigma-Aldrich), 0.6 g/LD, L-methionine (Sigma-Aldrich), 0.018 g/L vitamin Bl (Sigma-Aldrich), 0.09 g/L pyridoxine x HCl (vitamin B6, Sigma-Aldrich) and 15 mg/L tetracycline.

The pH value in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH ₄ OH solution. During fermentation, the pH value was maintained at 7.0 by automatic correction with 25% NH ₄ OH.

Foam control was achieved by automatic dosing of 4% v/v Struktol J673 in H ₂ O (Schill & Seilacher).

For inoculation, 100 ml of pre-culture 2 was pumped into the fermenter vessel. The initial volume was therefore about 1 L. The cultures were initially stirred at 400 rpm and gassed with compressed air disinfected via a sterile filter at an aeration rate of 2 vvm (vvm: introduction of compressed air into the fermentation mixture, given 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.

The target value for the _O2 saturation during fermentation was set to 30%. After the _O2 saturation dropped below the target value, a regulation cascade was started to bring the O2 saturation back to the target value. First, the gas supply was continuously increased (to max. 5 vvm) and then the stirring speed was continuously increased (to max. 1,500 rpm). Fermentation was carried out at a temperature of 30 ° C. After 2 h of fermentation, a sulfur source in the form of a sterile 60% (w/v) stock solution of sodium thiosulfate x 5 H ₂ 0 was added at a rate of 1.5 ml per hour.

As soon as the glucose content in the fermenter had dropped from an initial 15 g/L to approximately 2 g/L, a 56% (w/w) glucose solution was continuously added. The feeding rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g/L. Glucose was determined using a glucose analyzer from YS I (Yellow Springs, Ohio, USA).

The fermentation time was 65 hours. Afterwards, samples were taken from the fermenter broth and the content of hypotaurine and taurine in the culture supernatant was determined by HPLC. The results are summarized in Table 4.

Table 4 : Cell density, hypotaurine and taurine content of

Production strains after a fermentation period of 65 h

Example 5 : Separation of biomass

The processing of the

Fermenter broths from example 4 receive the assignments:

Approach 1 : Fermentation W3110 x pCys-CDOrn-CSADcc

Approach 2 : Fermentation W3110 x pCys-CSma-CSADcc

Approach 3 : Fermentation W3110 x pCys-CSma-CSADcc-CDOrn Approach 1: 720 ml of fermenter broth of the strain W3110 x pCys-CDOrn-CSADcc from Example 4 was titrated to pH 3.0 with 4 M H2SO4 and then incubated for 30 min at 80°C. After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm) and the fermentation supernatant was separated for further use. 630 ml of fermentation supernatant were obtained. The hypotaurine content was 56.3 g/L, corresponding to 35.5 g or 324.8 mmol hypotaurine.

Approach 2: 1000 ml of fermenter broth of the strain W3110 x pCys- CSma-CSADcc from example 4 was titrated to pH 3.0 with 25 ml of 4 M H2SO4 and then incubated for 20 min in an autoclave at 121°C (H+P Varioklav 135S). After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm) and the fermentation supernatant was separated for further use. 870 ml of fermentation supernatant were obtained. The taurine content was 13.0 g/L, corresponding to an amount of 11.3 g (90.3 mmol) of taurine used for further processing.

Approach 3: 880 ml of fermenter broth of the strain W3110 x pCys-CSma- CSADcc-CDOrn from example 4 was titrated to pH 3.0 with 4 M H2SO4 and then incubated for 30 min at 80°C. After cooling, the fermenter broth was centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm) and the fermentation supernatant was separated for further use. 830 ml of fermentation supernatant were obtained. The hypotaurine and taurine content of an aliquot of the fermentation supernatant was determined by HPLC. The hypotaurine content was 8.3 g/L, corresponding to 6.9 g or 63.1 mmol hypotaurine. The taurine content was 14.4 g/L, corresponding to 11.95 g or 95.4 mmol taurine. Example 6: Oxidation with H2O2

Mixture 1A: 630 ml of fermentation supernatant of the strain W3110 x pCys-CDOrn-CSADcc from example 5 was placed in a beaker on a magnetic stirrer and 55 ml of 30% (w/w) H2O2 (Sigma-Aldrich) was added dropwise in a dropping funnel with stirring over a period of 1 h so that the temperature of the mixture did not exceed 60°C. After addition of the H2O2, the mixture was stirred for a further 1 h. The mixture was then centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm). The content of hypotaurine and taurine was determined by HPLC from 1 ml of centrifugation supernatant (660 ml volume). Hypotaurine was no longer detectable. The taurine content was 62.7 g/L, corresponding to an amount of 41.4 g taurine in 660 ml centrifugation solution used for further processing.

Batch IB: 860 ml of fermenter broth from the fermentation of the strain W3110 x pCys-CDOrn-CSADcc analogous to Example 4 with 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 for 30 min at 80°C. After cooling, the fermenter broth was placed in a beaker on a magnetic stirrer and 70 ml of 30% (w/w) H2O2 (Sigma-Aldrich), corresponding to 0.62 mol H2O2, (molar ratio H2O2:hypotaurine of 1.22:1) was added dropwise in a dropping funnel with stirring over a period of 1 h so that the temperature of the batch did not exceed 60°C. After the H2O2 had been added, the batch was stirred for a further 1 h. A 1 ml aliquot of the mixture was centrifuged (Heraeus™ Fresco™ 21 centrifuge) and the hypotaurine and taurine content of the centrifugation supernatant was determined by HPLC. Hypotaurine was no longer detectable. The taurine content was 58.2 g/L.

Approach 3: 830 ml of fermentation supernatant of the strain W3110 x pCys-CSma-CSADcc-CDOrn from Example 5 was placed in a beaker on a magnetic stirrer and 12 ml of 30% (w/w) H2O2 (Sigma-Aldrich) was added in a dropping funnel with stirring. added dropwise over a period of 1 h so that the temperature of the mixture did not exceed 60°C. After addition of the H2O2, the mixture was stirred for a further 1 h. The mixture was then centrifuged (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm). The hypotaurine and taurine content of 1 ml of centrifugation supernatant (840 ml volume) was determined by HPLC. The taurine content was 21.7 g/L, corresponding to an amount of 18.2 g taurine in 840 ml of centrifugation supernatant used for further processing.

Example 7 : Concentration of fermentation supernatants

Approach 1: The centrifugation supernatant of strain W3110 x pCys-CDOrn-CSADcc after H ₂ 02 incubation from Example 6, Approach 1A, was evaporated to a volume of 130 ml in a rotary evaporator (Büchi Rotavapor R-205, bath temperature 80°C, vacuum 140 mbar).

Approach 2: The centrifugation supernatant after separation of the biomass from Example 5 was evaporated to a volume of 70 ml in a rotary evaporator (Büchi Rotavapor R-205, bath temperature 80°C, vacuum 140 mbar).

Approach 3: The centrifugation supernatant of the strain W3110 x pCys-CSma-CSADcc-CDOrn after H ₂ O2 treatment from Example 6 was evaporated to a volume of 86 ml in a rotary evaporator (Büchi Rotavapor R-205, bath temperature 80°C, vacuum 140 mbar).

Example 8: Ethanol extraction

Approach 1: The evaporated 130 ml volume of the W3110 x pCys-CDOrn-CSADcc strain from Example 7 was mixed with 530 ml ethanol at room temperature so that the final ethanol concentration in the mixture was 80% (v/v). The mixture was stirred for 30 min (magnetic stirrer), cooled to 0°C in an ice bath (overnight) and then centrifuged in a centrifuge pre-cooled to 4 °C (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm). The supernatant was separated, the precipitate was stirred with 720 ml 80% (v/v) ethanol for 30 min at RT and then centrifuged for 10 min at 6300 rpm. The supernatant was separated and the precipitate was used further.

Approach 2: The evaporated 70 ml batch from Example 7 was mixed with 280 ml ethanol at room temperature so that the final ethanol concentration in the mixture was 80% (v/v). The batch was stirred for 30 min (magnetic stirrer), incubated overnight in an ice bath at 0°C and then centrifuged in a centrifuge pre-cooled to 4°C (Thermo Scientific™ Multifuge X1R, TX-400 rotor, 10 min 6300 rpm). The supernatant was separated, the precipitate was stirred with 1000 ml 80% (v/v) ethanol for 30 min at RT and then centrifuged for 10 min at 6300 rpm. The supernatant was separated and the precipitate was reused.

Mixture 3: The evaporated mixture of the strain W3110 x pCys- CSma-CSADcc-CDOrn from Example 7 (86 ml volume) was mixed with 342 ml ethanol at room temperature so that the final ethanol concentration in the mixture was 80% (v/v). The mixture was stirred for 30 min (magnetic stirrer), cooled to 0°C in an ice bath and then filtered through a cellulose pleated filter (Fisherbrand™ QL 115; 185 mm diameter, 2-3 pm pore size) and the filter cake was washed with 880 ml 80% (v/v) ethanol. The filtrate was discarded. The undried filter cake was reused.

Example 9: Crystallization

Approach 1: The precipitate from Example 8 was dissolved in 200 ml H ₂ O by heating in a water bath at 90°C and then slowly cooled to RT (overnight) with gentle stirring (200 rpm) in the switched off water bath, then cooled to 0°C in an ice bath and then filtered through a pleated filter (Fisherbrand™ QL 115; 185 mm diameter, 2-3 pm pore size) filtered. The filtrate (mother liquor) was separated and the taurine content of an aliquot was determined by HPLC. The taurine content was 53.8 g/L (10.8 g in 200 ml filtrate). The crystals were washed with 100 ml ethanol and dried at 37°C. The dried crystals were weighed. The final weight of the crystals was 28.5 g. 21.3 mg of the crystals were weighed, dissolved in 1 ml H ₂ O (21.3 g/L concentration) and the taurine content was determined by HPLC. The taurine content was 20.7 g/L, corresponding to a taurine content of the crystals of 97.2%. The taurine yield in 28.5 g of crystals was therefore 27.7 g. The percentage taurine recovery based on the input amount of 41.4 g of taurine from Example 6, Batch 1A, was 66.9%. Including the taurine content of 10.8 g of the reusable mother liquor, the amount of taurine recovered was 38.5 g (92.9% of the input amount of 41.4 g of taurine from Batch 1A in Example 6).

Approach 2: The precipitate from Example 8 was dissolved in 40 ml H ₂ O by heating in a water bath at 90°C and then slowly cooled to RT in the switched off water bath with gentle stirring (200 rpm) (overnight), then cooled to 0°C in an ice bath and finally filtered through a pleated filter (Fisherbrand™ QL 115; 185 mm diameter, 2-3 pm pore size). The filtrate (mother liquor) was separated and the taurine content of an aliquot was determined by HPLC. The taurine content was 67.1 g/L (2.3 g in 35 ml filtrate). The crystals were washed with 50 ml ethanol and dried at 37°C. The dried crystals were weighed. The final weight of the crystals was 8.4 g. 20.0 mg of the crystals were weighed, dissolved in 1 ml H ₂ O (20 g/L concentration) and the taurine content was determined by HPLC. The taurine content was 19.2 g/L, corresponding to a taurine content of the crystals of 96.0%. The taurine yield in 8.4 g of crystals was therefore 8.1 g. The percentage taurine yield, based on the amount used of 11.3 g Taurine from Example 5 was 71.7%. Including the taurine content of 2.3 g of the reusable mother liquor, the recovered amount of taurine was 10.4 g (92.0% of the initial amount of 11.3 g of taurine).

Approach 3: The filter cake of the strain W3110 x pCys-CSma- CSADcc-CDOrn from Example 8 was dissolved in 50 ml H ₂ O by heating in a water bath at 90°C and then slowly cooled to RT in the switched off water bath with gentle stirring (200 rpm) (overnight), then cooled to 0°C in an ice bath and finally filtered through a pleated filter (Fisherbrand™ QL 115; 185 mm diameter, 2-3 pm pore size). The filtrate of 80 ml volume (mother liquor) was separated and the taurine content of an aliquot was determined by HPLC. The taurine content was 83.0 g/L (6.6 g in 80 ml filtrate). The crystals were washed with 100 ml ethanol and dried at 37°C. The dried crystals were weighed. The final weight of the crystals was 10.1 g. 20 mg of the crystals were weighed, dissolved in 1 ml H2O and the taurine content was determined by HPLC. The taurine content was 19.54 g/L, corresponding to a taurine content of the crystals of 97.7%. The taurine yield in 10.1 g of crystals was therefore 9.9 g. The percentage taurine yield, 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

Patent claims

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

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

3. Process according to one or more of claims 1 or 2, characterized in that before adding the solvent 1 (step ii), the addition of an oxidizing agent and incubation with it takes place.

4. Process according to claim 3, characterized in that the oxidizing agent is H2O2.

5. Process according to one or more of claims 3 or 4, characterized in that the incubation with the oxidizing agent takes place in a continuous process. Process according to one or more of claims 1 to 5, characterized in that the fermenter broth has an additive molar content of hypotaurine and taurine of at least 91.6 mM in the fermentation supernatant. Process according to one or more of claims 1 to 6, characterized in that before separating the 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 at most 6.0 and (2) raising the temperature of the fermenter broth to at least 50 °C and incubating for at least 5 minutes is carried out. Process according to claim 7, characterized in that at least one of the selected measures is carried out in continuous form. Process according to one or more of claims 1 to 8, characterized in that the water-miscible solvent 1 is a solvent selected from the group consisting of ethanol, isopropanol and mixtures thereof with H ₂ O. Process 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 the solvent 1 (step ii). Process according to one or more of claims 1 to 10, characterized in that the solvent 2 is water. Process according to one or more of claims 1 to 11, characterized in that the molar yield of taurine achieved is at least 50% based on the molar amount contained in the fermentation supernatant. Starting product consisting of at least one of the compounds selected from the group consisting of hypotaurine and taurine. 13. Process according to one or more of claims 1 to 12, characterized in that the production strain comprises a cds coding for a protein with SEQ ID No. 2.

14. The method according to one or more of claims 1 to 13, characterized in that the production strain comprises a cds encoding a protein having SEQ ID No. 4.

15. Method according to one or more of claims 1 to 14, characterized in that the production strain comprises a cds encoding a protein with SEQ ID No. 6.