Method for producing hypotaurine by fermentation

Abstract

The invention relates to a method for producing hypotaurine by fermentation. The invention is characterized in that in a step i, a microbial production strain is cultivated that is characterized in that 1. the production strain contains at least one expression vector, comprising a coding sequence (cds) a and a cds b and at least one of the cds selected from the group consisting of c, d, and e, wherein a is a cds which codes for a cysteine dioxygenase (CDO) belonging to the enzyme class EC 1.13.11.20, b is a cds which codes for a cysteine sulfinic acid decarboxylase (CSAD) belonging to the enzyme class EC 4.1.1.29, c is a cds which codes for a 3-phosphoglycerate dehydrogenase (SerA) with reduced feedback inhibition by means of serine, d is a cds which codes for a serine O-acetyl transferase (CysE) with reduced feedback inhibition by means of cysteine, and e is a cds which codes for a cysteine exporter, 2. the expression of the cds a and b in the expression vector is coordinated with the expression of at least one cds selected from the group consisting of c, d, and e in a polycistronic expression unit, and 3. hypotaurine is secreted from the production strain into the fermentation supernatant, and in a step ii, the fermentation supernatant is isolated. The hypotaurine content in the fermentation supernatant equals at least 10 g / L, and the molar ratio of hypotaurine : taurine in the fermentation supernatant equals at least 3:1.

Description

[0001] Process for the fermentative production of hypotaurine

[0002] The invention relates to a process for the fermentative production of hypotaurine, characterized in that i) a microbial production strain is cultivated which is characterized in that it

[0003] 1. contains at least one expression vector comprising a coding sequence (cds) a and a cds b and at least one of the cds selected from the group consisting of c, d and e, where a) a is a cds encoding a cysteine ​​dioxygenase (CDO) belonging to the enzyme class EC 1.13.11.20 and b) b is a cds encoding a cysteine ​​sulfinic acid decarboxylase (CSAD) belonging to the enzyme class EC 4.1.1.29 and c) c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with a feedback inhibition by serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme and d) d is a cds encoding a serine-O-acetyltransferase (CysE) with a feedback inhibition by cysteine ​​that is reduced by at least a factor of two compared to the corresponding wild-type enzyme and e) e is a cds encoding a cysteine ​​exporter,

[0004] 2. the expression of the cds a and b in the expression vector is coordinated with the expression of at least one cds selected from the group consisting of c, d and e in a polycistronic expression unit,

[0005] 3. Hypotaurine is secreted from the production strain into the fermentation supernatant and ii) the fermentation supernatant is isolated, whereby the hypotaurine content in the fermentation supernatant is at least 10 g / L and the molar ratio of hypotaurine to taurine in the fermentation supernatant is at least 3:1. Hypotaurine (2-aminoethanesulfinic acid, CAS number 300-84-5) is an aminosulfinic acid that occurs naturally as a breakdown product of the amino acid cysteine. As a sulfinic acid, hypotaurine acts as an antioxidant and is used in the food, feed, cosmetics, and pharmaceutical sectors. In addition, hypotaurine is a biosynthetic precursor of taurine (2-aminoethanesulfonic acid, CAS number 107-35-7).

[0006] For commercial use, hypotaurine is produced chemically, e.g., starting from 2-aminoethanethiol hydrochloride. With the trend away from chemically produced ingredients, driven by consumer demand, a biotechnological process for the production of hypotaurine is of interest. Known biosynthetic pathways to hypotaurine and further to taurine, starting from L-cysteine, can be found, for example, in the KEGG Pathway Database: "Taurine and hypotaurine metabolism." The most important synthesis steps leading from L-cysteine ​​to hypotaurine and further to taurine are shown in equations (1) to (5).

[0007] (1) L-Cysteine ​​+ O2 -> L-Cysteine ​​sulfinic acid

[0008] (2) L-cysteine ​​-> cysteamine + CO2

[0009] (3) L-cysteine ​​sulfinic acid -> hypotaurine + CO2

[0010] (4) Cysteamine + O2-> Hypotaurine

[0011] (5) Hypotaurine + ^ 02 -0 taurine

[0012] (1) : L-cysteine ​​is oxidized by the enzyme cysteine ​​dioxygenase (CDO, EC 1.13.11.20) to L-cysteine ​​sulfinic acid (3-sulf inoalanine, CAS number 207121-48-0).

[0013] (2): Cysteamine is formally formed by the decarboxylation of L-cysteine, but in mammals it is a reaction product of pantothenate biosynthesis, whereby the enzyme pantetheine hydrolase (EC 3.5.1.92) cleaves cysteamine from the precursor (R)-pantetheine. A cysteine ​​decarboxylase has been described for bacteria, but only acts on the C-terminal cysteine ​​of a precursor peptide as a partial reaction in the biosynthesis of a natural product and is not suitable for the production of cysteamine. The route (2) to hypotaurine, starting from cysteine ​​via cysteamine, is therefore technically irrelevant.

[0014] (3) : 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).

[0015] (4) : Cysteamine dioxygenase (EC 1.13.11.19) oxidizes cysteamine with atmospheric oxygen to hypotaurine.

[0016] (5) : The oxidation of hypotaurine to taurine has not yet been clearly clarified.

[0017] Known biotechnological processes primarily target the production of taurine, with hypotaurine being the main product in many cases due to the biosynthetic pathway used. The known biotechnological processes for producing hypotaurine are characterized by the fact that, as far as disclosed, hypotaurine yields are very low.

[0018] 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. The approach of Honjoh et al. (2010) thus corresponds to a biotransformation of L-cysteine ​​to hypotaurine. Intracellularly formed taurine was determined analytically, with and without H2O2 treatment (Table 1 in Honjoh et al.).

[0019] Based on the values ​​obtained after H2O2 treatment, it was concluded that a large portion of the intracellularly formed product was hypotaurine, although hypotaurine was not analytically quantified. The lack of direct quantification of hypotaurine is surprising, as hypotaurine analysis was included in the methods section of Honjoh et al. Thus, hypotaurine formation in baker's yeast has not been clearly demonstrated and was only indirectly inferred from the increased taurine values ​​after H2O2 oxidation.

[0020] WO 17 / 213142 A1 (Ajinomoto) describes taurine-producing strains by heterologous expression of genes encoding cysteine ​​dioxygenase (CDO) and L-cysteine ​​sulfinic acid decarboxylase (CSAD) in an originally cysteine-producing strain. Constructs of various CDO and CSAD genes were generated, each under the control of the known strong tac promoter. However, their suitability for hypotaurine or taurine production was not tested in strains optimized for cysteine ​​production, but in strains of E. coli (max. cysteine ​​production 2 pM, see Table 5 of WO 17 / 213142 A1, strain EcoT / pMW2ql 9 ) and Pantoea ananatis (max. cysteine ​​production 0 pM, see Table 5 of WO 17 / 213142 A1, strain PanT / pMW219), each with an inactivated tauABCD operon (named EcoT for E. coli and PanT for P. ananatis). The tauABCD operon is known to encode genes for the degradation of taurine. The main product was hypotaurine with a max.450 pM yield (49.1 mg / L hypotaurine at 109.2 g / mol mol. weight of hypotaurine). Thus, the optimization of cysteine ​​metabolism has been described as a possibility for improved hypotaurine production, but has not been experimentally proven. The low hypotaurine yields are not suitable for economical production.

[0021] US 2019 / 0062757 A1 (KnipBio) describes heterologous production strains for the manufacture of taurine or its precursor substances, with maximum yields of only 419 ng / ml hypotaurine being achieved. WT strains of E. coli and Methylobacterium extorquens were used as host strains. No measures to improve cysteine ​​production with the aim of higher hypotaurine yields were described. US 9, 267, 148 B2 and other publications by Plant Sensory Systems describe gene constructs that are said to be suitable for the heterologous expression of a cysteine ​​dioxygenase and an L-cysteine ​​sulfinic acid decarboxylase with the aim of producing taurine. In particular, the applications concern, on the one hand, gene constructs which express a CDO gene and a CSAD gene, each functionally linked to its own promoter, as individual expression cassettes (each monocistronic).On the other hand, gene constructs are also described that are intended to express a CDO and a CSAD gene in the form of a fusion protein, combined as a single expression cassette under the control of a single promoter (monocistronic expression cassette). The applications primarily target the production of taurine in plants, but no statements are made regarding yields. No measures for improving cysteine ​​production with the aim of higher hypotaurine yields were described. Overall, the applications do not disclose a applicable process for the fermentative production of hypotaurine.

[0022] Joo et al. (2018), J. Agric. Food Chem. 66: 13454–13463, describe a genetically modified strain of the bacterium Corynebacterium glutamicum with a production capacity of 0.5 g / L taurine when grown in shake flasks. Taurine was apparently accumulated intracellularly and not secreted into the culture medium. For the synthesis of taurine, the genes for L-cysteic acid synthase, cysteine ​​dioxygenase, and L-cysteine ​​sulfinic acid decarboxylase were heterologously expressed in the strain. Furthermore, a repressor gene for methionine and cysteine ​​biosynthesis was inactivated, which simultaneously resulted in improved sulfur uptake. Hypotaurine production was not analyzed.

[0023] The prior art thus discloses various metabolic engineering and biotransformation approaches for producing hypotaurine in heterologous production systems. No production strains with optimized cysteine ​​biosynthesis were used. None of the strains described in the prior art disclose the production of hypotaurine or taurine on an industrial scale using a fermentation process.

[0024] The object of the present invention was to provide a process for the fermentative production of hypotaurine with high yields in the fermentation supernatant. Since hypotaurine can be oxidized to taurine according to the state of the art, it was also necessary to achieve a yield of hypotaurine that significantly exceeds that of taurine.

[0025] The object is achieved by a process for the fermentative production of hypotaurine, characterized in that i) a microbial production strain is cultivated which is characterized in that it

[0026] 1. contains at least one expression vector comprising a coding sequence (cds) a and a cds b and at least one of the cds selected from the group consisting of c, d and e, where a) a is a cds encoding a cysteine ​​dioxygenase (CDO) belonging to the enzyme class EC 1.13.11.20 and b) b is a cds encoding a cysteine ​​sulfinic acid decarboxylase (CSAD) belonging to the enzyme class EC 4.1.1.29 and c) c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with a feedback inhibition by serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme and d) d is a cds encoding a serine-O-acetyltransferase (CysE) with a feedback inhibition by serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme cysteine ​​and e) e is a cds encoding a cysteine ​​exporter,

[0027] 2. the expression of the cds a and b in the expression vector is coordinated with the expression of at least one cds selected from the group consisting of c, d and e in a polycistronic expression unit,

[0028] 3 . Hypotaurine is secreted by the production strain into the fermentation supernatant and ii ) the fermentation supernatant is isolated, whereby the hypotaurine content in the fermentation supernatant is at least 10 g / L and the molar ratio of hypotaurine to taurine in the fermentation supernatant is at least 3 : 1 .

[0029] A major advantage of the present invention is that it discloses a fermentative process for producing hypotaurine, in which the expression vectors designed according to the principle of metabolic engineering combine the expression units for increased cysteine ​​production with expression units for hypotaurine production in the microbial production strain. In particular, the polycistronic arrangement of at least one eds of cysteine ​​metabolism with the eds for cysteine ​​dioxygenase (CDO) and the eds for cysteine ​​sulfinic acid decarboxylase (CSAD) in an artificial operon under the control of only one promoter allows the coordinated expression of cysteine ​​and hypotaurine metabolism genes in one production strain. This helps to avoid the accumulation of metabolic intermediates, such as L-cysteine, and maximizes the hypotaurine yield.

[0030] It is surprising that the product hypotaurine is secreted by the production strain into the fermentation supernatant at a concentration of >10 g / l (extracellular production) and accumulates extracellularly (Example 5). In contrast, the state of the art results in intracellular accumulation, as described for taurine by Joo et al. (2018, see above), or, depending on the production system, only partial secretion.

[0031] The use of the claimed production strain in the process according to the invention is therefore characterized in that no product accumulates in the biomass (fermenter cells) (see also Example 5). This extracellular production or accumulation, i.e. the biosynthesis of hypotaurine in the production strain followed by secretion (export, discharge) from the production strain into the culture medium, offers the great advantage that the volume in which hypotaurine can accumulate is not limited to the small volume of the cell contents (cystosol). This prevents high intracellular product concentrations from leading to toxic effects. Far higher hypotaurine yields can therefore be achieved than with intracellular production. Preferably, the biomass contains less than 1 g / l hypotaurine, based on the biomass contained in the fermenter broth, as described in Example 5.Particularly preferably, no hypotaurine is detectable in the biomass.

[0032] The extracellular production of hypotaurine also has the advantage of simplified product isolation, as hypotaurine can be isolated directly from the fermentation supernatant without the need for prior, laborious mechanical or chemical disruption of the cells. How hypotaurine is secreted from the production strain is unknown. It may be a passive mechanism, with hypotaurine diffusing through the cell membranes, or one or more transport proteins may be involved in the secretion.

[0033] A further advantage of the present invention is the low proportion of the by-product taurine, which is formed from hypotaurine through oxidation and reduces the hypotaurine yield. The process according to the invention is therefore very well suited for industrial use, as it enables the economical production of hypotaurine thanks to the high hypotaurine yields in fermentation. In the prior art, such as Joo et al. (2018, see above), however, there is a spontaneous oxidation of hypotaurine to taurine, which has the disadvantage that the hypotaurine content is low. Metabolism engineering (also called "pathway design"), in contrast to biotransformation, is a biotechnology method in which the metabolic pathways of an organism are modified by optimizing or altering genetic and regulatory processes. New or modified enzymes can be introduced into an organism by supplementing the genome with enzyme genes.Genes of endogenous enzymes are expressed in a more or less pronounced 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-derived metabolic product with an increased yield. In a metabolism engineering process, no starting materials specific to the metabolic product, such as an enzyme substrate, such as L-cysteine ​​as the starting compound for the production of hypotaurine, are used, 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 those skilled in the art from microbiological practice. The process disclosed in the present invention for the fermentative production of hypotaurine is a metabolic engineering process.

[0034] In contrast, biotransformation is defined as the conversion of one or more reactants into a product under enzymatic catalysis, whereby the enzyme substrate is added to a reaction mixture with the enzyme and converted enzymatically.

[0035] According to equation (1), L-cysteine ​​is enzymatically oxidized to L-cysteine ​​sulfinic acid. This reaction is catalyzed by a CDO enzyme (EC 1.13.11.20). Gene and protein sequences of CDO enzymes are accessible, for example, in the NCBI database under the search term "cysteine ​​dioxygenase." The invention encompasses genes encoding proteins with CDO activity, including preferably CDO enzymes selected from Rattus norvegicus (rat), Homo sapiens (human), Cyprinus carpio (carp), Bos taurus (cattle), Capra hircus (goat), Gallus gallus (rooster), or Synechococcus (algae), or homologous CDO enzymes. In a preferred embodiment, the method is characterized in that the CDO is the CDO from Rattus norvegicus (CDOrn) with the sequence given in SEQ ID NO: 2 or an enzyme with CDO activity that is at least 80%, particularly preferably at least 90% and especially preferably at least 95% identical thereto.Particularly preferably, the CDO is a protein having SEQ ID NO: 2. In particular, the CDOrn-cds has the sequence given in SEQ ID NO: 1.

[0036] The CDO enzyme activity test can be carried out as follows: i) Preparation of the enzyme to be tested:

[0037] 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 which 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.

[0038] The total protein concentration obtained can be determined, for example, using 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:

[0039] 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 used depends on the degree of purification. If culture broth, cell suspension of the reisolated cells, cell homogenate, or cell extract is used, at least 0.1 mg of the enzyme fractions prepared in i) is used. In the case of purified enzyme, at least 10 pg of the purified enzyme fraction is 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 L-cysteine ​​sulfinic acid content is determined by calibrated HPLC (see Example 3). The reference substances used for calibration are commercially available (Sigma-Aldrich).

[0040] 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 stated test conditions, then the mixture does not contain any active CDO.

[0041] According to equation (3), L-cysteine ​​sulfinic acid is enzymatically decarboxylated to hypotaurine. This reaction is catalyzed by a CSAD enzyme (EC 4.1.1.29) or, alternatively, with much lower enzyme activity, a GAD enzyme (EC 4.1.1.15). Gene and protein sequences of CSAD enzymes are available, for example, in the NCBI database under the search term "cysteine ​​sulfinic acid decarboxylase." Gene and protein sequences of GAD enzymes are available, for example, in the NCBI database under the search term "glutamic acid decarboxylase." The invention comprises genes which code for proteins with CSAD activity, including preferably CSAD enzymes selected from Cyprlnus carpio (carp), Rattus norvegicus (rat), Homo sapiens (human), Bos taurus (cattle), Capra hircus (goat), Gallus gallus (rooster), Eschericia coli or Synechococcus (algae) or CSAD enzymes homologous thereto.In a preferred embodiment, the method is characterized in that the CSAD is the CSAD from Cyprinus carpio (CSADcc) with the sequence given in SEQ ID NO: 6 or an enzyme with CSAD activity that is at least 80%, particularly preferably at least 90%, and especially preferably at least 95% identical thereto. The CSAD is particularly preferably a protein with SEQ ID NO: 6. Especially preferably, the CSADcc-cds has the sequence given in SEQ ID NO: 5 (nt 1-1503).

[0042] In an alternatively preferred embodiment, the method is characterized in that CSAD is the CSAD from Homo sapiens (CSADhs) with the sequence given in SEQ ID NO: 4 or an enzyme with CSAD activity that is at least 80%, particularly preferably at least 90% and especially preferably at least 95% identical thereto.

[0043] Particularly preferably, the CSAD is a protein with SEQ ID NO: 4. In particular, the CSADhs-cds has the sequence given in SEQ ID NO: 3 (nt 1-1509).

[0044] The CSAD enzyme activity test can be carried out as follows: i) Preparation of the enzyme to be tested:

[0045] 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 which 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 c) after mechanical disruption of the cell suspension or d) 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.

[0046] The total protein concentration obtained can be determined, for example, using 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:

[0047] 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 used depends on the degree of purification. If culture broth, cell suspension of the reisolated cells, cell homogenate, or cell extract is used, at least 0.1 mg of the enzyme fractions prepared in i) is used. In the case of purified enzyme, at least 10 pg of the purified enzyme fraction is 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 hypotaurine content is determined by calibrated HPLC (see Example 3). The reference substances used for calibration are commercially available (Sigma-Aldrich).

[0048] The detection limit of the HPLC method is 1 mg / L L-hypotaurine. If less than 1 mg / L hypotaurine is formed under the specified test conditions, then the mixture does not contain active CSAD.

[0049] 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 known as the coding region or structural gene. The DNA segment that contains all the basic information for producing biologically active RNA is called a gene, cistron or expression unit. A gene contains the DNA segment 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 comprise at least a promoter, a transcription start site, a translation start site and a ribosome binding site (RBS). Other possible expression signals include a terminator and one or more operators.

[0050] For the purposes of this invention, bacterial proteins such as SerA or CysE begin with a capital letter, while the sequences coding for these proteins (cds) are designated with a lowercase letter (e.g. serA or cysE). Likewise, the promoters controlling the expression of these cds are designated with a lowercase letter (e.g. serA promoter or cysE promoter). In contrast, proteins / enzymes and cds / genes of higher organisms such as Homo sapiens, Rattus norvegicus or Cyprinus carpio are designated with capital letters and, where appropriate, are each identified as protein / enzyme (e.g. CDO protein / enzyme or CSAD protein / enzyme) or cds / gene (e.g. CDO cds / gene or CSAD cds / gene).

[0051] A gene construct is a DNA molecule produced by cloning that contains at least one expression unit and may also include other genetic elements such as a selectable marker and replication origin. The gene construct can be a linear DNA molecule integrated into the genome or a circular DNA molecule in the form of a plasmid, also known as a vector. This vector is then referred to as an expression vector. When introduced into a suitable host strain (transformed), the genetic elements of the vector cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the plasmid.

[0052] So-called operons are often found in the genomes of microorganisms. An operon is characterized by a section of DNA from which several genes can be expressed in a coordinated manner. An operon is a section of DNA that contains all the basic information for the production of biologically active RNA. The operon contains the section of DNA from which a single-stranded RNA copy is produced by transcription and the expression signals that are involved in the regulation of this copying process. The RNA copy does not only comprise the cds of a single gene, but the cds of two or more genes. The expression signals of the operon comprise a promoter and a transcription start. Each cds of the operon comprises a translation start and a ribosome binding site. Furthermore, a terminator and one or more operators are possible as expression signals.

[0053] If only one gene is expressed from a promoter, it is called a monocistronic expression unit. If two, three, or more genes are expressed from a promoter, such an expression unit is called bicistronic, tricistronic, etc., or polycistronic.

[0054] Operons not only occur naturally in the genome of microorganisms, but can also be specifically produced by cloning ( artificial operons ).

[0055] The expression unit of an artificial operon also consists of a promoter functionally linked to two or more cds, each with an RBS in front of it (bi-, tri-, or polycistronic expression unit). In addition, an expression unit can also contain other genetic elements such as a terminator or operators. The term "functionally linked" means that the expression unit comprising promoter and cds, or promoter and two or more cds in the case of an operon, leads to the transcription and translation of the cds in the microorganism. A promoter is a nucleotide sequence upstream of the 5' end of the cds, which enables the expression of a cds. The promoter is located in the direction of synthesis upstream of the coding region.The promoter contains regions that determine the start of transcription of the gene by RNA polymerase and can also mediate the specific interaction with DNA-binding proteins (transcription factors) that influence the transcription strength.

[0056] In principle, all promoters active in the host strain are suitable as promoters for the aforementioned cds. These include all native promoters of the approximately 5,000 genes in E. coli, but also non-native (e.g., promoters from other species of the Enterobacteriaceae family) or "artificial" promoters such as the tac promoter. Preferred promoters in the polycistronic expression unit are the cysE, serA, and GAPDH promoters contained on pCys, particularly preferably the serA promoter, as described, for example, in Rex et al. (1991), J. Bacteriol. 173: 5944-5953, Fig. 2, nt 1 - nt 457.

[0057] Particularly preferred is the serA promoter having the sequence given in SEQ ID NO: 7.

[0058] An mRNA, also called messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for building a protein. With an mRNA, the blueprint for a specific protein is available in a cell. The mRNA molecule carries the message necessary for protein construction from the genetic information (DNA) to the protein-building ribosomes. In a cell, it is formed as a transcript of a section of DNA belonging to a gene. The genetic information stored in the DNA is not altered in the process.

[0059] Genes of eukaryotic organisms are predominantly so-called mosaic genes and, unlike prokaryotic genes, also contain non-coding regions called introns (intragenic regions). Coding sequences called exons (expressed regions) are DNA segments of a eukaryotic gene that, after transcription into RNA, are translated by the ribosomes into the amino acid sequence of a protein. The introns are spliced ​​from the primary transcript after transcription of DNA into RNA. The protein-coding RNA stripped of introns is called messenger RNA (mRNA), also known as "mature" mRNA. This undergoes further modifications such as capping and polyadenylation. The coding region of the mature mRNA is then translated into the protein sequence.If a eukaryotic gene with an exon / intron structure is to be expressed in prokaryotic organisms, it is necessary to retranslate the protein sequence or the coding region of the mature mRNA into intron-free DNA, since the processing of the exon / intron structure does not occur in prokaryotes. When reference is made to gene sequences derived from the protein sequence or from the mRNA in the context of this invention, precisely this process of retranslation is meant. It is preferred that sequence optimization, i.e., adaptation to the codon usage of the corresponding prokaryote (codon optimization), takes place simultaneously with the retranslation of the protein sequence or the mRNA sequence into DNA sequence.

[0060] Homologous genes or homologous DNA 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.

[0061] 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

[0062] Costs = Linear.

[0063] 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.

[0064] 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 aligning two or more protein sequences. The preset general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. The preset scoring parameters are: Matrix = BLOSUM62; Gap Costs = Existence: 11 Extension: 1; Compositional adjustments = Conditional compositional score matrix adjustment.

[0065] The abbreviation WT (Wt) stands for wild type. A wild-type gene is the form of a gene that has arisen naturally through evolution and is present in the wild-type genome. The DNA sequence of Wt genes is publicly available in databases such as NCBI.

[0066] 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. Fermentation is a process step in the production (cultivation) of cell cultures on an industrial scale. In this process, a preferably microbial production strain is induced to grow under defined conditions of culture medium, temperature, pH, oxygen supply, and medium mixing. If all components of the fermentation are determined at the beginning of the cultivation and then not changed again, it is referred to as batch fermentation.

[0067] If, after the start of fermentation, media components such as glucose (C source) or a complex amino acid mixture such as yeast extract (N source) are continuously added as a so-called feed, this is referred to as fed-batch fermentation (so-called feed process). Fed-batch fermentation can optimize the formation of biomass and target product. The aim of fermentation is, depending on the configuration (genetic makeup) of the production strain, the production of a protein / enzyme or a metabolite, in each case with the highest possible yield for further use. The product 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 of the biomass (fermentation supernatant), which has formed in the course of 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 product hypotaurine is produced by fermentation. In principle, the products of the fermentation can be found in the fermenter cells and / or in the fermentation medium. As disclosed in Example 5 of the present invention, hypotaurine is found within the detection limit exclusively in the fermentation medium.Therefore, only the hypotaurine produced by secretion into the fermentation medium is taken into account. The process is preferably characterized in that the hypotaurine content in the fermentation supernatant, based on the hypotaurine content in the fermentation mixture, is more than 70%, preferably more than 80% and particularly preferably more than 90%. The hypotaurine content in the fermentation supernatant corresponds to the extracellular (secreted) hypotaurine. The hypotaurine content in the fermentation mixture is the sum of the extracellular and intracellular hypotaurine content, also referred to as the total hypotaurine content. The extracellular proportion of hypotaurine is determined as described in Example 3 (Sample Preparation). The intracellular proportion of hypotaurine is determined as described in Example 5, with a detection limit of 1 mg / L hypotaurine applying in each case.

[0068] Shake flask culture is used to cultivate microorganisms on a laboratory scale, in contrast to production scale fermentation. Although shake flask culture also requires a specific medium and pH, and culture is carried out in the presence of oxygen and with constant agitation (shaking), more defined conditions regarding the medium, temperature, pH, oxygen supply, and medium mixing can be set and regulated in the fermenter. Culture on a smaller scale, e.g., in a shake flask, can also be used as a pre-culture for inoculating a larger-scale culture, e.g., in a fermenter.

[0069] The production scale in fermentation typically starts at 0.5 L batch volume and can reach 100,000 L or more. The production scale in shake flask culture typically ranges from 10 ml to 1000 ml batch volume.

[0070] Yield, within the meaning of the invention, is defined as the amount of product obtained by cultivating a production strain. The yield can be expressed as an absolute amount of product (mmol or g) or as a volume yield (concentration) in the amount of product relative to the volume (mM or g / L).

[0071] The process according to the invention comprises a fermentative process for producing hypotaurine using a microbial production strain. A production strain, by definition, comprises a microorganism, referred to as a host strain, and at least one gene construct. In the present invention, the production strain is characterized in that the gene construct is an expression vector.

[0072] Any microorganism that is amenable to recombinant DNA techniques and that is suitable for the fermentative production of recombinant proteins is suitable as a host 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).

[0073] The host strain used to prepare the production strain is preferably a prokaryotic microorganism, particularly preferably a bacterial strain selected from the group consisting of Coryn ebacteri um ssp. (such as particularly preferably C. glutami cum), Pantoea ssp. (such as particularly preferably P. anana tis) and Escherichia ssp. and especially preferably a microorganism of the species Escherichia coli. In a particularly preferred embodiment, the microorganism is the strain E. coli K12 W3110, commercially available under the strain number DSM 5911 from the DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH.

[0074] A production strain for the fermentative production of at least 10 g / l hypotaurine is characterized in that it contains a gene construct which (I) defines a metabolic pathway leading to hypotaurine by the expression of cds a (encoding CDO) and b (encoding CSAD) and

[0075] ( II ) a deregulated cysteine ​​biosynthesis pathway defined by the expression of at least one of the cds selected from the group consisting of c ( encoding SerA with reduced feedback inhibition by serine ), d ( encoding CysE with reduced feedback inhibition by cysteine ​​) and e ( encoding a cysteine ​​exporter ).

[0076] ( I ) The metabolic pathway leading to hypotaurine is characterized by the fact that it includes the reaction of L-cysteine ​​to hypotaurine according to equations ( 1 ) and ( 3 ).

[0077] The genes CDO and CSAD which constitute the hypotaurine metabolic pathway in the production strain can originate from the microorganism strain which is also the host strain (homologous expression) or they can be foreign genes (heterologous expression), either synthetically produced (including the resulting adaptation of the cds to expression in the host strain through so-called codon optimization) or isolated from strains other than the host strain, or there can be a combination of homologous and heterologous expression. Preference is given to heterologous expression of the genes of the hypotaurine metabolic pathway in the production strain, particular preference is given to heterologous expression of synthetically produced genes and especially preference is given to heterologous expression of codon-optimized, synthetically produced genes of the hypotaurine metabolic pathway in the production strain.

[0078] (II) A production strain containing a gene construct comprising expression units for a deregulated cysteine ​​biosynthesis pathway and thus suitable for cysteine ​​production is characterized in that it contains at least one of the cds selected from the group consisting of c, d and e: c) The production strain comprises at least one modified serA gene, ie it contains at least one cds c coding for a 3-phosphoglycerate dehydrogenase (SerA) with a feedback inhibition by L-serine reduced by at least a factor of two compared to the corresponding wild-type enzyme, it being possible for the SerA enzyme activity to 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. In addition, the production strain may additionally contain the unaltered WT serA gene.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.

[0079] 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:

[0080] - serA317 : Feedback-resistant SerA mutant, as used in the present invention in the vector pCys (Fig. 1). This 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).

[0081] - serA G349E: Mutation of glycine at position 349 of the WT SerA enzyme to glutamic acid.

[0082] - serA G349D: Mutation of glycine at position 349 of the WT SerA enzyme to aspartic acid.

[0083] - serA T372X: Mutation of threonine at position 372 of the WT SerA enzyme to X, where X is any natural

[0084] amino acid other than threonine. - serA mutated: serA gene in which 25% of the C-terminal amino acids are altered compared to WT, where the change is i) within the 50 C-terminal amino acids of the WT protein, ii) the change comprises a C-terminal deletion, and iii) the change comprises an insertion into the WT sequence. and / or d) The production strain comprises at least one modified cysE gene, ie it contains at least one cds d coding for a serine O-acetyl transferase (CysE) which, compared to the corresponding wild-type enzyme, has a feedback inhibition by cysteine ​​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. Furthermore, the production strain may additionally contain the unaltered WT cysE gene.

[0085] Particularly preferred variants of serine-O-acetyl transferase (CysE) have, compared to the corresponding wild-type enzyme, a feedback inhibition by cysteine ​​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.

[0086] Examples of cysE variants encoding CysE enzymes with reduced feedback inhibition and preferred embodiments within the scope of this invention are:

[0087] - cysE -mutated: Mutation in the sequence region position 97 up to and including 273.

[0088] - cysE-deleted: Deletion in the sequence region position 248 to 259 inclusive. Preferred specific mutations within the scope of this invention are:

[0089] - cysEII: G238S, mutation of glycine at position 238 to serine.

[0090] - cysEIII: G165D Mutation of glycine at position 165 to aspartic acid.

[0091] - cysEIV: A237V, mutation of alanine at position 237 to valine and G238S, mutation of glycine at position 238 to serine (double mutant).

[0092] - 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).

[0093] - cysEVI : G238S, mutation of glycine at position 238 to serine and M256I, mutation of methionine at position 256 to isoleucine (double mutant).

[0094] - cysEVII: A237V, mutation of alanine at position 237 to valine.

[0095] - cysEVIII: M256I, mutation of methionine at position 256 to isoleucine and A237V, mutation of alanine at position 237 to valine (double mutant).

[0096] - cysEX: T167A, mutation of threonine at position 167 to alanine (used in the vector pCys, example 1).

[0097] - cysEXI : T167A, mutation of threonine at position 167 to alanine and G245S, mutation of glycine at position 245 to serine (double mutant).

[0098] - 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).

[0099] - cysEXIII: V164A, mutation of valine at position 164 to alanine and F267L, mutation of phenylalanine at position 267 to leucine (double mutant).

[0100] - 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.

[0101] - cysEXVII : G165D, mutation of glycine at position 165 to aspartic acid and T167A, mutation of threonine at position 167 to alanine ( double mutant ) .

[0102] - 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) .

[0103] - 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.

[0104] - cysE Arabidopsis thaliana ( plant ) : A. thaliana contains three cysE genes , two of which are feedback-resistant to cysteine ​​.

[0105] - cysE M256I : Mutation of methionine at position 256 of the WT-CysE enzyme to I soleucine . and / or

[0106] Compared to the starting strain (host strain), the production strain comprises at least one increased expression of cds e (overexpression) or an additional cds e encoding a cysteine ​​exporter (-Ef fluxprotein), which leads to an increased expression of cysteine ​​exporters, so that the cell of the production strain has a cysteine ​​export from the cell that is at least two times higher than the corresponding starting strain (host strain), wherein 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 .

[0107] The increased expression of cysteine ​​exporters 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 the starting strain (host strain). 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 sequence that is at least 80%, particularly preferably at least 90% and especially preferably at least 95% identical thereto.

[0108] The cds that cause hypotaurine biosynthesis and the cds that cause the deregulated cysteine ​​biosynthesis pathway are present extrachromosomally on vectors in the microorganism strain, either combined on one vector or divided between several vectors.

[0109] In a preferred embodiment, the process for producing hypotaurine is characterized in that the cds encoding CDO (a) and the cds encoding CSAD (b) are present in the expression vector of the microbial production strain with the cds encoding SerA with reduced feedback inhibition by serine (c) in a tricistronic expression unit which is particularly preferably functionally linked to the serA promoter, which particularly preferably has the sequence given in SEQ ID NO: 7. This means that the expression of the three mentioned cds is regulated by only one promoter (i.e. there is a tricistronic operon). The three cds are present separately one after the other, each cds with its own RBS. Their expression occurs in a coordinated manner under the regulation of the same promoter, which is preferably the serA promoter, but results in 3 separate proteins, not fusion proteins.

[0110] Particularly preferred is the sequence in the operon such that in the tricistronic operon, the promoter, preferably the serA promoter, is followed by the serA-cds, then the CDO-cds, and finally the CSAD-cds. SerA is preferably serA317. SerA317 is disclosed in Bell et al., Eur. J.

[0111] Biochem. (2002) 269:4176-4184, referred to therein as "NSD:317" and encoding a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. The tricistronic expression unit of serA, CDO, and CSAD in the present invention allows the coupling of L-cysteine ​​and hypotaurine metabolism by their coordinated expression under the control of the serA promoter.

[0112] For example, in the expression vectors pCys-CDOrn-CSADhs (Fig. 2) and pCys-CDOrn-CSADcc (Fig. 3) disclosed in Example 1 of the present invention, the expression units serA317, CDOrn and CSADhs, or serA317, CDOrn and CSADcc, form an artificial tricistronic operon, wherein expression occurs under the control of the serA promoter. The serA promoter thus controls the expression of three cds in the gene constructs pCys-CDOrn-CSADhs and pCys-CDOrn-CSADcc, namely the expression of the cds encoding serA317 and the expression of the cds encoding the CDOrn enzyme, which was derived from the mRNA of the gene sequence present in Rattus norvegicus (rn), and in the gene construct pCys-CDOrn-CSADhs the expression of the cds encoding the CSADhs enzyme, which was derived from the mRNA of the gene sequence present in Homo sapiens (hs).in the gene construct pCys-CDOrn-CSADcc the expression of the cds encoding the CSADcc enzyme, which was derived from the mRNA of the gene sequence present in Cyprinus carpio (cc).

[0113] A particularly preferred gene construct, which comprises a cds encoding SerA with reduced feedback inhibition by serine and functionally linked to the serA promoter (c), cysE with reduced feedback inhibition by cysteine ​​and functionally linked to the cysE promoter (d), and a cysteine ​​exporter functionally linked to the promoter of the E. coli GAPDH gene (e) (GAPDH: glyceraldehyde-3-phosphate dehydrogenase), is the vector pCys disclosed in WO 2021 / 259491 (Wacker) and described in Example 1 (Fig. 1). Transforming pCys into a suitable host strain results in a production strain with a deregulated cysteine ​​biosynthesis pathway, which is thus suitable for cysteine ​​production (see, for example, WO 2021 / 259491, Wacker).Particularly preferably, the process for producing hypotaurine is characterized in that the CDs encoding CDO (a), CSAD (b), SerA with reduced feedback inhibition by serine (c), CysE with reduced feedback inhibition by cysteine ​​(d), and encoding the cysteine ​​exporter (e) are all present on a single expression vector. Especially preferably, the process for producing hypotaurine is characterized in that the expression vector of the microbial production strain is selected from the gene constructs pCys-CDOrn-CSADhs or pCys-CDOrn-CSADcc. These gene constructs are disclosed in Example 1, Fig. 2 and Fig. 3, respectively. In a particularly preferred embodiment, the expression vector of the microbial production strain is the gene construct pCys-CDOrn-CSADcc.

[0114] The gene construct according to the invention thus comprises feedback-resistant alleles of the cysE and / or serA gene and / or a gene for a cysteine ​​exporter, such as preferably the ydeD efflux gene, as well as genes coding for a CDO and a CSAD enzyme, wherein the CDO and CSAD genes, each with their own RBS, are linked in polycistronic expression units to the cysE, serA and / or the cysteine ​​efflux gene, such as preferably the ydeD gene. A tricistronic operon is preferred, the following configurations being conceivable, among others: a) serA-CDO-CSAD b) serA-CSAD-CDO c) cysE-CDO-CSAD d) cysE-CSAD-CDO e) ydeD-CDO-CSAD f) ydeD-CSAD-CDO.

[0115] In the tricistronic operon, a) and b) are preferably functionally linked to the serA promoter, c) and d) are preferably functionally linked to the cysE promoter, and e) and f) are preferably functionally linked to the promoter of the E. coli GAPDH gene in the tricistronic operon. A gene construct comprising a tricistronic operon comprising a) serA-CDO-CSAD or b) serA-CSAD-CDO is preferred.

[0116] Particularly preferred is a gene construct comprising a tricistronic operon comprising a) serA-CDO-CSAD.

[0117] As described in Example 1 of the present invention, the vector pCys (Fig. 1) described in WO 2021 / 259491 (Wacker) was expanded to include the genes for cysteine ​​dioxygenase from Rattus norvegicus (CDOrn), SEQ ID NO: 1, encoding a protein with the amino acid sequence SEQ ID NO: 2, for L-cysteine ​​sulfinic acid decarboxylase from Homo sapiens (CSADhs), SEQ ID NO: 3, nt 1 to nt 1509, encoding a protein with the amino acid sequence SEQ ID NO: 4 or for L-cysteine ​​sulfinic acid decarboxylase from Cyprinus carpio (CSADcc), SEQ ID NO: 5, nt 1 to nt 1503, encoding a protein with the amino acid sequence SEQ ID NO: 6. The vectors pCys-CDOrn-CSADhs (Fig. 2) and pCys-CDOrn-CSADcc (Fig. 3) were created for the production of hypotaurine. The gene constructs were produced according to the state of the art, preferably using conventional recombinant DNA techniques, as described in Example 1 and as are familiar to the skilled person.In pCys-CDOrn-CSADhs, the CDOrn and CSADhs genes are cloned in the form of an artificial tricistronic operon, each with its own ribosome binding site (RBS) but without its own promoter, behind the serA317 gene contained in pCys, so that their expression occurs under the control of the serA promoter. In pCys-CDOrn-CSADcc, the CDOrn and CSADcc genes are cloned in the form of an artificial tricistronic operon, each with its own ribosome binding site (RBS) but without its own promoter, behind the serA317 gene contained in pCys, so that their expression occurs under the control of the serA promoter.

[0118] The production of a production strain is carried out in a known manner by transformation of the gene construct according to the invention into the microorganism (host strain), preferably the gene construct pCys-CDOrn-CSADhs or the gene construct pCys-CDOrn-CSADcc, particularly preferably the gene construct pCys-CDOrn-CSADcc into the host strain E. coli K12 W3110.

[0119] As disclosed in the examples of the present invention, the fermentation of production strains according to the invention, which are characterized by the previously unknown coordinated expression of the CDO and CSAD genes from a polycistronic, preferably tricistronic operon under the control of the serA promoter derived from cysteine ​​metabolism, enables a surprisingly much higher production of hypotaurine than the prior art. The prior art, for example, discloses hypotaurine yields in E. coli of max. 450 pM in WO 17 / 213142 A1 (Ajinomoto), corresponding to 49.1 mg / L hypotaurine at 109.2 g / mol mol weight of hypotaurine. Honjoh et al. (2010) , Amino Acids 38: 173-1183, describe only the intracellular production of hypotaurine in yeast with yields of 85.3 pmol / g dry biomass, corresponding to 9.3 mg / g hypotaurine at 109.2 g / mol mol. weight of hypotaurine.However, L-cysteine ​​had to be added to the culture medium as a substrate for hypotaurine biosynthesis (see “Materials and Methods” in Hon oh et al . ).

[0120] Furthermore, the prior art only describes production yields for taurine, the oxidation product of hypotaurine. For example, US 2019 / 0062757 Al (KnipBio) discloses yields of 419 ng / ml taurine (E. coli), while Joo et al. (2018), J. Agric. Food Chem. 66: 13454–13463, describes taurine yields in C. glutamicum of max. 0.5 g / L taurine.

[0121] A preferred process for the fermentative production of hypotaurine is one characterized in that the fermentation volume is at least 1 L, with a production scale of at least 10 L being particularly preferred, of at least 1000 L being even more preferred, and a fermentation volume of at least 10,000 L being especially preferred. The hypotaurine content can be quantified from the culture broth. For this purpose, an aliquot of 1 ml is taken from the culture broth with a cell density OD 500 / ml of at least 1.0 / ml, for example, and incubated for 5 minutes at 80 °C. Subsequently, all solid components are separated off, for example by centrifugation for five minutes at maximum speed in a tabletop centrifuge, and the supernatant is quantified by HPLC calibrated for hypotaurine and taurine, as described, among others, in Example 3 for hypotaurine and taurine.

[0122] In the process according to the invention, the yield of the product hypotaurine at the end of the fermentation after a fermentation time of preferably a maximum of 65 h is preferably at least 10 g / L, particularly preferably at least 20 g / L and especially preferably at least 50 g / L, far more than known in the prior art (e.g. 49.1 mg / L hypotaurine in WO 17 / 213142 A1).

[0123] The process according to the invention thus enables the fermentative production of hypotaurine in previously unknown yields for applications in the food, feed and pharmaceutical sectors.

[0124] The fermentation process according to the invention is further characterized by the fact that it allows the production of hypotaurine in a high molar excess over the by-product taurine. The molar excess is defined as the quotient of the molar hypotaurine concentration in the fermentation supernatant at the end of fermentation divided by the molar taurine concentration in the fermentation supernatant at the same time. As disclosed in Example 4, the molar excess of hypotaurine:taurine is at least 3:1, preferably at least 5:1, particularly preferably at least 10:1, and especially preferably at least 20:1.

[0125] Media for culturing the production strain in shake flasks and by fermentation are familiar to those skilled in microbial cultivation. They typically consist of a carbon source (C source), a nitrogen source (N source), and additives such as vitamins, salts, and trace elements, as well as a sulfur source (S source), which optimize cell growth and hypotaurine production.

[0126] C sources are those that can be used by the production strain to form hypotaurine products. These include all forms of monosaccharides, including C6 sugars (hexoses) such as glucose, mannose, fructose, or galactose, and C5 sugars (pentoses) such as xylose, arabinose, or ribose, as well as all conceivable di- and polysaccharides formed from them, such as sucrose, lactose, maltose, maltodextrin, starch, or the monomers or oligomers released from them by hydrolysis (enzymatic or chemical). Other usable C sources other than sugars or carbohydrates are acetic acid (or acetate salts derived therefrom), ethanol, glycerol, citric acid (and their salts), or pyruvate (and its salts). Gaseous C sources such as carbon dioxide or carbon monoxide are also conceivable.

[0127] Preferred C sources for cultivating the production strain are glucose, fructose, sucrose, mannose, xylose and arabinose, among which glucose and sucrose are particularly preferred and glucose is especially preferred.

[0128] N sources are those that can be used by the production strain for biomass production. Examples include:

[0129] - Ammonia, gaseous or in aqueous solution as NH4OH or its salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate and / or

[0130] - the known nitrate salts such as KNO3, NaNO3, ammonium nitrate, Ca(NO3)2, Mg(NO3)2 and other N sources such as urea and / or

[0131] - complex amino acid mixtures such as yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor (corn steep liquor, liquid or dried as so-called CSD) and / or - NZ-amines and / or

[0132] - Yeast Nitrogen Base.

[0133] The addition of a sulfur source, either as a single addition in batch form or as a continuous feed, is necessary for the efficient production of hypotaurine. The continuous addition can take place as a pure feed solution or in a mixture with another feed component such as glucose. The process is preferably characterized in that it is carried out in the presence of at least one compound selected from the group consisting of a salt of sulfates, sulfites, dithionites, thiosulfates and sulfides, whereby the use of the respective acids is also conceivable given a given stability. Preferred sulfur sources are salts of sulfates, sulfites, thiosulfates and sulfides, particularly preferably salts of sulfates and thiosulfates. The process is particularly preferably characterized in that it is carried out in the presence of salts of thiosulfate.Particularly preferred as the salt of thiosulfate is a compound selected from the group consisting of sodium thiosulfate, ammonium thiosulfate and mixtures thereof.

[0134] The cultivation of microorganism cells can be carried out in so-called batch mode, i.e. in order to obtain biomass the cultivation medium (culture medium) is inoculated with a starter culture of the production strain (microorganism cells carrying one or more gene constructs) and then cell growth takes place without further feeding of nutrient sources. The cultivation can also be carried out in so-called fed-batch mode (also called cultivation in feed mode), i.e. a process for obtaining biomass in which, after an initial growth phase in batch mode, additional nutrient sources are added (feed). The feed can consist of the C source, the N source, the sulfur source, one or more vitamins or trace elements important for production or a combination of the above. The feed components can be added together as a mixture or separately in individual feed lines.Additionally, other media components as well as additives specifically designed to increase hypotaurine production can be added to the feed. The feed can be added continuously or in portions (discontinuously), or in a combination of continuous and discontinuous feed. The process according to the invention for the fermentative production of hypotaurine using a microbial production strain is preferably characterized in that the fermentative process is a fed-batch process.

[0135] Preferred C sources in feed are glucose, sucrose, and plant hydrolysates containing glucose or sucrose, as well as mixtures of the preferred C sources in any ratio. The most preferred C source in feed is glucose.

[0136] Preferably, the carbon source is added to the culture in such a way that the carbon source content in the fermenter during the production phase does not exceed 10 g / L. A maximum concentration of 2 g / L is preferred, particularly preferably 0.5 g / L, and especially preferably 0.1 g / L.

[0137] Preferred N sources in the feed are ammonia, gaseous or in aqueous solution as NH4OH and its salts ammonium sulfate, ammonium phosphate, ammonium acetate and ammonium chloride, as well as urea, KNO3, NaNO3 and ammonium nitrate, yeast extract, proteose peptone, malt extract, soy peptone, casamino acids, corn steep liquor as well as NZ amines and yeast nitrogen base, among which ammonia or ammonium salts, yeast extract, soy peptone or corn steep liquor (liquid or in dried form) are particularly preferred.

[0138] Preferred sulfur sources in the feed are salts of sulfates, sulfites, thiosulfates and sulfides, among which particularly preferred are salts of sulfates and thiosulfates and especially preferred are salts of thiosulfate, such as sodium thiosulfate and ammonium thiosulfate.

[0139] Further media additives include salts of the elements phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron, and, in trace amounts (i.e., in pM concentrations), salts of the elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel. Furthermore, organic acids (e.g., acetate, citrate), amino acids (e.g., isoleucine), and vitamins (e.g., vitamin B1, vitamin B6) can be added to the medium.

[0140] Cultivation takes place under pH and temperature conditions that favor the growth and hypotaurine production of the production strain. The pH range is preferably from pH 5 to pH 9. A pH range of pH 5.5 to pH 8 is particularly preferred. A pH range of pH 6.0 to pH 7.5 is especially preferred.

[0141] The preferred temperature range for growth of the production strain is 20°C to 40°C. The temperature range from 25°C to 37°C is particularly preferred, and from 28°C to 34°C is especially preferred.

[0142] The production strain can grow optionally without oxygen (anaerobic cultivation) or with oxygen (aerobic cultivation). Aerobic cultivation with oxygen is preferred.

[0143] During aerobic cultivation of the strain according to the invention for hypotaurine production, the oxygen saturation is preferably at least 10% (v / v), particularly preferably at least 20% (v / v), and especially preferably at least 30% (v / v). The oxygen saturation in the culture is regulated automatically according to the state of the art via a combination of gas supply and stirring speed.

[0144] The oxygen supply can be ensured by introducing compressed air or pure oxygen. Aerobic cultivation by introducing compressed air is preferred. The range of the compressed air supply during aerobic cultivation is preferably 0.05 vvm to 10 vvm (vvm: introduction of compressed air into the fermentation mixture expressed in liters of compressed air per liter of fermentation volume per minute). A compressed air introduction of 0.2 vvm to 8 vvm is particularly preferred, particularly preferably from 0.4 to 6 vvm and especially preferably from 0.8 to 5 vvm.

[0145] The maximum stirring speed is preferably 2500 rpm, particularly preferably a maximum of 2000 rpm and especially preferably a maximum of 1800 rpm.

[0146] The cultivation time is preferably between 10 h and 200 h. A cultivation time of 20 h to 120 h is particularly preferred. A cultivation time of 30 h to 100 h is especially preferred.

[0147] In the process according to the invention for the fermentative production of hypotaurine, the claimed microbial production strain is cultivated in step i, and the fermentation supernatant is isolated in step ii. This means that fermentation batches obtained by the described process contain hypotaurine accumulated extracellularly in the fermentation supernatant. Hypotaurine can be used directly or isolated from the fermentation medium. Various analytical methods for identifying, quantifying, and determining the purity of the hypotaurine are available, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectroscopy, gravimetry, or a combination of these analytical methods.

[0148] The figures show the plasmids used in the examples.

[0149] Fig. 1: pCys. Fig. 2: pCys-CDOrn-CSADhs.

[0150] Fig. 3: pCys-CDOrn-CSADcc .

[0151] Abbreviations used in the figures:

[0152] TetR: gene that confers resistance to tetracycline.

[0153] P15A LOCATION: 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

[0154] ORF306: ydeD (cysteine ​​efflux gene) cds

[0155] Seal: cleavage site for the restriction enzyme Seal

[0156] PpuMI : cleavage site for the restriction enzyme PpuMI

[0157] CDOrn: CDO (cysteine ​​dioxygenase) R. norvegicus cds

[0158] CSADhs: CSAD (cysteine ​​sulfinic acid decarboxylase) H. sapiens cds

[0159] CSADcc: CSAD (cysteine ​​sulfinic acid decarboxylase) C. carpio cds

[0160] RBS : Ribosome binding site

[0161] The following examples serve to further explain the

[0162] invention .

[0163] Example 1: Preparation of pCys-CDOrn-CSADhs and pCys-

[0164] CDOrn-CSADcc

[0165] The vectors pCys-CDOrn-CSADhs and pCys-CDOrn-CSADcc are derived from the vector pCys (Fig. 1). 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-

[0166] Allele) encoding a serine O-acetyltransferase with reduced feedback inhibition by cysteine, whose expression is controlled by the cysE promoter, and the efflux gene ydeD (ORF306), whose expression is controlled by the constitutive GAPDH promoter derived from the E. coli GAPDH gene. pCys also contains, cloned behind the ydeD (ORF306) efflux gene, the serA317 gene fragment encoding 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 according to SEQ ID NO: 7 is described in Rex et al. (1991) , J. Bacteriol. 173: 5944-5953, Fig.2, nt 1 - nt 457, revealed.

[0167] For cloning purposes, plasmid DNA of the vector pCys was cut with Seal and PpuMI and the 6.1 kb vector fragment was isolated after preparative agarose gel electrophoresis (QIAquick® Gel Extraction Kit, Qiagen), hereinafter referred to as pCys-Scal / PpuMI.

[0168] CDOrn: The amino acid sequence of cysteine ​​dioxygenase from Rattus norvegicus, derived from the mRNA of the gene, is disclosed in the NCBI (National Center for Biotechnology Information) database under Sequence ID: AAH70509.1. A codon-optimized DNA sequence for expression in E. coli was derived from the amino acid sequence (publicly available Eurofins Genomics GENEius software) and synthesized (Eurofins Genomics). The CDOrn DNA sequence is disclosed in SEQ ID NO: 1, encoding a protein with the amino acid sequence shown in SEQ ID NO: 2. The CDOrn DNA was synthesized (Eurofins Genomics). CSADhs-rrnB:

[0169] CSADhs: The amino acid sequence of cysteine ​​sulfinic acid decarboxylase (CSADhs) from Homo sapiens is disclosed in the NCBI (National Center for Biotechnology Information) database under Sequence ID: XP_016861786.1. There, CSADhs is deposited as "acidic amino acid decarboxylase GADL1 isoform XI." From the amino acid sequence, a DNA sequence codon-optimized for expression in E. coli was derived (publicly available Eurofins Genomics GENEius software). This DNA sequence, designated CSADhs-cds, is disclosed in SEQ ID NO: 3, nt 1 to 1509, encoding a protein with the amino acid sequence of SEQ ID NO: 4. The DNA sequence of the E. coli rrnB terminator (SEQ ID NO: 3, nt 1510 to 1842) was linked to nt 1509. The DNA sequence of the rrnB terminator is disclosed in Orosz et al., Eur. J. Biochem. (1991) 201: 653 -659.The DNA disclosed in SEQ ID NO: 3, consisting of the CSADhs-cds and the rrnB terminator, was produced synthetically (Eurofins Genomics) and designated CSADhs-rrnB .

[0170] CSADcc-rrnB: The cDNA gene (cDNA: complementary DNA, isolated from mRNA by reverse transcription) of cysteine ​​sulfinic acid decarboxylase (CSAD) from Cyprinus carpio (common carp) was isolated by Honjoh et al. (2010), Amino Acids 38: 1173-183, and the DNA sequence was disclosed in the NCBI database under Genbank Sequence ID: AB220585.1 (cds: nt 82-1584). A codon-optimized DNA sequence for expression in E. coli was derived from the amino acid sequence (publicly available Eurofins Genomics GENEius software). This DNA sequence, designated CSADcc-cds, is disclosed in SEQ ID NO: 5, nt 1 to 1503, encoding a protein with the amino acid sequence of SEQ ID NO: 6. The DNA sequence of the E. coli rrnB terminator (SEQ ID NO: 5, nt 1504 to 1834) was linked to the 3'-end of CSADcc-cds. The DNA sequence of the rrnB terminator is disclosed in Orosz et al. (1991), soThe CSADcc-rrnB DNA, consisting of the CSADcc-cds and the rrnB terminator, was synthesized (Eurofins Genomics). Primers used were: cdorn-lf (SEQ ID NO: 8), csadhs-2r (SEQ ID NO: 9), csadhs-3f (SEQ ID NO: 10), glf-2r (SEQ ID NO: 11), cdorn-3r (SEQ ID NO: 12), and csadcc-lf (SEQ ID NO: 13).

[0171] PCR products:

[0172] Table 1 summarizes the names of the PCR products used for cloning, their sizes, and the synthetic DNA (so-called template DNA) and primers used. PCR products PCRI to PCR4 were generated 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).

[0173] Table 1: PCR products for the production of gene constructs Preparation of the vector pCys-CDOrn-CSADhs :

[0174] The NEBuilder® cloning kit (NEB New England Biolabs) was used to generate the pCys-CDOrn-CSADhs vector. The 6.1 kb pCys-Scal / PpuMI vector fragment was ligated together using PCR1 and PCR2, following the manufacturer's instructions regarding the amounts of each DNA fragment 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 the transformed clones (Qiagen kit), and a correct clone was selected. The plasmid DNA of this clone was designated vector pCys-CDOrn-CSADhs (Fig. 2).

[0175] Preparation of the vector pCys-CDOrn-CSADcc :

[0176] The NEBuilder® cloning kit (NEB New England Biolabs) was used to generate the vector pCys-CDOrn-CSADcc. The 6.1 kb pCys-Scal / PpuMI vector fragment was ligated together with PCR3 and PCR4, following the manufacturer's instructions regarding the amounts of each DNA fragment 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. Plasmid DNA was isolated from the transformed clones (Qiagen kit), and a correct clone was selected. The plasmid DNA of the clone was designated vector pCys-CDOrn-CSADcc (Fig. 3).

[0177] Example 2: Production of product! on s strains

[0178] The starting strain (host strain) for the production of production strains was the microorganism strain Escherichia coli K12 W3110 (commercially available under strain number DSM 5911 from the DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). E. coli W3110 was transformed in a known manner with the vector pCys-CDOrn-CSADhs or the vector pCys-CDOrn-CSADcc, and transformants were selected on LBtet plates. One transformant in each case was selected as the production strain. The production strains were designated E. coli W3110 x pCys-CDOrn-CSADhs and E. coli W3110 x pCys-CDOrn-CSADcc and were used to produce hypotaurine.

[0179] Example 3: Production of hypotaurine in a shake flask

[0180] Preculture: A preculture of each of the production strains E. coli W3110 x pCys-CDOrn-CSADhs and E. coli W3110 x pCys-CDOrn-CSADcc was prepared in LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L tetracycline) (cultivation at 37 °C and 120 rpm overnight).

[0181] Main culture: 0.5 ml of the respective 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 Na2S2O3 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.

[0182] Composition of the SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L NH4 sulfate, 0.3 g / L MgSO4x 7 H2O, 0.015 g / L CaCl2x 2 H2O, 0.002 g / L FeSO4x 7 H2O, 1 g / L Na3Citrate x 2 H2O, 0.1 g / L NaCl;

[0183] 1 ml / L trace element solution.

[0184] Composition of the trace element solution: 0.15 g / L Na2Mo04x 2 H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2x 6 H2O, 0.25 g / L CuSO4x 5 H2O, 1.6 g / L MnCl2x 4 H2O, 0.3 g / L ZnSO4x 7 H2O.

[0185] The main cultures were incubated for 24 h at 30°C and 140 rpm in a chest shaker (Infors). After 24 h, 1 ml samples were taken and the cell density OD6oo / ml (optical density of the main culture, measured photometrically at 600 nm) was measured using a Genesys™ 10S UV-Vis spectrophotometer from Thermo-Scientific™, as well as the hypotaurine and taurine content by HPLC. For the E. coli W3110 x pCys-CDOrn-CSADhs strain, the hypotaurine content was 54.9 mg / L. The taurine content was 44.6 mg / L. For the E. coli W3110 x pCys-CDOrn-CSADcc strain, the hypotaurine content was 158.8 mg / L. The taurine content was 8.7 mg / L.

[0186] Sample preparation for quantification of hypotaurine and taurine by HPLC:

[0187] 1 ml of culture broth from shake flask culture or fermentation was incubated for 5 min at 80°C and then centrifuged for 5 min at 13,000 rpm (Heraeus™ Fresco™ 21 centrifuge). The cell culture supernatant was isolated. Cell culture supernatants from shake flask culture were directly analyzed for hypotaurine and taurine content by HPLC. Cell culture supernatants from fermentation were diluted 10-fold in H2O and analyzed for hypotaurine and taurine content by HPLC.

[0188] HPLC analysis of L-cysteine ​​sulfinic acid, hypotaurine, and taurine: For the quantitative determination of the compounds analyzed in the examples, as well as for L-cysteine ​​sulfinic acid to detect CDO enzyme activity, an HPLC method calibrated for L-cysteine ​​sulfinic acid, hypotaurine, and taurine was used. The reference substances used for calibration were commercially available (Sigma-Aldrich). An Agilent HPLC instrument, Model 1260 Infinity II, was used. It was equipped with a pre-column derivatization with o-phthaldialdehyde (OPA derivatization), a process known from the analysis of amino acids. The HPLC instrument was equipped with a fluorescence detector to detect the OPA-derivatized products of hypotaurine and taurine. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. An Accucore™ aQ column from the company was also used.Thermo Scientific™, length 100 mM, inner diameter 4.6 mm, particle size 2.6 pm, heated to 40°C in a column oven.

[0189] Mobile phase A: 25 mM Na phosphate, pH 6.0. Mobile phase B: methanol. The separation was performed in gradient mode: 0–25 min, 10% mobile phase B to 60% mobile phase B, followed by 2 min of 60% mobile phase B to 100% mobile phase B, followed by another 2 min of 100% mobile phase B, at a flow rate of 0.5 ml / min. Retention time of L-cysteine ​​sulfinic acid: 4.1 min. Retention time of taurine: 14.8 min. Retention time of hypotaurine: 15.7 min.

[0190] Example 4 : Production of hypotaurine by fermentation

[0191] Pre-culture 1 :

[0192] 20 ml of LBtet medium were inoculated into a 100 ml Erlenmeyer flask with the production strain E. coli W3110 x pCys-CDOrn-CSADhs or the production strain E. coli W3110 x pCys-CDOrn-CSADcc and incubated for 7 h on a shaker (150 rpm, 30°C).

[0193] Pre-culture 2 :

[0194] Subsequently, each preculture was completely transferred to 100 ml of SMI medium supplemented with 5 g / L glucose, 5 mg / L vitamin B1, and 15 mg / L tetracycline (for the composition of SMI medium, see Example 3). The cultures were each incubated in a 1-L Erlenmeyer flask at 30°C for 17 h at 150 rpm (Infors chest shaker). After this incubation, the cell densities (OD 600 / ml) were between 3 and 5.

[0195] Main crop:

[0196] The fermentations were carried out in a Biostat B fermenter (2 1 working volume) from Sartorius BBI Systems GmbH.

[0197] The culture medium (900 ml) contained 15 g / L glucose, 10 g / L tryptone (Difco), 5 g / L yeast extract (Difco), 2.4 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.25 g / L NaCl, 0.6 g / L MgSO4x 7 H2O, 0.03 g / L CaCl2x 2 H2O, 0.15 g / L FeSO4x 7 H2O, 1 g / L Na3Citrate x 2 H2O 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 in the fermenter was initially adjusted to 7.0 by pumping in a 25% NH4OH solution. During fermentation, the pH was maintained at 7.0 by automatic correction with 25% NH4OH or 4 M H3PO4. Foam control was achieved by automatically dosing in 4% v / v Struktol J673 in H2O (Schill & Seilacher).

[0198] For inoculation, 100 ml of preculture 2 was pumped into each fermenter vessel. The initial volume was thus 1 L. The cultures were initially stirred at 400 rpm and aerated with compressed air purified through a sterile filter at an aeration rate of 2 vvm (vvm: compressed air introduced into the fermentation mixture, expressed in liters of compressed air per liter of fermentation volume per minute). Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0199] The target value for O2 saturation during fermentation was set at 30%. Once the O2 saturation fell below the target value, a regulation cascade was initiated to bring the O2 saturation back to the target value. First, the gas supply was continuously increased (to a maximum of 5 vvm) and then the stirring speed was continuously increased (to a maximum of 1,500 rpm).

[0200] The 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 H2O was added at a rate of 1.5 ml per hour.

[0201] Once 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 feed rate was adjusted so that the glucose concentration in the fermenter no longer exceeded 2 g / L. Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0202] The fermentation duration was 65 h. Samples were taken from the fermentation mixture 22 h, 40 h, and 65 h after the start of fermentation, and the cell density (OD 600 / ml) and the hypotaurine and taurine content in the culture supernatant were determined by HPLC. The results are summarized in Tables 2 and 3.

[0203] For strain W3110 x pCys-CDOrn-CSADhs, after a fermentation time of 65 h, the hypotaurine yield was 20.8 g / L (190.5 mM at a hypotaurine molecular weight of 109.2 g / mol) and the taurine yield was 7.4 g / L (59.1 mM at a taurine molecular weight of 125.2 g / mol). The molar ratio of hypotaurine to taurine was 3.2:1.

[0204] For strain W3110 x pCys-CDOrn-CSADcc, after a fermentation time of 65 h, the hypotaurine yield was 62.5 g / L (572.2 mM at a hypotaurine molecular weight of 109.2 g / mol) and the taurine yield was 1.2 g / L (9.6 mM at a taurine molecular weight of 125.2 g / mol). The molar ratio of hypotaurine to taurine was 59.6:1.

[0205] Table 2: Time course of cell density, hypotaurine and taurine content of the fermentation of the production strain W3110 x pCys-CDOrn-CSADhs Table 3: Time course of cell density, hypotaurine and taurine content of the fermentation of the production strain W3110 x pCys-CDOrn-CSADcc

[0206] Example 5: Extraction of hypotaurine and taurine from

[0207] Fermenter cells

[0208] The fermentation batch of the production strain E. coli W3110 x pCys-CDOrn-CSADcc from Example 4 with a hypotaurine content of 62.5 g / L and a taurine content of 1.2 g / L (Table 3) was used. Two 1 ml portions of the fermenter broth (2 ml total volume) were centrifuged for 5 min at 13,000 rpm (Heraeus™ Fresco™ 21 centrifuge), and the supernatant was discarded. The cell pellets were resuspended in 1 ml of H2O each, centrifuged for 5 min at 13,000 rpm, and the supernatant was discarded. The cell pellets were suspended in 1 ml of H2O each, corresponding to a total volume of 2 ml and the volume of fermenter broth used above. A cell extract was prepared from the cell suspension. For this purpose, the FastPrep-24TM 5G cell homogenizer from MP Biomedicals was used.The cell pellets, each suspended in 1 ml of H2O, were lysed in 1.5 ml tubes containing glass beads (“Lysing Matrix B”) prepared by the manufacturer (3 x 20 seconds at a shaking frequency of 6000 rpm with a 30 seconds break between each interval). The resulting cell homogenates were combined and centrifuged for 5 minutes at 13000 rpm to produce a cell extract. The cell extract was analyzed by HPLC for hypotaurine and taurine content. Neither hypotaurine nor taurine could be detected, with a detection limit of the HPLC analysis of 1 mg / L for hypotaurine and taurine, respectively.

Claims

Patent claims:

1. A process for the fermentative production of hypotaurine, characterized in that i) a microbial production strain is cultivated which is characterized in that it 1. contains at least one expression vector comprising a coding sequence (cds) a and a cds b and at least one of the cds selected from the group consisting of c, d and e, where a) a is a cds encoding a cysteine ​​dioxygenase (CDO) belonging to the enzyme class EC 1.13.11.20 and b) b is a cds encoding a cysteine ​​sulfinic acid decarboxylase (CSAD) belonging to the enzyme class EC 4.1.1.29 and c) c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with a feedback inhibition by serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme and d) d is a cds encoding a serine-O-acetyltransferase (CysE) with a feedback inhibition by serine that is reduced by at least a factor of two compared to the corresponding wild-type enzyme cysteine ​​and e) e is a cds encoding a cysteine ​​exporter, 2. the expression of cds a and b in the expression vector is coordinated with the expression of at least one cds selected from the group consisting of c, d and e in a polycistronic expression unit, 3. Hypotaurine is secreted from the production strain into the fermentation supernatant and ii) the fermentation supernatant is isolated, wherein the hypotaurine content in the fermentation supernatant is at least 10 g / L and the molar ratio of hypotaurine to taurine in the fermentation supernatant is at least 3:

1. The process according to claim 1, characterized in that the host strain used to produce the microbial production strain is a microorganism of the species Escherichia coli. The process according to one or more of claims 1 or 2, characterized in that the CDO is the CDO from Rattus norvegicus (CDOrn) having the sequence given in SEQ ID NO: 2 or an enzyme with CDO activity that is at least 80% identical thereto. Method according to one or more of claims 1 to 3, characterized in that the CSAD is the CSAD from Cyprinus carpio (CSADcc) with the sequence given in SEQ ID NO: 6 or an enzyme with CSAD activity which is at least 80% identical thereto.The method according to one or more of claims 1 to 3, characterized in that the CSAD is the CSAD from Homo sapiens (CSADhs) with the sequence given in SEQ ID NO: 4 or an enzyme with CSAD activity that is at least 80% identical thereto. The method according to one or more of claims 1 to 5, characterized in that the cds encoding CDO (a) and the cds encoding CSAD (b) are present in the expression vector of the microbial production strain with the cds encoding SerA with reduced feedback inhibition by serine (c) in a tricistronic expression unit that is functionally linked to the serA promoter. Process according to one or more of claims 1 to 6, characterized in that the CDs encoding CDO (a), CSAD (b), SerA with reduced feedback inhibition by serine (c), CysE with reduced feedback inhibition by cysteine ​​(d) and encoding the cysteine ​​exporter (e) are all present on a single expression vector. Process according to one or more of claims 1 to 7, characterized in that the expression vector of the microbial production strain is selected from the gene constructs pCys-CDOrn-CSADhs or pCys-CDOrn-CSADcc. Process according to one or more of claims 1 to 8, characterized in that the fermentation volume is at least 1 L. Process according to one or more of claims 1 to 9, characterized in that it is carried out in the presence of salts of thiosulfate.Process according to one or more of claims 1 to 10, characterized in that the fermentation process is a fed-batch process. Process according to one or more of claims 1 to 11, characterized in that the hypotaurine content in the fermentation supernatant, based on the hypotaurine content in the fermentation batch, is over 70%.

Images