Method for producing hypotaurine by fermentation

Abstract

The present invention relates to a method for producing hypotaurine by fermentation. The invention is characterized in that, in step i, a microbial production strain is cultured, characterized in that: 1. The production strain comprises at least one expression vector comprising the coding sequences (cds) a and cds b and at least one cds selected from the group consisting of c, d and e, where a is the cds encoding a cysteine dioxygenase (CDO) belonging to the enzyme class EC 1.13. 11.20, and d is the cds encoding a cysteine dioxygenase (CDO) belonging to the enzyme class EC 1.13. 11.20; wherein, b is a cds encoding a cysteine sulfinate decarboxylase (CSAD) belonging to the enzyme class EC 4.1. 1.29, c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with reduced feedback inhibition of serine, d is a cds encoding a serine O-acetyltransferase (CysE) with reduced feedback inhibition of cysteine, and e is a cds encoding a cysteine efflux protein, c is a cds encoding a cysteine sulfinate decarboxylase (CSAD) belonging to the enzyme class EC 4.1. 1.29, c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) with reduced feedback inhibition of cysteine, d is a cds encoding a cysteine O-acetyltransferase (CysE) with reduced feedback inhibition of cysteine. 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 the polycistron expression unit, and 3. Hypotaurine is secreted into the fermentation supernatant from the producing strain, and in step ii, the fermentation supernatant is isolated. The sub-taurine content in the fermentation supernatant is equal to at least 10 g / L, and the molar ratio of sub-taurine: taurine in the fermentation supernatant is equal to at least 3: 1.

Description

Technical Field

[0001] The present invention relates to a method for fermentatively producing hypotaurine, characterized in that:

[0002] i) Culturing a microbial production strain, the microbial production strain being characterized in that it:

[0003] 1. Contains at least one expression vector, the expression vector containing coding sequences (cds) a and cds b and at least one cds selected from the group consisting of c, d, and e,

[0004] a) a is a cds encoding a cysteine dioxygenase (CDO) belonging to enzyme class EC 1.13.11.20, and

[0005] b) b is a cds encoding a cysteine sulfinate decarboxylase (CSAD) belonging to enzyme class EC 4.1.1.29, and

[0006] c) c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) in which the feedback inhibition of serine is reduced by at least 2-fold relative to the corresponding wild-type enzyme,

[0007] d) d is a cds encoding a serine O-acetyltransferase (CysE) in which the feedback inhibition of cysteine is reduced by at least 2-fold relative to the corresponding wild-type enzyme, and

[0008] e) e is a cds encoding a cysteine efflux protein,

[0009] 2. The expression of cds a and cds 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,

[0010] 3. Hypotaurine is secreted by the production strain into the fermentation supernatant, and

[0011] ii) Separating the fermentation supernatant,

[0012] The content of hypotaurine in the fermentation supernatant is at least 10 g / L, and the molar fraction of hypotaurine:taurine in the fermentation supernatant is at least 3:1. Background Art

[0013] Hypotaurine (2-aminoethanesulfinic acid, CAS No. 300-84-5) is an amino sulfinic acid that exists in nature as a degradation product of the amino acid cysteine. As a sulfinic acid, hypotaurine acts as an antioxidant and is used in the food, feed, cosmetic, and pharmaceutical industries. In addition, hypotaurine is a biosynthetic precursor of taurine (2-aminoethanesulfonic acid, CAS No. 107-35-7).

[0014] Taurine is produced, for example, starting from 2-aminoethanethiol hydrochloride for commercial use. Due to the trend of consumers moving away from chemically produced ingredients, biotechnological methods for producing taurine are of interest. Starting from L-cysteine, the known biosynthetic pathways to taurine and forward to taurine can be found, for example, in the KEGG Pathway Database: "Taurine and hypoaurine metabolism". The most important synthetic steps from L-cysteine to taurine and forward to taurine are shown in equations (1) to (5).

[0015] (1) L-cysteine + O2 → L-cysteine sulfinic acid

[0016] (2) L-cysteine → cysteamine + CO2

[0017] (3) L-cysteine sulfinic acid → taurine + CO2

[0018] (4) Cysteamine + O2 → taurine

[0019] (5) Taurine + 1 / 2 O2 → taurine

[0020] (1): L-cysteine is oxidized by the enzyme cysteine dioxygenase (CDO, EC 1.13.11.20) to L-cysteine sulfinic acid (3-sulfopropanoic acid, CAS No. 207121-48-0).

[0021] (2): Cysteamine is formally formed by the decarboxylation of L-cysteine, but in mammals, it is a reaction product of pantothenic acid biosynthesis, in which the enzyme pantetheinase (EC 3.5.1.92) cleaves cysteamine from the precursor -pantetheine. Bacterial cysteine decarboxylase has been described, but it only acts on the C-terminal cysteine of the precursor peptide as an auxiliary role in the biosynthesis of natural substances and is not applicable to the production of cysteamine. Therefore, the pathway (2) from cysteine to taurine via cysteamine has no technical significance.

[0022] (3): Cysteine sulfinic acid decarboxylase (CSAD, EC 4.1.1.29) decarboxylates L-cysteine sulfinic acid to taurine (2-aminoethanesulfonic acid, CAS No. 300-84-5).

[0023] (4): Cysteamine dioxygenase (EC 1.13.11.19) oxidizes cysteamine to taurine with atmospheric oxygen.

[0024] (5): The oxidation of taurine to taurine is not fully understood.

[0025] Known biotechnological methods are mainly aimed at producing taurine, but the pathway of the biosynthetic route means that hypotaurine is the main product in many cases. The known biotechnological methods for producing hypotaurine are characterized by a very low yield of hypotaurine disclosed therein.

[0026] Honjoh et al. (2010), Amino Acids 38:173 - 1183 described a genetically engineered yeast strain that heterologously expressed the CDO gene and the CSAD gene from carp (Cyprinus carpio). When L-cysteine was added to the growth of the genetically engineered strain, it was observed that hypotaurine was produced as the main product and also a relatively low proportion of taurine. Although Saccharomyces cerevisiae itself is able to produce cysteine from its own metabolism, the production of hypotaurine requires the external addition of L-cysteine to the growth medium. Therefore, the method of Honjoh et al. (2010) is equivalent to the bioconversion of L-cysteine to hypotaurine. Analytical determinations of taurine formed intracellularly with and without H2O2 treatment were carried out (Table 1 in Honjoh et al.). It was inferred from the significantly higher taurine values after H2O2 treatment that most of the intracellularly formed product was in the form of hypotaurine, although no analytical quantification of hypotaurine was performed. The indirect quantification of hypotaurine was unexpected because the analysis of hypotaurine was listed in the method section of Honjoh et al. Therefore, the formation of hypotaurine in baker's yeast was not clearly demonstrated and was only indirectly inferred from the elevated taurine values after H2O2 oxidation.

[0027] WO 17 / 213142 A1 (Ajinomoto) describes strains that produce taurine due to the heterologous expression of genes encoding cysteine dioxygenase (CDO) and L-cysteine sulfinate decarboxylase (CSAD) in strains that initially produce cysteine. Constructs consisting of different CDO and CSAD genes are generated, each under the control of the known strong tac promoter. However, in strains optimized for cysteine production, their suitability for hypotaurine or taurine production was not investigated, but in strains of Escherichia coli (maximally producing 2 μM cysteine; see Table 5 of WO 17 / 213142 A1, strain EcoT / pMW2q19) and Pantoea ananatis (maximally producing 0 μM cysteine; see Table 5 of WO 17 / 213142 A1, strain PanT / pMW219), each has an inactivated tauABCD operon (designated EcoT for E. coli and PanT for Pantoea ananatis). The tauABCD operon is known to encode genes for taurine degradation. The main product is hypotaurine with a maximum yield of 450 μM (49.1 mg / L hypotaurine, where the molecular weight of hypotaurine is 109.2 g / mol). Thus, optimization of cysteine metabolism is described as an option for improved hypotaurine production, but not experimentally demonstrated. The low hypotaurine yield does not allow for economically viable production.

[0028] US 2019 / 0062757 A1 (KnipBio) describes heterologous production strains for the production of taurine or its precursors, with a maximum yield achieved of only 419 ng / ml hypotaurine. The host strains used are WT strains of Escherichia coli and Methylobacterium extorquens. No measures for improving cysteine production targeting higher hypotaurine yields are described.

[0029] US 9,267,148 B2 and other publications of Plant Sensory Systems describe gene constructs designed for the heterologous expression of cysteine dioxygenase and L-cysteine sulfinate decarboxylase, with the goal of producing taurine. In particular, these applications first relate to gene constructs that express (in each case monocistronically) the CDO gene and the CSAD gene as a single expression cassette, each CDO gene and CSAD gene functionally linked to its own promoter. Second, gene constructs designed to express the CDO gene and the CSAD gene in the form of a fusion protein are also described, which are combined into a single expression cassette under the control of only one promoter (monocistronic expression cassette). The main focus of these applications is the production of taurine in plants, although no description of the yield is provided. No measures for improving cysteine production targeting higher hypotaurine yields are described. In summary, these applications do not disclose any methods applicable to the fermentative production of hypotaurine.

[0030] Joo et al. (2018), J. Agric. Food Chem. 66:13454 - 13463 described a genetically engineered strain of Corynebacterium glutamicum with an output of 0.5 g / L taurine when grown in shake flasks. Apparently, taurine accumulates intracellularly and is not secreted into the growth medium. The synthesis of taurine was achieved by heterologous expression of the genes for L - cysteine synthase, cysteine dioxygenase, and L - cysteine sulfinate decarboxylase in the strain. In addition, the repressor genes related to methionine and cysteine biosynthesis were inactivated, which simultaneously achieved improved sulfur uptake. No analysis of hypotaurine production was performed.

[0031] Thus, the prior art discloses various metabolic engineering and biotransformation methods for producing hypotaurine in heterologous production systems. Production strains with optimized cysteine biosynthesis are not used. Regarding the production of hypotaurine or taurine on an industrial scale by fermentation methods, no strains described in the prior art are disclosed. Summary of the Invention

[0032] The object of the present invention is to provide a method for fermentative production of hypotaurine with a high yield in the fermentation supernatant. According to the prior art, since hypotaurine can be oxidized to taurine, a further object is to achieve a yield of hypotaurine that substantially exceeds the yield of taurine.

[0033] This object is achieved by a method for fermentative production of hypotaurine, characterized in that

[0034] i) cultivating a microbial production strain, which is characterized in that it:

[0035] 1. contains at least one expression vector, the expression vector containing coding sequences (cds) a and cds b and at least one cds selected from the group consisting of c, d, and e,

[0036] a) a is a cds encoding a cysteine dioxygenase (CDO) belonging to enzyme class EC 1.13.11.20, and

[0037] b) b is a cds encoding a cysteine sulfinate decarboxylase (CSAD) belonging to enzyme class EC 4.1.1.29, and

[0038] c) c is a cds encoding a 3 - phosphoglycerate dehydrogenase (SerA) in which the feedback inhibition of serine is reduced by at least 2 - fold relative to the corresponding wild - type enzyme,

[0039] d) d is a cds encoding a serine O - acetyltransferase (CysE) in which the feedback inhibition of cysteine is reduced by at least 2 - fold relative to the corresponding wild - type enzyme, and

[0040] e) e is the cds encoding a cysteine exporter,

[0041] 2. The expression of cds a and cds 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 the polycistronic expression unit,

[0042] 3. hypotaurine is secreted by the production strain into the fermentation supernatant, and

[0043] ii) the fermentation supernatant is separated,

[0044] The content of hypotaurine in the fermentation supernatant is at least 10 g / L, and the molar fraction of hypotaurine:taurine in the fermentation supernatant is at least 3:1.

[0045] The main advantage of the present invention is to disclose a fermentation method for producing hypotaurine, in which an expression vector designed according to the principles of metabolic engineering combines an expression unit for increasing cysteine production with an expression unit for hypotaurine production in a microbial production strain. In particular, the polycistronic arrangement of at least one cds of cysteine metabolism, the cds of cysteine dioxygenase (CDO), and the cds of cysteine sulfinic acid decarboxylase (CSAD) in an artificial operon under the control of only one promoter allows for the coordinated expression of cysteine and hypotaurine metabolism genes in the production strain. This helps to avoid the accumulation of metabolic intermediates (such as L-cysteine) and maximize the yield of hypotaurine.

[0046] Surprisingly, the product hypotaurine is secreted by the production strain into the fermentation supernatant (extracellular production) at a concentration of ≥10 g / l and accumulates extracellularly (Example 5). In contrast, in the prior art, intracellular enrichment occurs, such as described by Joo et al. (2018, supra) for taurine, or depending on the production system, only partial secretion occurs.

[0047] Thus, the use of the production strain according to the claims in the method according to the invention is characterized by the lack of product accumulation in the biomass (fermenter cells) (see also Example 5). This extracellular production or enrichment (i.e., biosynthesis of hypotaurine in the production strain followed by secretion (export, transfer) from the production strain into the growth medium) has the following main advantages: the volume in which hypotaurine can accumulate is not limited to the small volume of the cell contents (cytosol). This avoids toxic effects due to high intracellular product concentrations. Thus, much higher yields of hypotaurine can be achieved compared to intracellular production. Preferably, based on the biomass present in the fermentation broth, the biomass contains less than 1 g / l of hypotaurine, as described in Example 5. Particularly preferably, no detectable hypotaurine is present in the biomass.

[0048] In addition, the extracellular production of hypotaurine has the advantage of simplifying product separation, since hypotaurine can be directly separated from the fermentation supernatant without the need for prior elaborate mechanical or chemical cell disruption. It is not known how hypotaurine is secreted from the production strain. The mechanism may be passive, accompanying the diffusion of hypotaurine across the cell membrane, or one or more transporters may be involved in the secretion.

[0049] Another advantage of the present invention is the low proportion of the by-product taurine, which is formed by the oxidation of hypotaurine and reduces the yield of hypotaurine. Thus, the method according to the invention is very suitable for industrial applications, since it allows the economically viable production of hypotaurine, which is attributed to the high yield of hypotaurine in the fermentation. In contrast, in the prior art such as Joo et al. (2018, see above), spontaneous oxidation of hypotaurine to taurine occurs, which has the disadvantage of low hypotaurine content.

[0050] In contrast to biotransformation, metabolic engineering (also known as "pathway design") is a biotechnological method for modifying the metabolic pathways of organisms by optimizing or modifying genetic and regulatory processes. New or modified enzymes can be introduced into an organism by supplementing the genome with the gene of the enzyme, or the genes of endogenous enzymes can be expressed at enhanced or reduced levels, thereby establishing new metabolic pathways or enhancing or reducing existing metabolic pathways in the organism. The aim of metabolic engineering is for the organism to produce new metabolites or intracellular endogenous metabolites in increased yields. Metabolic engineering methods do not use starting materials specific for the metabolite, such as enzyme substrates, e.g., L-cysteine as a starting compound for the production of hypotaurine; instead, it uses only a nutrient medium, also known as a growth medium, which is required for the growth of the organism in question and consists of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts 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 method for the fermentative production of hypotaurine disclosed in the present invention is a metabolic engineering method.

[0051] In contrast, biotransformation is defined as the conversion of one or more reactants into products under enzyme catalysis, where the enzyme substrate is added to the reaction together with the enzyme and the enzyme conversion is carried out.

[0052] According to equation (1), L-cysteine is enzymatically oxidized to L-cysteine sulfinic acid. This reaction is catalyzed by the CDO enzyme (EC1.13.11.20). The gene and protein sequences of the CDO enzyme are available, for example, in the NCBI database under the search term "cysteine dioxygenase". The present invention encompasses genes encoding proteins having CDO activity, preferably including CDO enzymes selected from Rattus norvegicus (rat), Homo sapiens (human), Cyprinus carpio (carp), Bos taurus (cow), Capra hircus (goat), Gallus gallus (chicken) 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) having the sequence specified in SEQ ID NO: 2, or an enzyme having at least 80%, particularly preferably at least 90% and most preferably at least 95% identity with CDO activity. Particularly preferably, the CDO is the protein of SEQ ID NO: 2. Particularly preferably, the CDOrn cds has the sequence specified in SEQ ID NO: 1.

[0053] The CDO enzyme activity assay can be carried out as follows:

[0054] i) Production of the enzyme to be tested:

[0055] The enzyme produced by growing in shake flasks or in fermentation can be used in the following reaction:

[0056] - as an aliquot from the culture broth without further treatment, or

[0057] - as an aliquot of the cell suspension after re-separating the cells from the culture broth by, for example, centrifugation, or

[0058] - in the form of an aliquot of the cell homogenate

[0059] a) after mechanically disrupting the cell suspension, or

[0060] b) in the form of chemically permeabilized cells (e.g., by chloroform)

[0061] or

[0062] - as a cell extract after removing particulate components from the cell homogenate or

[0063] - an enzyme purified, for example, by chromatography.

[0064] The total protein concentration obtained in each case can be determined, for example, using the commercially available Qubit 3.0 fluorometer from Thermo Fisher Scientific with the “ Protein Assay Kit” according to the manufacturer's instructions.

[0065] ii) Determination of CDO enzyme activity:

[0066] A solution buffered to pH 7 with potassium phosphate is initially charged with L-cysteine (10 mM final concentration), and the reaction is started by adding the enzyme from i). The assay volume is 10 ml. The temperature at which the assay is carried out is 30 °C. The amount of enzyme used depends on the purity. If culture broth, cell suspension of re-separated cells, cell homogenate or cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of the purified enzyme, at least 10 μg of the purified enzyme fraction is used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the assay is removed in each case and the assay is centrifuged for 10 min, and the content of L-cysteine sulfinic acid is determined by calibrated HPLC (see Example 3); the reference substance for calibration is commercially available (Sigma-Aldrich).

[0067] 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 above assay conditions, the assay does not contain any active CDO.

[0068] According to equation (3), L-cysteine sulfinic acid is enzymatically decarboxylated to hypotaurine. This reaction is catalyzed by CSAD enzyme (EC 4.1.1.29), or by GAD enzyme (EC 4.1.1.15) with much lower enzyme activity. The gene and protein sequences of CSAD enzyme are available, for example, in the NCBI database under the search term "cysteine sulfinic acid decarboxylase". The gene and protein sequences of GAD enzyme are available, for example, in the NCBI database under the search term "glutamic acid decarboxylase". The present invention encompasses genes encoding proteins having CSAD activity, preferably including CSAD enzymes selected from the group consisting of: Cyprinus carpio (carp), Rattus norvegicus (rat), Homo sapiens (human), Bos taurus (cow), Capra hircus (goat), Gallus gallus (chicken), Escherichia coli or Synechococcus (algae) or homologous CSAD enzymes. In a preferred embodiment, the method is characterized in that CSAD is CSAD from carp (CSADcc) having the sequence specified in SEQ ID NO: 6, or an enzyme having at least 80%, particularly preferably at least 90% and most preferably at least 95% identity to CSAD having activity. Particularly preferably, CSAD is the protein having SEQ ID NO: 6. Particularly preferably, the CSADcc cds has the sequence specified in SEQ ID NO: 5 (nt 1-1503).

[0069] In an alternative preferred embodiment, the method is characterized in that CSAD is CSAD from Homo sapiens (CSADhs) having the sequence specified in SEQ ID NO: 4, or an enzyme having at least 80%, particularly preferably at least 90% and most preferably at least 95% identity to CSAD having activity.

[0070] Particularly preferably, CSAD is the protein having SEQ ID NO: 4. Particularly preferably, the CSADhs cds has the sequence specified in SEQ ID NO: 3 (nt 1-1509).

[0071] The CSAD enzyme activity can be assayed as follows:

[0072] i) Production of the enzyme to be tested:

[0073] The enzyme produced by growing in shake flasks or in fermentation can be used in the following reactions:

[0074] - As an aliquot from the culture broth without further treatment, or

[0075] - As an aliquot of the cell suspension after re-separating the cells from the culture broth by, for example, centrifugation, or

[0076] - In the form of an aliquot of the cell homogenate

[0077] c) after mechanical disruption of the cell suspension, or

[0078] d) in the form of chemically permeabilized cells (e.g., by chloroform)

[0079] or

[0080] - as a cell extract after removal of particulate components from the cell homogenate or

[0081] - an enzyme purified, for example, by chromatography.

[0082] The total protein concentration obtained in each case can be determined, for example, using a commercially available Qubit 3.0 fluorometer from Thermo Fisher Scientific with the “ Protein Assay Kit” according to the manufacturer's instructions.

[0083] ii) Determination of CSAD enzyme activity:

[0084] A solution buffered to pH 7 with potassium phosphate is initially charged with L-cysteine sulfinic acid (10 mM final concentration), and the reaction is started by adding the enzyme from i). The assay volume is 10 ml. The temperature at which the assay is carried out is 30 °C. The amount of enzyme used depends on the purity. If culture broth, cell suspension of re-isolated cells, cell homogenate or cell extract is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of a purified enzyme, at least 10 μg of the purified enzyme fraction is used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the assay is removed in each case and the assay is centrifuged for 10 min, and the taurine content is determined by calibrated HPLC (see Example 3); the reference substance for calibration is commercially available (Sigma-Aldrich).

[0085] The detection limit of the HPLC method is 1 mg / L taurine. If less than 1 mg / L of taurine is formed under the above assay conditions, the assay does not contain any active CSAD.

[0086] An open reading frame (ORF, synonymous with cds or coding sequence) refers to a region of DNA or RNA that starts with a start codon and ends with a stop codon and encodes the amino acid sequence of a protein. The ORF is also referred to as the coding region or structural gene.

[0087] A gene, cistron or expression unit refers to a DNA segment that contains all the essential information for the production of a biologically active RNA. A gene contains a DNA segment from which a single-stranded RNA copy is produced by transcription and expression signals that participate in regulating the copy process. The expression signals include at least a promoter, transcription initiation, translation initiation and ribosome binding site (RBS). Terminators and one or more operators are additional possible expression signals.

[0088] In the context of the present invention, bacterial proteins, such as SerA or CysE, start with a capital letter, while the sequences encoding said proteins (cds) are denoted by lowercase letters (e.g., serA or cysE). Similarly, the promoters controlling the expression of said cds are denoted by lowercase letters (e.g., serA promoter or cysE promoter). In contrast, proteins / enzymes and cds / genes of higher organisms (e.g., Homo sapiens, Rattus norvegicus or Cyprinus carpio) are denoted by capital letters and, if necessary, are labeled in each case as protein / enzymes (e.g., CDO protein / enzymes or CSAD protein / enzymes) or cds / genes (e.g., CDO cds / genes or CSAD cds / genes).

[0089] A gene construct refers to a DNA molecule produced by cloning that contains at least one expression unit and may also contain other genetic elements together with it, such as selection markers and origins of replication. A gene construct can be a linear DNA molecule integrated into the genome, or it can be a circular DNA molecule in the form of a plasmid, also synonymously referred to as a vector. The said vector is then called an expression vector. After insertion (transformation) into a suitable host strain, the genetic elements of the vector cause extrachromosomal inheritance of the vector during cell growth and cause the production of the protein encoded by the cds.

[0090] In the genomes of microorganisms, so-called operons can be found in many cases. An operon is characterized by a DNA segment that expresses multiple genes in a coordinated manner. An operon refers to a DNA segment that contains all the essential information for the production of a biologically active RNA. An operon contains a DNA segment from which a single-stranded RNA copy is produced by transcription and expression signals that participate in regulating the copy process. However, the RNA copy includes not only the cds of a single gene, but also the cds of two or more genes. The expression signals of an operon include a promoter and transcription initiation. Each cds of an operon includes translation initiation and ribosome binding site. Terminators and one or more operators are additional possible expression signals.

[0091] If only one gene is expressed by a promoter, this is called a monocistronic expression unit. If two, three or more genes are expressed by a promoter, such an expression unit is called dicistronic, tricistronic, etc. or polycistronic.

[0092] Operons not only occur naturally in the genomes of microorganisms, but can also be produced specifically by cloning (artificial operons).

[0093] The expression unit of an artificial operon also consists of a promoter (bicistronic, tricistronic or polycistronic expression unit) that is functionally linked to two or more cds using an upstream RBS in each case. In addition, the expression unit can also contain other genetic elements, such as terminators or operators. The term "functionally linked" means that an expression unit containing a promoter and a cds, or in the case of an operon a promoter and two or more cds, results in the transcription and translation of the cds in a microorganism.

[0094] A promoter is a nucleotide sequence that allows the expression of a cds and is located upstream of the 5' end of the cds. In the synthetic direction, the promoter appears before the coding region. The promoter contains regions that define the initiation of transcription of the gene by RNA polymerase and additionally mediate specific interactions with DNA-binding proteins (transcription factors) that affect the transcription level.

[0095] All promoters that are active in the host strain are generally suitable as promoters for the above-mentioned cds. These include all natural promoters of approximately 5000 genes in Escherichia coli, but also include non-natural promoters (e.g., promoters from other species of the Enterobacteriaceae) or "artificial" promoters such as the tac promoter. Preferred promoters in polycistronic expression units are the cysE, serA and GAPDH promoters present on pCys, particularly preferably the serA promoter, as described, for example, in Rex etal. (1991), J. Bacteriol. 173:5944 - 5953, Fig 2, nt 1 - nt 457.

[0096] Particularly preferred is the serA promoter having the sequence specified in SEQ ID NO: 7.

[0097] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries genetic information for the synthesis of proteins. mRNA provides the instructions for the assembly of a specific protein in the cell. The mRNA molecule transfers the necessary information for protein synthesis from the genetic information (DNA) to the ribosome responsible for protein synthesis. In the cell, it is formed as a transcript of the DNA segment corresponding to the gene. In this way, the genetic information stored in the DNA remains unchanged.

[0098] Genes of eukaryotes are mainly so-called chimeric genes, which, different from prokaryotic genes, also contain non-coding segments called introns (intragenic regions). The coding sequences, called exons (expressed regions), are DNA segments of eukaryotic genes, which are translated into the amino acid sequence of a protein by ribosomes after being transcribed into RNA. After DNA is transcribed into RNA, introns are spliced from the primary transcript. The protein-coding RNA from which introns have been removed is called messenger RNA (mRNA) or “mature” mRNA. This undergoes further modifications such as capping and polyadenylation. Then the coding region of the mature mRNA is translated into a protein sequence. If a eukaryotic gene containing an exon / intron structure is to be expressed in a prokaryote, it is necessary to back-translate the protein sequence or the coding region of the mature mRNA into intronless DNA, because the processing of the exon / intron structure does not occur in prokaryotes. Whenever an expression gene sequence derived from this protein sequence or a gene sequence derived from mRNA is used in the context of the present invention, it exactly means this process of back-translation. Preferably, sequence optimization, i.e., adaptation to the codon usage of the corresponding prokaryote (codon optimization), accompanies the back-translation of the protein sequence or mRNA sequence into a DNA sequence.

[0099] Homologous genes or homologous DNA sequences should be understood to mean that the DNA sequences or DNA segments of these genes are at least 80% identical, preferably at least 90% identical and particularly preferably at least 95% identical.

[0100] The degree of DNA identity is determined by the “nucleotide blast” program, which can be found at http: / / blast.ncbi.nlm.nih.gov / and is based on the blastn algorithm. The algorithm parameters for aligning two or more nucleotide sequences are default parameters. The default general parameters are: maximum target sequences = 100; short query = “automatically adjust parameters for short input sequences”; expect threshold = 10; word size = 28; automatically adjust parameters for short input sequences = 0. The corresponding default scoring parameters are: match / mismatch scores = 1, -2; gap cost = linear.

[0101] Homologous protein sequences should be understood to mean that the protein sequences of these proteins or protein segments are at least 80% identical, preferably at least 90% identical and particularly preferably at least 95% identical.

[0102] In http: / / blast.ncbi.nlm.nih.gov / The protein sequences were compared using the "protein blast" program. This program uses the blastp algorithm. The algorithm parameters for aligning two or more protein sequences are the default parameters. The default general parameters are: maximum target sequences = 100; short query = "automatically adjust parameters for short input sequences"; expectation threshold = 10; word size = 3; automatically adjust parameters for short input sequences = 0. The default scoring parameters are: Matrix = BLOSUM62; gap costs = existence: 11 extension: 1; composition adjustment = conditional composition score matrix adjustment.

[0103] The abbreviation WT (Wt) refers to wild type. A wild type gene refers to the form of a gene that occurs naturally through evolution and is present in the wild type genome. The DNA sequence of the Wt gene is publicly available in databases such as NCBI.

[0104] Allelic restriction can be the state of genes that can be transformed into each other by mutation (i.e., by changes in the nucleotide sequence of DNA). Genes that occur naturally in microorganisms are called wild type alleles, and variants derived from them are called mutant alleles of the gene.

[0105] Fermentation is a method step for producing (culturing) cell cultures on an industrial scale, in which a preferred microbial production strain is grown under defined conditions of medium, temperature, pH, oxygen supply, and mixing of the medium. If all the fermentation components are defined at the start of the culture and then not changed, the fermentation is called batch fermentation.

[0106] If, after the start of fermentation, medium components such as glucose (carbon source) or a complex amino acid mixture such as yeast extract (nitrogen source) are continuously supplied as so-called feeds, the fermentation is called fed-batch fermentation (so-called feeding process). Fed-batch fermentation allows optimization of the formation of biomass and the target product. Depending on the construction (genetic composition) of the production strain, the aim of the fermentation is to produce the highest possible yield of protein / enzyme or metabolite for further use. The product hypotaurine can be produced by fermentation. The final product of fermentation is the fermenter broth consisting of the biomass of the cells of the production strain (fermenter cells) and the cell-free fermentation medium (fermentation supernatant) formed during fermentation by the growth medium and metabolites secreted by the fermenter cells. Compared with the growth medium defined by its chemical composition, the composition of the fermentation medium is not clearly defined because the formation of metabolites is unpredictable. In the present invention, the product hypotaurine is produced by fermentation. In principle, the fermentation product can be present in the fermenter cells and / or the fermentation medium. As disclosed in Example 5 of the present invention, hypotaurine was found only in the fermentation medium within the detection limit. Therefore, only hypotaurine produced by secretion into the fermentation medium is considered.

[0107] Preferably, the method is characterized in that, based on the taurine content in the fermentation batch, the taurine content in the fermentation supernatant is higher than 70%, preferably higher than 80% and particularly preferably higher than 90%. The taurine content in the fermentation supernatant corresponds to the extracellular (secreted) taurine. The taurine content in the fermentation batch is the sum of the extracellular and intracellular taurine contents, also referred to as the total taurine content. Determine the extracellular portion of taurine as described in Example 3 (“Sample Preparation”). Determine the intracellular portion of taurine as described in Example 5, and the detection limit of taurine is 1 mg / L.

[0108] Flask growth is used to culture microorganisms on a laboratory scale, as opposed to fermentation production scale. Although flask cultivation also involves specifying a particular medium and pH and culturing under constant motion (shaking) in the presence of oxygen, more defined conditions regarding the medium, temperature, pH, oxygen supply, and mixing of the medium can be established and regulated in a fermenter. Cultivation on a smaller scale (e.g., in a flask) can also be used as a pre-culture for inoculation of a larger scale cultivation (e.g., a fermenter).

[0109] The production scale in fermentation typically starts with a batch volume of 0.5 L and can be up to 100,000 L or higher. The production scale of flask growth is usually in the batch volume range of 10 ml - 1000 ml.

[0110] The yield in the context of the present invention is defined as the amount of product obtained by growing a production strain. This yield can be specified as the absolute amount of the product (mmol or g) or as the volumetric yield (concentration) of the product based on volume (mM or g / L).

[0111] The method according to the present invention comprises a fermentation method using a microbial production strain for the production of taurine. By definition, the production strain comprises a microorganism (referred to as the 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.

[0112] Suitable host strains are any microorganisms that are amenable to recombinant DNA technology and are suitable for the fermentative production of recombinant proteins. Suitable microbial strains include bacterial strains selected from the Corynebacteriaceae or Enterobacteriaceae, yeasts (e.g., Saccharomyces cerevisiae, Yarrowia lipolytica) or fungi (e.g., Aspergillus niger).

[0113] The host strain for producing the production strain is preferably a prokaryotic microorganism, particularly preferably a bacterial strain selected from the group consisting of Corynebacterium ssp. (such as Corynebacterium glutamicum, which is particularly preferred), Pantoea ssp. (such as Pantoea ananatis, which is particularly preferred), and Escherichia ssp., and particularly preferably a microorganism of the species Escherichia coli. In a particularly preferred embodiment, the microorganism is the strain Escherichia coli K12W3110, which is commercially available from DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the strain number DSM 5911.

[0114] The production strain for the fermentative production of at least 10 g / l taurine is characterized in that it comprises a gene construct, which gene construct comprises

[0115] (I) A metabolic pathway leading to taurine and defined by the expression of cds a (encoding CDO) and cds b (encoding CSAD), and

[0116] (II) A dysregulated cysteine biosynthesis pathway, defined by the expression of at least one 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 efflux protein).

[0117] (I) The metabolic pathway leading to taurine is characterized in that it comprises the conversion of L - cysteine to taurine according to equations (1) and (3).

[0118] The genes CDO and CSAD that constitute the taurine metabolic pathway in the production strain can be derived from a microbial strain that is also the host strain (homologous expression), or they can be foreign genes (heterologous expression), which are produced by synthesis (which allows, in particular, the modification of cds by so - called codon optimization for expression in the host strain) or isolated from a strain different from the host strain, or a combination of homologous and heterologous expression can be carried out.

[0119] It is preferred to heterologously express the genes that constitute the taurine metabolic pathway in the production strain, particularly preferably to heterologously express genes produced by synthesis, and particularly preferably to heterologously express genes that constitute the taurine metabolic pathway and are produced by synthesis and codon - optimized in the production strain.

[0120] (II) The production strain containing a gene construct (including an expression unit for a dysregulated cysteine biosynthesis pathway) and thus suitable for cysteine production is characterized in that it contains at least one cds selected from the group consisting of c, d, and e:

[0121] c) The production strain contains at least one modified serA gene, i.e., it contains at least one cds c encoding 3-phosphoglycerate dehydrogenase (SerA), wherein the feedback inhibition of L-serine is reduced by at least 2-fold relative to the corresponding wild-type enzyme, and wherein the SerA enzyme activity can be determined, for example, by the oxidative photometry 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 an unmodified WT serA gene.

[0122] In a particularly preferred variant of 3-phosphoglycerate dehydrogenase (SerA), the feedback inhibition of L-serine is reduced by at least 5-fold, more preferably by at least 10-fold, and in a more preferred embodiment, by at least 50-fold relative to the corresponding wild-type enzyme.

[0123] Examples of serA variants encoding SerA enzymes with reduced feedback inhibition are known from the prior art and are preferred embodiments in the context of the present invention:

[0124] - serA317: A feedback-resistant SerA mutant used in the present invention in vector pCys ( Figure 1 ). It is a C-terminal deletion mutant of SerA, including 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, where it is called "NSD:317"; see also Example 1).

[0125] - serA G349E: The glycine at position 349 of the WT SerA enzyme is mutated to glutamate.

[0126] - serA G349D: The glycine at position 349 of the WT SerA enzyme is mutated to aspartate.

[0127] - serA T372X: The threonine at position 372 of the WT SerA enzyme is mutated to X, where X can be any natural amino acid other than threonine.

[0128] - serA mutant: A serA gene in which 25% of the C-terminal amino acids are modified compared to WT, and the modification is

[0129] i) among the 50 C-terminal amino acids of the WT protein, and

[0130] ii) modifications including C-terminal deletions, and

[0131] iii) modifications including insertions into the WT sequence.

[0132] and / or

[0133] d) The production strain contains at least one modified cysE gene, i.e., it contains at least one cds d encoding serine O-acetyltransferase (CysE), wherein the feedback inhibition of cysteine is reduced by at least 2-fold relative to the corresponding wild-type enzyme (as described, for example, in Nakamori et al., Appl. Env. Microbiol. (1998) 64:1607-1611), wherein the CysE enzyme activity can be determined photometrically, for example, by consuming the CysE substrate acetyl-CoA produced by reacting with L-serine to form O-acetyl-L-serine, as described, for example, by "Nakamori et al., Appl. Env. Microbiol. (1998) 64:1607-1611". In addition, the production strain can additionally contain an unmodified WT cysE gene.

[0134] In a particularly preferred variant of serine O-acetyltransferase (CysE), the feedback inhibition of cysteine is reduced by at least 5-fold, particularly preferably by at least 10-fold, and in a more preferred embodiment, by at least 50-fold, relative to the corresponding wild-type enzyme.

[0135] Examples of cysE variants encoding CysE enzymes with reduced feedback inhibition and preferred embodiments in the context of the present invention are:

[0136] - cysE mutations: mutations in the sequence region from position 97 to and including position 273.

[0137] - cysE deletions: deletions in the sequence region from position 248 to and including position 259.

[0138] Specific mutations that are preferred embodiments in the context of the present invention are:

[0139] - cysEII: G238S, a glycine at position 238 is mutated to serine.

[0140] - cysEIII: G165D, a glycine at position 165 is mutated to aspartic acid.

[0141] -cysEIV: A237V, where alanine at position 237 is mutated to valine, and G238S, where glycine at position 238 is mutated to serine (double mutant).

[0142] -cysEV: A237V, where alanine at position 237 is mutated to valine, and G238S, where glycine at position 238 is mutated to serine, and M256I, where methionine at position 256 is mutated to isoleucine (triple mutant).

[0143] -cysEVI: G238S, where glycine at position 238 is mutated to serine, and M256I, where methionine at position 256 is mutated to isoleucine (double mutant).

[0144] -cysEVII: A237V, where alanine at position 237 is mutated to valine.

[0145] -cysEVIII: M256I, where methionine at position 256 is mutated to isoleucine, and A237V, where alanine at position 237 is mutated to valine (double mutant).

[0146] -cysEX: T167A, where threonine at position 167 is mutated to alanine (used in vector pCys, Example 1).

[0147] -cysEXI: T167A, where threonine at position 167 is mutated to alanine, and G245S, where glycine at position 245 is mutated to serine (double mutant).

[0148] -cysEXII: K97Q, where lysine at position 97 is mutated to glutamine, and G238S, where glycine at position 238 is mutated to serine, and F267L, where phenylalanine at position 267 is mutated to leucine (triple mutant).

[0149] -cysEXIII: V164A, where valine at position 164 is mutated to alanine, and F267L, where phenylalanine at position 267 is mutated to leucine (double mutant).

[0150] -cysEXIV: T167A, where threonine at position 167 is mutated to alanine, and M256Stop, where methionine at position 256 is mutated to Stop (double mutant).

[0151] -cysEXVI: D250G, where aspartic acid at position 250 is mutated to glycine.

[0152] -cysEXVII: G165D, where glycine at position 165 is mutated to aspartic acid, and T167A, where threonine at position 167 is mutated to alanine (double mutant).

[0153] -cysEXXIII: T167A, where threonine at position 167 is mutated to alanine, A237V, where alanine at position 237 is mutated to valine, and G238S, where glycine at position 238 is mutated to serine (triple mutant).

[0154] -cysE M256X: methionine at position 256 of the WT CysE enzyme is mutated to X, where X can be any natural amino acid other than methionine.

[0155] -cysE Arabidopsis (plant): Arabidopsis contains three cysE genes, two of which are feedback resistant to cysteine.

[0156] -cysE M256I: methionine at position 256 of the WT CysE enzyme is mutated to isoleucine.

[0157] and / or

[0158] e) Compared to the parental strain (host strain), the production strain includes at least one highly expressed cds e (overexpressed) or an additional cds e encoding a cysteine efflux protein (-efflux protein), which results in an increased expression of the cysteine efflux protein, such that in the cells of the production strain, the cysteine exported from the cells is increased by at least 2-fold relative to the corresponding parental strain (host strain), where the cysteine export can be determined by photometric measurement of the extracellular cysteine content (including cysteine, cystine, and the adduct (R)-2-methylthiazolidine-2,4-dicarboxylic acid formed from cysteine and pyruvate) according to "Gaitonde, Biochem. J. (1967) 104:627 - 633", as described, for example, in US 5,972,663B.

[0159] The increased expression of the cysteine efflux protein results in the cysteine exported from the cells being preferably increased by at least 5-fold, particularly preferably by at least 10-fold, and especially preferably by at least 20-fold relative to the parental strain (host strain).

[0160] The cds encoding the cysteine efflux protein is preferably one or more sequences selected from the group consisting of ydeD, yfiK, cydDC, bcr, and emrAB of Escherichia coli, or sequences that are at least 80%, particularly preferably at least 90%, and especially preferably at least 95% identical.

[0161] The cds that causes biosynthesis of hypotaurine and the cds that causes deregulation of the cysteine biosynthesis pathway are located extrachromosomally on a vector in a microbial strain, combined on one vector, or separated on multiple vectors.

[0162] In a preferred embodiment, the method for producing hypotaurine is characterized in that the cds (a) encoding CDO and the cds (b) encoding CSAD are present together with the cds (c) encoding SerA with reduced feedback inhibition of serine in a tricistronic expression unit in an expression vector of a microbial production strain. The tricistronic expression unit is particularly preferably functionally linked to a serA promoter, and the serA promoter particularly preferably has the sequence specified in SEQ ID NO: 7. This means that the expression of the above three cds is regulated by only one promoter (i.e., there is a tricistronic operon). The 3 cds are separate and follow each other, and each cds has its own RBS. They are expressed in a coordinated manner under the regulation of the same promoter (preferably the serA promoter), but the result is 3 separate proteins rather than a fusion protein.

[0163] Particularly preferably, the order in the operon is such that after the promoter (preferably the serA promoter) is the serA cds in the tricistronic operon, then the CDO cds, and finally the CSAD cds. SerA is preferably serA317. SerA317 is disclosed in Bell et al., Eur. J. Biochem. (2002) 269:4176 - 4184, where it is called "NSD:317" and encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. The tricistronic expression unit consisting of serA, CDO, and CSAD in the present invention allows coupling of L-cysteine and hypotaurine metabolism through their coordinated expression under the control of the serA promoter.

[0164] For example, the expression vectors pCys-CDOrn-CSADhs( Figure 2 ) and pCys-CDOrn-CSADcc( Figure 3) In which, the expression units serA317, CDOrn and CSADhs, and serA317, CDOrn and CSADcc respectively form artificial tricistronic operons, in which the 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 cds encoding the CDOrn enzyme, which is derived from the mRNA of the gene sequence obtainable from Rattus norvegicus (rn), and in the gene construct pCys-CDOrn-CSADhs, the expression of the cds encoding the CSADhs enzyme, which is derived from the mRNA of the gene sequence obtainable from Homo sapiens (hs), and in the gene construct pCys-CDOrn-CSADcc, the expression of the cds encoding the CSADcc enzyme, which is derived from the mRNA of the gene sequence obtainable from Cyprinus carpio (cc).

[0165] A particularly preferred gene construct comprising a cds (c) encoding a SerA having reduced feedback inhibition of serine and functionally linked to a serA promoter, a cds (d) encoding a cysE having reduced feedback inhibition of cysteine and functionally linked to a cysE promoter, and a cds (e) encoding a cysteine efflux protein functionally linked to the promoter of the Escherichia coli GAPDH gene (GAPDH: glyceraldehyde 3-phosphate dehydrogenase) is the vector pCys disclosed in WO 2021 / 259491 (Wacker) and described in Example 1 Figure 1 ). If pCys is transformed into a suitable host strain, a production strain with a deregulated cysteine biosynthesis pathway and thus suitable for cysteine production is obtained (see, for example, WO 2021 / 259491, Wacker).

[0166] Particularly preferably, the method for producing hypotaurine is characterized in that the cds (a) encoding CDO, the cds (b) encoding CSAD, the cds (c) encoding SerA having reduced feedback inhibition of serine, the cds (d) encoding CysE having reduced feedback inhibition of cysteine, and the cds (e) encoding a cysteine efflux protein are all present on a single expression vector. Particularly preferably, the method for producing hypotaurine is characterized in that the expression vector of the microbial production strain is selected from the gene constructs pCys-CDOrn-CSADhs and pCys-CDOrn-CSADcc. The gene constructs are respectively disclosed in Example 1 Figure 2 and Figure 3 ). In a particularly preferred embodiment, the expression vector of the microbial production strain is the gene construct pCys-CDOrn-CSADcc.

[0167] Thus, the gene construct according to the invention comprises a feedback-resistant allele of the cysE gene and / or the serA gene and / or a gene for a cysteine efflux protein such as preferably the ydeD efflux gene, and also a gene encoding a CDO enzyme and a CSAD enzyme, the CDO gene and the CSAD gene, each having its own RBS, linked in a polycistronic unit to the cysE gene, the serA gene and / or the cysteine efflux gene, such as preferably the ydeD gene. A preferred tricistronic operon, possible constructs include the following:

[0168] a) serA-CDO-CSAD

[0169] b) serA-CSAD-CDO

[0170] c) cysE-CDO-CSAD

[0171] d) cysE-CSAD-CDO

[0172] e) ydeD-CDO-CSAD

[0173] f) ydeD-CSAD-CDO.

[0174] a) and b) are preferably functionally linked to the serA promoter in the tricistronic operon, c) and d) are preferably functionally linked to the cysE promoter in the tricistronic operon, and e) and f) are preferably functionally linked to the promoter of the Escherichia coli GAPDH gene in the tricistronic operon, although a) to f) can be functionally linked to any other promoter active in Escherichia coli.

[0175] A gene construct preferably comprising a tricistronic operon, the tricistronic operon comprising

[0176] a) serA-CDO-CSAD or

[0177] b) serA-CSAD-CDO.

[0178] Particularly preferred is a gene construct comprising a tricistronic operon, the tricistronic operon comprising

[0179] a) serA-CDO-CSAD.

[0180] As described in Example 1 of the present invention, the vector pCys described in WO 2021 / 259491 (Wacker) Figure 1)Extended from the gene of cysteine dioxygenase (CDOrn) from Rattus norvegicus, SEQ ID NO: 1, encoding a protein with amino acid sequence SEQ ID NO: 2, for L-cysteine sulfinic acid decarboxylase (CSADhs) from Homo sapiens, SEQ ID NO: 3, nt 1 to nt 1509, encoding a protein with amino acid sequence SEQ ID NO: 4, or for L-cysteine sulfinic acid decarboxylase (CSADcc) from Cyprinus carpio, SEQ ID NO: 5, nt 1 to nt 1503, encoding a protein with amino acid sequence SEQ ID NO: 6. The result is the vectors pCys-CDOrn-CSADhs ( Figure 2 ) and pCys-CDOrn-CSADcc ( Figure 3 ). Gene constructs are produced according to the prior art and preferably by means of conventional recombinant DNA techniques, as described in Example 1 and familiar to those skilled in the art. In pCys-CDOrn-CSADhs, the CDOrn gene and the CSADhs gene are cloned in the form of an artificial tricistronic operon, each having its own ribosome binding site (RBS) after the serA317 gene present in pCys, but not having its own promoter, such that their expression occurs under the control of the serA promoter. In pCys-CDOrn-CSADcc, the CDOrn gene and the CSADcc gene are cloned in the form of an artificial tricistronic operon, each having its own ribosome binding site (RBS) after the serA317 gene present in pCys but not having its own promoter, such that their expression occurs under the control of the serA promoter.

[0181] Production strains are generated in a known manner by transforming the gene constructs according to the invention into a microorganism (host strain). Preferably, the gene construct pCys-CDOrn-CSADhs or the gene construct pCys-CDOrn-CSADcc, and particularly preferably the gene construct pCys-CDOrn-CSADcc, is transformed into the host strain Escherichia coli K12 W3110.

[0182] As disclosed in the embodiments of the present invention, compared with the prior art, it is characterized in that the fermentation of the production strain according to the present invention by the coordinated expression of the previously unknown CDO gene and CSAD gene under the control of the serA promoter derived from cysteine metabolism through a polycistronic operon, preferably a tricistronic operon, allows unexpectedly much higher hypotaurine production. In the prior art, for example, as disclosed in WO 17 / 213142A1 (Ajinomoto), the hypotaurine production in Escherichia coli is up to 450 μM, corresponding to 49.1 mg / L of hypotaurine, where the molecular weight of hypotaurine is 109.2 g / mol. Honjo et al. (2010), Amino Acids 38: 173 - 1183 only described the intracellular production of hypotaurine in yeast, with a yield of 85.3 μmol / g dry biomass, corresponding to 9.3 mg / g of hypotaurine, where the molecular weight of hypotaurine is 109.2 g / mol. However, the growth medium must receive the addition of L-cysteine as a substrate for hypotaurine biosynthesis (see "Materials and Methods" in Honjoh et al.).

[0183] Moreover, the prior art only described the production yields of taurine (the oxidation product of hypotaurine). For example, US2019 / 0062757 A1 (KnipBio) disclosed a taurine production of 419 ng / ml (Escherichia coli), while Joo et al. (2018), J. Agric. Food Chem. 66: 13454 - 13463 mentioned a taurine production of up to 0.5 g / L of taurine in Corynebacterium glutamicum.

[0184] Preferably, the method for fermentative production of hypotaurine is characterized in that the fermentation volume is at least 1 L, a production scale of at least 10 L is particularly preferred, a production scale of at least 1000 L is especially preferred, and a fermentation volume of at least 10,000 L is specifically preferred.

[0185] The content of hypotaurine can be quantified from the culture broth. This is accomplished by, for example, taking 1 ml aliquots of the culture broth with a cell density OD 600 / ml of at least 1.0 / ml, and incubating it at 80 °C for 5 min, then removing all solid components, for example, by centrifuging at maximum speed in a tabletop centrifuge for 5 minutes, and quantifying the supernatant by HPLC calibrated for hypotaurine and taurine, as described for hypotaurine and taurine especially in Example 3.

[0186] In the process according to the invention, at the end of fermentation after a fermentation time of preferably at most 65 h, the yield of the product hypotaurine is preferably at least 10 g / L, particularly preferably at least 20 g / L and very particularly preferably at least 50 g / L, far greater than that known in the prior art (e.g., 49.1 mg / L hypotaurine in WO 17 / 213142A1). Thus, for applications in the food sector, feed sector and pharmaceutical sector, the process according to the invention allows the fermentative production of hypotaurine at yields previously unknown.

[0187] Another feature of the fermentation process according to the invention is that it allows the production of a high molar excess of hypotaurine relative to the by-product taurine. The molar excess is defined as the quotient of the molar concentration of hypotaurine in the fermentation supernatant at the end of fermentation divided by the molar concentration of taurine in the simultaneous fermentation supernatant. 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 very particularly preferably at least 20:1.

[0188] The media for the production strain in shake flasks and by fermentative growth are familiar to the person skilled in the art from the practice of microbial cultivation. They generally consist of a carbon source, a nitrogen source and additives such as vitamins, salts and trace elements, as well as a sulfur source which optimizes cell growth and hypotaurine production.

[0189] The carbon sources are those which can be used by the production strain for the formation of the hypotaurine product. 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, and all conceivable disaccharides and polysaccharides formed therefrom, such as sucrose, lactose, maltose, maltodextrin, starch, and monomers or oligomers released therefrom (enzymatically or chemically) by hydrolysis. In addition to sugars or carbohydrates, other available carbon sources are acetic acid (or acetates derived therefrom), ethanol, glycerol, citric acid (and its salts) or pyruvic acid (and its salts). However, gaseous carbon sources such as carbon dioxide or carbon monoxide are also conceivable.

[0190] Preferred carbon sources for the growth of the production strain are glucose, fructose, sucrose, mannose, xylose and arabinose, with glucose and sucrose being particularly preferred and glucose being very particularly preferred.

[0191] The nitrogen sources are those which can be used by the production strain for the formation of biomass. Examples of those which can be used for this purpose include:

[0192] - ammonia in gaseous form or in aqueous solution such as NH4OH, or its salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate or ammonium nitrate, and / or

[0193] - Known nitrates such as KNO3, NaNO3, ammonium nitrate, Ca(NO3)2, Mg(NO3)2 and other nitrogen sources such as urea, and / or

[0194] - A complex mixture of amino acids, such as yeast extract, peptone, malt extract, soy peptone, casein amino acids, corn steep liquor (in liquid form or in dry form, so-called CSD), and / or

[0195] - NZ amine, and / or

[0196] - Yeast nitrogen base.

[0197] For the effective production of hypotaurine, it is necessary to meter in a sulfur source, either as a one-time addition in batch form or as a continuous feed. The continuous metering can be carried out as a pure feed solution or as a mixture with other feed components (such as glucose). Preferably, the method is characterized in that it is carried out in the presence of at least one compound selected from the group consisting of: sulfates, sulfites, dithionites, thiosulfates, and salts of sulfides, and the use of the corresponding acids can also be envisaged under given stability conditions. Preferred sulfur sources are sulfates, sulfites, thiosulfates, and salts of sulfides, with sulfates and thiosulfates being particularly preferred. Especially preferably, the method is characterized in that it is carried out in the presence of thiosulfate. Particularly preferred as thiosulfate are compounds selected from the group consisting of: sodium thiosulfate, ammonium thiosulfate, and mixtures thereof.

[0198] Growth (culturing of microbial cells) can be carried out in a so-called batch mode, i.e., by inoculating a growth medium (culture medium) with a starter culture of a production strain (microbial cells carrying one or more gene constructs) and then allowing the cells to grow without further supply of a nutrient source to obtain biomass. Growth can also be carried out in a so-called fed-batch mode (also referred to as growth in fed mode), i.e., a method for obtaining biomass in which an additional supply (feed) of a nutrient source follows the initial stage of growth in batch mode. The feed can consist of a carbon source, a nitrogen source, a sulfur source, one or more vitamins or trace elements important for production, or a combination of the foregoing. The feed components can be metered together as a mixture or alternatively separately in separate feed sections. In addition, other culture medium components, as well as additives that particularly increase the production of hypotaurine, can be added to the feed. The feed can be provided continuously or partially (discontinuously), or as a combination of continuous feed and discontinuous feed. Preferably, the method for fermentatively producing hypotaurine using a microbial production strain according to the present invention is characterized in that the fermentation method is a fed-batch method.

[0199] Preferred carbon sources in the feed are glucose, sucrose, and plant hydrolysates containing glucose or sucrose, as well as mixtures of the preferred carbon sources in any mixing ratio. A particularly preferred carbon source in the feed is glucose.

[0200] Preferably, the carbon source is metered into the culture such that the content of the carbon source during the production phase in the fermenter 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.

[0201] Preferred nitrogen sources in the feed are ammonia in gaseous form or in the form of an aqueous NH4OH solution, and its salts: ammonium sulfate, ammonium phosphate, ammonium acetate, and ammonium chloride, as well as additionally urea, KNO3, NaNO3, and ammonium nitrate, yeast extract, peptone, malt extract, soy peptone, casein amino acids, corn steep liquor, and also NZ amine and yeast nitrogen base, among which ammonia or ammonium salts, yeast extract, soy peptone, or corn steep liquor (in liquid form or in dry form) are particularly preferred.

[0202] Preferred sulfur sources in the feed are salts of sulfates, sulfites, thiosulfates, and sulfides, among which sulfates and thiosulfates are particularly preferred, and thiosulfates such as sodium thiosulfate and ammonium thiosulfate are particularly preferred.

[0203] Additional medium supplements that can be added are salts of elemental phosphorus, chlorine, sodium, magnesium, nitrogen, potassium, calcium, and iron, as well as salts of trace (i.e., at μM concentration) elements molybdenum, boron, cobalt, manganese, zinc, copper, and nickel. In addition, organic acids (e.g., acetic acid, citric acid), amino acids (e.g., isoleucine), and vitamins (e.g., vitamin B1, vitamin B6) can be added to the medium.

[0204] Growth is carried out under pH and temperature conditions that promote the growth of the production strain and taurine production. The pH range is preferably in the range of pH 5 to pH 9. A pH range from pH 5.5 to pH 8 is particularly preferred. A pH range from pH 6.0 to pH 7.5 is especially preferred.

[0205] The preferred temperature range for the growth of the production strain is 20°C to 40°C. The temperature range is particularly preferably 25°C to 37°C, and especially preferably 28°C to 34°C.

[0206] The growth of the production strain can optionally occur without oxygen supply (anaerobic culture) or with oxygen supply (aerobic culture). Aerobic culture with oxygen is preferred.

[0207] In the case of aerobic cultivation of the strains according to the invention for hypotaurine production, a saturation of preferably at least 10% (v / v), particularly preferably at least 20% (v / v) and very particularly preferably at least 30% (v / v) is set for the oxygen content. According to the prior art, the oxygen saturation in the culture is automatically regulated by a combination of gas supply and stirring speed.

[0208] The supply of oxygen 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 in aerobic cultivation preferably ranges from 0.05 vvm to 10 vvm (vvm: specified as liters of compressed air introduced into the fermentation batch per minute per liter of fermentation volume). Particularly preferably, 0.2 vvm - 8 vvm of compressed air is introduced, very particularly preferably 0.4 - 6 vvm of compressed air is introduced, and specifically preferably 0.8 - 5 vvm of compressed air is introduced.

[0209] The maximum stirring speed is preferably 2500 rpm, particularly preferably at most 2000 rpm and very particularly preferably at most 1800 rpm.

[0210] 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 very particularly preferred.

[0211] In the process for the fermentative production of hypotaurine according to the invention, in step i, the microbial production strain according to the claims is cultivated and in step ii, the fermentation supernatant is separated. This means that the fermentation batch obtained by the described process contains hypotaurine that has accumulated extracellularly in the fermentation supernatant. Hypotaurine can be used further directly or can be separated from the fermentation medium. Various analytical methods are available for the identification, quantification and determination of the purity of hypotaurine, including spectrophotometry, NMR, gas chromatography, HPLC, mass spectrometry, gravimetry or combinations of these analytical methods. Description of the Drawings

[0212] The drawings show the plasmids used in the examples.

[0213] Figure 1 : pCys.

[0214] Figure 2 : pCys - CDOrn - CSADhs.

[0215] Figure 3 : pCys - CDOrn - CSADcc.

[0216] Abbreviations used in the drawings:

[0217] TetR: gene conferring tetracycline resistance

[0218] P15A ORI: Replication origin

[0219] serA317: serA (3 - phosphoglycerate dehydrogenase gene encoding amino acids 1 to 317) cds

[0220] cysE X: cysE (serine O - acetyltransferase gene, feedback resistant) cds

[0221] ORF306: ydeD (cysteine efflux gene) cds

[0222] ScaI: Cleavage site of restriction enzyme ScaI

[0223] PpuMI: Cleavage site of restriction enzyme PpuMI

[0224] CDOrn: CDO (cysteine dioxygenase) Rattus norvegicus cds

[0225] CSADhs: CSAD (cysteine sulfinic acid decarboxylase) Homo sapiens cds

[0226] CSADcc: CSAD (cysteine sulfinic acid decarboxylase) Cyprinus carpio cds

[0227] RBS: Ribosome binding site Detailed implementation manners

[0228] The following examples are used to further illustrate the present invention:

[0229] Example 1: The production vectors pCys - CDOrn - CSADhs and pCys - CDOrn - CSADcc of pCys - CDOrn - CSADhs and pCys - CDOrn - CSADcc are derived from the vector pCys( Figure 1 ).

[0230] 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 not only a replication origin and a tetracycline resistance gene (parent vector pACYC184), but also a cysEX allele (synonym: cysE allele) that encodes a serine O - acetyltransferase with reduced feedback inhibition of cysteine and whose expression is controlled by the cysE promoter, and also contains an efflux gene ydeD (ORF306) whose expression is controlled by a constitutive GAPDH promoter derived from the Escherichia coli GAPDH gene.

[0231] In addition, pCys additionally contains the serA317 gene fragment, which was cloned after the ydeD (ORF306) efflux gene and encodes the N-terminal 317 amino acids of the SerA protein (total length: 410 amino acids). The Escherichia 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, where it is called "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 disclosed in Rex et al. (1991), J. Bacteriol. 173:5944-5953, Fig. 2, nt 1-nt 457.

[0232] For cloning purposes, the plasmid DNA of vector pCys was cut with ScaI and PpuMI and the 6.1 kb vector fragment (hereinafter referred to as pCys-ScaI / PpuMI) was isolated by preparative agarose gel electrophoresis ( Gel Extraction Kit, Qiagen).

[0233] CDOrn: The amino acid sequence of cysteine dioxygenase from Rattus norvegicus of the mRNA derived from this gene is disclosed in the NCBI (National Center for Biotechnology Information) database with sequence ID: AAH70509.1. This amino acid sequence was used to derive a DNA sequence codon-optimized for expression in Escherichia coli (the publicly available Eurofins Genomics GENEius software), and said DNA sequence was synthesized and produced (Eurofins Genomics). The CDOrn DNA sequence is disclosed in SEQ ID NO:1 and encodes a protein with the amino acid sequence of SEQ ID NO:2. The CDOrn DNA was synthesized and produced (Eurofins Genomics).

[0234] CSADhs-rrnB:

[0235] 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. CSADhs is deposited there as "acidic amino acid decarboxylase GADL1 isoform X1". This amino acid sequence was used to derive a codon-optimized DNA sequence for expression in Escherichia coli (the publicly available Eurofins Genomics GENEius software). This DNA sequence, named CSADhs cds, is disclosed in SEQ ID NO: 3, nt 1 to 1509, and encodes a protein with the amino acid sequence of SEQ ID NO: 4. The DNA sequence of the Escherichia coli rrnB terminator (SEQ ID NO: 3, nt 1510 to 1842) was ligated 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 consisting of CSADhs cds and the rrnB terminator, disclosed in SEQ ID NO: 3, was synthetically produced (Eurofins Genomics) and named CSADhs-rrnB.

[0236] CSADcc-rrnB: The cDNA gene of cysteine sulfinic acid decarboxylase (CSAD) from common carp (Cyprinus carpio) was isolated by Honjoh et al. (2010), Amino Acids 38:1173-183 (cDNA: complementary DNA, isolated from mRNA by reverse transcription), and this DNA sequence is disclosed in the NCBI database under GenBank sequence ID: AB220585.1 (cds: nt 82-1584). This amino acid sequence was used to derive a codon-optimized DNA sequence for expression in Escherichia coli (the publicly available Eurofins Genomics GENEius software). This DNA sequence, named CSADcc cds, is disclosed in SEQ ID NO: 5, nt 1 to 1503, and encodes a protein with the amino acid sequence of SEQ ID NO: 6. The 3' end of CSADcc cds was ligated to the DNA sequence of the Escherichia coli rrnB terminator (SEQ ID NO: 5, nt 1504 to 1834). The DNA sequence of the rrnB terminator is disclosed in Orosz et al. (1991); see above. The CSADcc-rrnB DNA consisting of CSADcc cds and the rrnB terminator was synthetically produced (Eurofins Genomics).

[0237] Primers used:

[0238] cdorn-1f (SEQ ID NO: 8)

[0239] csadhs-2r (SEQ ID NO: 9)

[0240] csadhs-3f (SEQ ID NO: 10)

[0241] glf-2r (SEQ ID NO: 11)

[0242] cdorn-3r (SEQ ID NO: 12)

[0243] csadcc-1f (SEQ ID NO: 13)

[0244] PCR products:

[0245] Table 1 outlines the PCR products used for cloning in terms of the name and size of the PCR products and the synthetic DNA (so-called template DNA) and primers used. Using appropriate primers, PCR products PCR1 to PCR4 were produced from the corresponding template DNA by PCR (“Phusion TM High-Fidelity” DNA polymerase, Thermo Scientific TM ) and separated by agarose gel electrophoresis ( Gel Extraction Kit, Qiagen).

[0246] Table 1: PCR products for the production of gene constructs

[0247]

[0248] Production of vector pCys-CDOrn-CSADhs:

[0249] By using a cloning kit (NEB New England Biolabs), vector pCys-CDOrn-CSADhs was produced. According to the manufacturer's instructions regarding the amounts of the individual DNA fragments used in the ligation mixture, the 6.1 kbp Cys-ScaI / PpuMI vector fragment was ligated together with PCR1 and PCR2. Thereafter, the ligation mixture was transformed into Escherichia coli according to the manufacturer's instructions In 10-β (NEB New England Biolabs). Select clones from the transformation on LBtet plates. The LBtet plates contain 10 g / L tryptone (GIBCO TM ), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl, 15 g / L agar, and 15 mg / L tetracycline (Sigma-Aldrich). Isolate plasmid DNA (Qiagen Kit) from the transformed clones and select the correct clones. Name the cloned plasmid DNA as vector pCys-CDOrn-CSADhs( Figure 2 ).

[0250] Production of vector pCys-CDOrn-CSADcc:

[0251] By using a cloning kit (NEB New England Biolabs) to produce vector pCys-CDOrn-CSADcc. Link the 6.1 kbp Cys-ScaI / PpuMI vector fragment with PCR3 and PCR4 according to the manufacturer's instructions regarding the amounts of the individual DNA fragments used in the ligation mixture. Thereafter, transform the ligation mixture into Escherichia coli 10-β (NEB New England Biolabs) according to the manufacturer's instructions. Select clones from the transformation on LBtet plates. Isolate plasmid DNA (Qiagen Kit) from the transformed clones and select the correct clones. Name the cloned plasmid DNA as vector pCys-CDOrn-CSADcc( Figure 3 ).

[0252] Example 2: Production of the production strain

[0253] The parental strain (host strain) used for producing the production strain is the microbial strain Escherichia coli K12 W3110 (commercially available from DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the strain number DSM 5911). Transform Escherichia coli W3110 with vector pCys-CDOrn-CSADhs or vector pCys-CDOrn-CSADcc in a known manner and select the transformants on LBtet plates. Select one transformant each as the production strain. Name these production strains as Escherichia coli W3110 x pCys-CDOrn-CSADhs and Escherichia coli W3110 x pCys-CDOrn-CSADcc, and use them for the production of hypotaurine.

[0254] Example 3: Production of hypotaurine in shake flasks

[0255] Pre-cultures: Pre-cultures of the production strain Escherichia coli W3110 x pCys - CDOrn - CSADhs and the production strain Escherichia coli W3110 x pCys - CDOrn - CSADcc were prepared in LBtet medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L tetracycline) (grown overnight at 37 °C and 120 rpm).

[0256] Main cultures: 0.5 ml of each pre-culture was transferred to a 300 ml baffled conical flask containing 30 ml of SM1 medium supplemented with 15 g / L glucose, 2 g / L Na2S2O3·5H2O, 0.1 g / L L-isoleucine, 0.1 g / L D,L-methionine, 0.1 g / L L-threonine, 5 mg / L vitamin B1 and 15 mg / L tetracycline.

[0257] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L NH4 sulfate, 0.3 g / L MgSO4·7H2O, 0.015 g / L CaCl2·2H2O, 0.002 g / L FeSO4·7H2O, 1 g / L sodium citrate·2H2O, 0.1 g / L NaCl; 1 ml / L trace element solution.

[0258] Composition of the trace element solution: 0.15 g / L Na2MoO4·2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2·6H2O, 0.25 g / L CuSO4·5H2O, 1.6 g / L MnCl2·4H2O, 0.3 g / L ZnSO4·7H2O.

[0259] The main cultures were incubated for 24 h at 30 °C and 140 rpm in an incubator shaker (Infors). After 24 h, 1 ml of sample was taken and the cell density OD was measured using a Genesys 10S UV / visible spectrophotometer from Thermo Scientific TM Genesys TM 10S UV / visible spectrophotometer 600OD / ml (optical density of the main culture, measured photometrically at 600 nm), and the contents of hypotaurine and taurine were determined by HPLC. For the strain Escherichia coli W3110 x pCys-CDOrn-CSADhs, the content of hypotaurine was 54.9 mg / L. The content of taurine was 44.6 mg / L. For the strain Escherichia coli W3110 x pCys-CDOrn-CSADcc, the content of hypotaurine was 158.8 mg / L. The content of taurine was 8.7 mg / L.

[0260] Sample preparation for the quantification of hypotaurine and taurine by HPLC:

[0261] 1 ml of the culture broth from shake flask growth or fermentation was incubated at 80 °C for 5 minutes and then centrifuged at 13,000 rpm for 5 minutes (Heraeus TM Fresco TM 21 centrifuge). The cell culture supernatant was separated. The contents of hypotaurine and taurine in the cell culture supernatant from shake flask growth were directly analyzed by HPLC. The cell culture supernatant from fermentation was diluted 10-fold in H2O and the contents of hypotaurine and taurine were analyzed by HPLC.

[0262] HPLC analysis of L-cysteine sulfinic acid, hypotaurine and taurine: For the quantitative determination of the compounds quantitatively analyzed in the examples and for L-cysteine sulfinic acid used for the detection of CDO enzyme activity, an HPLC method calibrated for L-cysteine sulfinic acid, hypotaurine and taurine respectively was employed; the reference substances for calibration were commercially available (Sigma-Aldrich). An Agilent 1260 Infinity II HPLC system was used, which was equipped with units from the same manufacturer for pre-column derivatization with o-phthalaldehyde (OPA derivatization), as known from amino acid analysis. To detect the OPA-derived products of hypotaurine and taurine, the HPLC system was equipped with a fluorescence detector. The detector was set at an excitation wavelength of 330 nm and an emission wavelength of 450 nm. An Accucore TM aQ column from Thermo Scientific TM with a length of 100 mm, an inner diameter of 4.6 mm and a particle size of 2.6 μm was thermally equilibrated in a column oven at 40 °C.

[0263] Eluent A: 25 mM sodium phosphate, pH 6.0. Eluent B: methanol. Separation was carried out in gradient mode: 10% Eluent B to 60% Eluent B over 0 - 25 minutes, then 60% Eluent B to 100% Eluent B over 2 minutes, then 100% Eluent B for an additional 2 minutes, at a flow rate of 0.5 ml / min. Retention time of L-cysteine sulfinic acid: 4.1 minutes. Retention time of taurine: 14.8 min. Retention time of hypotaurine: 15.7 minutes.

[0264] Example 4: Production of hypotaurine by fermentation

[0265] Pre-culture 1:

[0266] The production strains Escherichia coli W3110 x pCys-CDOrn-CSADhs and Escherichia coli W3110 x pCys-CDOrn-CSADcc were each inoculated into 20 ml of LBtet medium in a 100 ml conical flask and incubated on a shaker (150 rpm, 30 °C) for 7 hours.

[0267] Pre-culture 2:

[0268] Thereafter, each of the pre-cultures 1 was completely transferred to 100 ml of SM1 medium supplemented with 5 g / L glucose, 5 mg / L vitamin B1 and 15 mg / L tetracycline (for the composition of the SM1 medium, see Example 3). The cultures were each shaken in a conical flask (1 L volume) at 30 °C at 150 rpm for 17 h (Infors incubator shaker). After this incubation, the cell density OD 600 / ml was each 3 to 5.

[0269] Main culture:

[0270] Fermentation was carried out in a Biostat B fermenter (2 l working volume) from Sartorius BBI Systems GmbH.

[0271] The growth medium (900 ml) contains 15 g / L glucose, 10 g / L tryptone (Difco), 5 g / L yeast extract (Difco), 2.4 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.25 g / L NaCl, 0.6 g / L MgSO4 x 7H2O, 0.03 g / L CaCl2 x 2H2O, 0.15 g / L FeSO4 x 7H2O, 1 g / L sodium citrate x 2H2O, 1 ml of trace element solution (see Example 3), 0.9 g / L L-isoleucine (Sigma-Aldrich), 0.6 g / L D,L-methionine (Sigma-Aldrich), 0.018 g / L vitamin B1 (Sigma-Aldrich), 0.09 g / L pyridoxine x HCl (vitamin B6, Sigma-Aldrich), and 15 mg / L tetracycline.

[0272] The pH in the fermenter was initially adjusted to 7.0 by pumping in 25% NH4OH solution. During fermentation, the pH was maintained at a value of 7.0 by automatic correction with 25% NH4OH or 4M H3PO4. Foam control was achieved by automatically metering in 4% v / v Struktol J673 (Schill&Seilacher) in H2O.

[0273] For inoculation, 100 ml of preculture 2 was pumped into the fermenter vessel in each case. Thus, the initial volume was 1 L. The culture was initially stirred at 400 rpm and aerated with compressed air sterilized by a sterile filter at an aeration rate of 2 vvm (vvm: specifies the compressed air introduced into the fermentation batch 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.

[0274] The target value for O2 saturation during fermentation was set at 30%. After the O2 saturation had dropped below the target value, the regulatory cascade was initiated to restore the O2 saturation to the target value. This involved first continuously increasing the gas supply (up to a maximum of 5 vvm) and then continuously increasing the stirring speed (up to a maximum of 1500 rpm).

[0275] Fermentation was carried out at a temperature of 30 °C. After 2 h of fermentation time, the sulfur source in the form of a sterile 60% (w / v) stock solution of sodium thiosulfate x 5H2O was fed at a rate of 1.5 ml / hour.

[0276] Once the glucose content in the fermenter decreased from the initial 15 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was metered in continuously. The feed rate was adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L from then on. Glucose was determined using a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0277] After fermentation times of 65 h, 22 h, 40 h, and 65 h after the start of fermentation, samples were taken from the fermentation batch, and then the cell density OD 600 / ml was determined by HPLC and the contents of hypotaurine and taurine in the culture supernatant were determined. The results are summarized in Tables 2 and 3.

[0278] For the strain W3110 x pCys-CDOrn-CSADhs, after 65 h of fermentation time, the hypotaurine production was 20.8 g / L (190.5 mM, the molecular weight of hypotaurine is 109.2 g / mol), and the taurine production was 7.4 g / L (59.1 mM, the molecular weight of taurine is 125.2 g / mol). The molar ratio of hypotaurine:taurine was 3.2:1.

[0279] For the strain W3110 x pCys-CDOrn-CSADcc, after 65 h of fermentation time, the hypotaurine production was 62.5 g / L (572.2 mM, the molecular weight of hypotaurine is 109.2 g / mol), and the taurine production was 1.2 g / L (9.6 mM, the molecular weight of taurine is 125.2 g / mol). The molar ratio of hypotaurine:taurine was 59.6:1.

[0280] Table 2: Time course of cell density and hypotaurine and taurine contents in the fermentation of the production strain W3110x pCys-CDOrn-CSADhs

[0281]

[0282] Table 3: Time course of cell density and hypotaurine and taurine contents in the fermentation of the production strain W3110 x pCys-CDOrn-CSADcc

[0283]

[0284] Example 5: Extraction of hypotaurine and taurine from fermenter cells

[0285] Use the fermentation batch of the production strain Escherichia coli W3110 x pCys-CDOrn-CSADcc with a hypotaurine content of 62.5 g / L and a taurine content of 1.2 g / L from Example 4 (Table 3). Centrifuge 2 x 1 ml of the fermentation broth (2 ml total volume) at 13,000 rpm for 5 min (Heraeus TM Fresco TM 21 centrifuge) and discard the supernatant. Resuspend each cell pellet in 1 ml of H2O and centrifuge at 13,000 rpm for 5 min, and discard the supernatant. Suspend each cell pellet in 1 ml of H2O, corresponding to a total volume of 2 ml and corresponding to the volume of the fermentation broth used above. The cell suspension was used to prepare cell extracts. This was done using a FastPrep-24TM 5G cell homogenizer from MP Biomedicals. Disrupt the cell pellet suspended in 1 ml of H2O in a 1.5 ml tube ("Lysing Matrix B") assembled by the manufacturer containing glass beads (3 x 20 s, at a shaking frequency of 6000 rpm, with a pause between each case of 30 s). Combine the obtained cell homogenates and centrifuge at 13,000 rpm for 5 minutes in order to prepare cell extracts. Analyze the contents of hypotaurine and taurine in the cell extracts by HPLC. In the HPLC analysis, no hypotaurine and taurine were detected at the detection limit of 1 mg / L of hypotaurine / taurine.

Claims

1. A method for fermentative production of hypotaurine, characterized in that, i) Culturing a microbial production strain, the microbial production strain being characterized in that it:

1. Comprising at least one expression vector, said expression vector comprising coding sequences (cds) a and cds b and at least one cds selected from the group consisting of c, d, and e, a) a is a cds encoding a cysteine dioxygenase (CDO) belonging to enzyme class EC 1.13.11.20, and b) b is a cds encoding a cysteine sulfinic acid decarboxylase (CSAD) belonging to enzyme class EC 4.1.1.29, and c) c is a cds encoding a 3-phosphoglycerate dehydrogenase (SerA) in which the feedback inhibition by serine is reduced by at least 2-fold relative to the corresponding wild-type enzyme, d) d is a cds encoding a serine O-acetyltransferase (CysE) in which the feedback inhibition by cysteine is reduced by at least 2-fold relative to the corresponding wild-type enzyme, and e) e is a cds encoding a cysteine efflux protein, 2. The expression of cds a and cds b in the said 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. Taurine is secreted by the said production strain into the fermentation supernatant, and ii) separating the said fermentation supernatant, wherein the content of taurine in the said fermentation supernatant is at least 10 g / L, and the molar fraction of taurine:taurine in the said fermentation supernatant is at least 3:

1.

2. The method according to claim 1, characterized in that the host strain used for producing the said microbial production strain is a microorganism of the species Escherichia coli.

3. The method according to claim 1 or 2, characterized in that The CDO is the CDO from Rattus norvegicus (CDOrn) having the sequence specified in SEQ ID NO: 2 or an enzyme having at least 80% identity with CDO activity.

4. The method according to one or more of claims 1 to 3, characterized in that, The CSAD is the CSAD from Cyprinus carpio (CSADcc) having the sequence specified in SEQ ID NO: 6 or an enzyme having at least 80% identity with CSAD activity.

5. The method according to one or more of claims 1 to 3, characterized in that The CSAD is the CSAD from Homo sapiens (CSADhs) having the sequence specified in SEQ ID NO: 4 or an enzyme having at least 80% identity with CSAD activity.

6. The method according to one or more of claims 1 to 5, characterized in that The cds (a) encoding CDO and the cds (b) encoding CSAD in the expression vector of the said microbial production strain are present together with the cds (c) encoding SerA with reduced feedback inhibition by serine in a tricistronic expression unit functionally linked to the serA promoter.

7. The method according to one or more of claims 1 to 6, characterized in that The cds (a) encoding CDO, the cds (b) encoding CSAD, the cds (c) encoding SerA with reduced feedback inhibition by serine, the cds (d) encoding CysE with reduced feedback inhibition by cysteine, and the cds (e) encoding a cysteine efflux protein are all present on a single expression vector.

8. The method 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 and pCys-CDOrn-CSADcc.

9. The method according to one or more of claims 1 to 8, characterized in that, The fermentation volume is at least 1 L.

10. The method according to one or more of claims 1 to 9, characterized in that, The method is carried out in the presence of thiosulfate.

11. The method according to one or more of claims 1 to 10, characterized in that, The fermentation method is a fed-batch method.

12. The method according to one or more of claims 1 to 11, characterized in that, Based on the taurine content in the fermentation batch, the taurine content in the fermentation supernatant is higher than 70%.

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