Method for producing L-sulfoalanine and use thereof

Abstract

The invention relates to a method for producing L-sulfoalanine, comprising the reaction of O-phosphate-L-serine (OPS) with a salt of sulphurous acid (sulfite) and a sulfoalanine synthase (CS enzyme) belonging to the enzyme class EC 2.5. 1.76 by a biotransformation method. The L-sulfoalanine according to the invention may be decarboxylated to form taurine. The invention also relates to the use of the produced L-sulfoalanine for the production of taurine.

Description

[0001] The present invention relates to a method for producing L-cysteic acid, which comprises the reaction of O-phospho-L-serine (OPS) with a salt of sulfurous acid (sulfite) and a cysteic acid synthase (CS enzyme) belonging to the enzyme class EC 2.5.1.76 in a bioconversion. The L-cysteic acid produced according to the present invention can be decarboxylated to form taurine. The present invention also relates to the use of the produced L-cysteic acid for producing taurine.

[0002] L-Sulfoalanine ((R)-2-amino-3-sulfopropionic acid, 3-sulfo-L-alanine, CAS 498-40-8) is a non-proteinogenic L-amino acid that can be detected in nature as an oxidation product of the proteinogenic amino acid L-cysteine, for example in wool. Sulfoalanine is also an intermediate in the biosynthesis of coenzyme M (CoM, 2-mercaptoethanesulfonic acid, CAS 3375-50-6) in methanogenic archaea. In methanogens, the biosynthetic pathway of L-sulfoalanine starts from O-phospho-L-serine (OPS, L-serine-O-phosphate, L-2-amino-3-hydroxypropionic acid 3-phosphate, CAS 407-41-0), which reacts with sulfite under the catalysis of sulfoalanine synthase (CS enzyme) to produce L-sulfoalanine, as shown in formula (1):

[0003] (1) OPS+SO3 2- ->L-Cysteine+HPO4 2-

[0004] Graham et al., Biochem. J. (2009) 424: 467-478, disclose a gene product from Methanoscarcina acetivorans, which is related to threonine synthase, produced recombinantly in E. coli and enriched as enzymatically inactive protein in so-called inclusion bodies, and detectable enzyme activity can only be measured after renaturation of the complex inclusion bodies. After renaturation, in analytical assays (HPLC and mass spectrometry) of the renatured protein, it was possible to demonstrate the production of L-cysteic acid from the reaction of commercially available (chemically synthesized) OPS with sulfite according to formula (1). These findings indicate that the denatured protein from M. acetivorans is related to the activity of cysteic acid synthase and determine its genetic relationship to threonine synthase.

[0005] L-Cysteine ​​can be produced by chemical methods, such as oxidation of cysteine ​​with chlorine in alcoholic solution, bromine in hydrochloric acid or iodine in DMSO, or by oxidative cleavage of cystine. Alternatively, L-Cysteine ​​can also be produced by oxidation of L-cysteic acid. Known chemical production methods for L-Cysteine ​​are not considered sustainable, using environmentally harmful chemicals and have low consumer acceptance, especially for applications in the food, cosmetic and pharmaceutical fields.

[0006] For example, L-cysteic acid can be used in fish farming or in the cosmetics industry, for example as a (DSM) component. In peptide chemistry, L-cysteic acid can be used as a water-soluble protective group. In addition, L-cysteic acid can also be converted into taurine by decarboxylation.

[0007] Graham et al. (2009, supra) showed that when the CS enzyme with a molecular weight of 44 kDa was heterologously expressed in E. coli, it could not be produced in an active form, but was only enriched in inclusion bodies in an inactive form. Dissolution of the inclusion bodies and refolding to form the active enzyme resulted in a very low yield of 3 mg of refolded protein per liter of E. coli culture, and was therefore not suitable as a preparative bioconversion method for the industrial-scale production of L-cysteic acid.

[0008] According to Graham et al. (2009, supra), only when the CS enzyme was expressed as a 103 kDa CS fusion protein, the enzyme activity could be detected without refolding, but the enzyme yield and the specific activity of the enzyme (0.015 U / mg protein) were very low. This prior art shows that active CS enzyme can only be produced in E. coli, which has high production costs and low yields.

[0009] In a metabolic engineering approach, Joo et al. (2018) described a genetically modified and sulfur utilization optimized Corynebacterium glutamicum strain for taurine production in J. Agric. Food Chem. 66:13454–13463 using the CS gene from M. acetivorans. The CS activity of the gene expressed in C. glutamicum was detected by enzyme assay analysis. The activity of the CS enzyme in the taurine-producing strain was inferred indirectly. As shown in Fig. S2 in the Supporting Information of Joo et al. (2018, supra), C. glutamicum cells expressing CS accumulated mainly OPS, the substrate for the CS reaction, intracellularly, in addition to serine, cysteine, and taurine. No details were given about the production of L-cysteic acid. Joo et al. (2018, supra) showed that the simultaneous presence of OPS and CS enzymes in the cell was not sufficient for efficient production of L-cysteic acid, even though other sulfur-containing compounds such as cysteine ​​and taurine were produced intracellularly in measurable quantities in this strain.

[0010] Tevatia et al., Algal Research (2015) 9:21-26, describe the natural production of taurine in microalgae, in which L-cysteic acid was also detected as an intermediate. As shown in Fig. 1 in Tevatia et al. (2015, see above), the biosynthetic pathway of algae is from L-serine to L-cysteic acid ("Cysteate" in Fig. 1). However, this synthesis pathway does not include the enzymatic reaction of the CS enzyme according to formula (1). None of the biosynthetic pathways synthesizes L-cysteic acid through OPS. The intracellular content of L-cysteic acid is very low and is accompanied by a variety of by-products that can increase the difficulty of post-processing, such as methionine, cysteine, cysteinesulfinic acid, hypotaurine and taurine, so the growth of microalgae is not suitable for the production of L-cysteic acid.

[0011] In metabolic engineering methods, US2019 / 0062757A1 (KnipBio) describes strains for heterologous production of taurine or its precursors. Strains expressing CS enzymes and other biosynthetic genes from taurine metabolism of various configurations are described. The yields achieved by hypotaurine and taurine are very low, up to 419 ng / ml. The yield of L-cysteic acid is not mentioned. In this prior art, a CS gene construct is used, but no enzyme assay for detecting CS enzyme activity is performed. In addition, it is not shown that L-cysteic acid can be produced biotechnologically in preparative quantities by this metabolic engineering method. Similarly, the literature does not disclose whether the strains used to produce OPS or CS enzymes are suitable for bioconversion to produce L-cysteic acid according to formula (1).

[0012] Steinfeld et al. describe in ACS Chem. Biol. (2014) 9: 1104-1112 that the intracellular production of OPS is increased in an E. coli strain lacking the serB gene. Increased extracellular production of OPS is not described (see FIG. 5 of Steinfeld et al.).

[0013] EP 2 444 481 B1 of CJ CheilJedang (KR) describes a method for producing L-cysteine ​​using a strain with reduced SerB activity to produce OPS, which is then enzymatically reacted with sulfide or thiosulfate to form L-cysteine. It is not clear whether the enzymes used here, O-phosphoserine sulfhydrylase (OPSS, EC 2.5.1.65), can also utilize sulfite as a substrate in the sense of CS enzymes. In fact, Steiner et al. studied the reaction mechanism of the OPSS enzyme from Mycobacterium tuberculosis in J. Bacteriol. (2014), 196: 3410-3420 and found that, unlike Na2S (sulfide) and Na2S2O3 (thiosulfate), sulfites such as Na2S2O5 and Na2SO3 are not suitable as S donors for the reaction of aminoacrylate intermediates bound to the enzyme, which is derived from the binding of OPS to the OPSS enzyme and the removal of phosphorylation (see Fig. 2 of Steiner et al., 2014, supra). Therefore, the inactivation of the serB gene is disclosed in EP 2 444 481 B1, as is also known from Steinfeld et al. (2014, supra), thereby producing a strain capable of producing OPS. OPS is then used for the bioconversion of the OPSS enzyme to produce cysteine.

[0014] The biotechnological production of OPS is known in the prior art. However, the prior art does not provide a method for producing a CS enzyme which is suitable for providing a sufficiently active CS enzyme for a hitherto unknown industrially viable bioconversion process of OPS with sulfite, wherein L-cysteic acid is produced on a preparative scale for further use.

[0015] Therefore, there is a need for an environmentally friendly and sustainable process for producing L-cysteic acid on a preparative scale, for which a biotechnological process is suitable. Due to the consumer-driven trend away from chemically produced ingredients, the present invention is directed to a biotechnological process for producing L-cysteic acid by bioconversion, which is suitable for use on an industrial scale.

[0016] The object of the present invention is to provide an industrially applicable method for economically and efficiently producing L-cysteic acid by bioconversion of OPS rather than chemical methods and avoiding the use of L-cysteic acid producing strains produced by metabolic engineering, and to use the L-cysteic acid produced thereby for further uses, such as for the production of taurine.

[0017] The above object is achieved by a method for producing L-cysteic acid, which comprises the reaction of O-phospho-L-serine (OPS) with a salt of sulfurous acid (sulfite) and cysteic acid synthase (CS enzyme) in a bioconversion, the cysteic acid synthase belonging to the enzyme class EC 2.5.1.76.

[0018] In the context of the present invention, a distinction is made between production methods:

[0019] 1. Chemical method

[0020] 2. Biotechnology methods:

[0021] a) Through metabolic engineering

[0022] Different from biotransformation, metabolic engineering (also referred to as "pathway design") is a biotechnological method that changes the metabolic pathways of an organism by optimization or modification of genetic or regulatory methods. New or modified enzymes can be introduced into an organism by supplementing enzyme genes in the genome, or genes of endogenous enzymes can be expressed at enhanced or weakened levels, thereby establishing new metabolic pathways in an organism or enhancing or weakening existing metabolic pathways. The goal of metabolic engineering is to make an organism produce new metabolites or cell endogenous metabolites with increased yields. The method of metabolic engineering does not use metabolite-specific starting materials, such as enzyme substrates, such as OPS in the present invention; instead, it only uses the nutrient medium required for the growth of the relevant organism, which is composed of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts or complex amino acid mixtures, such as peptone or yeast extract) and other salts required for growth. This nutrient medium is known to those skilled in the art in microbiological practice.

[0023] b) Through biotransformation

[0024] Biotransformation is defined as the conversion of one or more reactants into products under enzyme catalysis, and the enzyme substrate is added to the reaction batch together with the enzyme. In the present invention, in the reaction batch, in the presence of sulfite, the added enzyme substrate (e.g., OPS of the present invention) is enzymatically converted by an enzyme selected from the enzyme class of cysteic acid synthase (CS enzyme, EC 2.5.1.76). The reactants can come from chemical or biotechnological production. For example, the OPS used in the method of the present invention can come from chemical synthesis, or from biotechnological production by culturing a production strain. The enzyme used for enzyme catalysis is preferably from biotechnological production carried out by fermentation of a production strain of Enterobacteriaceae, and the Enterobacteriaceae heterologously expresses the CS enzyme.

[0025] One advantage of the present invention is that the process of the present invention for producing L-cysteic acid from OPS and sulfite with the help of CS enzyme is a bioconversion process. This means that it is a highly targeted specific reaction that does not require, for example, complex cultivation steps or purification steps from microbial cultures. In addition, bioconversion can achieve high space-time yields in a simple manner by optimizing the reaction conditions and optimizing the amounts of reactants and enzymes used, which is much more difficult in the case of metabolic engineering by strain optimization. In summary, the process of the present invention is implementable and controllable in a low-cost manner.

[0026] Reaction (1) of the present invention is catalyzed by cysteic acid synthase (CS enzyme) belonging to enzyme class EC 2.5.1.76. CS enzyme in enzymatically active form refers to a protein capable of catalyzing the synthesis of L-cysteic acid from OPS and sulfite, as described in the following CS enzyme activity assay.

[0027] CS enzyme activity assay can be performed as follows:

[0028] i) CS enzyme produced in shake flasks or fermentation culture can be used in the following forms:

[0029] - as an aliquot from culture without further workup, or

[0030] - as an aliquot of a cell suspension after re-isolation of the cells from the culture (e.g. by centrifugation), or

[0031] - in the form of aliquots of cell homogenate

[0032] a) the cell homogenate is formed after mechanical disruption of a cell suspension, or

[0033] b) in the form of chemically permeabilized cells (e.g. by chloroform permeabilization), or

[0034] - as a cell extract after removal of particulate components from a cell homogenate, or

[0035] - As an enzyme purified, for example by chromatography.

[0036] As described in Example 3 of the present invention, the fluorescence intensity can be measured by the Qubit 3.0 fluorometer from Thermo Fisher Scientific, for example using the " The total protein concentration obtained in each case was determined by PCR using the Protein Assay Kit” according to the manufacturer’s instructions.

[0037] ii) A solution buffered to pH 7 with potassium dihydrogen phosphate is initially added with OPS (final concentration of 10 mM) and sodium sulfite (final concentration of 20 mM), and then the CS enzyme is added to start the reaction. The assay volume is 10 ml. The assay temperature is 30° C. The amount of CS enzyme used depends on the purity. If a culture medium, a cell suspension of re-isolated cells or a cell homogenate is used, at least 0.1 mg of the enzyme fraction prepared in i) is used. In the case of a purified CS enzyme, at least 10 μg of the purified enzyme component is used. 1 h, 2 h and 4 h after the start of the reaction, 1 ml of the assay is centrifuged for 10 min each time and the content of OPS and L-cysteic acid is determined by calibrated HPLC (see Example 4); the reference substances used for calibration are commercially available (Sigma-Aldrich).

[0038] A preferred feature of the method of the present invention is that the CS enzyme is produced by culturing a microbial strain from the Enterobacteriaceae family, and preferably the CDS encoding the CS enzyme is heterologously expressed in the Enterobacteriaceae family microbial strain, particularly preferably expressed in an enzymatically active form.

[0039] Heterologous expression refers to the expression of the CDS of a gene or a portion of a gene in a host organism that does not essentially possess the gene or gene fragment. The CDS of a heterologous gene is introduced into a host organism using recombinant DNA technology. The CDS of a heterologous gene can be introduced into a host organism extrachromosomally by integration into its genome or in the form of an autonomously replicating gene construct (plasmid, vector).

[0040] The CDS of the heterologous gene is preferably introduced into the host organism in the form of an autonomously replicating gene construct (plasmid, vector).

[0041] There is a difference between constitutive expression and inducible expression, depending on the genetic factor (promoter) used to control the expression of the heterologous gene CDS. In the case of constitutive expression, gene expression is active at all stages of cell culture (unregulated). In the case of inducible expression (regulated), gene expression is stimulated by adding an inducer molecule to the cell culture, such as the inducing molecule IPTG added in Examples 3 and 7 of the present invention for inducing the tac promoter. Inducible expression is preferred, wherein gene expression is stimulated by adding an inducer molecule to the cell culture.

[0042] In contrast, homologous expression involves the overexpression of the CDS of a gene in a host organism from the genome from which the gene originally originated.

[0043] For example, if the heterologously expressed protein is an enzyme, it may be present in an enzymatically active form or it may be enriched in inclusion bodies or the like in an enzymatically inactive form.

[0044] Therefore, heterologous expression of the CS enzyme in enzymatically active form means that: i) the CDS of the gene encoding the CS enzyme introduced into the host strain is not encoded in the genome of the host strain, ii) at least the CDS of the gene encoding the CS enzyme is chromosomally integrated into the genome of the host organism by recombinant DNA technology, or is preferably introduced into the host organism extrachromosomally by an autonomously replicating vector, and iii) the CS enzyme is expressed in enzymatically active form via said CDS.

[0045] Particularly preferably, the heterologously expressed CS enzyme is expressed in an enzymatically active form by a microbial strain. This means that the heterologously introduced CDS expresses a CS enzyme that is present in an enzymatically active form in the microorganism after protein biosynthesis and any post-translational modifications, such as the addition of a cofactor such as pyridoxal phosphate (see the entry under entry number EC2.5.1.76 in the KEGG enzyme database) in the case of a CS enzyme. "Expressed in an enzymatically active form" does not mean that the protein is initially produced in the form of an inactivated protein in inclusion bodies and is present in an enzymatically active form only after renaturation.

[0046] In summary, in a particularly preferred embodiment, the method is characterized in that the CS enzyme is produced by culturing a microbial strain from the family Enterobacteriaceae, which strain heterologously expresses the CS enzyme in enzymatically active form.

[0047] In the context of the present invention, a reaction batch is defined as a mixture of reactants (starting materials), enzyme and optionally further reactants, wherein the reactants are converted into products.

[0048] Reaction yield in the sense of the present invention is defined as the amount of reactant converted to product under reaction conditions. Yield can be expressed as the absolute yield of product (mmol or g), expressed as the volumetric yield of the absolute amount of product per unit volume (mM or g / L), or expressed as the relative yield of product, which is expressed as the percentage of reactant used (taking into account the molecular weight of reactant and product), also referred to as percentage yield.

[0049] In the context of the present invention, the term "growth" of microbial cells or its synonym "culture" includes shake flask culture methods and fermentation methods. The culture medium used for the growth or cultivation of microorganisms is called growth medium or culture medium, or in the case of fermentation, it is also called fermentation medium. In the growth, culture or fermentation medium, the growth / culture / fermentation of the production strain produces a culture fluid or fermentation tank liquid. The culture fluid / fermentation tank liquid is composed of the biomass of the cells of the production strain and the culture supernatant / fermentation supernatant without biomass, and the culture supernatant / fermentation supernatant without biomass is formed by the growth medium and the metabolites secreted by the cells during the growth process.

[0050] Fermentation is a process step for producing (culturing) a cell culture on an industrial scale (production scale), in which a preferred microbial production strain is grown under conditions such as a defined medium, temperature, pH value, oxygen supply and medium mixing. Depending on the configuration (genetic composition) of the production strain, the purpose of the fermentation is to produce proteins / enzymes or metabolites, which are produced in each case with the highest possible yield for further use. The components of the method of the present invention, OPS and CS enzymes, can be produced by fermentation. The final product of the fermentation is a fermentation tank liquid, which consists of the cell biomass of the production strain (fermentation tank cells) and a biomass-free fermentation supernatant, which is formed during the fermentation process by the growth medium and metabolites secreted by the fermentation tank cells. The target product of the fermentation can be present in the fermentation tank cells or in the fermentation supernatant. For example, OPS is present in the fermentation supernatant, while the CS enzyme is present in the fermentation tank cells.

[0051] Compared to the production scale carried out by fermentation, shake flask culture is used for laboratory-scale microbial cultivation. Although shake flask culture also involves specifying a specific culture medium and pH value, and cultivating with continuous movement (shaking) in the presence of oxygen, more specific conditions regarding culture medium, temperature, pH, oxygen supply and culture medium mixing can be established and adjusted in a fermentor. Smaller-scale cultivation, such as shake flask culture, can also be used as a pre-culture for inoculation of larger-scale cultivation (e.g., fermentor).

[0052] The production strain is defined as a microbial strain suitable for producing (e.g., producing by fermentation) a certain product. The difference between the production strain is that it can produce products by genetic modification (improvement). Genetic modification can be due to the transformation of the genome (chromosome transformation), due to the introduction of an autonomously replicating extrachromosomal genetic element (e.g., plasmid), and due to the combination of chromosomal modification and extrachromosomal modification. The example of a production strain with chromosomal modification is the E. coli W3110-ΔserB strain related to OPS described in Example 1. The example of a production strain produced by introducing a plasmid is the E. coli JM105 x pCSma-pKKj strain related to the production of CS enzyme described in Example 3. The microbial strain carrying an extrachromosomal genetic element refers to a host strain or a host organism, and the extrachromosomal genetic element refers to a gene construct, a plasmid, a vector or an expression vector.

[0053] An open reading frame (ORF, synonymous with CDS or coding sequence) refers to a region of DNA or RNA that begins with a start codon, ends with a stop codon, and encodes the amino acid sequence of a protein. An ORF is also called a coding region or a structural gene.

[0054] A gene refers to a DNA segment containing all the basic information for producing RNA with biological activity. A gene comprises a portion of DNA that produces a single-stranded RNA copy by transcription, and an expression signal that participates in regulating this replication process. The expression signal comprises, for example, at least one promoter, a transcription start point, a translation start point, and a ribosome binding site (RBS). Terminators and one or more operators are other possible expression signals.

[0055] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries the genetic information for protein synthesis. mRNA provides the assembly instructions for a specific protein in the cell. The mRNA molecule transfers the information required for protein synthesis from the genetic information (DNA) to the ribosomes responsible for protein synthesis. In the cell, it is formed as a transcript of the DNA segment corresponding to the gene. The genetic information stored in the DNA remains unchanged during this process.

[0056] The genes of eukaryotes are mainly chimeric genes. Unlike the genes of prokaryotes, the genes of eukaryotes also contain introns ( Intragenic region ) of the genome. The coding sequences are called exons ( Expression region ), is a DNA segment of a eukaryotic gene that, after being transcribed into RNA, is translated by the ribosome into the amino acid sequence of a protein. After DNA is transcribed into RNA, introns are sheared off from the primary transcript. The RNA encoding a protein without introns is called messenger RNA (mRNA), or "mature" mRNA. It also needs to undergo further modifications, such as capping and polyadenylation. The coding region of the mature mRNA is then translated into a protein sequence. If a eukaryotic gene containing an exon / intron structure is to be expressed in a prokaryotic organism, the protein sequence or coding region of the mature mRNA must be reversely translated into DNA without introns, because the processing of the exon / intron structure does not occur in prokaryotes. In the context of the present invention, when it comes to a gene sequence derived from a protein sequence or a gene sequence derived from mRNA, it is this reverse translation process that is referred to. Sequence optimization, i.e., codon usage adapted to the corresponding prokaryotic organism (codon optimization), is preferably performed simultaneously with the reverse translation of the protein sequence or mRNA sequence into a DNA sequence.

[0057] Genetic construct refers to a DNA molecule that is linked to a gene and other genetic elements (e.g., promoter, terminator, selection marker, origin of replication). In the context of the present invention, a genetic construct is a circular DNA molecule, referred to as a plasmid, vector or expression vector. The genetic elements of a genetic construct cause extrachromosomal inheritance during cell growth and cause the production of the protein encoded by the gene.

[0058] Abbreviation WT (Wt) refers to wild type. Wild-type gene refers to the gene form that is naturally produced by evolution and present in the wild-type genome. The DNA sequence of the wild-type gene can be publicly obtained in databases such as NCBI (National Center for Biotechnology Information). Microbial strains with Wt genomes are called wild-type strains.

[0059] In the present invention, the L-cysteic acid obtained from the bioconversion of OPS with sulfite can be used directly without further post-treatment steps, or it can be enriched or purified by known methods. These methods are amino acid separation methods known to those skilled in the art. Examples of the methods include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization, etc.

[0060] Preferably, the process is characterized in that the reaction batch containing L-cysteic acid can be used further without further work-up, purification or isolation steps.

[0061] In another preferred embodiment, the method is characterized in that the produced L-cysteic acid is separated from the reaction batch.

[0062] In the context of the present invention, denaturation refers to a structural change of a molecule, such as a protein, which is associated with a loss of biological function in said molecule, even if its primary structure remains unchanged.

[0063] The characteristic of a denatured protein is that it has no enzymatic activity, i.e. in the context of the present invention, the CS enzyme cannot catalyze the synthesis of L-cysteic acid from OPS and sulfite. Denaturation can be caused by physical or chemical influences. Incorrectly or incompletely folded, enzymatically inactive proteins aggregate into protein aggregates (called inclusion bodies) in cells, which can be regarded as naturally denatured proteins. Inclusion bodies are particularly easy to observe when high levels of expression are present, when the concentration of newly synthesized protein chains is very high, meaning that aggregation of proteins is favored rather than folding into a three-dimensional form with enzymatic activity. Whether a heterologously expressed protein appears in the form of insoluble inclusion bodies or in an active form is unpredictable, and depends not only on the primary structure of the protein chain (continuity of the amino acid sequence), but also on the expression system used and the parameters used, by which the biosynthesis rate of the protein can be controlled (e.g. growth temperature, induction strength of inducible promoters). However, in the context of the present invention, it was surprising that the sulfalanine synthase from Methanosarcina acetica was produced in enzymatically active form in E. coli, since according to the prior art (Graham et al., 2009, supra) this protein could only be produced in an inactive form in inclusion bodies when heterologously expressed in E. coli.

[0064] Renaturation refers to the conversion of denatured proteins back to their biologically active spatial structure. Renaturation in protein biochemistry refers to the use of chaotropic compounds to bring denatured proteins in inclusion bodies back into solution, and then removing the chaotropic compounds to renature the proteins. In protein chemistry, urea and guanidine hydrochloride are particularly used for this purpose.

[0065] Preferably, the method is characterized in that the CS enzyme is used in the reaction without a prior renaturation step.

[0066] Renaturation stepThe method comprises the following steps: dissolving the denatured protein in a medium containing a chaotropic compound and then removing the chaotropic compound. The extent of removal of the chaotropic compound depends on the specific protein. For example, the chaotropic compound can be removed by dialysis, selectively binding the chaotropic compound to a support material, selectively binding the protein to be renatured to a support material and then eluting under renaturation conditions, or diluting the chaotropic compound to below a critical concentration, below which the denaturation effect is no longer produced (see Graham et al., 2009, supra). The prior art has described methods for protein renaturation. For example, renaturation conditions include dissolving the denatured protein in a 6M urea aqueous solution or a 6M guanidine hydrochloride aqueous solution.

[0067] Dilution or removal of the chaotropic compound below a critical concentration depends on the specific protein and means reducing the concentration of the chaotropic compound (such as urea or guanidine) below a concentration at which the protein of interest can resume its three-dimensional structure in its active form.

[0068] Chaotropic compounds are chemical substances that can disrupt the ordered hydrogen bonds in water. Chaotropic compounds include barium salts (such as barium chloride or barium acetate), guanidine hydrochloride, thiocyanates (such as guanidine thiocyanate), perchlorates, iodides, butanol, phenol, thiourea, urea and / or surfactants.

[0069] Surfactants (also called detergents or soaps) are organic compounds that act as surface-active substances, i.e., due to their structure, they are arranged at the interface between two phases, thereby reducing the surface tension, thereby achieving effects such as wetting. By reducing the surface tension, they can promote the mixing of the two phases and even the formation of emulsions. Surfactants are characterized by the polarity of their molecular structure, with one part of the molecule being hydrophilic and mediating dissolution in water, and another part of the molecule being hydrophobic, which allows the surfactant to solubilize hydrophobic compounds and promote dissolution in water. The surfactants used include nonionic surfactants (polyalkylene glycol ethers, fatty alcohol propoxylates, alkyl glycosides, alkyl polyglycosides, octylphenol ethoxylates (such as Triton X-100), nonylphenol ethoxylates), anionic surfactants (alkyl carboxylates, alkyl benzene sulfonates, alkyl sulfonates, fatty alcohol sulfates (such as sodium lauryl sulfate), alkyl ether sulfates, sulfoacetates), cationic surfactants based on quaternary ammonium compounds (distearyldimethylammonium chloride, "Esterquat") and / or zwitterionic (amphoteric) surfactants based on betaines (such as "cocoamidopropyl betaine") or sulfobetaines (such as cocoamidopropyl hydroxysulfobetaine).

[0070] In a preferred embodiment, the method is characterized in that the CS enzyme is used in the reaction, but no prior renaturation step is required. Since the above-mentioned complex, costly and environmentally harmful renaturation step is not required, the method without the renaturation step is economically very advantageous. In the case of producing the CS enzyme by culturing the production strain, especially in the case of producing the CS enzyme by fermentation, for the host cell expressing the CS enzyme, it is not necessary to first mechanically or chemically destroy it in a complex, costly and environmentally harmful (e.g. due to waste) process, and for inclusion bodies, such as the inclusion bodies in which the enzyme is enriched in an inactive form as described by Graham et al. (2009), it is not necessary to separate from the cell lysate and renature it so that the protein can be used in the enzymatic reaction.

[0071] The method for producing L-cysteic acid by bioconversion according to the invention requires the presence of OPS. OPS can be produced chemically or biotechnologically, for example by fermentation of an OPS producing strain. Possible methods for chemical production of OPS are, for example, phosphorylation of L-serine, or production of the racemate O-phospho-D / L-serine, which can be used directly, or OPS can be obtained in advance from the racemate, for example by resolution.

[0072] Preferably, the method for producing L-cysteic acid is characterized in that the OPS used in the reaction is produced biotechnologically. This is achieved by culturing an OPS producing strain. Particularly preferably, OPS is produced biotechnologically by culturing an OPS producing strain, wherein OPS accumulates in the cell culture supernatant (extracellularly).

[0073] Those skilled in the art can use isotope analysis to determine whether the substance (e.g., OPS) they wish to use as a reactant in the method is from chemical production or biotechnological production (e.g., fermentation production). For example, Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: A distinguishable isotope analysis method described in 2521-2527 is based on, for example, the determination of carbon or nitrogen isotope ratios, which differ depending on whether the product is from chemical (petroleum) production or biotechnological production (e.g., fermentation (plant-based) production). For example, if the glucose used to cultivate the production strain is from plant-based production, the method for producing OPS is a biotechnological production method, such as a fermentation (plant-based) production method, which also applies to the method described in Example 2.

[0074] For example, in the cysteine ​​metabolism of Escherichia coli, OPS is used as a biosynthetic precursor for L-serine. L-serine is formed by the dephosphorylation of OPS. The reaction is enzymatically catalyzed by O-phospho-L-serine phosphatase (SerB, EC3.1.3.3). It is known from the prior art that Escherichia coli strains with inhibited SerB activity can accumulate OPS. Therefore, a microbial strain with inhibited SerB activity is characterized in that it can no longer produce L-serine by dephosphorylation of OPS and can therefore accumulate OPS.

[0075] Preferably, the method is characterized in that the OPS used in the reaction is produced using a microbial strain having an inhibited O-phospho-L-serine phosphatase (SerB enzyme) activity belonging to the enzyme class EC3.1.3.3. This means that, in this case, a microbial strain having inhibited SerB activity is used as a production strain of OPS, and the inhibition of SerB activity includes genetic modification of the microbial strain. The SerB activity of the genetically unmodified microbial strain (Wt strain) is set to 100%, and the definition of the microbial strain having inhibited SerB activity is that it contains a lower SerB activity compared to the 100% activity of the Wt strain, preferably not more than 20% relative to the wild-type strain activity set to 100%, particularly preferably not more than 10%, and particularly preferably 0% (serB gene inactivation) activity. The SerB activity based on the percentage of the Wt strain activity that can still be measured in the modified microbial strain is called residual activity.

[0076] Microbial strains with inhibited SerB activity are characterized by having one or more of the following genetic modifications:

[0077] -Deletion of the chromosomal gene encoding the SerB enzyme.

[0078] -Introducing mutations in the chromosomal gene encoding the SerB enzyme to reduce the activity of the endogenous gene.

[0079] -Replace the chromosomal gene encoding the SerB enzyme with a mutated gene to reduce the activity of the endogenous gene.

[0080] - Introducing mutations into the regulatory region of the gene encoding the SerB enzyme to reduce endogenous enzyme activity.

[0081] - Introducing antisense oligonucleotides complementary to the transcript of the gene encoding the SerB enzyme to inhibit the translation of the mRNA.

[0082] As described in the prior art, the enzymatic activity of L-3-phosphoserine phosphatase (SerB activity) can be determined by enzymatic release of phosphate from L-3-phosphoserine, which is quantitatively determined photometrically in the form of a molybdate complex at a wavelength of 340 nm. The SerB activity of the WT strain determined using this enzyme assay is set to 100%, and the residual activity of the microbial strain having inhibited SerB activity is determined under the same conditions.

[0083] Particularly preferred is the method for producing L-cysteic acid according to the present invention, characterized in that the OPS used in the reaction is produced using a microbial strain in which the chromosomal gene encoding the SerB enzyme is deleted. This is also referred to as knockout of the serB gene. In this case, the microbial strain having inhibited SerB activity is preferably characterized in that the chromosomal nucleotide sequence of the serB gene is deleted, the sequence comprising the complete serB CDS encoding the SerB enzyme and flanking sequences, the flanking sequences being up to 1000 nt at the 5' upstream of the serB CDS, including the sequence of the serB promoter, and up to 1000 nt at the 3' downstream of the serB CDS, including the sequence of the serB terminator. Particularly preferred deletions are the deletions described in Example 1, i.e., deletions of the serB CDS encoding the SerB enzyme.

[0084] The microbial strain with suppressed SerB activity is preferably selected from Corynebacteriaceae and Enterobacteriaceae, particularly preferably selected from Corynebacterium, Pantoea and Escherichia, especially preferably selected from Pantoea ananatis and Escherichia coli. Very preferably, the microbial strain with suppressed SerB activity to produce OPS for the reaction of the present invention is Escherichia coli K12 W3110 strain.

[0085] The microbial strain with inhibited SerB activity is preferably characterized by a serine auxotrophy, i.e. the strain itself cannot form the amino acid L-serine for growth. The malnutrition can be overcome by adding serine or glycine to the culture medium (growth medium), in each case as a pure substance or as a component of a complex medium component (such as yeast extract, peptone or tryptone), or as a mixture of a pure substance and a complex medium component. Preference is given to mixtures of pure substances selected from glycine and L-serine and complex medium components, including particularly preferred mixtures of glycine and complex medium components. The addition of glycine to the growth medium is particularly preferred.

[0086] The glycine content of the growth medium as a pure substance is preferably 0.1 g / L to 10 g / L, particularly preferably 0.2 g / L to 5 g / L, especially preferably 0.3 g / L to 2 g / L.

[0087] According to the prior art, the serB gene or a part of the gene can be isolated and foreign DNA cloned into the serB gene, thereby interrupting the protein-defining open reading frame of the serB gene. Thus, a DNA construct suitable for targeted inactivation of a serB gene can consist of a 5' DNA fragment homologous to the genomic serB gene, followed by a gene fragment containing the foreign DNA, followed by a 3' DNA fragment also homologous to the genomic serB gene.

[0088] Therefore, the possible regions in the serB gene for homologous recombination to occur include not only the region encoding O-phospho-L-serine phosphatase. Possible regions may also include the flanking DNA sequences of the serB gene, i.e., the 5' region before the start of the coding region (gene transcription promoter) and the 3' region after the end of the coding region (gene transcription terminator), which modification by homologous recombination can lead to the inactivation of the serB gene, just like the modification of the coding region.

[0089] The exogenous DNA is preferably an expression cassette of a selection marker. It consists of a gene transcription promoter functionally connected to the actual selection marker gene, optionally followed by a gene transcription terminator. In this case, the selection marker also comprises the 5' and 3' flanking homologous sequences of the serB gene.

[0090] Preferably, the selection marker comprises 5' and 3' flanking homologous sequences of the serB gene, each sequence having a length of at least 30 nucleotides, particularly preferably at least 50 nucleotides.

[0091] Thus, a DNA construct for inactivating a serB gene may consist, starting from the 5' end, of sequences homologous to the serB gene, followed by an expression cassette for a selectable marker (eg selected from antibiotic resistance genes), followed by other sequences homologous to the serB gene.

[0092] In a preferred embodiment, the DNA construct for inactivating the serB gene consists, starting from the 5′ end, of a sequence homologous to the serB gene, which has a length of at least 30 nucleotides, particularly preferably a length of at least 50 nucleotides, followed by a selection marker expression cassette selected from antibiotic resistance genes, followed by further sequences homologous to the serB gene, which have a length of at least 30 nucleotides, particularly preferably a length of at least 50 nucleotides.

[0093] Selectable marker genes are generally genes whose gene product enables a parent strain to grow under selective conditions where the original parent strain cannot grow.

[0094] Preferred selection marker genes are selected from antibiotic resistance genes, such as ampicillin resistance gene, tetracycline resistance gene, kanamycin resistance gene, chloramphenicol resistance gene or neomycin resistance gene. Other preferred selection marker genes allow parent strains with metabolic defects (such as amino acid auxotrophy) to grow under selective conditions, which is the result of correcting the metabolic defect by expressing the selection marker gene. Finally, another possibility is such selection marker genes, whose gene products change compounds that are inherently toxic to the parent strain through chemical reactions, thereby inactivating the compounds (for example, the gene for acetamidase, which decomposes the compound acetamide, which is toxic to many microorganisms, into non-toxic products acetic acid and ammonia).

[0095] Among the selection marker genes, ampicillin resistance gene, tetracycline resistance gene, kanamycin resistance gene and chloramphenicol resistance gene are particularly preferred. Tetracycline resistance gene and kanamycin resistance gene are particularly preferred.

[0096] In addition to targeted gene inactivation, there are also systems based on homologous recombination that offer the option of removing selectable markers from the genome, thereby generating double and multiple mutants. An example of such a system is the so-called Lambda Red technology, commercially available as the "Quick and Easy E. coli Gene Deletion Kit", which is based on the DNA sequence of Gene Bridges GmbH. Technology (see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Recombination, Cat. No. K006, Version 2.3, June 2012" and references cited therein, such as Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645).

[0097] Example 1 of the present invention describes an example of producing a microbial strain having suppressed SerB activity by deleting the serB gene.

[0098] use Microbial strains with inhibited SerB activity produced by the technology can be suitable for extracellular production of OPS, such as described in the second embodiment of the present invention for the Escherichia coli W3110-ΔserB strain. OPS can accumulate both intracellularly and extracellularly, and the amount of OPS accumulated intracellularly depends on the growth conditions (Steinfeld et al., see above). The culture conditions selected in Example 2 of the present invention allow OPS to accumulate extracellularly. The content of extracellular OPS is preferably at least 1 g / L, particularly preferably at least 3 g / L, and even more preferably at least 6 g / L.

[0099] The advantage of the described method for the biotechnological production of OPS is that the OPS is present extracellularly in the culture broth, and the OPS obtained, for example, in the culture carried out in the second example, can be used directly as a source of OPS in the method for the production of L-cysteic acid according to the invention after removal of the particulate biomass (for example by centrifugation or filtration), preferably without further post-treatment, purification or separation steps, for example including extraction, adsorption, ion exchange chromatography, precipitation and crystallization. This method is particularly economical and avoids the isolation of OPS. Therefore, it is particularly preferred that the method for the production of L-cysteic acid is characterized in that the OPS used comes from the cell culture supernatant of a microbial strain grown, which has an inhibited activity of an O-phospho-L-serine phosphatase (SerB enzyme) belonging to the enzyme class EC3.1.3.3.

[0100] In a particularly preferred embodiment, the L-cysteic acid production method is characterized in that, in addition to the CS enzyme used in the reaction, the OPS used in the reaction is also produced by biotechnology, particularly preferably by fermentation.

[0101] Preferably, the CS enzyme is produced by culturing an Enterobacteriaceae microbial strain heterologously expressing the CS enzyme.

[0102] The microbial strain is also referred to as a CS enzyme producing strain, which consists of a host strain and a gene construct for expressing a CS gene.

[0103] Preferably, the method is characterized in that the CS enzyme is produced by culturing a microbial strain of the genus Escherichia, particularly preferably a microbial strain of the species Escherichia coli, especially preferably the Escherichia coli K12 JM105 strain, which heterologously expresses the CS enzyme and very preferably in enzymatically active form.

[0104] The preferred gene construct for expressing the CS gene is an expression vector in the form of a plasmid, including the expression vector pCSma-pKKj ( Figure 3 ).

[0105] Cysteic acid alanine synthase is known as an enzyme for the biosynthesis of coenzyme M of, for example, methanogens. Preferably, the method for producing L-cysteic acid alanine is characterized in that the CS enzyme is derived from Methanosarcina acetica or the CS enzyme is a homologous sequence thereof, and particularly preferably the CS enzyme is derived from Methanosarcina acetica. Particularly preferably, the encoding DNA sequence is SEQ ID NO: 3, which encodes a protein having the amino acid sequence of SEQ ID NO: 4, or the encoding DNA sequence is a nucleotide sequence homologous to SEQ ID NO: 3.

[0106] Homologous nucleotide sequences are understood to mean that the DNA sequences of these genes or DNA segments are at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical. Preferred homologous nucleotide sequences are gene sequences from Methanocella paludicola (NCBI gene ID: 8682885), Methanosarcina barkeri (NCBI gene ID: 24822660), Methanoculleus marisnigri (NCBI gene ID: 4845938) or nucleotide sequences homologous thereto.

[0107] DNA identity was determined by the "nucleotide blast" program, which is based on the blastn algorithm and can be used in http: / / blast.ncbi.nlm.nih.gov / Found at . The algorithm parameters used to align two or more nucleotide sequences are the default parameters. The default general parameters are: Maximum target sequences = 100; Short sequences = "Automatically adjust parameters for short input sequences"; Expected threshold = 10; Field length = 28; Automatically adjust parameters for short input sequences = 0. The corresponding default scoring parameters are: Match / mismatch score = 1, -2; Gap penalty = linear.

[0108] In a preferred embodiment, the method is characterized in that the CS enzyme has the amino acid sequence specified in SEQ ID NO: 4 or an amino acid sequence homologous thereto, wherein the amino acid sequence homologous to SEQ ID NO: 4 has a sequence identity of at least 50%, preferably at least 70%, particularly preferably at least 80% with SEQ ID NO: 4, and has the enzymatic activity of cysteic acid synthase. The enzymatic activity of cysteic acid synthase can be detected in a CS activity assay as defined above. The amino acid sequence of the homologous CS enzyme can be searched in the NCBI database (National Center for Biotechnology Information, USA) using the search term "cysteic acid synthase" or using the auxiliary program "Protein BLAST" by inputting the amino acid sequence of SEQ ID NO: 4. The enzyme homologous to the CS enzyme from Methanosarcina acetica is preferably selected from Methanocella paludicola (NCBI No.: WP_012900738.1), Methanolinea mesophila (NCBI No.: WP_245249687.1), Methanosarcina barkeri (NCBI No.: WP_011308449.1), Methanosarcina nigricans (NCBI No.: WP_011842967.1).

[0109] By searching the gene of CS enzyme by "Protein BLAST", homologous protein sequences can be found from many bacteria in the Archaea domain, which also include thermophilic organisms (growth temperature>50°C, up to 110°C). Their enzymes usually also have optimal activity>50°C. The present invention also includes CS enzymes from the Archaea domain, whose optimal activity is greater than 50°C.

[0110] Protein sequences were compared using the "Protein BLAST" program at http: / / blast.ncbi.nlm.nih.gov / . The program uses the blastp algorithm. The algorithm parameters used to align two or more protein sequences were the default parameters. The default general parameters were: maximum target sequences = 100; short sequences = "Automatically adjust parameters for short input sequences"; expected threshold = 10; field length = 3; Automatically adjust parameters for short input sequences = 0. The default scoring parameters were: matrix = BLOSUM62; gap penalty = presence: 11 extension: 1; composition adjustment = conditional composition scoring template adjustment.

[0111] Preferably, the method is characterized in that the CS enzyme is not a fusion protein. The term "fusion protein" refers to a DNA sequence encoding a protein or a portion of a protein that is fused in the laboratory with one or more DNA sequences encoding another protein or a portion of another protein in the framework, thereby encoding a modified (extended) protein that does not exist in nature. The fused DNA sequence can be connected at the 5' end, the 3' end, or both the 5' end and the 3' end. It is also possible to insert a fusion DNA sequence into the sequence encoding the protein (for example, as a connection between two domains of the protein). Fusion proteins are also referred to as hybrid proteins or hybrid enzymes. This means that the protein having cysteic acid synthase activity according to the present invention is only encoded by the CDS of the relevant gene, and the CDS of CS is not extended by the sequence added to the CDS of CS. Proteins having a CDS fused to a nucleotide sequence encoding a protein sequence that will be excised during protein biosynthesis or post-translational modification (for example, an output signal sequence that mediates protein secretion) do not belong to the category of "fusion proteins". In the context of the present invention, the term "fusion protein" always refers to mature proteins.

[0112] In the context of the present invention, "producing the CS enzyme by culturing a CS enzyme producing strain" means that the CS enzyme is produced as an enzymatically active protein without refolding and preferably not expressed as a fusion protein. Particularly preferred is the production of the CS enzyme from Methanosarcina acetica in the form of an enzymatically active protein without refolding and without expression as a fusion protein by culturing a CS enzyme producing strain, wherein the producing strain is produced by using a host strain of E. coli. Particularly preferred is the production of the CS enzyme from Methanosarcina acetica by culturing the E. coli JM105 x pCSma-pKKj producing strain described in Example 3.

[0113] In the method of the present invention, the CS enzyme obtained by culturing the production strain can be used in the form of a culture solution without further treatment, or in the form of a cell suspension after re-separation from the culture solution, for example, by centrifugation or filtration. In addition, the CS enzyme can be used in the form of a cell homogenate after mechanical disruption of the cell suspension, or in the form of chemically permeabilized cells (such as by chloroform permeabilization), or in the form of a cell extract after removing particulate components in the cell homogenate, or in the form of an enzyme purified by, for example, chromatography.

[0114] The CS enzyme is preferably used in the form of a culture broth, in particular a fermentation broth, without further treatment; or in the form of a cell suspension after re-isolation from the culture broth, or in the form of a cell homogenate after mechanical disruption of the cell suspension, or in the form of chemically permeabilized cells (eg by chloroform).

[0115] It is particularly preferred to use the CS enzyme in the form of a cell suspension separated again from a culture medium, or in the form of a cell homogenate. It is particularly preferred to use the CS enzyme in the form of a cell homogenate.

[0116] In a preferred embodiment, after culturing the CS enzyme producing strain, the CS enzyme is prepared in the form of a cell homogenate, and the homogenate is directly used as the CS enzyme in the bioconversion process according to the present invention. Example 3 of the present invention discloses a possible preferred embodiment.

[0117] In general, all conceivable sulfites are suitable for the reaction in the process for producing L-cysteic acid, including the known salts such as Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its anhydride Na2S2O5) or KHSO3. It is also possible to use gaseous sulfur dioxide, which is the anhydride of sulfurous acid, which can be introduced into the reaction batch, where it is hydrated to form sulfurous acid H2SO3 and, depending on the pH value, reacts with the deprotonated form HSO3. - and SO3 2- In a state of balance.

[0118] Preferably, Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its anhydride Na2S2O5), KHSO3 are used, particularly preferably Na2SO3, NaHSO3 (or its anhydride Na2S2O5) and (NH4)2SO3, particularly preferably Na2SO3 and NaHSO3 (or its anhydride Na2S2O5). In a very preferred embodiment, the sulfite used in the process for producing L-cysteic acid is Na2SO3 or NaHSO3 (or its anhydride Na2S2O5).

[0119] According to formula (1), when OPS reacts to generate L-cysteic acid, a stoichiometric amount of phosphoric acid is released, which may cause the pH value in the batch to decrease as the reaction proceeds. Since too low a pH value will affect the activity of the CS enzyme, it is necessary to prevent the pH value from dropping too much. This can be achieved passively by using an appropriate high-concentration buffer in the batch, or actively by using a measurement and control unit.

[0120] The pH value is preferably actively controlled by a measuring and control unit (as described in Example 6) and when the pH value deviates from the target value, the desired pH value is restored by quantitative addition of alkaline or acidic solution (the so-called pH stabilization method).

[0121] The reaction temperature can be selected between 5° C. and 80° C. The reaction temperature is preferably selected between 10° C. and 60° C., particularly preferably between 15° C. and 50° C., and particularly preferably between 20° C. and 40° C.

[0122] The reaction can be carried out at a pH value between 4.0 and 9.0, preferably at a pH value between 5.0 and 8.5, particularly preferably at a pH value between 5.5 and 8.0, and particularly preferably at a pH value between 6.0 and 7.5.

[0123] The solvent used in the method for producing L-cysteic acid is preferably water.

[0124] The method for producing L-cysteic acid of the present invention can be carried out in a discontinuous operation or a continuous operation. In a discontinuous operation (intermittent operation), all reactants are added to the batch during the reaction, and the batch is processed after the reaction is completed. In a continuous operation, the CS enzyme is added as a stationary phase, for example, fixed on a membrane reactor or a support, and the substrate OPS and sulfite are metered in as a mobile phase. The contact time of the mobile phase with the stationary phase is set to a time that allows the substrate OPS to completely react with the sulfite to generate the product L-cysteic acid. Discontinuous operation (intermittent operation) is preferred.

[0125] The concentration of sulfite in the batch is preferably at least equimolar to OPS, preferably at least 1.5-fold molar excess of sulfite, particularly preferably at least 2-fold molar excess, especially preferably at least 5-fold molar excess.

[0126] The OPS concentration in the batch is preferably at least 80 mg / L, particularly preferably at least 1 g / L, and especially preferably at least 5 g / L.

[0127] Preferably, the process for producing L-cysteic acid is characterized in that the molar yield of L-cysteic acid, based on the molar amount of OPS used, is at least 60%, particularly preferably at least 80%, especially preferably at least 90%.

[0128] The method of the invention is suitable for industrial scale. The batch volume is preferably >10 ml, particularly preferably >1 L, and especially preferably >100 L.

[0129] In contrast to the prior art, surprisingly, the CS enzyme produced by heterologous recombinant production in a CS enzyme producing strain has enzymatic activity in the cell homogenate, does not require refolding (renaturation), preferably does not require the aid of a fusion partner, and is suitable for previously unknown biotransformations for the efficient production of L-cysteic acid. To this end, OPS produced by cultivation (e.g., by fermentation), OPS from the cell culture supernatant of a culture of an OPS producing strain preferably having inhibited SerB enzyme activity, or commercially available OPS can be reacted with sulfite according to formula (1).

[0130] Furthermore, the present invention also preferably provides a method for producing taurine, characterized in that the L-cysteic acid produced according to the present invention is decarboxylated.

[0131] L-Cysteine ​​is decarboxylated to generate taurine according to formula (2):

[0132] (2) L-Cysteine->Taurine+CO2

[0133] Particularly preferably, the process for producing taurine is characterized in that the L-cysteic acid produced according to the process of the invention is directly further used for producing taurine, i.e. without further post-treatment, purification or separation steps, as disclosed in Examples 8 and 9 of the present invention.

[0134] The process of decarboxylating L-cysteic acid to taurine can be achieved by chemical methods or by bioconversion under enzyme catalysis. It is known that thermal decarboxylation at high temperature under metal catalysis is considered to be unsustainable, and its disadvantages are high energy consumption and high proportion of by-products. Preferably, the decarboxylation reaction is a bioconversion of L-cysteic acid to taurine by enzymatic decarboxylation.

[0135] For the production of taurine by enzymatic decarboxylation, the L-cysteic acid produced by the process according to the invention can be used directly in the form of a reaction batch without further post-treatment steps. However, it is also possible to remove particulate biomass from the reaction batch before enzymatic decarboxylation, for example by centrifugation, or to separate the L-cysteic acid from the reaction batch beforehand.

[0136] It is preferred to use the reaction batch containing L-cysteic acid directly, or to use the reaction batch containing L-cysteic acid after removing the particulate biomass.

[0137] Particular preference is given to using the reaction batch containing L-cysteic acid directly without further workup steps, as disclosed in Example 8 for the production of taurine, for example.

[0138] Enzymes suitable for enzymatically decarboxylating L-cysteic acid to taurine according to formula (2) include L-cysteine ​​sulfenic acid decarboxylase (CSAD, EC 4.1.1.29), aspartate 1-decarboxylase (EC 4.1.1.11) or glutamate decarboxylase (EC 4.1.1.15).

[0139] Preferably, the taurine production method is characterized in that the decarboxylation is achieved by cysteine ​​sulfenic acid decarboxylase (CSAD enzyme) belonging to the enzyme class EC 4.1.1.29. It is known that CSAD enzyme can decarboxylate L-cysteine ​​sulfenic acid to hypotaurine according to formula (3).

[0140] (3) L-cysteine ​​sulfinic acid->hypotaurine+CO2

[0141] To varying degrees, these enzymes are also able to decarboxylate L-cysteic acid as substrate to taurine. In Examples 8 and 9, in particular the CSADcc enzyme from carp (Cyprinus carpio) is suitable for decarboxylating L-cysteic acid to taurine according to formula (2).

[0142] CSAD enzymes are mainly found in metazoans (multicellular animals), including mammals, such as humans (Homosapiens), cattle (Bos taurus), rats (Rattus norvegicus), mice (Mus musculus), and also in fish, such as carp (Cyprinus carpio). Enzymes with CSAD activity are also found in unicellular organisms, such as algae, such as from Synechococcus, and in bacteria or fungi.

[0143] Preferred are CSAD enzymes from mammals including humans (Homo sapiens), cattle (Bostaurus), rats (Rattus norvegicus) or mice (Mus musculus), and fish such as carp (Cyprinus carpio).

[0144] CSAD enzymes from human, rat or carp are particularly preferred.

[0145] Particularly preferred CSAD enzymes are from carp, referred to as CSADcc. The DNA sequence underlying the CSADcc amino acid sequence is available from the NCBI database, GenBank sequence ID: AB220585.1 (CDS: nt 82-1584). Preferably, the CSADcc CDS DNA sequence derived from the corresponding amino acid sequence is codon-optimized for expression in a specific selected microorganism such as E. coli (e.g., as described in SEQ ID NO: 5, nt 31-1530 for E. coli, which encodes a protein having the amino acid sequence of SEQ ID NO: 6). Codon optimization can be performed using publicly available software programs, such as the Eurofins Genomics GENEius software used in Example 7.

[0146] Preferably, the taurine production method is characterized in that the amino acid sequence of the CSAD enzyme is SEQ ID NO:6.

[0147] Preferably, the CSAD enzyme, in particular CSADcc, is recombinantly produced by a microbial production strain. For example, the CSAD enzyme is recombinantly produced in the E. coli production strain disclosed in Example 7. The CDS of CSAD is cloned into an expression vector, such as the vector pKKj (see Example 3) in a known manner, and a gene construct, such as pCSADcc-pKKj ( Figure 4 ). A production strain is produced by transforming a gene construct containing the CDS of CSAD (e.g., pCSADcc-pKKj) into a microbial host strain (e.g., E. coli JM105) in a similarly known manner, and using the production strain thus produced (e.g., E. coli JM105 x pCSADcc-pKKj) to produce the CSAD enzyme in a similarly known manner. The CSAD enzyme can be produced on a shake flask scale for laboratory purposes (as described in Example 7), or fermented in a known manner.

[0148] The CSAD enzyme contains pyridoxal phosphate (PLP, CAS No. 54-47-7) as a cofactor. Therefore, the addition of PLP to the growth medium or bioconversion batch for the conversion of L-cysteic acid to taurine is one way to achieve method improvements. Since PLP belongs to the vitamin B6 family, supplementation with other members of the vitamin B6 family, such as pyridoxine (CAS No. 65-23-6), pyridoxal (CAS No. 66-72-8) or pyridoxamine (CAS No. 85-87-0), are also suitable alternatives for improving the method.

[0149] The CSAD enzyme obtained by shaking flask culture or fermentation, preferably CSADcc, can be used in the form of a culture solution without further treatment, or in the form of a cell suspension after re-separating the cells by, for example, centrifugation. In addition, the CSAD enzyme, preferably CSADcc, can be used in the form of a cell homogenate after mechanically destroying the cell suspension, or in the form of chemically permeabilized cells (e.g., by chloroform), or in the form of a cell extract after removing the particulate components in the cell homogenate, or in the form of an enzyme purified by, for example, chromatography. As described in Examples 7 and 8, the CSAD enzyme is preferably used in the form of a cell suspension after re-separating the cells from the culture solution.

[0150] The bioconversion of L-cysteic acid to taurine by the CSAD enzyme is carried out under pH and temperature conditions that allow L-cysteic acid to be effectively decarboxylated to taurine. The pH range for the bioconversion is preferably between 5.0 and 9.0, and the temperature range is preferably between 20°C and 70°C.

[0151] The bioconversion of taurine from L-cysteic acid can be carried out in a discontinuous operation or a continuous operation. In a discontinuous operation (intermittent operation), all reactants are added to the batch during the reaction, and the batch is post-processed after the reaction is completed. In a continuous operation, the CSAD enzyme is added as a stationary phase, for example fixed to a membrane reactor or a support, and the substrate L-cysteic acid is metered in as a mobile phase. The contact time of the mobile phase with the stationary phase is set to a time that allows the substrate L-cysteic acid to completely react to generate the product taurine. Discontinuous operation (intermittent operation) is preferred.

[0152] In the bioconversion process for producing taurine, the concentration of L-cysteic acid is preferably at least 80 mg / L, particularly preferably at least 1 g / L, and especially preferably at least 5 g / L.

[0153] Preferably, the taurine production process is characterized in that the molar yield of taurine, based on the molar amount of L-cysteic acid used, is at least 60%, preferably at least 80%, particularly preferably at least 90%, especially preferably at least 95%.

[0154] Preferably, the process steps for producing L-cysteic acid (bioconversion 1) and for producing taurine are carried out sequentially, ie one after the other. In another preferred embodiment, the taurine production process is characterized in that all process steps are carried out in one reaction batch.

[0155] If all process steps are carried out in one reaction batch, the process is also called a one-pot process or a one-pot reaction.

[0156] Example 9 of the present invention discloses a method for carrying out such a one-pot reaction, in which the bioconversion of OPS into L-sulfoalanine according to formula (1) and the bioconversion of L-sulfoalanine into taurine according to formula (2) are carried out simultaneously, that is, in one reaction batch, wherein OPS reacts with sulfite (salt of sulfurous acid) in the presence of CS enzyme and CSAD enzyme. In the first reaction, L-sulfoalanine is formed, which is decarboxylated to taurine "in situ" by the CSAD enzyme. The product distribution of L-sulfoalanine and taurine depends on the activity relationship of the CS enzyme and the CSAD enzyme. In the case of sufficient quantitative addition of the CSAD enzyme, L-sulfoalanine can be quantitatively converted into taurine. The following method is preferred: wherein the OPS used is converted into L-sulfoalanine and taurine, and the total molar yield of L-sulfoalanine and taurine is greater than 60%, particularly preferably greater than 70%, and especially preferably greater than 80%.

[0157] The process of producing L-cysteic acid and decarboxylating it to taurine in one reaction batch is of particular interest when considering economic feasibility.

[0158] It is also conceivable that, in the context of a metabolic engineering approach, the genes for cysteic acid synthase (preferably CSma) and L-cysteine ​​sulfenic acid decarboxylase (preferably CSADcc) are expressed in an OPS production strain, and taurine is produced by culturing this production strain in the presence of a sulfur source, preferably sulfite (sulfurous acid salt). Similarly, it is conceivable that the genes for cysteic acid synthase (preferably CSma) and L-cysteine ​​sulfenic acid decarboxylase (preferably CSADcc) are co-expressed in one strain, and in the presence of sulfite, cells cultured from the strain react with OPS to produce taurine as a final product.

[0159] Preferably, the bioconversion process for producing taurine is one in which the process components OPS, CS enzyme and CSAD enzyme are produced separately.

[0160] Taurine can be used directly without further processing steps, or it can be enriched or purified by known methods. These methods are all known to those skilled in the art, such as methods for separating amino acids. Examples include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization, etc.

[0161] The present invention also provides the use of L-cysteic acid formed during the production of taurine.

[0162] Compared to known chemical methods for producing taurine from fossil raw materials, the use of L-cysteic acid produced in the method of the present invention to produce taurine allows a biotechnological method starting from plant raw materials to be realized. The biotechnological method for producing taurine is particularly beneficial in applications in the food, feed or cosmetics field because it is a sustainable production method.

[0163] The present invention will be further illustrated by the following examples, but is not limited thereto:

[0164] Example 1 : Production of serB deletion mutants in Escherichia coli

[0165] The strain used was Escherichia coli K12 W3110 (available from DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) under strain number DSM 5911). The target of gene inactivation was the coding sequence of the serB gene in Escherichia coli. The DNA sequence of the CDS of the serB gene from Escherichia coli K12 (SEQ ID NO: 1, nt 67-nt 1032) encodes a protein having the amino acid sequence of SEQ ID NO: 2, which can be found in the NCBI (National Center for Biotechnology Information) gene database under gene ID: 948913.

[0166] Using Gene Bridges GmbH The serB gene of E. coli was inactivated by the technique as described in the user manual of the "Quick and Easy E. coli Gene Deletion Kit" (see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Recombination, Cat. No. K006, Version 2.3, June 2012" and references cited therein, such as Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645). For this purpose, plasmids pKD13, pKD46 and pCP20 were used:

[0167] -3.4kb plasmid pKD13 ( Figure 1 ) has been published in the "GenBank" gene database with the accession number AY048744.1.

[0168] -6.3kb plasmid pKD46 ( Figure 2 ) has been published in the "GenBank" gene database with the accession number AY048746.1.

[0169] The 9.4 kb plasmid pCP20 has been disclosed in Cherepanov and Wackernagel, Gene 158 (1995): 9-14.

[0170] To inactivate the serB gene in E. coli W3110 by homologous recombination using the Lambda Red system, the following steps were performed:

[0171] 1. E. coli W3110 was transformed with plasmid pKD46 (the so-called "red recombinase" plasmid, Fig. 2) and an ampicillin-resistant clone was isolated, designated W3110 x pKD46.

[0172] 2. Use plasmid pKD13 ( Figure 1 ) DNA and primers serb-1f (SEQ ID NO: 7) and serb-2r (SEQ ID NO: 8) were used in a PCR reaction ("Phusion TM High-Fidelity DNA Polymerase, Thermo Scientific TM) to produce a serB-specific DNA fragment suitable for inactivating serB. In the PCR reaction, a 1.4 kb PCR product is formed, which contains a 30 nt DNA fragment at both the 5' and 3' ends, which is specific for the serB gene in E. coli W3110. In addition, the PCR product contains the expression cassette of the kanamycin resistance gene in pKD13, as well as the flanking sequences at the 5' and 3' ends of the kanamycin expression cassette, the so-called "FRT direct repeat sequence" (in Figure 1 These short fragments of DNA were used in subsequent work steps as recognition sequences for the "FLP recombinase" (contained on the plasmid pCP20) to remove the antibiotic marker kanamycin.

[0173] Primer serb-1f contains 30 nucleotides (nt) from the 5' region of the serb gene (nt67-96 in SEQ ID NO: 1), followed by 20 nt specific for plasmid pKD13 ( Figure 1 referred to as “pr-1” in the literature).

[0174] Primer serb-2r contained 30 nt from the 3' region of the serb gene (nt 1006-1035 in SEQ ID NO: 1, in reverse complement form) followed by 20 nt specific for plasmid pKD13 ( Figure 1 referred to as "pr-2" in the literature).

[0175] 3. Isolate the 1.4 kb PCR product and treat it with the restriction endonuclease Dpn I, which is familiar to those skilled in the art and only cuts methylated DNA, to remove the residual pKD13 plasmid DNA. Non-methylated DNA from the PCR reaction will not be degraded.

[0176] 4. The 1.4 kb PCR product was transformed into E. coli W3110 x pKD46, which is specific for the serB gene and contains an expression cassette for the kanamycin resistance gene, and kanamycin-resistant clones were isolated on LBkan-glycine plates at 30°C. LBkan-glycine plates contain LB medium (10 g / L tryptone (GIBCO TM ), 5 g / L yeast extract (BD Biosciences), 5 g / L NaCl), 1.5% agar, 15 mg / L kanamycin (Sigma-Aldrich), and 1 g / L glycine (Sigma-Aldrich).

[0177] 5. The four kanamycin-resistant clones obtained were purified on LBkan-glycine plates (ie, clones were isolated by single colonization) and checked in a PCR reaction to determine whether the kanamycin resistance cassette had been correctly integrated into the serB gene.

[0178] Genomic DNA for PCR reaction was isolated using a DNA isolation kit (Qiagen) from cells of a kanamycin-resistant clone of E. coli W3110 x pKD46 grown in Lbkan-glycine medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin, and 1 g / L glycine) ("Phusion DNA Isolation Kit"). TM High-Fidelity DNA Polymerase, Thermo Scientific TM ). Genomic DNA of the wild-type strain of Escherichia coli W3110 was used as a control. The primers used for the PCR reaction were serb-3f (SEQ ID NO: 9, 5' flanking sequence of the serB gene, nt 1-22 in SEQ ID NO: 1) and serb-4r (SEQ ID NO: 10, 3' flanking sequence of the serB gene, nt 1066-1085 in SEQ ID NO: 1, in reverse complement form).

[0179] E. coli W3110 wild-type DNA produced a 1.1 kb DNA fragment in the PCR reaction, which is consistent with the expected result for an intact gene. In contrast, the four kanamycin-resistant clones studied produced a DNA fragment of approximately 1.6 kb in the PCR reaction, which is the expected result of a 1.4 kb PCR product integrated into the serB gene at the site defined by primers serB-1f and serB-2r. This result indicated that the kanamycin resistance gene had been successfully integrated into the site of the serB gene and that the serB gene had been inactivated. The clone with the inactivated serB gene was selected and treated at 42°C to remove the temperature-sensitive plasmid pKD46, thereby making the strain sensitive to ampicillin again. This strain was named W3110-ΔserB::kan.

[0180] 6. To eliminate the kanamycin selection marker, W3110-ΔserB::kan was transformed with plasmid pCP20 and transformants were selected at 30°C. The 9.4 kb vector pCP20 was disclosed in Cherepanov and Wackernagel (1995), Gene 158:9-14. The FLP recombinase gene is present on the vector pCP20. The FLP recombinase recognizes the FRT sequences flanking the kanamycin resistance gene expression cassette, thereby being able to remove the kanamycin expression cassette. For this purpose, the clones obtained at 30°C were cultured at 37°C. Under these conditions, the expression of the FLP recombinase is induced and the replication of the pCP20 vector is inhibited.

[0181] This step results in clones in which the serB gene is inactivated and sensitive to kanamycin again (so-called "clearance" of the antibiotic selection marker). Removal of the kanamycin cassette from the genome of the ΔserbB mutant allows the introduction of further mutations to generate double or multiple mutants.

[0182] W3110-ΔserB::kan regained sensitivity to kanamycin after treatment with the pCP20 plasmid and was examined as follows:

[0183] - By plating on LB-glycine plates and LBkan-glycine plates:

[0184] Growth on LB-glycine plates was positive, whereas growth was no longer observed on LBkan-glycine plates, indicating that the kanamycin cassette had been successfully removed from the genome.

[0185] -Check by PCR reaction:

[0186] To this end, genomic DNA was isolated from a kanamycin-sensitive clone (Qiagen DNA Isolation Kit) and a PCR reaction was performed using primers serb-3f (SEQ ID NO: 9) and serb-4r (SEQ ID NO: 10) ("Phusion TM High-Fidelity DNA Polymerase, Thermo Scientific TM ). The E. coli W3110 wild-type DNA produced a 1.1 kb DNA fragment in the PCR reaction, which is consistent with the expected result for an intact serB gene. In contrast, the kanamycin-sensitive clone produced a DNA fragment of approximately 250 nt in the PCR reaction, which corresponds to the expected size of the 5' and 3' fragments of the inactivated serB gene remaining after homologous recombination.

[0187] The strain isolated from this step was named Escherichia coli W3110-ΔserB. The strain is characterized in that it contains an inactivated serB gene and the strain regains sensitivity to the antibiotic kanamycin.

[0188] Example 2 :Generation of OPS

[0189] Production in shake flasks:

[0190] OPS was produced by E. coli W3110-ΔserB strain cultured in shake flasks. For comparison, the production of OPS in WT strain E. coli W3110 was analyzed. As a pre-culture for shake flask culture, E. coli W3110 and E. coli W3110-ΔserB were inoculated in 3 ml of LB-glycine medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 0.1 g / L glycine) and cultured on a shaker at 30°C and 135 rpm for 16 h.

[0191] Main culture: After this, a portion of each preculture was transferred to a 300 ml conical flask (with baffles) containing 30 ml of SM1 medium containing 15 g / L glucose, 5 mg / L vitamin B1 (Sigma-Aldrich), 0.1 g / L amino acids (L-isoleucine, D,L-methionine and L-threonine), and 0.5 g / L glycine (all Sigma-Aldrich).

[0192] Composition of SM1 medium: 12g / L K2HPO4, 3g / L KH2PO 4、 5g / L(NH4)2SO4, 0.3g / L MgSO4x7H2O, 0.015g / L CaCl2 x 2H2O, 0.002g / L FeSO4 x 7H2O, 1g / L sodium citrate x 2H2O, 0.1g / LNaCl; 1ml / L trace element solution.

[0193] The composition of the trace element solution: 0.15g / L Na2MoO4·2H2O, 2.5g / L H3BO3, 0.7g / L CoCl2·6H2O, 0.25g / L CuSO4·5H2O, 1.6g / L MnCl2·4H2O, 0.3g / L ZnSO4·7H2O.

[0194] Inoculate the main culture medium with enough preculture to establish an initial cell density of 0.3 / ml in each case OD 600 / ml (optical density of the main culture, measured at 600 nm). On this basis, batches of 30 ml were cultivated at 30° C. and 135 rpm for 24 h.

[0195] After 24 h, samples were taken and the cell density OD in the culture supernatant and cell pellet was determined. 600 / ml and OPS content. For this purpose, 2 ml of the cell culture in each case were centrifuged at 13000 rpm for 5 minutes (Heraeus TM Fresco TM 21 centrifuge). The cell culture supernatant was directly analyzed for OPS content by HPLC. The cell pellet in each case was resuspended in 2 ml of H2O and a cell extract was prepared. This was done by using the FastPrep-24 TM 5G cell homogenizer. In each case, 2x 1ml cell pellets suspended in H2O were broken in a 1.5ml test tube ("Lysing Matrix B") containing glass beads assembled by the manufacturer (3x 20 seconds at an oscillation frequency of 6000rpm, with an interval of 30 seconds between each case). In each case, the obtained cell homogenates were combined and centrifuged at 13000rpm for 5min to prepare cell extracts. The content of OPS in the cell extracts was analyzed by HPLC. The results are summarized in Table 1.

[0196] Table 1: Growth of E. coli W3110 strain and E. coli W3110-ΔserB strain and HPLC analysis of OPS content in cell culture supernatant and cell extract

[0197]

[0198] HPLC analysis of OPS, L-cysteic acid and taurine: For the quantitative determination of the compounds analyzed in the examples, an HPLC method calibrated for OPS, L-cysteic acid and taurine, respectively, was used; all reference substances used for calibration were commercially available (Sigma-Aldrich). An Agilent 1260 Infinity II HPLC system was used, which was equipped with a device from the same manufacturer for pre-column derivatization of o-phthalaldehyde (OPA derivatization) as known in amino acid analysis. For the detection of the OPA-derivatized products of OPS, L-cysteic acid and taurine, the HPLC system was equipped with a fluorescence detector. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm. A Phenomenex HPLC system was also used. A C18(2) column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm was thermally equilibrated at 40 °C in a column oven.

[0199] Eluent A: 25 mM sodium phosphate, pH 6.0. Eluent B: methanol. Separation was performed in gradient mode: 1%-15% eluent B at 0-10 min, then 15% eluent B for 15 min, flow rate 1.0 ml / min. Retention time of L-cysteic acid: 6.95 min. Retention time of OPS: 7.65 min. Retention time of taurine: 21.9 min.

[0200] Production of OPS by fermentation:

[0201] OPS was produced by fermentation of E. coli W3110-ΔserB strain.

[0202] Pre-culture 1:

[0203] In a 100 ml conical flask, 20 ml of LB-glycine medium was inoculated with E. coli W3110-ΔserB strain and cultured on a shaker (150 rpm, 32° C.) for 7 h.

[0204] Pre-culture 2:

[0205] Thereafter, the entire pre-culture 1 was transferred to 100 ml of SM1 medium supplemented with 10 g / L glucose, 10 g / L yeast extract, 0.3 g / L D,L-methionine, 1 g / L glycine, and 5 mg / L vitamin B1. The culture was shaken at 150 rpm for 17 h at 32°C in a conical flask (1 L volume) (Infors shaking incubator). After cultivation, the cell density OD 600 / ml is 5.7 / ml.

[0206] Main training:

[0207] Fermentation in the " The fermentation was carried out in a "Microbiology Parallel Bioreactor System" fermentor. A culture vessel with a total volume of 1.8 L was used. The fermentation medium (600 ml) contained 10 g / L glucose, 5 g / L yeast extract, 5 g / L (NH4)2SO4, 5 g / L KH2PO4, 0.5 g / L NaCl, 0.225 g / L CaCl2 x 2H2O, 1.2 g / L MgSO4x7H2O, 0.075 g / L FeSO4 x 7H2O, 1 g / L sodium citrate x 2H2O, 1 g / L glycine, 1 g / L L-threonine, 0.018 g / L vitamin B1, 0.09 g / L vitamin B6 and 10 ml of trace element solution (see shake flask growth section).

[0208] First, the pH value in the fermentation tank was adjusted to 7.0 by pumping in 25% NH4OH solution. During the fermentation process, the pH value was automatically corrected with 25% NH4OH or 4M H3PO4 to maintain it at 7.0. Foam control was achieved by automatically metering in 4% v / v Struktol J673 H2O solution (Schill & Seilacher). At the time of inoculation, 60 ml of preculture 2 was pumped into the fermentation tank. Therefore, the initial volume was about 660 ml. The culture was initially stirred at 400 rpm and aerated with compressed air sterilized by a sterile filter at a rate of 2 vvm (air volume per minute per volume of culture medium). Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0209] The target value of O2 saturation during fermentation was set to 30%. After the O2 saturation fell below the target value, the regulation cascade was opened to restore the O2 saturation to the target value. This consisted of first continuously increasing the gas supply (maximum 5 vvm) and then continuously increasing the stirring speed (maximum 1600 rpm). The fermentation was carried out at a temperature of 32°C.

[0210] Once the glucose content in the fermenter dropped from the initial 10 g / L to about 2 g / L, a 56% (w / w) glucose solution was continuously metered in. The feed rate was adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L from then on. Glucose was measured using a glucose analyzer from YSI (Yellow Springs, Ohio, USA). 23 h after the start of fermentation, 3.5 ml of a 200 g / L glycine solution in H2O was added to the fermentation batch.

[0211] The fermentation time was 53 hours. Samples were taken from the fermentation batch 23h, 30h, 47h and 53h after the start of fermentation, and the cell density OD was measured from an aliquot. 600Another aliquot in each case was incubated at 80°C for 5 min and centrifuged, and the OPS content in the cell culture supernatant was then determined by HPLC. Table 2 summarizes the cell density and OPS content.

[0212] Table 2: Time course of cell density and OPS concentration in fermentation of E. coli W3110-ΔserB strain

[0213]

[0214] Example 3 :Production of CSma enzyme

[0215] The sulfalanine synthase from Methanosarcina acetica (CSma) was used. The amino acid sequence of the CSma enzyme is available in the NCBI database under the accession ID WP_048066469. The amino acid sequence was used to obtain a codon-optimized DNA sequence for expression in E. coli (publicly available Eurofins Genomics GENEius software), which was prepared synthetically (Eurofins Genomics). The synthetically prepared DNA has the sequence disclosed in SEQ ID NO: 3 and contains the CDS of the gene, hereinafter referred to as CSma CDS (SEQ ID NO: 3), which encodes a protein having the amino acid sequence disclosed in SEQ ID NO: 4, hereinafter referred to as CSma. For cloning purposes, the synthetically prepared DNA contains an EcoRI cleavage site at the 5' end and a HindIII cleavage site at the 3' end.

[0216] Vector pCSma-pKKj (suitable for recombinant expression of CSma CDS Figure 3 ) is produced by cutting the synthetically prepared DNA with EcoRI and HindIII and cloning it as an EcoRI / HindIII fragment into a vector pKKj cut with EcoRI and HindIII in a known manner. The expression vector pKKj is disclosed in EP2670837A1 (Wacker application) and is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database and its accession number is M77749.1. About 1.7 kb (bp 262-1947 of the DNA sequence disclosed in M77749.1) is removed from the 4.6 kb plasmid to obtain the 2.9 kb expression vector pKKj.

[0217] The CSma CDS was expressed in E. coli by transforming the vector pCSma-pKKj into the E. coli K12 JM105 strain in a known manner. The E. coli K12 JM105 strain can be purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH under the strain number DSM 3949.

[0218] Transformed clones were screened on LBamp plates. LBamp contained 10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl, 15 g / l agar and 100 mg / l ampicillin (Sigma-Aldrich). One clone was screened and cultured in a shake flask. The CSma producing strain was named E. coli JM105 x pCSma-pKKj. CSma CDS was expressed in a known manner in E. coli JM105 x pCSma-pKKj under the control of an IPTG-induced tac promoter (IPTG: isopropyl β-thiogalactopyranoside, Sigma-Aldrich) functionally linked to CSma.

[0219] Cultivation in shake flasks:

[0220] A preculture of E. coli JM105 x pCSma-pKKj strain was prepared in LBamp medium (37°C, 120 rpm, Infors shaking incubator, overnight growth).

[0221] 2 ml of pre-culture solution (OD 600 3.4 / ml) was used as inoculum for a main culture (0.3 L conical flask) of 50 ml SM1 medium (Example 2), to which 15 g / L glucose, 5 g / L peptone (Oxoid), 2.5 g / L yeast extract, 0.005 g / L vitamin B1 (Sigma-Aldrich), 5 g / L pyridoxal phosphate (PLP, Sigma-Aldrich) and 100 mg / L ampicillin were added. The main culture was shaken in a shaking incubator (Infors) at 30°C and 140 rpm. After 4 hours of cultivation, an OD of 0.05 was reached. 600 The cell density was 2.0. Then the inducer IPTG (Sigma-Aldrich, final concentration of 0.4 mM) was added and culture was continued for 20 h in a shaking incubator (Infors) at 30°C and 140 rpm. At the end of the culture, the cell density OD 600 It is 3.1 / ml.

[0222] Cells were isolated from shake flask cultures by centrifugation (10 min at 15000 rpm, Sorvall RC5C centrifuge, equipped with SS34 rotor). 50 ml shake flask grown cell pellets were resuspended in 2 ml of 100 mM potassium phosphate (pH 7.0); 100 mM KCl (KPi 7.0 buffer) to prepare a cell suspension and used to prepare a cell homogenate. As described in Example 2, a FastPrep-24TM 5G cell homogenizer from MP Biomedicals was used to prepare a cell homogenate. The obtained cell homogenate (2 ml volume) was used for the bioconversion of OPS to L-cysteic acid (Example 5) without further post-treatment.

[0223] The protein content of the cell homogenate was measured by Qubit 3.0 Fluorometer from Thermo Fisher Scientific using the Protein assay kit" was used according to the manufacturer's instructions. The protein content of the cell homogenate from the shake flask culture was 5.3 mg / ml.

[0224] Example 4 : Bioconversion using CSma enzyme to produce L-cysteic acid from commercially available OPS and Na2SO3 Two batches were carried out simultaneously:

[0225] Batch 1: In a 100 ml conical flask, 8.15 ml of KPi7.0 buffer was first added, followed by 1 ml of 0.2 M Na2SO3 solution in KPi7.0 buffer, 0.5 ml of CSma cell homogenate from shake flask culture (Example 3), and 350 μl of 0.2 M OPS (Sigma-Aldrich) solution in KPi7.0 buffer. The batch volume was 10 ml.

[0226] Batch 2: This batch (control batch without Na2SO3) had the same composition as Batch 1. Batch 2 contained 1 ml of KPi7.0 buffer as a replacement for Na2SO3 solution.

[0227] Both batches were cultured at 30°C and 140 rpm in a shaking incubator (Infors). After 1 h, 2 h and 4 h, 1 ml was taken from each batch and incubated at 80°C for 5 min and centrifuged, and the supernatant was analyzed by HPLC. Table 3 shows the time course of the production of L-cysteic acid from OPS.

[0228] Table 3: Time course of OPS and Na2SO3 bioconversion to produce L-cysteic acid using CSma cell homogenate

[0229]

[0230] Example 5 : Production of L-cysteic acid by bioconversion of culture supernatant containing OPS and Na2SO3 from shake flask culture by CSma enzyme

[0231] In a 100 ml conical flask, 9 ml of the cell culture supernatant of the shake flask culture of the Escherichia coli W3110-ΔserB strain (Example 2) was first added, wherein the OPS content was 113.6 mg / L, and then 0.3 ml of 3M KCl, 0.5 ml of 0.2M Na2SO3 in KPi7.0 buffer and 1 ml of the shake flask cultured CSma cell homogenate (Example 3) were added. The batch volume was 10.8 ml. The batch was cultured in an oscillating incubator (Infors) at 30°C and 140 rpm. At the beginning and after 6 h, 1 ml was taken in each case of each batch and incubated at 80°C for 5 min and centrifuged, and the content of OPS and L-sulfoalanine in the supernatant was analyzed by HPLC. The process of the reaction changing over time is summarized in Table 4. Based on the molar amount of OPS used, the molar yield of L-sulfoalanine is 97.6%.

[0232] Table 4: Time course of L-cysteic acid production catalyzed by cell culture supernatant containing OPS and Na2SO3 using cell homogenate containing CSma

[0233]

[0234] Example 6 : Preparative-scale production of L-cysteic acid by biotransformation of OPS at constant pH

[0235] OPS substrate: 10 ml of fermentation broth produced by fermentation of E. coli W3110-ΔserB strain (Example 2) was centrifuged (15000 rpm for 10 min, Sorvall RC5C centrifuge, equipped with SS34 rotor), and the OPS content in the fermentation supernatant was determined by HPLC. The OPS content was 5.6 g / L.

[0236] CSma homogenate: A cell homogenate of E. coli JM105xpCSma-pKKj strain was prepared from 2 x 50 ml shake flask cultures as described in Example 3. 15 μl of 500 mg / L PLP was added to the homogenate. The total volume of the homogenate was 4 ml.

[0237] Biotransformation batches: A 50 ml thermostattable double-walled lower conical reaction vessel (accessory for the TitratorTitroLine alpha, Schott) was connected to a thermostat (Lauda) via a hose connection and the temperature was adjusted to 30°C.

[0238] The reaction batch contained 6 ml of fermentation supernatant containing OPS (wherein the OPS content was 5.6 g / L), 4 ml of CSma homogenate, 0.2 ml of 3 M KCl, 0.1 ml of 0.1 M DTE (dithiothreitol, Sigma-Aldrich) and 0.3 ml of 1 M Na2SO3 solution in KPi7.0 buffer. The batch volume was 10.6 ml. The OPS concentration in the batch was 17.1 mM (0.18 mmol of OPS in the 10.6 ml batch volume). The batch was stirred using a magnetic stirrer. The batch was also equipped with a pH electrode (Mettler Toledo) connected to a pH control unit (TitroLine alpha titrator, Schott), which was operated in pH steady-state mode according to the manufacturer's instructions. Under pH steady-state conditions, the pH value in the reaction vessel was kept constant at a set pH of 7.0 over the entire reaction duration by metering 0.5 M NaOH from a burette connected to the control unit.

[0239] The reaction time was 4 h. Since 0.5 M NaOH was added to the batch from the burette to maintain a constant pH of 7.0, the batch volume after 4 h of reaction was 13.0 ml. 2 h and 4 h after the start of the reaction, aliquots of 50 μl were taken in each case of each batch, and the content of OPS and L-cysteic acid in the supernatant was analyzed by HPLC. The course of the reaction over time is summarized in Table 5. After a reaction time of 4 h, the content of L-cysteic acid in the batch was 2399.6 mg / L (14.2 mM), which corresponds to an absolute molar yield of L-cysteic acid of 0.18 mmol for a batch volume of 13.0 ml. The OPS used was completely consumed. Based on the amount of 0.18 mmol of OPS used, this is equivalent to a yield of 100%.

[0240] Table 5: OPS and L-cysteic acid content detected by HPLC according to reaction time using fermentation supernatant containing OPS, Na2SO3 and cell homogenate containing CSma from shake flask culture

[0241]

[0242] Example 7 : Recombinant production of CSADcc from Cyprinus carpio in Escherichia coli

[0243] CSADcc gene:

[0244] The mRNA-derived cDNA sequence of cysteine ​​sulfenic acid decarboxylase (CSAD) from carp has been published in the NCBI database (National Center for Biotechnology Information, USA) with Genbank sequence ID: AB220585.1 (CDS: nt 82–1584). The corresponding amino acid sequence was used to obtain a codon-optimized DNA sequence expressed in Escherichia coli (publicly available Eurofins Genomics GENEius software), which was prepared synthetically (Eurofins Genomics). The synthetically prepared DNA has the sequence disclosed in SEQ ID NO: 5 and contains the CDS of the gene, hereinafter referred to as CSADcc CDS (SEQ ID NO: 5, nt 31-1530), which encodes a protein having the amino acid sequence disclosed in SEQ ID NO: 6, hereinafter referred to as CSADcc. For cloning purposes, the synthetically prepared DNA contained an EcoRI cleavage site at the 5' end (SEQ ID NO: 5, nt 25-30) and a HindIII cleavage site at the 3' end (SEQ ID NO: 5, nt 1532-1537).

[0245] Vector pCSADcc-pKKj:

[0246] The vector pCSADcc-pKKj ( Figure 4 ) was produced by cutting the synthetically prepared DNA with EcoRI and HindIII and cloning it as an EcoRI / HindIII fragment into the vector pKKj cut with EcoRI and HindIII in a known manner (see Example 3). This produced the vector pCSADcc-pKKj. The CSADcc CDS was expressed in E. coli by transforming the vector pCSADcc-pKKj into the E. coli K12 JM105 strain in a known manner. The transformed clones were screened on LBamp plates. One clone was screened and cultured in a shake flask. The strain producing CSADcc was named E. coli JM105 x pCSADcc-pKKj. The CSADcc CDS was expressed in E. coli JM105 xpCSADcc-pKKj, which was cultured in a shake flask similar to the E. coli JM105 x pCSma-pKKj described in Example 3.

[0247] Cultivate in shake flasks: Add 10 ml of pre-culture solution (OD 6003.1 / ml) was used as inoculum for a main culture (1 L conical flask) of 100 ml SM1 medium supplemented with 15 g / L glucose, 5 g / L peptone, 2.5 g / L yeast extract, 0.005 g / L vitamin B1, 5 g / L pyridoxal phosphate and 100 mg / L ampicillin. The main culture was grown and induced as described in Example 3. Cells (OD 2.004 / 0.004) were isolated from the 100 ml shake flask culture by centrifugation. 600 7.1 / ml), and the cell pellet was resuspended in 4 ml of KPi7.0 buffer. This cell suspension was directly used for biotransformation experiments.

[0248] Example 8 : Production of taurine from L-cysteic acid by bioconversion

[0249] In a 100 ml conical flask, 9 ml of the batch from Example 5 was first added, wherein the content of L-sulfoalanine was 84.5 mg / L (Table 4), and then 1 ml of the cell suspension of the CSADcc enzyme from Example 7 was added. The batch volume was 10 ml. The batch was incubated in an oscillating incubator (Infors) at 37 ° C and 140 rpm. After 2 h, 1 ml of the batch was incubated at 80 ° C for 5 min and centrifuged, and the supernatant was analyzed by HPLC. The L-sulfoalanine used was completely consumed. The amount of taurine formed was 66.4 mg / L.

[0250] Example 9 : Production of taurine from OPS by bioconversion

[0251] In a 100 ml conical flask, 9 ml of a shake flask cultured batch of Escherichia coli W3110-ΔserB strain (Example 2) was first added, wherein the OPS content was 94.7 mg / L, and then 0.5 ml of a 0.2 M Na2SO3 solution in a KPi7.0 buffer and 1 ml of a shake flask cultured CSma cell homogenate (Example 3) and 1 ml of a cell suspension of CSADcc enzyme (Example 7) were added. The batch volume was 11.5 ml. The batch was incubated in an oscillating incubator (Infors) at 30°C and 140 rpm. After 4 h, 1 ml of the batch was incubated at 80°C for 5 min and centrifuged, and the supernatant was analyzed by HPLC. The OPS used was completely consumed. At the same time, 34.4 mg / L of L-sulfoalanine and 40.4 mg / L of taurine were formed.

[0252] Abbreviations used in the figure:

[0253] bla: gene conferring resistance to ampicillin (β-lactamase)

[0254] kanR: a gene that confers resistance to kanamycin

[0255] ORI: origin of replication

[0256] pr-1: Primer binding site 1

[0257] pr-2: Primer binding site 2

[0258] FRT1: FLP recombinase recognition sequence 1

[0259] FRT2: FLP recombinase recognition sequence 2

[0260] araC: araC gene (suppressor gene)

[0261] P araC: promoter of araC gene

[0262] P araB: promoter of araB gene

[0263] Gam: Gam recombination gene of λ phage

[0264] Bet: lambda phage Bet recombination gene

[0265] Exo: λ phage Exo recombination gene

[0266] ORI101: a temperature-sensitive origin of replication

[0267] RepA: Gene for plasmid replication protein A

[0268] Ptac: tac promoter

[0269] EcoRI: cleavage site of restriction endonuclease EcoRI

[0270] HindIII: cleavage site of restriction endonuclease HindIII

[0271] CSma: CDS of sulfalanine synthase gene from Methanosarcina aceticum

[0272] CSADcc: CDS of cysteine ​​sulfenic acid decarboxylase gene from common carp Sequence Listing <110> Wacker Chemie AG <120> Method for producing L-cysteic acid and use thereof <130> OPS <160> 10 <170> PatentIn Version 3.5 <210> 1 <211> 1085 <212> DNA <213> Escherichia coli <220> <221> gene <222> (1)..(1085) <220> <221> gene <222> (1)..(1085) <223> serB <400> 1 tgagacttaa tgttgccaga agcaatggtt attttccctg cttcgaacga ttttacagga 60 gccttaatgc ctaacattac ctggtgcgac ctgcctgaag atgtctcttt atggccgggt 120 ctgcctcttt cattaagtgg tgatgaagtg atgccactgg attaccacgc aggtcgtagc 180 ggctggctgc tgtatggtcg tgggctggat aaacaacgtc tgacccaata ccagagcaaa 240 ctgggtgcgg cgatggtgat tgttgccgcc tggtgcgtgg aagattatca ggtgattcgt 300 ctggcaggtt cactcaccgc acgggctaca cgcctggccc acgaagcgca gctggatgtc 360 gccccgctgg ggaaaatccc gcacctgcgc acgccgggtt tgctggtgat ggatatggac 420 tccaccgcca tccagattga atgtattgat gaaattgcca aactggccgg aacgggcgag 480 atggtggcgg aagtaaccga acgggcgatg cgcggcgaac tcgattttac cgccagcctg 540 cgcagccgtg tggcgacgct gaaaggcgct gacgccaata ttctgcaaca ggtgcgtgaa 600 aatctgccgc tgatgccagg cttaacgcaa ctggtgctca agctggaaac gctgggctgg 660 aaagtggcga ttgcctccgg cggctttact ttctttgctg aatacctgcg cgacaagctg 720 cgcctgaccg ccgtggtagc caatgaactg gagatcatgg acggtaaatt taccggcaat 780 gtgatcggcg acatcgtaga cgcgcagtac aaagcgaaaa ctctgactcg cctcgcgcag 840 gagtatgaaa tcccgctggc gcagaccgtg gcgattggcg atggagccaa tgacctgccg 900 atgatcaaag cggcagggct ggggattgcc taccatgcca agccaaaagt gaatgaaaag 960 gcggaagtca ccatccgtca cgctgacctg atgggggtat tctgcatcct ctcaggcagc 1020 ctgaatcaga agtaattgct cgcccgccat cctgcgggcg gcacagcatt aacgaggtac 1080 accgt 1085 <210> 2 <211> 322 <212> PRT <213> Escherichia coli <220> <221> Peptide <222> (1)..(322) <220> <221> Peptide <222> (1)..(322) <223> SerB protein <400> 2 Met Pro Asn Ile Thr Trp Cys Asp Leu Pro Glu Asp Val Ser Leu Trp 1 5 10 15 Pro Gly Leu Pro Leu Ser Leu Ser Gly Asp Glu Val Met Pro Leu Asp 20 25 30 Tyr His Ala Gly Arg Ser Gly Trp Leu Leu Tyr Gly Arg Gly Leu Asp 35 40 45 Lys Gln Arg Leu Thr Gln Tyr Gln Ser Lys Leu Gly Ala Ala Met Val 50 55 60 Ile Val Ala Ala Trp Cys Val Glu Asp Tyr Gln Val Ile Arg Leu Ala 65 70 75 80 Gly Ser Leu Thr Ala Arg Ala Thr Arg Leu Ala His Glu Ala Gln Leu 85 90 95 Asp Val Ala Pro Leu Gly Lys Ile Pro His Leu Arg Thr Pro Gly Leu 100 105 110 Leu Val Met Asp Met Asp Ser Thr Ala Ile Gln Ile Glu Cys Ile Asp 115 120 125 Glu Ile Ala Lys Leu Ala Gly Thr Gly Glu Met Val Ala Glu Val Thr 130 135 140 Glu Arg Ala Met Arg Gly Glu Leu Asp Phe Thr Ala Ser Leu Arg Ser 145 150 155 160 Arg Val Ala Thr Leu Lys Gly Ala Asp Ala Asn Ile Leu Gln Gln Val 165 170 175 Arg Glu Asn Leu Pro Leu Met Pro Gly Leu Thr Gln Leu Val Leu Lys 180 185 190 Leu Glu Thr Leu Gly Trp Lys Val Ala Ile Ala Ser Gly Gly Phe Thr 195 200 205 Phe Phe Ala Glu Tyr Leu Arg Asp Lys Leu Arg Leu Thr Ala Val Val 210 215 220 Ala Asn Glu Leu Glu Ile Met Asp Gly Lys Phe Thr Gly Asn Val Ile 225 230 235 240 Gly Asp Ile Val Asp Ala Gln Tyr Lys Ala Lys Thr Leu Thr Arg Leu 245 250 255 Ala Gln Glu Tyr Glu Ile Pro Leu Ala Gln Thr Val Ala Ile Gly Asp 260 265 270 Gly Ala Asn Asp Leu Pro Met Ile Lys Ala Ala Gly Leu Gly Ile Ala 275 280 285 Tyr His Ala Lys Pro Lys Val Asn Glu Lys Ala Glu Val Thr Ile Arg 290 295 300 His Ala Asp Leu Met Gly Val Phe Cys Ile Leu Ser Gly Ser Leu Asn 305 310 315 320 Gln Lys <210> 3 <211> 1251 <212> DNA <213> Methanosarcina acetivorans <220> <221> gene <222> (1)..(1251) <220> <221> gene <222> (1)..(1251) <223> CSma <400> 3 atgggccgct ttatcctgaa atgcctgaaa tgtggccgtg aatatagtca ggagtatcgc 60 ttgacttgcg aaaacgacga tagcttcctg cgtgccgaat acctggaaaa gaaacttgag 120 ctgcgtaaac aacctgggat tggacgtttc cattcgtggc tgccggtaca ggaagagctg 180 accacggaag cgggtcccat tacgtacaaa tcggaggcat tagcccgcga attagggctg 240 tcaaacttgt acatcggctt tagcggctat tggccggaga aaggcgcctt tatcaaaacc 300 tgtagcttca aagagttaga agcgcatcct acaatgcagc tgctgaagga aagcggcggg 360 aaggccattg tgcttgcttc tgcaggcaac actggacggg cgtttgcgca tgtgagtgcc 420 ctcaccggaa ccgacgtgta cattgtggtt ccagactcag gcattccgaa actgtggttg 480 ccagaagaac cgaccgactc tattcacctt atcagcatga ccccagggaa cgactatacc 540 gatgccatta atctggccgg acgcattgcg aaattaccgg gtatggttcc tgaaggcggt 600 gcacgcaacg tggctcgtcg cgaaggcatg ggcactgtga tgctggatgc agcggtaacg 660 atcggtaaaa tgccggatca ctactttcaa gccgttggca gtggcacagg cggtatttca 720 gcctgggaag ctagcttacg cctgcgtgaa gatggtcgct ttgggtccaa actgccgaaa 780 ctccagctta ctcagaacct cccgtttgtc cccatgtata acgcgtggca agagggtcgc 840 cgggatatta tcccggaaat tgacatgaaa gatgcgaaga agcgtatcga ggaaacgtat 900 gcaaccgtcc tgaccaatcg tgcaccgccg tattccgtga caggtggtct gtacgatgcg 960 ttggtcgata ccgatggcat catgtatgcg gtttccaaag aagaagcctt ggatgcgaaa 1020 gcgctgttcg aaagtctcga aggtatcgat attctgccgc catctgctgt tgctgcagct 1080 tcgttgctca aagcagtcga agcgggtaat gtcgggaaag acgacacgat tctgctgaat 1140 atcgcgggtg gcggttcaa acgccttaag gaagatttca ccctgtttca gattgagccg 1200 gagattacgg tgtcgaatcc cgatgtacct ctggaagagc tgaaattata a 1251 <210> 4 <211> 416 <212> PRT <213> Methanosarcina acetivorans <220> <221> Peptide <222> (1)..(416) <220> <221> Peptide <222> (1)..(416) <223> CSma protein <400> 4 Met Gly Arg Phe Ile Leu Lys Cys Leu Lys Cys Gly Arg Glu Tyr Ser 1 5 10 15 Gln Glu Tyr Arg Leu Thr Cys Glu Asn Asp Asp Ser Phe Leu Arg Ala 20 25 30 Glu Tyr Leu Glu Lys Lys Leu Glu Leu Arg Lys Gln Pro Gly Ile Gly 35 40 45 Arg Phe His Ser Trp Leu Pro Val Gln Glu Glu Leu Thr Thr Glu Ala 50 55 60 Gly Pro Ile Thr Tyr Lys Ser Glu Ala Leu Ala Arg Glu Leu Gly Leu 65 70 75 80 Ser Asn Leu Tyr Ile Gly Phe Ser Gly Tyr Trp Pro Glu Lys Gly Ala 85 90 95 Phe Ile Lys Thr Cys Ser Phe Lys Glu Leu Glu Ala His Pro Thr Met 100 105 110 Gln Leu Leu Lys Glu Ser Gly Gly Lys Ala Ile Val Leu Ala Ser Ala 115 120 125 Gly Asn Thr Gly Arg Ala Phe Ala His Val Ser Ala Leu Thr Gly Thr 130 135 140 Asp Val Tyr Ile Val Val Pro Asp Ser Gly Ile Pro Lys Leu Trp Leu 145 150 155 160 Pro Glu Glu Pro Thr Asp Ser Ile His Leu Ile Ser Met Thr Pro Gly 165 170 175 Asn Asp Tyr Thr Asp Ala Ile Asn Leu Ala Gly Arg Ile Ala Lys Leu 180 185 190 Pro Gly Met Val Pro Glu Gly Gly Ala Arg Asn Val Ala Arg Arg Glu 195 200 205 Gly Met Gly Thr Val Met Leu Asp Ala Ala Val Thr Ile Gly Lys Met 210 215 220 Pro Asp His Tyr Phe Gln Ala Val Gly Ser Gly Thr Gly Gly Ile Ser 225 230 235 240 Ala Trp Glu Ala Ser Leu Arg Leu Arg Glu Asp Gly Arg Phe Gly Ser 245 250 255 Lys Leu Pro Lys Leu Gln Leu Thr Gln Asn Leu Pro Phe Val Pro Met 260 265 270 Tyr Asn Ala Trp Gln Glu Gly Arg Arg Asp Ile Ile Pro Glu Ile Asp 275 280 285 Met Lys Asp Ala Lys Lys Arg Ile Glu Glu Thr Tyr Ala Thr Val Leu 290 295 300 Thr Asn Arg Ala Pro Pro Tyr Ser Val Thr Gly Gly Leu Tyr Asp Ala 305 310 315 320 Leu Val Asp Thr Asp Gly Ile Met Tyr Ala Val Ser Lys Glu Glu Ala 325 330 335 Leu Asp Ala Lys Ala Leu Phe Glu Ser Leu Glu Gly Ile Asp Ile Leu 340 345 350 Pro Pro Ser Ala Val Ala Ala Ala Ser Leu Leu Lys Ala Val Glu Ala 355 360 365 Gly Asn Val Gly Lys Asp Asp Thr Ile Leu Leu Asn Ile Ala Gly Gly 370 375 380 Gly Phe Lys Arg Leu Lys Glu Asp Phe Thr Leu Phe Gln Ile Glu Pro 385 390 395 400 Glu Ile Thr Val Ser Asn Pro Asp Val Pro Leu Glu Glu Leu Lys Leu 405 410 415 <210> 5 <211> 1566 <212> DNA <213> Carp <220> <221> Gene <222> (1)..(1566) <220> <221> Gene <222> (1)..(1566) <223> CSADcc <400> 5 gttaacaatt tcacacagga aacagaattc atgagttcgt ccaaggaata tatgaatggc 60 cacgtgcatc tggaggagag tgatatgtat gaatccgatg gcaaactgtt tctgacggaa 120 gcctttaacg ttatcatgga agaaattttg cacaaaggta ccgattcgga agagaaagtg 180 tgtgagtgga aagatcccga tcagctgcgt gccctcttgg atctggatct gcgtgaacat 240 ggggaatcac ataaacagct gcttcaacgc gtgcgtgacg ttgcacgcta ttctgtcaaa 300 acctgtcatc cgcgcttctt caatcagctg tttgccggcg tagactatca cgctcttacc 360 ggacgcttgc tgaccgaaac cctgaatacg agtcagtaca cttacgaagt cgctccggtg 420 tttgttctta tggaagagga agtgctttgc aaattacgca gcctcgtcgg atggtctgaa 480 ggcgacggta ttttctgtcc tggcggcagc atgtccaaca tgtatgcgat gaatgtggct 540 cgttattggg ctttccctca ggttaaaacg cagggtttat gggcaacgcc acgtatggcg 600 atttcacct ctcaacaggg cattacagc atgaagaaag cggcagcctt tctgggtatt 660 ggcacagaca atgtgttcat cgtacaggtc gatgaaagcg ggagtatgcg tcctgaggat 720 ctcgaagcga aaatcgtaca agcgaaaagc caggatgccg ttcccttct tgtgaacgca 780 acagccggta caaccgttca aggtgcctttt gatccgctga atcgtattgc ggacatctca 840 gaacgcaacg gcatgtggat gcacgtcgat gcggcttggg gtggttcagt gctgtttagc 900 aagaagcaca aacatctggt tgcgggcatt gaacgtgcaa acagcgtgac ttggaatccg 960 cacaaaatgc tgctggccgg gttacagtgt tctgtcgtac tgtttcgcga taccacgaat 1020 ttactgatgc actgccattc ggccaaagcg acgtacctct tccaacaaga caagttttac 1080 gacacttcgc ttgatactgg ggacaaaagc attcagtgcg gtcgcaaggt ggattgcttg 1140 aaactgtggc tgatgtggaa agcgattggc acccatgggt tatccgagcg tgtagagaaa 1200 gcgtttgctt tagcacgcta tctggtcgaa gaaatggaaa agcgcgacaa cttcaagctg 1260 gtgtgcaaag gtccgtttgt aaacgtgtgc ttttggttta tcccgccgtc actccgccgc 1320 aaagaaaact ccgcggatta ccaagaacgg ttgtcgaaag tggcaccggt tatcaaagag 1380 cggatgatga aacgtggaac gatgatggtg ggttatcagc caatggatgg ccatgtcaac 1440 ttctttcgca tggttgttgt cagtccacag ttgaccacca aagacatgga cttcttcctg 1500 gatgagattg aagatctggg caaagacctg taagcttggc tgttttggcg gatgagagaa 1560 gatatc 1566 <210> 6 <211> 500 <212> PRT <213> Carp <220> <221> Peptide <222> (1)..(500) <220> <221> Peptide <222> (1)..(500) <223> CSADcc protein <400> 6 Met Ser Ser Ser Lys Glu Tyr Met Asn Gly His Val His Leu Glu Glu 1 5 10 15 Ser Asp Met Tyr Glu Ser Asp Gly Lys Leu Phe Leu Thr Glu Ala Phe 20 25 30 Asn Val Ile Met Glu Glu Ile Leu His Lys Gly Thr Asp Ser Glu Glu 35 40 45 Lys Val Cys Glu Trp Lys Asp Pro Asp Gln Leu Arg Ala Leu Leu Asp 50 55 60 Leu Asp Leu Arg Glu His Gly Glu Ser His Lys Gln Leu Leu Gln Arg 65 70 75 80 Val Arg Asp Val Ala Arg Tyr Ser Val Lys Thr Cys His Pro Arg Phe 85 90 95 Phe Asn Gln Leu Phe Ala Gly Val Asp Tyr His Ala Leu Thr Gly Arg 100 105 110 Leu Leu Thr Glu Thr Leu Asn Thr Ser Gln Tyr Thr Tyr Glu Val Ala 115 120 125 Pro Val Phe Val Leu Met Glu Glu Glu Val Leu Cys Lys Leu Arg Ser 130 135 140 Leu Val Gly Trp Ser Glu Gly Asp Gly Ile Phe Cys Pro Gly Gly Ser 145 150 155 160 Met Ser Asn Met Tyr Ala Met Asn Val Ala Arg Tyr Trp Ala Phe Pro 165 170 175 Gln Val Lys Thr Gln Gly Leu Trp Ala Thr Pro Arg Met Ala Ile Phe 180 185 190 Thr Ser Gln Gln Gly His Tyr Ser Met Lys Lys Ala Ala Ala Phe Leu 195 200 205 Gly Ile Gly Thr Asp Asn Val Phe Ile Val Gln Val Asp Glu Ser Gly 210 215 220 Ser Met Arg Pro Glu Asp Leu Glu Ala Lys Ile Val Gln Ala Lys Ser 225 230 235 240 Gln Asp Ala Val Pro Phe Phe Val Asn Ala Thr Ala Gly Thr Thr Val 245 250 255 Gln Gly Ala Phe Asp Pro Leu Asn Arg Ile Ala Asp Ile Ser Glu Arg 260 265 270 Asn Gly Met Trp Met His Val Asp Ala Ala Trp Gly Gly Ser Val Leu 275 280 285 Phe Ser Lys Lys His Lys His Leu Val Ala Gly Ile Glu Arg Ala Asn 290 295 300 Ser Val Thr Trp Asn Pro His Lys Met Leu Leu Ala Gly Leu Gln Cys 305 310 315 320 Ser Val Val Leu Phe Arg Asp Thr Thr Asn Leu Leu Met His Cys His 325 330 335 Ser Ala Lys Ala Thr Tyr Leu Phe Gln Gln Asp Lys Phe Tyr Asp Thr 340 345 350 Ser Leu Asp Thr Gly Asp Lys Ser Ile Gln Cys Gly Arg Lys Val Asp 355 360 365 Cys Leu Lys Leu Trp Leu Met Trp Lys Ala Ile Gly Thr His Gly Leu 370 375 380 Ser Glu Arg Val Glu Lys Ala Phe Ala Leu Ala Arg Tyr Leu Val Glu 385 390 395 400 Glu Met Glu Lys Arg Asp Asn Phe Lys Leu Val Cys Lys Gly Pro Phe 405 410 415 Val Asn Val Cys Phe Trp Phe Ile Pro Pro Ser Leu Arg Arg Lys Glu 420 425 430 Asn Ser Ala Asp Tyr Gln Glu Arg Leu Ser Lys Val Ala Pro Val Ile 435 440 445 Lys Glu Arg Met Met Lys Arg Gly Thr Met Met Val Gly Tyr Gln Pro 450 455 460 Met Asp Gly His Val Asn Phe Phe Arg Met Val Val Val Ser Pro Gln 465 470 475 480 Leu Thr Thr Lys Asp Met Asp Phe Phe Leu Asp Glu Ile Glu Asp Leu 485 490 495 Gly Lys Asp Leu 500 <210> 7 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primers <220> <221> misc_feature <222> (1)..(50) <220> <221> misc_feature <222> (1)..(50) <223> serb-1f <400> 7 atgcctaaca ttacctggtg cgacctgcct gtgtaggctg gagctgcttc 50 <210> 8 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Primers <220> <221> misc_feature <222> (1)..(50) <220> <221> misc_feature <222> (1)..(50) <223> serb-2r <400> 8 ttacttctga ttcaggctgc ctgagaggat ctgtcaaaca tgagaattaa 50 <210> 9 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Primers <220> <221> misc_feature <222> (1)..(22) <220> <221> misc_feature <222> (1)..(22) <223> serb-3f <400> 9 tgagacttaa tgttgccaga ag 22 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Primers <220> <221> misc_feature <222> (1)..(20) <220> <221> misc_feature <222> (1)..(20) <223> serb-4r <400> 10 acggtgtacc tcgttaatgc 20

Claims

1. A method for producing L-cysteic acid, the method comprising: The reaction of O-phospho-L-serine (OPS) with salts of sulfurous acid (sulfite) and cysteic acid synthase (CS enzyme) belonging to the enzyme class EC 2.5.1.76 is carried out in a biotransformation.

2. The method according to claim 1, characterized in that The CS enzyme is produced by culturing an Enterobacteriaceae microbial strain which heterologously expresses the CS enzyme in an enzymatically active form.

3. The method according to any one of claims 1 and 2, characterized in that CS enzyme was used in the reaction without a prior renaturation step.

4. The method according to any one or more of claims 1 to 3, characterized in that The CS enzyme is produced by culturing a microbial strain of the species Escherichia coli which heterologously expresses the CS enzyme in enzymatically active form.

5. The method according to any one or more of claims 1 to 4, characterized in that The OPS used in the reaction was produced by biotechnology.

6. The method according to any one or more of claims 1 to 5, characterized in that The OPS used in the reaction is produced by a microbial strain in which the activity of O-phospho-L-serine phosphatase (SerB enzyme) belonging to the enzyme class EC 3.1.3.3 is inhibited.

7. The method according to any one or more of claims 1 to 6, characterized in that The CS enzyme has the amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence homologous thereto, wherein the amino acid sequence homologous to SEQ ID NO: 4 has at least 50% sequence identity with SEQ ID NO: 4 and has sulfaalanine synthase activity.

8. The method according to any one or more of claims 1 to 7, characterized in that The molar yield of L-cysteic acid is at least 60%, based on the molar amount of OPS used.

9. A method for producing taurine, characterized in that Production and decarboxylation of L-cysteic acid according to the process according to any one or more of claims 1 to 8.

10. The method according to claim 9, characterized in that The decarboxylation is achieved by cysteine ​​sulfenate decarboxylase (CSAD enzyme) belonging to enzyme class EC 4.1.1.

29.

11. The method according to claim 10, characterized in that The amino acid sequence of the CSAD enzyme is SEQ ID No:

6.

12. The method according to any one or more of claims 9 to 11, characterized in that The molar yield of taurine is at least 60%, based on the molar amount of L-cysteic acid used.

13. The method according to any one or more of claims 9 to 12, characterized in that All process steps are carried out in one reaction batch (one-pot process).

14. Use of L-cysteic acid produced according to any one or more of the methods of claims 1 to 8 for producing taurine.

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