Method for producing L-cysteic acid and its use

Abstract

The present invention relates to a method for producing L-cysteic acid, which includes a reaction of O-phospho-L-serine (OPS) with a salt of sulfurous acid (sulfite) and cysteic acid synthase (CS enzyme) belonging to the enzyme class EC 2.5.1.76 in a bioconversion process. The L-cysteic acid 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 the production of taurine.

Description

Technical Field

[0001] The present invention relates to a method for producing L-cysteic acid, which includes a reaction of O-phospho-L-serine (OPS) and a sulfite salt (sulfite) and cysteic acid synthase (CS enzyme) belonging to enzyme class EC2.5.1.76 in in vivo conversion. The L-cysteic acid produced by the present invention can be decarboxylated to form taurine. The present invention further relates to the use of the produced L-cysteic acid for the production of taurine.

Background Art

[0002] L-cysteic acid ((R)-2-amino-3-sulfopropanoic acid, 3-sulfo-L-alanine, CAS 498-40-8) is an amino acid that does not constitute a protein and is detected in nature, for example, in wool, as an oxidation product of L-cysteine, which is an amino acid that constitutes a protein. Cysteic acid is also an intermediate in the biosynthesis of coenzyme M (CoM, 2-mercaptoethanesulfonic acid, CAS 3375-50-6) by methanogenic archaea. The biosynthetic pathway of L-cysteic acid in methanogenic bacteria starts from O-phospho-L-serine (OPS, L-serine-O-phosphate, L-2-amino-3-hydroxypropanoic acid 3-phosphate, CAS 407-41-0) and reacts with sulfite in a reaction catalyzed by cysteic acid synthase (CS enzyme) to form L-cysteic acid. (1) OPS + SO3 2- -> L-cysteic acid + HPO4 2-

[0003] In Graham et al., Biochem. J. (2009) 424: 467-478, it was disclosed that for gene products derived from Methanosarcina acetivorans, which are related to threonine synthase, recombinantly produced in Escherichia coli (E. coli) and concentrated as enzymatically inactive proteins in so-called inclusion bodies, detectable enzyme activity could only be measured after complex reconstitution of the inclusion bodies. After reconstitution, the generation of L-cysteic acid could be demonstrated by reacting commercially available (chemically synthesized) OPS with sulfite according to formula (1) in an analytical assay (HPLC and mass spectrometry) with the reconstituted protein. As a result of these studies, the reconstituted protein derived from Methanosarcina acetivorans was associated with the activity of cysteic acid synthase, and the genetic relationship with threonine synthase was established.

[0004] L-Cysteic acid can be produced chemically, for example, by oxidation of cysteine with chlorine in an alcohol solution, bromine in hydrochloric acid, or iodine in DMSO HCl, or by oxidative cleavage of cystine. Furthermore, L-cysteic acid can also be produced by oxidation of L-cysteine sulfinic acid. The known methods for the chemical production of L-cysteic acid are not considered sustainable, use environmentally harmful chemicals, and have low consumer acceptance, especially for applications in the fields of food, cosmetics, and pharmaceuticals.

[0005] L-Cysteic acid can be used, for example, in fish farming or in the cosmetics field, for example, as a component of Regu®-Slim (DSM) for skin care. In peptide chemistry, L-cysteic acid is used as a water-soluble protecting group. Furthermore, L-cysteic acid can also be converted to taurine by decarboxylation.

[0006] 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 was not produced in an active form but only concentrated in an inactive form in inclusion bodies. In the solubilization of inclusion bodies and refolding to form active enzymes, a very low yield of only 3 mg of refolded protein per liter of E. coli culture was obtained, and thus it was not suitable for a preparative in vivo conversion process for producing L-cysteine acid on an industrial scale.

[0007] According to Graham et al. (2009, supra), enzyme activity without refolding was only detectable when the CS enzyme was expressed as a 103 kDa CS fusion protein, but both the enzyme yield and the specific enzyme activity of 0.015 U / mg protein were extremely low. This prior art shows that the active CS enzyme can only be produced in low yields in a high-cost process involving great labor in E. coli.

[0008] In the metabolic engineering approach, Joo et al. (2018), J. Agric. Food Chem. 66: 13454 - 13463 described genetically modified strains of Corynebacterium glutamicum, a bacterium for taurine production using the CS gene from Methanosarcina acetivorans, and strains optimized for sulfur utilization. For the genes expressed in C. glutamicum, CS activity was analytically detected by enzyme assay. The activity of the CS enzyme in the taurine-producing strain was inferred indirectly. As shown in Figure S2 of the "Supporting Information" of Joo et al. (2018, supra), cells of CS-expressing C. glutamicum accumulate OPS, which is mainly the substrate of the CS reaction, intracellularly, and further accumulate serine, cysteine, and taurine. The details of the production of L-cysteic acid have not been clarified. Joo et al. (2018, supra) showed that the simultaneous presence of OPS and CS enzyme in cells is insufficient for the effective production of L-cysteic acid, even when the intracellular production of other sulfur-containing compounds such as cysteine and taurine occurs at measurable yields in the same strain.

[0009] Tevatia et al., Algal Research (2015) 9: 21-26 described the natural production of taurine in microalgae, involving the detection of L-cysteic acid as an intermediate. As described in Figure 1 of Tevatia et al. (2015, supra), the biosynthetic pathway in algae obtains L-cysteic acid ( "cysteic acid" in Figure 1) from L-serine. However, this synthetic pathway does not include the enzyme reaction of the CS enzyme of formula (1). None of the described biosynthetic pathways result in L-cysteic acid via OPS. The intracellular content of L-cysteic acid is extremely low, and the growth of microalgae is not suitable for the production of L-cysteic acid because it is accompanied by multiple by-products that make post-treatment difficult, such as methionine, cysteine, cysteine sulfinic acid, hypotaurine, and taurine.

[0010] In a metabolic engineering approach, U.S. Patent Application Publication No. 2019 / 0062757 (KnipBio) describes heterologous production strains for the production of taurine or its precursors. Described are strains that express the CS enzyme and other biosynthetic genes derived from taurine metabolism in various configurations. The yields of hypotaurine and taurine were extremely low, at most 419 ng / mL. No mention is made of the yield of the production of L-cysteine acid. Although a CS gene construct is used in this prior art, no enzyme assay has been performed to detect CS enzyme activity. Furthermore, this metabolic engineering approach does not show that L-cysteine acid can be produced in a fractionated amount by biotechnology. Similarly, it is not disclosed whether the production strains of OPS or CS enzyme described in this document are suitable for in vivo conversion use to produce L-cysteine acid according to formula (1).

[0011] Steinfeld et al., ACS Chem. Biol. (2014) 9: 1104-1112 describes an increase in the intracellular production of OPS in an E. coli strain lacking the serB gene. No description is made of an increase in the extracellular production of OPS (see Figure 5 of Steinfeld et al.).

[0012] European Patent No. 2444481 of CJ CheilJedang Corporation (KR) describes a method for producing L-cysteine, which uses a strain with reduced SerB activity to produce OPS, and then reacts this with sulfide or thiosulfate in an enzymatic reaction to form L-cysteine. It is also not known whether the enzyme class of O-phosphoserine sulfhydrylase (OPSS, EC 2.5.1.65) used here can utilize sulfite as a substrate for the CS enzyme. In fact, Steiner et al. J. Bacteriol. (2014), 196: 3410-3420 investigated the reaction mechanism of the OPSS enzyme from Mycobacterium tuberculosis and found that, in contrast to Na2S (sulfide) and Na2S2O3 (thiosulfate), sulfites such as Na2S2O5 and Na2SO3 are not suitable as S donors for the reaction with the enzyme-bound aminoacrylate intermediate formed by the binding of OPS to the OPSS enzyme and the elimination of phosphate (Steiner et al., 2014, Figure 2, see above). Thus, European Patent No. 2444481 discloses, as is also known from Steinfeld et al. (2014, see above), the production of a strain that inactivates the serB gene to produce OPS. Subsequently, OPS was used in the in vivo conversion by the OPSS enzyme to produce cysteine.

[0013] The production of OPS by biotechnology is well known in the prior art. However, the prior art has not provided a method for producing a CS enzyme suitable for providing a CS enzyme with sufficient activity for the industrially applicable method of in vivo conversion of OPS by sulfite sulfite, which produces L-cysteine acid on a preparative scale for further use.

[0014] Therefore, there is a need for an environmentally friendly and sustainable method for producing L-cysteic acid on a fractional scale, for which biotechnology methods are suitable. As a consumer-driven trend, there is a move away from chemically produced ingredients, and the object of the present invention is a biotechnology method for producing L-cysteic acid by in vivo conversion, which is suitable for industrial-scale use.

Summary of the Invention

[0015] The object of the present invention is to provide an industrially applicable method for producing L-cysteic acid in a cost-effective manner by in vivo conversion of OPS, while avoiding the production of L-cysteic acid-producing strains by metabolic engineering, instead of chemical methods, and to use the L-cysteic acid thus produced for further applications such as taurine production.

[0016] This 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) belonging to the enzyme class EC 2.5.1.76 in in vivo conversion.

Embodiments for Carrying Out the Invention

[0017] In the context of the present invention, the production methods are distinguished as follows: 1. Chemical methods 2. Biotechnology methods (a) Biotechnology methods by metabolic engineering Metabolic engineering (also called "pathway design") is a biotechnology method that modifies the metabolic pathways of organisms by optimizing or modifying genes and regulatory processes, as opposed to in vivo conversion. By supplementing the genome with genes for novel or modified enzymes, the enzyme can be introduced into the organism, or the genes for endogenous enzymes can be expressed at enhanced or attenuated levels, thereby establishing new metabolic pathways in the organism or enhancing or attenuating existing metabolic pathways. The goal of metabolic engineering is for the organism to produce new metabolic products or endogenous cellular metabolites in high yields. In metabolic engineering methods, enzyme substrates, such as OPS in the present invention, characteristic starting materials for metabolic products are not used; instead, only the nutrient medium required for the growth of the target organism, composed of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts, or a complex amino acid mixture such as peptone or yeast extract), and other salts necessary for growth, is used. Such nutrient media are known to those skilled in the art from microbiological practice. (b) Biotechnological method by in vivo conversion In vivo conversion is defined as the conversion of one or more reactants to products under enzymatic catalysis, and the enzyme substrate is added to the reaction batch together with the enzyme. In the reaction batch, the added enzyme substrate such as OPS in the present invention is enzymatically converted in the present invention by an enzyme selected from the class of cysteine synthase (CS enzyme, EC 2.5.1.76) in the presence of a salt of sulfurous acid. The reactants can be derived from chemical or biotechnological production. The OPS used in the method of the present invention may be obtained, for example, by chemical synthesis or by biotechnological production by the growth of a production strain. The enzyme used for enzymatic catalysis preferably originates from biotechnological production by fermentation of a production strain of the family Enterobacteriaceae that heterologously expresses the CS enzyme.

[0018] An advantage of the present invention is that the method of the present invention for producing L-cysteic acid from OPS and sulfite with the aid of CS enzyme is an in vivo conversion method. This means that the in vivo conversion method is a highly targeted specific reaction and does not require, for example, complex culture processes or purification processes from microbial cultures. Furthermore, by optimizing the reaction conditions and the amounts of reactants and enzymes used, in vivo conversion can easily achieve a high space-time yield that is much more difficult in the optimization of strains in the case of metabolic engineering. Generally, the method of the present invention can be carried out and controlled in an economically simple manner.

[0019] Reaction (1) in the present invention is catalyzed by the enzyme cysteic acid synthase (CS enzyme) belonging to the enzyme class EC2.5.1.76. The enzymatically active form of the CS enzyme refers to a protein that can catalyze the synthesis of L-cysteic acid from OPS and salts of sulfurous acid, as described in the following CS enzyme activity assay.

[0020] The CS enzyme activity assay can be carried out as follows: (i) The CS enzyme produced by growth in a shaking flask or during fermentation can be used in the reaction as follows: · As an aliquot from the culture broth without further post-treatment; or · As an aliquot of the cell suspension after re-isolating the cells from the culture broth by, for example, centrifugation; or · In the form of an aliquot of the cell homogenate; (a) After mechanically disrupting the cell suspension, or (b) In the form of cells that have been chemically permeabilized (e.g., by chloroform); Or · As a cell extract after removing particulate components from the cell homogenate; or · As an enzyme purified, for example, by chromatography. As described in Example 3 of the present invention, the total protein concentration obtained in each case can be determined, for example, using the "Qubit (registered trademark) Protein Assay Kit" with a Qubit 3.0 Fluorometer manufactured by Thermo Fisher Scientific according to the manufacturer's instructions. (ii) First, OPS (final concentration 10 mM) and sodium sulfite (final concentration 20 mM) are added to a solution buffered to pH 7 with potassium phosphate, and CS enzyme is added to initiate the reaction. The assay volume is 10 mL. The temperature at which the assay is performed is 30°C. The amount of CS enzyme used varies depending on its purity. When using culture broth, cell suspension of re-isolated cells, or cell homogenate, at least 0.1 mg of the enzyme fraction prepared in (i) is used. In the case of purified CS enzyme, at least 10 μg of the purified enzyme fraction is used. One hour, two hours, and four hours after the start of the reaction, 1 mL of the assay solution is taken out in each case, centrifuged for 10 minutes, and the contents of OPS and L-cysteic acid are determined by calibrated HPLC (see Example 4). The reference substances used for calibration are commercially available (Sigma-Aldrich).

[0021] The method according to the present invention is preferably characterized in that the CS enzyme is produced by the growth of a microbial strain of the family Enterobacteriaceae. The cds encoding the CS enzyme is heterologously expressed in a microbial strain of the family Enterobacteriaceae, and particularly preferably expressed in an enzymatically active form.

[0022] Heterologous expression is understood to mean expressing the cds of a gene or a part of the cds of a gene in a host organism that does not originally have the gene or gene fragment. Introducing the cds of a heterologous gene into a host organism involves the use of recombinant DNA technology. The cds of a heterologous gene can be introduced into a host organism by integration into the genome of the host organism or by extrachromosomal integration in the form of an autonomously replicating gene construct (plasmid, vector).

[0023] It is preferable to introduce the cds of a heterologous gene into a host organism in the form of a self-replicating gene construct (plasmid, vector).

[0024] Depending on the genetic element (promoter) used to control the expression of the cds of a heterologous gene, constitutive expression and inducible expression are distinguished. In the case of constitutive expression, gene expression is activated (uncontrolled) at all stages of cell culture. In the case of inducible expression (controlled), gene expression is stimulated by adding an inducer molecule to the cell culture, for example, by adding the inducer molecule IPTG for inducing the tac promoter in Examples 3 and 7 of the present invention. Inducible expression in which gene expression is stimulated by adding an inducer molecule to the cell culture is preferred.

[0025] In contrast, homologous expression involves overexpressing the cds of a gene from the genome from which the gene originally derived in a host cell.

[0026] When the heterologous expressed protein is, for example, an enzyme, this may be, for example, in an enzymatically active form, or may be concentrated in an enzymatically inactive form, for example, in inclusion bodies.

[0027] Therefore, heterologous expression of the CS enzyme in an 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, preferably, introduced extracellularly into the host organism by a self-replicating vector, and (iii) the CS enzyme is expressed in an enzymatically active form by the said cds.

[0028] Particularly preferably, the heterologously expressed CS enzyme is expressed in an enzymatically active form by the microbial strain. This means that the heterologously introduced cds expresses a CS enzyme that is in an enzymatically active form in the microorganism following protein biosynthesis and any post-translational modifications, such as the incorporation of cofactors like pyridoxal phosphate in the case of the CS enzyme (see entry number EC2.5.1.76 in the KEGG enzyme database). "Expressed in an enzymatically active form" does not mean that the protein is initially produced as an inactive protein in inclusion bodies and only becomes enzymatically active after refolding.

[0029] In summary, the method in a particularly preferred embodiment is produced by growing a microbial strain of the Enterobacteriaceae family that heterologously and in an enzymatically active form expresses the CS enzyme.

[0030] In the context of the present invention, a reaction batch is defined as a mixture of reactants (starting materials), enzymes, and optionally other reactants, in which the reactants are converted into products in the reaction batch.

[0031] The yield of a reaction within the meaning of the present invention is defined as the amount of reactant used that is converted into a product under the reaction conditions. The yield can be expressed as the absolute yield of the product (mmol or g), the volumetric yield (mM or g / L) as the absolute amount of product per unit volume, or the relative yield of the product as a proportion of the reactants used (taking into account the molecular weights of the reactants and products), and the relative yield is also referred to as the percent yield.

[0032] In the context of the present invention, the term "growth" or, synonymously, "cultivation" of microbial cells encompasses both the shake flask culture method and the fermentation method. The medium used for the growth or cultivation of microorganisms is called a growth medium or a culture medium, and in the case of fermentation, it is also called a fermentation medium. By growing / cultivating / fermenting the production strain in the growth medium, culture medium or fermentation medium, a culture broth / fermentation broth is obtained. The culture broth / fermentation broth consists of the biomass of the cells of the production strain and a cell-free culture supernatant / fermentation supernatant formed during growth from the growth medium and during growth from the metabolites secreted by the cells.

[0033] Fermentation is a method step for the production (cultivation) of cell cultures on an industrial scale (manufacturing scale), preferably growing a microbial production strain under defined conditions of culture medium, temperature, pH, oxygen supply and mixing of the medium. Depending on the constitution (genetic constitution) of the production strain, the aim of fermentation is to produce proteins / enzymes or metabolites in the highest possible yield for further use in any case. The OPS and CS enzymes, which are components of the method of the present invention, can be produced by fermentation. The final product of fermentation is a fermentation broth consisting of the biomass of the cells of the production strain (fermentation cells) and a cell-free fermentation supernatant formed from the growth medium and metabolites secreted by the fermentation cells during fermentation. The target product of fermentation can be present in the fermentation cells or in the fermentation supernatant. For example, OPS is present in the fermentation supernatant and the CS enzyme is present in the fermentation cells.

[0034] In contrast to the production scale by fermentation, shake flask culture is used for culturing microorganisms on a laboratory scale. In shake flask culture, it is necessary to specify a particular medium and pH and culture in the presence of oxygen under a certain movement (shaking), but more defined conditions regarding the medium, temperature, pH, oxygen supply and mixing of the medium can be established and regulated in a fermenter. A smaller scale culture, for example in a shake flask, can also be used as a pre-culture for a larger scale culture, for example for inoculating a fermenter.

[0035] A production strain is defined, for example, as a microbial strain suitable for the production of products by fermentation. A production strain is distinguished by the fact that, as a result of genetic modification, (improved) production of the product is possible. The genetic modification may be due to modification of the genome (chromosomal modification), introduction of autonomously replicating extrachromosomal genetic elements such as plasmids, or a combination of chromosomal modification and extrachromosomal modification. Examples of chromosomally modified production strains include the Escherichia coli W3110-ΔserB strain described in Example 1 with respect to the production of OPS. Examples of production strains produced by the introduction of plasmids include the Escherichia coli JM105×pCSma-pKKj strain described in Example 3 with respect to the production of CS enzyme. A microbial strain carrying an extrachromosomal genetic element is called a host strain or host organism, and the extrachromosomal genetic element is called a gene construct, plasmid, vector, or expression vector.

[0036] An open reading frame (ORF, synonymous with cds or coding sequence), refers to a region of DNA or RNA that encodes the amino acid sequence of a protein, starting with a start codon and ending with a stop codon. An ORF is also called a coding region or structural gene.

[0037] A gene refers to the part of DNA that contains all the basic information for generating biologically active RNA. A gene includes the part of DNA where a single-stranded RNA copy is generated by transcription and the expression signals involved in the regulation of this copying process. Expression signals include, for example, at least one promoter, transcription start point, translation start point, and ribosome binding site (RBS). Terminators and one or more operators are additional possible expression signals.

[0038] mRNA, also known as messenger RNA, is a single-stranded ribonucleic acid (RNA) that carries genetic information for protein synthesis. mRNA presents the assembly instructions for specific proteins within a cell. mRNA molecules convey the message necessary for protein synthesis from genetic information (DNA) to ribosomes responsible for protein synthesis. Inside the cell, mRNA molecules are formed as transcripts of the DNA portion corresponding to genes. The genetic information stored in DNA does not change through this process.

[0039] Eukaryotic genes are mainly known as mosaic genes and, unlike prokaryotic genes, also contain non-coding regions called introns (intragenic regions). Coding sequences called exons (expressed regions) are a part of the DNA of eukaryotic genes and, after being transcribed into RNA, are translated by ribosomes into the amino acid sequence of proteins. After transcription from DNA to RNA, introns are spliced out of the primary transcript. Protein-coding RNA without introns is called messenger RNA (mRNA), or "mature" mRNA. This undergoes further modifications such as capping and polyadenylation. Subsequently, the coding region of mature mRNA is translated into a protein sequence. When a eukaryotic gene containing an exon / intron structure is expressed in a prokaryote, since the prokaryote does not process the exon / intron structure, it is necessary to reverse translate the protein sequence or the coding region of mature mRNA into intron-free DNA. In the context of the present invention, when referring to a gene sequence derived from a protein sequence or a gene sequence derived from mRNA, this exactly means this reverse translation process. Optimization of the sequence, i.e., adaptation to the codon usage of the corresponding prokaryote (codon optimization), is preferably carried out simultaneously with the reverse translation of the protein sequence or mRNA sequence into a DNA sequence.

[0040] A gene construct refers to a DNA molecule in which a gene is linked to other genetic elements (e.g., promoter, terminator, selection marker, origin of replication). The gene construct in the context of the present invention is a circular DNA molecule and is called a plasmid, vector or expression vector. The genetic elements of the gene construct cause its extrachromosomal inheritance during cell growth and produce the protein encoded by the gene.

[0041] The abbreviation WT (Wt) refers to the wild type. A wild-type gene refers to the form of a gene that occurs naturally during the process of evolution and exists in the wild-type genome. The DNA sequence of the Wt gene is publicly available in databases such as the NCBI (National Center for Biotechnology Information) database. A microbial strain having a Wt genome is called a Wt strain.

[0042] L-cysteic acid obtained from the in vivo conversion of OPS using sulfite according to the present invention can be directly further used without any further post-treatment steps or concentration or purification by known methods. Such methods are known to those skilled in the art from methods for isolating amino acids. Examples include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization.

[0043] Preferably, the method of the present invention is characterized in that a reaction batch containing L-cysteic acid is further used without performing any further post-treatment, purification or isolation steps.

[0044] In another preferred embodiment, the method is characterized in that the produced L-cysteic acid is isolated from the reaction batch.

[0045] Denaturation in the context of the present invention refers to a structural change of a molecule such as a protein, and refers to a structural change related to the loss of the biological function in the molecule while its primary structure remains unchanged.

[0046] The denatured protein is enzymatically inactive, i.e., in the case of the CS enzyme in the context of the present invention, it is characterized by the inability to catalyze the synthesis of L-cysteate from OPS and sulfite. Denaturation can be caused by physical or chemical influences. Enzymatically inactive proteins that are not correctly folded or are incompletely folded may accumulate intracellularly as protein aggregates (known as inclusion bodies), which can be regarded as naturally denatured proteins. Inclusion bodies are observed especially at high expression levels, and as a result, the fact that newly synthesized protein chains are obtained at high concentrations means that their aggregation is prioritized over folding into an enzymatically active three-dimensional form. Whether a heterologously expressed protein occurs in the form of insoluble inclusion bodies or in an active form cannot be predicted and depends not only on the primary structure of the protein chain (sequence of amino acids) but also on the expression system and usage parameters (e.g., growth temperature, induction intensity of the inducible promoter) that can control the biosynthesis rate of the protein. However, in the case of the present invention, it is surprising that the cysteate synthase derived from Methanosarcina acetivorans was produced in an enzymatically active form in Escherichia coli, because according to the prior art (Graham et al., 2009, cited above), the protein was only produced in an inactive form in inclusion bodies heterologously expressed in E. coli.

[0047] Renaturation refers to the reverse conversion of a denatured protein into a biologically active spatial structure. Renaturation in protein biochemistry involves returning the denatured protein in inclusion bodies into solution using chaotropic compounds and then removing the chaotropic compounds to renature the protein. In particular, urea and guanidine hydrochloride are used in protein chemistry for this purpose.

[0048] Preferably, the method of the present invention is characterized in that the CS enzyme is used in the reaction without undergoing a prior renaturation step.

[0049] Regeneration processIt includes the following method steps: dissolving a denatured protein in a medium containing a chaotropic compound and then removing the chaotropic compound. The degree of removal of the chaotropic compound depends on the specific protein. The chaotropic compound can be removed, for example, by dialysis, by selective binding of the chaotropic compound to a support material, by selective binding of the regenerated protein to a support material and subsequent elution under regeneration conditions, or by diluting the chaotropic compound to a concentration below the critical concentration at which it has no denaturing effect (see, for example, Graham et al., 2009, supra). Methods for protein regeneration are described in the prior art. Examples of regeneration conditions include dissolving the denatured protein in an aqueous solution of 6 M urea or in an aqueous solution of 6 M guanidine hydrochloride.

[0050] Dilution or removal of the chaotropic compound below the critical concentration depends on the specific protein and means reducing the concentration of the chaotropic compound, such as urea or guanidine, to a concentration below which the protein of interest can refold into its active three-dimensional structure.

[0051] A chaotropic compound refers to a chemical substance that disrupts the regular hydrogen bonds in water. Examples of chaotropic compounds include barium salts such as barium chloride or barium acetate, thiocyanates such as guanidine hydrochloride or guanidine thiocyanate, perchlorates, iodides, butanol, phenol, thiourea, urea, and / or surfactants.

[0052] A surfactant (also called a detergent or soap) is an organic compound that acts as a surface-active substance. That is, due to its structure, the surfactant is arranged between the interfaces of two phases so as to lower the surface tension and, as a result, become wettable, for example. By lowering the surface tension, the surfactant promotes the mixing of the two phases and, in some cases, leads to the formation of an emulsion. A surfactant Distinguished by the polarity of the molecular structure, a part of the molecule has hydrophilicity that mediates solubility in water, another part of the molecule has hydrophobicity, and the surfactant solubilizes the hydrophobic compound and contributes to solubilization in water. The surfactants used are nonionic surfactants (polyalkylene glycol ethers, fatty alcohol propoxylates, alkyl glucosides, alkyl polyglucosides, octylphenol ethoxylates such as Triton X-100, nonylphenol ethoxylates), anionic surfactants (alkyl carboxylates, alkylbenzene sulfonates, alkyl sulfonates, fatty alcohol sulfates such as sodium lauryl sulfate, alkyl ether sulfates, sulfacetates), cationic surfactants based on quaternary ammonium compounds (distearyldimethylammonium chloride, "Esterquat") and / or zwitterionic (amphoteric) surfactants based on betaines (e.g., "cocamidopropyl betaine") or sulfobetaines (e.g., cocamidopropyl hydroxysultaine).

[0053] In a preferred embodiment, the method of the present invention is characterized in that the CS enzyme is used in the reaction without undergoing a prior regeneration step. Since the complex, costly, and environmentally harmful regeneration step as described above is not required, working without a regeneration step is extremely economically advantageous. In the case of the production of the CS enzyme by the growth of a production strain, especially when the CS enzyme is produced by fermentation, for the host cells expressing the CS enzyme, it is further complex and costly, and in a method that is, for example, environmentally harmful due to waste, it is not necessary to first mechanically or chemically disrupt it, and for example, for inclusion bodies in which the enzyme is concentrated in an inactive form as described in Graham et al. (2009), it is not necessary to isolate and regenerate it from the cell lysate to make the protein usable in the enzyme reaction.

[0054] The method of the present invention for producing L-cysteic acid by in vivo conversion requires the availability of OPS. OPS can be produced chemically or biotechnologically, for example, by fermentation of an OPS-producing strain. Possible methods for the chemical production of OPS are, for example, the phosphorylation of L-serine, or the production of racemic O-phospho-D / L-serine that can be used directly, or OPS can be obtained in advance from the racemate, for example, by decomposition.

[0055] Preferably, the method for producing L-cysteic acid is characterized in that the OPS used in the reaction is produced by biotechnology. This is achieved by the growth of an OPS-producing strain. Particularly preferably, it is the biotechnological production of OPS by the growth of an OPS-producing strain in which OPS accumulates in the cell culture supernatant (extracellular).

[0056] A person skilled in the art can determine whether a substance such as OPS to be used as a reactant in the method of the present invention is derived from chemical production or from biotechnological production, for example, production by fermentation, using isotope analysis. Distinguishable isotope analysis methods are described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527, and are based on the determination of the proportion of different isotopes, for example, the ratio of carbon or nitrogen isotopes, depending on whether the product is derived from chemical production (petroleum-based) production or from biotechnological, for example, fermentative (plant-based) production. The method for producing OPS is, for example, a biotechnological production method, for example, a fermentative (plant-based) production method when the glucose used for the growth of the production strain is derived from plant-based production, and this is also applicable to the method described in Example 2.

[0057] For example, in the cysteine metabolism of Escherichia coli, OPS acts as a biosynthetic precursor of L-serine. The latter is formed by dephosphorylation of OPS. This reaction is enzymatically catalyzed by O-phospho-L-serine phosphatase (SerB, EC3.1.3.3). It is known from the prior art that an Escherichia coli strain with suppressed SerB activity can accumulate OPS. Therefore, a microbial strain with suppressed SerB activity is characterized in that it cannot produce L-serine by dephosphorylation of OPS and, as a result, can accumulate OPS.

[0058] Preferably, this method is characterized in that the OPS used in the reaction is produced using a microbial strain in which the activity of O-phospho-L-serine phosphatase (SerB enzyme) belonging to enzyme class EC3.1.3.3 is suppressed. This means that, in this case, a microbial strain with suppressed SerB activity is used as an OPS-producing strain, and the suppression of this SerB activity involves genetic modification of the microbial strain. Taking the SerB activity of an unmodified microbial strain (Wt strain) as 100%, a microbial strain with suppressed SerB activity has a lower SerB activity compared to the 100% activity of the Wt strain, and preferably has a SerB activity of 20% or less, particularly preferably 10% or less, and most preferably 0% (inactivation of the SerB gene) relative to the activity with the wild strain taken as 100%. The SerB activity that can still be measured in the modified microbial strain and is expressed as a percentage based on the activity in the Wt strain is called the residual activity.

[0059] A microbial strain with suppressed SerB activity is characterized by one or more of the following genetic modifications: · Deletion of the chromosomal gene encoding enzyme SerB. · Introduction of a mutation into the chromosomal gene encoding enzyme SerB to reduce the activity of the endogenous gene. · Substitution of the chromosomal gene encoding enzyme SerB with a mutated gene to reduce the activity of the endogenous gene. · Introduction of a mutation into the regulatory region of the gene encoding enzyme SerB to reduce the endogenous enzyme activity. ·Introduction of an antisense oligonucleotide complementary to the transcript of the gene encoding the enzyme SerB to inhibit the translation of mRNA.

[0060] As described in the prior art, the L-3-phosphoserine phosphatase enzyme activity (SerB activity) can be determined by the enzymatic release of phosphate from L-3-phosphoserine, and the released phosphate is determined photometrically at 340 nm as a molybdate complex. Using this enzyme assay method, the SerB activity determined for the WT strain is taken as 100% activity, and the residual activity of the microbial strain with suppressed SerB activity is measured under the same conditions.

[0061] Particularly preferably, the method for producing L-cysteic acid of the present invention is characterized in that the OPS used in the reaction is produced using a microbial strain lacking the chromosomal gene encoding the enzyme SerB. This is also called knockout of the SerB gene. Preferably, the microbial strain with suppressed SerB activity, in this case, lacks the chromosomal nucleotide sequence of the SerB gene including the complete SerB cds encoding the enzyme SerB, the adjacent sequence up to 1000 nt upstream in the 5' direction of the SerB cds including the sequence of the SerB promoter, and the adjacent sequence up to 1000 nt downstream in the 3' direction of the SerB cds including the sequence of the SerB terminator. A particularly preferred deletion is the deletion of the serB coding region encoding the enzyme SerB, which is the deletion described in Example 1.

[0062] Microbial strains having suppressed SerB activity are preferably selected from the family Corynebacteriaceae and the family Enterobacteriaceae, particularly preferably selected from the genus Corynebacterium, the genus Pantoea, and the genus Escherichia, and most preferably selected from the species Pantoea ananatis and the species Escherichia coli. Very preferably, the microbial strain with suppressed SerB activity for producing OPS for the reaction of the present invention is the Escherichia coli K12 W3110 strain.

[0063] Microbial strains with suppressed SerB activity are preferably auxotrophic for serine, i.e., the strain itself cannot form the amino acid L-serine for growth. The auxotrophy can be overcome by adding serine or glycine to the culture medium (growth medium), and in each case, it can be added as a pure substance, or as a constituent of complex medium components such as yeast extract, peptone, or tryptone, and as a mixture of a pure substance and complex medium components. A mixture of a pure substance selected from glycine and L-serine and complex medium components is preferred, and a mixture of glycine and complex medium components is particularly preferred. It is particularly preferred to add glycine to the growth medium.

[0064] The content of glycine as a pure substance in the growth medium is preferably 0.1 g / L to 10 g / L, particularly preferably 0.2 g / L to 5 g / L, and most preferably 0.3 g / L to 2 g / L.

[0065] According to the prior art, by isolating the serB gene or a part of the gene and cloning foreign DNA into the serB gene, the open reading frame that defines the protein of the serB gene can be interrupted. Therefore, a DNA construct suitable for targeted inactivation of the serB gene can be composed of a 5' section of DNA homologous to the genomic serB gene, followed by a gene segment consisting of foreign DNA, and then followed by a 3' section of DNA homologous to the genomic serB gene again.

[0066] Therefore, the region where homologous recombination may occur in the serB gene may include not only the region encoding O-phospho-L-serine phosphatase but also other regions. The possible regions can also include DNA sequences adjacent to the serB gene, that is, the 5' region (gene transcription promoter) before the start of the coding region and the 3' region (gene transcription terminator) after the end of the coding region. These modifications by homologous recombination can lead to inactivation of the serB gene in the same way as modifications of the coding region.

[0067] The foreign DNA is preferably a selection marker expression cassette. This consists of a gene transcription promoter functionally linked to the actual selection marker gene, followed by a gene transcription terminator if necessary. In this case, the selection marker also includes the 5' and 3' adjacent homologous sequences of the serB gene.

[0068] Preferably, the selection marker includes the 5' and 3' adjacent homologous sequences of the serB gene, each having a length of at least 30 nucleotides, particularly preferably at least 50 nucleotides.

[0069] Therefore, the DNA construct for inactivating the serB gene can start from the 5' end, consist of a sequence homologous to the serB gene, followed by an expression cassette of a selection marker selected from, for example, the class of antibiotic resistance genes, and then followed by a further sequence homologous to the serB gene.

[0070] In a preferred embodiment, the DNA construct for inactivating the serB gene consists of, starting from the 5'-end, a sequence homologous to the serB gene that is at least 30 nucleotides in length, particularly preferably at least 50 nucleotides in length, followed by an expression cassette for a selection marker selected from the class of antibiotic resistance genes, and then a further sequence homologous to the serB gene that is at least 30 nucleotides in length, particularly preferably at least 50 nucleotides in length.

[0071] A selection marker gene is generally a gene whose gene product enables the growth of the parental strain under selective conditions where the original parental strain cannot grow.

[0072] Preferred selection marker genes are selected from the group of antibiotic resistance genes such as, for example, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, the chloramphenicol resistance gene, or the neomycin resistance gene. Another preferred selection marker gene enables a parental strain having a metabolic defect (e.g., amino acid auxotrophy) to grow under selective conditions as a result of the correction of the metabolic defect by the expression of the selection marker gene. Finally, as another possibility, a selection marker gene for a gene product that chemically changes a compound that is essentially toxic to the parental strain and thereby inactivates the compound (e.g., the gene for the acetamidase enzyme that decomposes acetamide, a compound toxic to many microorganisms, into acetic acid and ammonia, non-toxic products).

[0073] Among the selection marker genes, the ampicillin resistance gene, the tetracycline resistance gene, the kanamycin resistance gene, and the chloramphenicol resistance gene are particularly preferred. The tetracycline resistance gene and the kanamycin resistance gene are especially preferred.

[0074] Systems based on homologous recombination also exist, and in addition to inactivating the target gene, they offer the option of removing the selection marker from the genome, thereby enabling the production of double and multiple mutants. Such systems include, for example, the so-called Lambda Red technology, which is commercially available as the "Quick and Easy E. coli Gene Deletion Kit" based on the Red® / ET® technology of Gene Bridges GmbH ("Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and the references cited therein, see, for example, Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645).

[0075] In Example 1 of the present invention, an example of producing a microbial strain in which SerB activity is suppressed by deleting the serB gene will be described.

[0076] The microbial strain with suppressed SerB activity produced using the Red® / ET® technology may be suitable for the extracellular production of OPS, as described for the Escherichia coli W3110-ΔserB strain in Example 2 of the present invention, for example. OPS can accumulate either intracellularly or extracellularly, and the amount of OPS accumulating intracellularly depends on the growth conditions (Steinfeld et al., see above). The culture conditions selected in Example 2 of the present invention enable the extracellular accumulation of OPS. The extracellular OPS content is preferably at least 1 g / L, particularly preferably at least 3 g / L, and most preferably at least 6 g / L.

[0077] The advantage of the described method for the biotechnological production of OPS is that the OPS present extracellularly in the culture broth, as obtained for example in the growth carried out in the second embodiment, can be directly used in the method of the present invention for producing L-cysteic acid as an OPS source, without, for example, undergoing further post-treatment, purification or isolation steps such as extraction, adsorption, ion exchange chromatography, precipitation and crystallization, after removal of the particulate biomass by centrifugation or filtration. This procedure is particularly economical and avoids the isolation of OPS. Thus, particularly preferably, the method for producing L-cysteic acid is characterized by using OPS obtained from the cell culture supernatant of the growth of 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 suppressed.

[0078] In a particularly preferred embodiment, the method for producing L-cysteic acid is characterized in that, in addition to the CS enzyme used in the reaction, the OPS used in the reaction is produced by biotechnology, particularly preferably by fermentation.

[0079] Preferably, the CS enzyme is produced by culturing a microbial strain of the family Enterobacteriaceae that heterologously expresses the CS enzyme.

[0080] The microbial strain, also called a CS enzyme-producing strain, consists of a host strain and a gene construct for the expression of the CS gene.

[0081] Preferably, this method is characterized in that the CS enzyme is produced by the growth of a microbial strain of the genus Escherichia, particularly preferably a microbial strain of the Escherichia coli species, and most preferably a microbial strain of Escherichia coli K12 JM105, which heterologously expresses the CS enzyme, very preferably in an enzymatically active form.

[0082] A preferred gene construct for expressing the CS gene is an expression vector in plasmid form, particularly preferably comprising the expression vector pCSma-pKKj disclosed in Example 3 (Figure 3).

[0083] Cysteate synthase is known, for example, for the biosynthesis of coenzyme M by methanobacteria. Preferably, the method for producing L-cysteate is characterized in that the CS enzyme is derived from Methanosarcina acetivorans or has a sequence homologous thereto, and particularly preferably the CS enzyme is derived from Methanosarcina acetivorans. Particularly preferably, the coding DNA sequence is SEQ ID NO: 3, which encodes a protein having the amino acid sequence of SEQ ID NO: 4 or a nucleotide sequence homologous thereto.

[0084] Homologous nucleotide sequences are understood to mean that the DNA sequences of these genes or DNA portions are at least 80% identical, preferably at least 90% identical, and particularly preferably at least 95% identical. Preferred homologous nucleotide sequences are sequences of genes derived from Methanocella paludicola (NCBI Gene ID: 8682885), Methanosarcina barkeri (NCBI Gene ID: 24822660), Methanoculleus marisnigri (NCBI Gene ID: 4845938), or nucleotide sequences homologous thereto.

[0085] The degree of DNA identity is determined by the "nucleotide blast" program based on the blastn algorithm at http: / / blast.ncbi.nlm.nih.gov / . The algorithm parameters used for the alignment of two or more nucleotide sequences are the default parameters. The default general parameters are as follows: Max target sequences = 100; Short queries = “Automatically adjust parameters for short input sequences”; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding default scoring parameters are as follows: Match / Mismatch Scores = 1, -2; Gap Costs = Linear.

[0086] In a preferred embodiment, in the method of the present invention, the CS enzyme has an amino acid sequence defined by SEQ ID NO: 4 or an amino acid sequence homologous thereto, and the amino acid sequence homologous to SEQ ID NO: 4 has at least 50%, preferably at least 70%, particularly preferably at least 80% sequence identity to SEQ ID NO: 4, and at the same time, has cysteate synthase activity. The cysteate synthase activity can be detected as defined above in the CS activity assay. The amino acid sequence of the homologous CS enzyme can be found using the search term "cysteate synthase" in the NCBI database (National Center for Biotechnology Information, USA), or by inputting the amino acid sequence of SEQ ID NO: 4 and using the auxiliary program "Protein BLAST". Enzymes homologous to the CS enzyme derived from Methanosarcina acetivorans are preferably selected from Methanocella paludicola (NCBI number: WP_012900738.1), Methanolinea mesophila (NCBI number: WP_245249687.1), Methanosarcina barkeri (NCBI number: WP_011308449.1), Methanoculleus marisnigri (NCBI number: WP_011842967.1).

[0087] When searching for the gene of the CS enzyme with "Protein BLAST", homologous protein sequences are found from a number of bacteria derived from the archaeal domain, including thermophilic organisms (growing at temperatures from over 50 °C to 110 °C). Those enzymes also usually show optimal activity at over 50 °C. The present invention also includes CS enzymes derived from the archaeal domain that have optimal activity at over 50 °C.

[0088] Protein sequences are compared using the "Protein BLAST" program at http: / / blast.ncbi.nlm.nih.gov / . This program uses the blastp algorithm. The algorithm parameters used for the alignment of two or more protein sequences are the default parameters. The default general parameters are as follows: Max target sequences = 100; Short queries = “Automatically adjust parameters for short input sequences”; Expect Threshold = 10; Word size = 3; Automatically adjust parameters for short input sequences = 0. The default scoring parameters are as follows: Matrix = BLOSUM62; Gap Costs = Existence: 11 Extension: 1; Compositional adjustments = Conditional compositional score template adjustment.

[0089] Preferably, this method is characterized in that the CS enzyme is not a fusion protein. The term "fusion protein" means that a DNA sequence encoding a protein or a part of a protein is fused in-frame in the laboratory with one or more DNA sequences encoding an additional protein or an additional part of a protein, resulting in a modified (extended) protein that does not exist in nature. The fusion DNA sequences can be joined at the 5'-end, at the 3'-end, or at both the 5'-end and the 3'-end. It is also conceivable to insert a fusion DNA sequence within the sequence encoding the protein (for example, as a connection between two domains of a protein). A fusion protein is also called a hybrid or hybrid enzyme. This means that the protein of the present invention having cysteine synthase activity is encoded only by the cds of the gene of interest, and the CS cds is not extended by a sequence further added to the CS cds. A protein having a coding region fused with a nucleotide sequence encoding a protein sequence that is cleaved again during protein biosynthesis or post-translational modification, for example, a translocation signal sequence that mediates protein secretion, is not included in the term "fusion protein". The term "fusion protein" in the context of the present invention always refers to the mature protein.

[0090] In the context of the present invention, "production of the CS enzyme by growth of a CS enzyme-producing strain" refers to producing the CS enzyme as an enzymatically active protein without refolding and preferably without relying on expression as a fusion protein. Particularly preferably, it is the production of the CS enzyme from Methanosarcina acetivorans by growth of a CS enzyme-producing strain as an enzymatically active protein without refolding and without relying on expression as a fusion protein, and this production strain is produced using a host strain of the Escherichia coli species. Particularly preferably, it is the production of the CS enzyme from Methanosarcina acetivorans by growth of the E. coli production strain JM105×pCSma-pKKj described in Example 3.

[0091] In the method of the present invention, the CS enzyme obtained by the growth of the production strain can be used as a culture broth without further post-treatment, or as a cell suspension after re-separating cells from the culture broth by, for example, centrifugation or filtration. Furthermore, the CS enzyme can be in the form of a cell homogenate after mechanical disruption of the cell suspension, or in the form of chemically permeabilized cells (e.g., by chloroform), or as a cell extract after removing particulate components from the cell homogenate, or as an enzyme purified, for example, by chromatography.

[0092] The CS enzyme is preferably used as a culture broth, particularly preferably as a fermentation broth, without further post-treatment, as a cell suspension after re-separating cells from the culture broth, or as a cell homogenate after mechanically disrupting the cell suspension, or in the form of chemically permeabilized cells (e.g., by chloroform).

[0093] Particularly preferably, the CS enzyme is used as a cell suspension after re-separating cells from the culture broth, or as a cell homogenate, particularly preferably as a cell homogenate.

[0094] In a preferred embodiment, following the growth of the CS enzyme-producing strain, the CS enzyme is produced as a cell homogenate, and the homogenate is directly used as the CS enzyme in the in vivo conversion method of the present invention. One possible preferred embodiment is disclosed in Example 3 of the present invention.

[0095] In principle, for the reaction in the method for producing L-cysteine acid, all possible sulfites, including the known salts Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its Na2S2O5 anhydride), or KHSO3, are suitable. It is also conceivable to use gaseous sulfur dioxide, which is an anhydride of sulfurous acid, and this can be introduced into the reaction batch, where it hydrates to H2SO3 of sulfurous acid and, depending on the pH, is in the deprotonated form of HSO3 - and SO3 2- and is in equilibrium.

[0096] It is preferable to use Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its Na2S2O5 anhydride), KHSO3. It is particularly preferable to use Na2SO3, NaHSO3 (or its Na2S2O5 anhydride) and (NH4)2SO3. It is especially preferable to use Na2SO3 and NaHSO3 (or its Na2S2O5 anhydride). In a highly preferred embodiment, the sulfite used in the method for producing L-cysteic acid is Na2SO3 or NaHSO3 (or its Na2S2O5 anhydride).

[0097] According to formula (1), the reaction of OPS to form L-cysteic acid releases a stoichiometric amount of phosphoric acid and may lower the pH in the batch as the reaction progresses. If the pH is too low, it will affect the activity of the CS enzyme, so it is necessary to prevent the pH from dropping too much. This can be done passively by an appropriate high-concentration buffer in the batch or actively by a measurement and control unit.

[0098] When the pH deviates from the target value, it is preferable to restore the desired pH by quantitative addition of an alkaline solution or an acid (so-called pH-stat method), or by active pH control by a measurement and control unit (for example, as described in Example 6).

[0099] The reaction temperature is selected between 5°C and 80°C. A reaction temperature of 10°C to 60°C is preferable, a reaction temperature of 15°C to 50°C is particularly preferable, and a reaction temperature of 20°C to 40°C is especially preferable.

[0100] The reaction can be carried out at a pH of 4.0 to 9.0, preferably at a pH of 5.0 to 8.5, particularly preferably at a pH of 5.5 to 8.0, and especially preferably at a pH of 6.0 to 7.5.

[0101] The solvent used in the method for producing L-cysteic acid is preferably water.

[0102] The method for producing L-cysteic acid according to the present invention can be carried out in a discontinuous operation or a continuous operation. In the discontinuous operation (batch operation), all reactants are added in batches during the reaction, and the batch is post-treated after the reaction is completed. In the continuous operation, the CS enzyme is introduced as a stationary phase (for example, immobilized on a membrane reactor or a carrier), and the substrates OPS and sulfite are metered and supplied as a mobile phase. The contact time between the mobile phase and the stationary phase is set so that the substrate OPS can completely react with the sulfite to form the product L-cysteic acid. The discontinuous (batch) operation is preferred.

[0103] The concentration of sulfite in the batch is preferably selected such that it is at least equimolar, preferably at least 1.5-fold molar excess, particularly preferably at least 2-fold molar excess, and most preferably at least 5-fold molar excess, relative to OPS.

[0104] The concentration of OPS in the batch is preferably at least 80 mg / L, particularly preferably at least 1 g / L, and most preferably at least 5 g / L.

[0105] Preferably, the production method of L-cysteic acid is characterized in that the molar yield of L-cysteic acid relative to the molar amount of OPS used is at least 60%, particularly preferably at least 80%, and most preferably at least 90%.

[0106] The method according to the present invention is suitable for industrial-scale use, and the batch volume is preferably more than 10 mL, particularly preferably more than 1 L, and most preferably more than 100 L.

[0107] Surprisingly, contrary to the prior art, the CS enzyme recombinantly produced heterologously in a CS enzyme-producing strain is enzymatically active in the cell homogenate without refolding (regeneration), preferably without relying on a fusion partner, and has been found to be suitable for the efficient production of L-cysteic acid in in vivo conversion, which was not previously known. For this purpose, OPS produced from the cell culture supernatant of the growth of an OPS-producing strain, preferably with the activity of the SerB enzyme suppressed, by culture, for example fermentation, or commercially available OPS, can be reacted with sulfite according to formula (1).

[0108] Furthermore, a method for producing taurine, characterized in that the L-cysteic acid produced according to the present invention is decarboxylated, is preferred.

[0109] L-cysteic acid is decarboxylated to taurine according to formula (2). (2) L-cysteic acid -> taurine + CO2

[0110] Particularly preferably, the method for producing taurine is characterized in that the L-cysteic acid produced by the method of the present invention is further directly used for the production of taurine, for example as disclosed in Examples 8 and 9 of the present invention, that is, used without further post-treatment, purification, or isolation steps.

[0111] The decarboxylation of L-cysteic acid to taurine can be carried out chemically or under the enzymatic catalysis of in vivo conversion. Thermal decarboxylation at high temperature under a metal catalysis that is not considered sustainable is known, but this has the disadvantages of consuming a large amount of energy and having a high proportion of by-products. Preferably, the decarboxylation reaction is an in vivo conversion by the enzymatic decarboxylation of L-cysteic acid to taurine.

[0112] For the production of taurine by decarboxylation of an enzyme, the L-cysteic acid produced by the method of the present invention can be used directly in the form of a reaction batch without further post-treatment steps. However, before enzymatic decarboxylation, it is also possible to remove particulate biomass from the reaction batch, for example by centrifugation, or to isolate L-cysteic acid from the reaction batch in advance.

[0113] Direct use of the L-cysteic acid-containing reaction batch or use of the L-cysteic acid-containing reaction batch after removing particulate biomass is preferred.

[0114] For taurine production, as disclosed in Example 8 for example, it is particularly preferred to use the L-cysteic acid-containing reaction batch directly without further post-treatment steps.

[0115] For the enzymatic catalytic decarboxylation of L-cysteic acid to taurine according to formula (2), enzymes from the class of cysteine sulfinic acid decarboxylase (CSAD, EC 4.1.1.29), aspartate-1-decarboxylase (EC 4.1.1.11), or glutamate decarboxylase (EC 4.1.1.15) are suitable.

[0116] Preferably, the method for producing taurine is characterized in that the decarboxylation is carried out by cysteine sulfinic acid decarboxylase (CSAD enzyme) belonging to the enzyme class EC 4.1.1.29. The CSAD enzyme is known to decarboxylate L-cysteine sulfinic acid to hypotaurine according to formula (3) (3) L-cysteine sulfate -> hypotaurine + CO2

[0117] Although there are differences to some extent, these enzymes are also capable of decarboxylating L-cysteic acid to taurine as a substrate. In particular, as shown in Example 8 and Example 9, for example, the CSADcc enzyme derived from Cyprinus carpio is suitable for decarboxylating L-cysteic acid to taurine according to formula (2).

[0118] CSAD enzyme is mainly found in metazoans (multicellular animals) including mammals such as, for example, humans (Homo sapiens), cows (Bos taurus), rats (Rattus norvegicus), mice (Mus musculus), etc., but is also found in fish such as, for example, Cyprinus carpio. Enzymes having CSAD activity are also found in algae, for example, unicellular organisms of the genus Synechococcus, bacteria or fungi.

[0119] CSAD enzymes derived from mammals selected from humans (Homo sapiens), cows (Bos taurus), rats (Rattus norvegicus) or mice (Mus musculus), and CSAD enzymes derived from fish such as, for example, carp (Cyprinus carpio) are preferred.

[0120] CSAD enzymes derived from humans (Homo sapiens), rats (Rattus norvegicus) or carp (Cyprinus carpio) are particularly preferred.

[0121] Most preferably, the CSAD enzyme is derived from carp (Cyprinus carpio) and is called CSADcc. The DNA sequence that forms the basis of the amino acid sequence of CSADcc is accessible with GenBank sequence ID: AB220585.1 (coding region: nt82 - 1584) in the NCBI database. From the corresponding amino acid sequence, it is preferred that a CSADcc coding region DNA sequence codon-optimized for expression in a particularly selected microorganism (such as Escherichia coli, etc.) is derived (for example, in Escherichia coli, it is defined by nt31 - 1530 of SEQ ID NO: 5, which encodes a protein having the amino acid sequence of SEQ ID NO: 6). For codon optimization, generally available software programs such as the Eurofins Genomics GENEius software used in Example 7 can be utilized.

[0122] Preferably, the method for producing taurine is characterized in that the amino acid sequence of the CSAD enzyme is SEQ ID NO: 6.

[0123] Preferably, the CSAD enzyme, particularly preferably CSADcc, is recombinantly produced by a microbial production strain. The production of the CSAD enzyme by recombinant production in an E. coli production strain is disclosed, for example, in Example 7. The CSAD cds is cloned in a known manner into an expression vector, for example vector pKKj (see Example 3), and a gene construct, for example pCSADcc-pKKj (Figure 4) is produced. The production strain is likewise transformed in a known manner with a gene construct containing the CSAD cds, for example pCSADcc-pKKj, into a microbial host strain, for example E. coli strain JM105, and the resulting production strain, for example E. coli JM105 × pCSADcc-pKKj, is used to produce the CSAD enzyme in a known manner as well. The CSAD enzyme can be produced on a shake flask scale for laboratory purposes (for example, as described in Example 7), or can be produced by fermentation in a known manner.

[0124] The CSAD enzyme contains pyridoxal phosphate (PLP, CAS number 54-47-7) as a cofactor. Thus, supplementing the growth medium or in vivo conversion batch for the conversion from L-cysteic acid to taurine with PLP provides one way to achieve method improvement. Since PLP belongs to the vitamin B6 family, supplementing with other members of the vitamin B6 family, such as pyridoxine (CAS number 65-23-6), pyridoxal (CAS number 66-72-8) or pyridoxamine (CAS number 85-87-0), etc., is a suitable alternative means for method improvement.

[0125] The CSAD enzyme, preferably CSADcc, obtained by growth or fermentation in a shaking flask can be used as a culture broth without further post-treatment, or as a cell suspension after re-isolating the cells by, for example, centrifugation. Furthermore, the CSAD enzyme, preferably CSADcc, 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 (e.g., by chloroform), or as a cell extract after removing the particulate components from the cell homogenate, or as an enzyme purified, for example, by chromatography. The CSAD enzyme is preferably used as a cell suspension after re-isolating the cells from the culture broth as described, for example, in Examples 7 and 8.

[0126] The in vivo conversion of L-cysteic acid to taurine by the CSAD enzyme is carried out under pH and temperature conditions under which decarboxylation of L-cysteic acid to taurine is efficiently performed. The pH at which the in vivo conversion is carried out is preferably in the range of 5.0 to 9.0, and the temperature is preferably in the range of 20°C to 70°C.

[0127] The in vivo conversion for producing taurine from L-cysteic acid can be carried out in a discontinuous or continuous operation. In a discontinuous operation (batch operation), all the reactants are added in batches during the reaction, and the batch is post-treated after the reaction is completed. In a continuous operation, the CSAD enzyme is introduced as a stationary phase (e.g., immobilized on a membrane reactor or a carrier), and the substrate L-cysteic acid is metered and supplied as a mobile phase. The contact time between the mobile phase and the stationary phase is set so that the substrate L-cysteic acid can completely react to form the product taurine. A discontinuous (batch) operation is preferred.

[0128] The concentration of L-cysteic acid in the in vivo conversion for producing taurine is preferably at least 80 mg / L, particularly preferably at least 1 g / L, and most preferably at least 5 g / L.

[0129] Preferably, the method for producing taurine 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%, and most preferably at least 95%.

[0130] Preferably, the method steps for L-cysteic acid production (in vivo conversion 1) and taurine production proceed continuously, i.e., one after another in sequence. In an alternatively preferred embodiment, the method for producing taurine is characterized in that all method steps are carried out in a single reaction batch.

[0131] When all method steps are carried out in a single reaction batch, this method is also referred to as a one-pot method or a one-pot reaction.

[0132] Example 9 of the present invention discloses a method for carrying out such a one-pot reaction, in which the in vivo conversion of OPS to L-cysteic acid according to formula (1) and the in vivo conversion of L-cysteic acid to taurine according to formula (2) are carried out simultaneously, i.e., in a single reaction batch, and OPS reacts with sulfite (salt of sulfurous acid) in the presence of CS enzyme and CSAD enzyme. L-cysteic acid is formed in the first reaction and is decarboxylated "in situ" to taurine by the CSAD enzyme. The product distribution of L-cysteic acid and taurine is determined by the CS enzyme activity relative to the CSAD enzyme activity. By a sufficient metered addition of the CSAD enzyme, L-cysteic acid can be quantitatively converted to taurine. A method is preferred in which the OPS used is converted to L-cysteic acid and taurine, and the total molar yield of L-cysteic acid and taurine is 60% or more, particularly preferably 70% or more, and most preferably 80% or more.

[0133] Carrying out the method for producing L-cysteic acid and the decarboxylation step to taurine in a single reaction batch is particularly interesting in terms of economic feasibility.

[0134] In the context of metabolic engineering approaches, it is also conceivable that the genes for cysteic acid synthase, preferably CSma, and L-cysteine sulfinic acid decarboxylase, preferably CSADcc, are expressed in an OPS-producing strain, and taurine is produced by the growth of such a producing strain in the presence of a sulfur source, preferably a sulfite (salt of sulfurous acid). Similarly, it is also conceivable that the genes for cysteic acid synthase, preferably CSma, and L-cysteine sulfinic acid decarboxylase, preferably CSADcc, are co-expressed in one strain, and cells from the growth of the strain react with OPS in the presence of sulfite to produce taurine as the final product.

[0135] For the production of taurine, an in vivo conversion method is preferred, in which the components OPS, CS enzyme, and CSAD enzyme are produced individually.

[0136] Taurine can be used directly without further post-treatment steps, or it can be concentrated or purified by known methods. Such methods are known to those skilled in the art, for example, from methods for isolating amino acids. Examples include filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, and crystallization.

[0137] The present invention further provides the use of L-cysteic acid formed by the method for producing taurine.

[0138] Compared with known chemical methods for producing taurine from fossil raw materials, producing taurine using L-cysteic acid produced by the method of the present invention enables a biotechnology method starting from plant raw materials. The biotechnology method for producing taurine is particularly interesting for applications in the food, feed, or cosmetics fields due to its sustainable production method.

[0139] The present invention is further illustrated by the following examples, but is not limited by these examples.

Examples

[0140] Example 1: Production of serB deletion mutants in Escherichia coli The strain used was Escherichia coli K12 W3110 (commercially available under strain number DSM 5911 from DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures]). The target for gene inactivation was the coding sequence of the serB gene derived from Escherichia coli (E. coli). The DNA sequence (SEQ ID NO: 1, nt67 to nt1032) of the coding region of the serB gene derived from Escherichia coli K12, which encodes a protein having the amino acid sequence of SEQ ID NO: 2, is accessible in the NCBI (National Center for Biotechnology Information) gene database under gene ID 948913.

[0141] The E. coli serB gene was inactivated using the Red® / ET® technology of Gene Bridges GmbH as described in detail below (see the user manual of the "Quick and Easy E. coli Gene Deletion Kit", "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and the references cited therein, for example, Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640 - 6645). For this purpose, plasmids pKD13, pKD46, and pCP20 were used: · The 3.4 kb plasmid pKD13 (Figure 1) is disclosed under accession number AY048744.1 in the gene database "GenBank". ·The 6.3 kb plasmid pKD46 (Figure 2) is disclosed under accession number AY048746.1 in the gene database "GenBank". ·The 9.4 kb plasmid pCP20 is disclosed in Cherepanov and Wackernagel, Gene 158 (1995): 9-14.

[0142] In order to inactivate the serB gene of Escherichia coli (E. coli) W3110 by homologous recombination using the Lambda Red system, the following steps were carried out:

[0143] 1. Escherichia coli (E. coli) W3110 was transformed with the plasmid pKD46 (so-called "Red Recombinase" plasmid, Figure 2), and ampicillin-resistant clones were isolated and designated as W3110×pKD46.

[0144] 2. In a PCR reaction (using "Phusion (trademark) High-Fidelity" DNA polymerase, Thermo Scientific (trademark)) with the DNA of plasmid pKD13 (Figure 1) and primers serb-1f (SEQ ID NO: 7) and serb-2r (SEQ ID NO: 8), a serB-specific DNA fragment suitable for its inactivation was produced. In the PCR reaction, a 1.4 kb PCR product was formed that contained a 30 nt DNA portion specific to the serB gene from Escherichia coli (E. coli) W3110 at both the 5' and 3' ends. Furthermore, this PCR product contained an expression cassette for the kanamycin resistance gene contained in pKD13, and short portions of DNA that flanked the 5' and 3' ends of the kanamycin expression cassette (so-called "FRT direct repeats", designated as "FRT1" and "FRT2" in Figure 1) and were used as recognition sequences for the "FLP recombinase" (contained in plasmid pCP20) in a subsequent post-treatment step to remove the antibiotic marker kanamycin.

[0145] Primer serb-1f contained 30 nucleotides (nt) from the 5' region of the serB gene (nt67 - 96 of SEQ ID NO: 1), followed by 20 nt specific to plasmid pKD13 (referred to as "pr-1" in Figure 1).

[0146] Primer serb-2r contained 30 nt from the 3' region of the serB gene (nt1006 - 1035 of SEQ ID NO: 1 in reverse complementary form), followed by 20 nt specific to plasmid pKD13 (referred to as "pr-2" in Figure 1).

[0147] To remove the remaining pKD13 plasmid DNA, the 1.4 kb PCR product was isolated and treated with the restriction endonuclease Dpn I, which is familiar to those skilled in the art and cleaves only methylated DNA. Unmethylated DNA from the PCR reaction is not degraded.

[0148] 4. The 1.4 kb PCR product containing the expression cassette of the kanamycin resistance gene specific to the serB gene was transformed into Escherichia coli (E. coli) W3110×pKD46, and kanamycin-resistant clones were isolated on LB kanamycin - glycine plates at 30°C. The LB kanamycin - glycine plates contained LB medium (10 g / L tryptone from GIBCO (trademark), 5 g / L yeast extract from BD Biosciences, 5 g / L NaCl), 1.5% agar, 15 mg / L kanamycin (Sigma - Aldrich), and 1 g / L glycine (Sigma - Aldrich).

[0149] 5. Four of the obtained kanamycin - resistant clones were purified (i.e., isolated by singularization) on LB kanamycin - glycine plates, and it was determined whether the kanamycin resistance cassette was correctly integrated into the serB gene in the PCR reaction.

[0150] The genomic DNA used in the PCR reaction (「Phusion(™) High-Fidelity」 DNA Polymerase, Thermo Scientific(™)) was isolated from cells that had grown a kanamycin-resistant clone of Escherichia coli (E. coli) W3110×pKD46 in LB kanamycin-glycine medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 mg / L kanamycin, 1 g / L glycine) using a DNA isolation kit (Qiagen). Genomic DNA of the Escherichia coli (E. coli) W3110 wild-type strain was used as a control. The primers used in the PCR reaction were serb-3f (SEQ ID NO: 9, 5'-adjacent to the serB gene, nt 1-22 of SEQ ID NO: 1) and serb-4r (SEQ ID NO: 10, 3'-adjacent to the serB gene, nt 1066-1085 of SEQ ID NO: 1, reverse complementary form).

[0151] As expected for the intact gene, the Escherichia coli (E. coli) W3110 wild-type DNA yielded a 1.1 kb DNA fragment in the PCR reaction. On the other hand, for the four kanamycin-resistant clones under study, a DNA fragment of approximately 1.6 kb was obtained in the PCR reaction, as expected if the kanamycin resistance gene had been successfully integrated into the serB gene at the site defined by the primers serb-1f and serb-2r. This result indicated that the kanamycin resistance gene had been successfully integrated into the locus of the serB gene and that the serB gene had been inactivated. Clones with the inactivated serB gene were selected and treated at 42°C to remove the temperature-sensitive plasmid pKD46, thereby restoring the strain's ampicillin sensitivity again. This strain was designated W3110-ΔserB::kan.

[0152] 6. To remove the kanamycin selection marker, W3110-ΔserB::kan was transformed with plasmid pCP20, and the transformants were selected at 30°C. The 9.4 kb vector pCP20 is 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 adjacent to the expression cassette of the kanamycin resistance gene and causes the removal of the kanamycin expression cassette. For this purpose, the clones obtained at 30°C were incubated at 37°C. Under these conditions, the expression of the FLP recombinase was induced and the replication of the pCP20 vector was suppressed.

[0153] As a result of this step, clones were obtained in which the serB gene was inactivated and the sensitivity to kanamycin was restored (so-called "curing" of the antibiotic selection marker). By removing the kanamycin cassette from the genome of the ΔserbB mutant, further mutations can be introduced to produce double or multiple mutants.

[0154] W3110-ΔserB::kan regained kanamycin sensitivity after treatment with the pCP20 plasmid. This was confirmed as follows:

[0155] · By plating on LB-glycine and LB-kanamycin-glycine plates: Growth on the LB-glycine plates was positive, while growth was no longer observed on the LB-kanamycin-glycine plates. This indicates that the kanamycin cassette was successfully removed from the genome.

[0156] · By PCR reaction: For this purpose, genomic DNA was isolated from the kanamycin-sensitive clone (Qiagen DNA isolation kit) and used in a PCR reaction (with "Phusion™ High-Fidelity" DNA Polymerase, Thermo Scientific™) using the primers serb-3f (SEQ ID NO: 9) and serb-4r (SEQ ID NO: 10). E. coli W3110 wild-type DNA produced a 1.1 kb DNA fragment in the PCR reaction, as expected for the intact serB gene. In contrast, the kanamycin-sensitive clone produced a DNA fragment of approximately 250 nt in the PCR reaction. This corresponds to the expected sizes of the 5' and 3' fragments of the inactivated serB gene remaining after homologous recombination.

[0157] The strain isolated in this step was designated E. coli W3110-ΔserB. This strain was distinguished by the presence of the inactivated serB gene and the regained sensitivity of the strain to the antibiotic kanamycin.

[0158] Example 2: Production of OPS Production in shake flasks: OPS was produced by growing the E. coli W3110-ΔserB strain in a shaking flask. For comparison, OPS production in the wild-type strain E. coli W3110 was analyzed. As a preculture for shaking flask culture, the E. coli W3110 strain and the E. coli W3110-ΔserB strain were each inoculated into 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 incubated for 16 h on a shaker at 30 °C and 135 rpm.

[0159] Main culture: Subsequently, a portion of each preculture was transferred into a 300 mL Erlenmeyer flask (with baffles) containing 30 mL of SM1 medium supplemented with 15 g / L glucose, 5 mg / L vitamin B1 (manufactured by Sigma-Aldrich), 0.1 g / L each of the amino acids L-isoleucine, D,L-methionine, and L-threonine, and 0.5 g / L glycine (all manufactured by Sigma-Aldrich).

[0160] Composition of SM1 medium: 12 g / L K2HPO4, 3 g / L KH2PO4, 5 g / L (NH4)2SO4, 0.3 g / L MgSO4·7H2O, 0.015 g / L CaCl2·2H2O, 0.002 g / L FeSO4·7H2O, 1 g / L Na3 citrate·2H2O, 0.1 g / L NaCl; 1 mL / L of trace element solution.

[0161] Composition of trace element solution: 0.15 g / L Na2MoO4·2H2O, 2.5 g / L H3BO3, 0.7 g / L CoCl2·6H2O, 0.25 g / L CuSO4·5H2O, 1.6 g / L MnC12·4H2O, 0.3 g / L ZnSO4·7H2O.

[0162] For the main culture, a sufficient amount of the preculture was inoculated in each case to achieve an initial cell density of OD 600 / mL (optical density of the main culture measured at 600 nm) of 0.3 / mL. Using this as the starting material, 30 mL batches were incubated at 30 °C and 135 rpm for 24 hours.

[0163] After 24 hours, samples were taken, and the cell density OD was measured in both the culture supernatant and the cell pellet 600The OPS content per mL and the OPS content were measured. For this purpose, 2 mL of the cell culture medium was centrifuged at 13,000 rpm for 5 minutes in each case (Heraeus (trademark) Fresco (trademark) 21 centrifuge). The cell culture supernatant was directly analyzed for the OPS content by HPLC. The cell pellet was resuspended in 2 mL of H2O in each case to prepare a cell extract. This was achieved using the MP Biomedicals FastPrep-24 (trademark) 5G cell homogenizer. In each case, 2 × 1 mL of the cell pellet suspended in H2O was disrupted in a manufacturer-assembled 1.5 mL tube containing glass beads ("Lysing Matrix B") (3 × 20 seconds at a shaking frequency of 6,000 rpm at intervals of 30 seconds in each case). In each case, the obtained cell homogenates were combined and centrifuged at 13,000 rpm for 5 minutes to prepare a cell extract. This cell extract was analyzed for the OPS content by PLC. The results are summarized in Table 1.

[0164]

Table 1

[0165] HPLC analysis of OPS, L-cysteic acid, and taurine: For the quantification of the compounds analyzed in the examples, calibrated HPLC methods were employed for OPS, L-cysteic acid, and taurine, respectively; all the standard substances used for calibration were commercially available products (Sigma-Aldrich). The Agilent 1260 Infinity II HPLC system was used. This system was equipped with the manufacturer's unit for pre-column derivatization (OPA derivatization) with o-phthalaldehyde, as known in the analysis of amino acids. The HPLC system was equipped with a fluorescence detector to detect the production of OPA derivatives of OPS, L-cysteic acid, and taurine. The detector was set at an excitation wavelength of 330 nm and an emission wavelength of 450 nm. Also used was a Phenomenex Luna (registered trademark) C18(2) column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm, which was thermally equilibrated to 40 °C in a column oven.

[0166] Eluent A: 25 mM Na phosphate, pH 6.0. Eluent B: Methanol. Separation was carried out in gradient mode: 1% - 15% Eluent B for 0 - 10 minutes, followed by 15% Eluent B for 15 minutes, flow rate 1.0 mL / min. Retention time of L-cysteic acid: 6.95 minutes. Retention time of OPS: 7.65 minutes. Retention time of taurine: 21.9 minutes.

[0167] Production of OPS by fermentation: OPS was produced by fermentation of Escherichia coli (E. coli) W3110-ΔserB strain.

[0168] Pre-culture 1: In 20 mL of LB-glycine medium, the Escherichia coli (E. coli) W3110-ΔserB strain was inoculated in a 100 mL Erlenmeyer flask and incubated for 7 hours on a shaker (150 rpm, 32 °C).

[0169] Pre-culture 2: Then, 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. This culture solution was shaken at 150 rpm for 17 hours in a 32 °C Erlenmeyer flask (volume 1 L) (Infors incubator shaker). After this culture, the cell density OD 600 / mL was 5.7 / mL.

[0170] Main culture: Fermentation was carried out in an Eppendorf "DASGIP® Parallel Bioreactor System for Microbiology" fermenter. 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 CaCl2×2H2O, 1.2 g / L MgSO4×7H2O, 0.075 g / L FeSO4×7H2O, 1 g / L Na3 citrate×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 the section on shake flask culture).

[0171] A 25% NH4OH solution was pumped in to adjust the pH of the fermenter to 7.0 at the start. The pH during fermentation was maintained at a value of 7.0 by automatic correction using 25% NH4OH or 4M H3PO4. Foam control was achieved by the automatic metered addition of 4% (v / v) Struktol J673 (Schill & Seilacher) in H2O. For inoculation, preculture 2 (60 mL) was pumped into the fermenter vessel. Thus, the initial volume was approximately 660 mL. The culture was first stirred at 400 rpm and aerated at an aeration rate of 2 vvm (volume of air per volume of culture per minute) with sterile filtered compressed air. Under these initial conditions, the oxygen probe was calibrated to 100% saturation before inoculation.

[0172] The target value for O2 saturation during fermentation was adjusted to 30%. After the O2 saturation dropped below the target value, the adjustment cascade was started to restore the O2 saturation to the target value. This was done by first continuously increasing the gas supply rate (up to a maximum of 5 vvm) and then continuously increasing the stirring speed (up to a maximum of 1,600 rpm). Fermentation was carried out at a temperature of 32°C.

[0173] When the glucose content in the fermenter decreased from an initial 10 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was added continuously. Subsequently, the feed rate was adjusted so that the glucose concentration in the fermenter did not exceed 2 g / L. Glucose was measured using a glucose analyzer obtained from YSI (Yellow Springs, Ohio, USA). 23 hours after the start of fermentation, 3.5 mL of a 200 g / L glycine solution in H2O was added to the fermentation batch.

[0174] The fermentation time was 53 hours. Samples were taken from the fermentation batch 23 hours, 30 hours, 47 hours, and 53 hours after the start of fermentation, and the cell density OD 600 / mL was measured from one aliquot. In each case, a further aliquot was incubated at 80 °C for 5 minutes, centrifuged, and the OPS content in the cell culture supernatant was measured by HPLC. The cell density and OPS content are summarized in Table 2.

[0175]

Table 2

[0176] Example 3: Production of CSma enzyme Cysteine synthase (CSma) derived from Methanosarcina acetivorans (M. acetivorans) was used. The amino acid sequence of the CSma enzyme is accessible with the accession ID WP_048066469 in the NCBI database. Using this amino acid sequence, a DNA sequence optimized for codons for expression in Escherichia coli (E. coli) was derived (using the generally available Eurofins Genomics GENEius software), and this was produced synthetically (Eurofins Genomics). The synthetically produced DNA had the sequence disclosed in SEQ ID NO: 3, which had the amino acid sequence disclosed in SEQ ID NO: 4 and contained the coding region of the gene encoding a protein designated CSma (hereinafter referred to as the CSma coding region (SEQ ID NO: 3)). For cloning purposes, the synthetically produced DNA contained an EcoRI cleavage site at the 5' end and a HindIII cleavage site at the 3' end.

[0177] The vector pCSma-pKKj suitable for recombinant expression of the CSma coding region (Figure 3) was produced by cleaving the synthetically produced DNA with EcoRI and HindIII and cloning it as an EcoRI / HindIII fragment into the vector pKKj cleaved with EcoRI and HindIII by a known method. The expression vector pKKj disclosed in EP2670837A1 (Wacker's application) is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database with the accession number M77749.1. Approximately 1.7 kb (bp262 to 1947 of the DNA sequence disclosed in M77749.1) was removed from the 4.6 kb plasmid, thereby obtaining the 2.9 kb expression vector pKKj.

[0178] The vector pCSma-pKKj was transformed into Escherichia coli K12 JM105 strain in a known manner, and the CSma coding region was expressed in Escherichia coli. Escherichia coli K12 JM105 strain is commercially available from DSMZ - German Collection of Microorganisms and Cell Cultures GmbH under the strain number DSM 3949.

[0179] The clones obtained by transformation were selected on LB ampicillin plates. LB ampicillin 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). The clones were selected and cultured in a shaking flask. The CSma - producing strain was designated as Escherichia coli JM105×pCSma - pKKj. The CSma coding region was expressed in Escherichia coli JM105×pCSma - pKKj in a known manner under the control of an IPTG - inducible tac promoter (IPTG: isopropyl β - thiogalactoside, Sigma - Aldrich) functionally linked to the CSma coding region.

[0180] Growth in a shaking flask: A preculture of Escherichia coli JM105×pCSma - pKKj strain was prepared in LB ampicillin medium (grown overnight at 37°C and 120 rpm, Infors chest shaker).

[0181] 2 mL of the preculture (OD of 3.4 / mL) 600 ) was used as the inoculum for the main culture (0.3 L Erlenmeyer flask) of 50 mL of SM1 medium (Example 2) supplemented with 15 g / L glucose; 5 g / L peptone (Oxoid); 2.5 g / L yeast extract; 0.005 g / L vitamin B1 (Sigma - Aldrich); 5 mg / L pyridoxal phosphate (PLP, Sigma - Aldrich), and 100 mg / L ampicillin. The main culture was shaken in a chest shaker (Infors) at 30°C and 140 rpm. After 4 hours of culture, the cell density OD600 reached 2.0. Subsequently, the inducer IPTG (Sigma-Aldrich, final concentration 0.4 mM) was added, and growth was continued for an additional 20 hours at 30 °C and 140 rpm in a chest shaker (Infors). At the end of growth, the cell density OD 600 was 3.1 / mL.

[0182] Cells from the shake flask culture were isolated by centrifugation (10 minutes at 15,000 rpm using a Sorvall RC5C centrifuge equipped with an SS34 rotor). The cell pellet from a 50 mL shake flask culture was suspended in 2 mL of 100 mM potassium phosphate, pH 7.0; 100 mM KCl (KPi7.0 buffer) to prepare a cell suspension, which was used for the preparation of cell homogenates. The cell homogenates were prepared as described in Example 2 using a FastPrep-24TM 5G cell homogenizer from MP Biomedicals. The resulting cell homogenates (volume 2 mL) were used without further post-treatment for the in vivo conversion of OPS to L-cysteic acid (Example 5).

[0183] The protein content of the cell extracts was determined using the "Qubit(R) Protein Assay Kit" according to the manufacturer's instructions with a Qubit 3.0 Fluorometer from Thermo Fisher Scientific. The protein content of the cell homogenates from the shake flask culture was 5.3 mg / mL.

[0184] Example 4: Production of L-cysteic acid from commercially available OPS and Na 2 SO 3 by in vivo conversion using CSma enzyme Two batches were carried out in parallel:

[0185] Batch 1: First, 8.15 mL of KPi 7.0 buffer was added to a 100 mL Erlenmeyer flask, followed by 1 mL of a 0.2 M solution of Na2SO3 in KPi 7.0 buffer, 0.5 mL of CSma cell homogenate from shaken flask culture (Example 3), and 350 μL of a 0.2 M solution of OPS (Sigma - Aldrich) in KPi 7.0 buffer in sequence. The batch volume was 10 mL.

[0186] Batch 2: The batch (comparative batch without Na2SO3) had the same composition as Batch 1. Instead of the Na2SO3 solution, 1 mL of KPi 7.0 buffer was added to Batch 2.

[0187] Both batches were incubated in a chest shaker (Infors) at 30 °C and 140 rpm. After 1 hour, 2 hours, and 4 hours, 1 mL of the batch was incubated at 80 °C for 5 minutes in each case, centrifuged, and the supernatant was analyzed by HPLC. The time course of L - cysteic acid production from OPS is shown in Table 3.

[0188]

Table 3

[0189] Example 5: Production of L-cysteic acid from OPS-containing culture supernatant from shake flask culture and Na 2 SO 3 by in vivo conversion using CSma enzyme First, 9 mL of cell culture supernatant from the shake flask culture of Escherichia coli (E. coli) W3110-ΔserB strain (Example 2) with an OPS content of 113.6 mg / L was added to a 100 mL Erlenmeyer flask, followed by the addition of 0.3 mL of 3M KC, 0.5 mL of 0.2M Na2SO3 in KPi 7.0 buffer, and 1 mL of CSma cell homogenate from the shake flask culture (Example 3). The batch volume was 10.8 mL. The batch was incubated in a chest shaker (Infors) at 30 °C and 140 rpm. At the start and after 6 hours, 1 mL of the batch was incubated at 80 °C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC for the contents of OPS and L-cysteic acid. The progress of the reaction over time is summarized in Table 4. The molar yield of L-cysteic acid based on the molar amount of OPS used was 97.6%.

[0190]

Table 4

[0191] Example 6: Preparation and production of L-cysteic acid by in vivo conversion of OPS at a constant pH OPS substrate: 10 mL of fermentation broth derived from the fermentation of Escherichia coli (E. coli) W3110-ΔserB strain (Example 2) was centrifuged (15,000 rpm for 10 minutes, equipped with a Sorvall RC5C centrifuge and an SS34 rotor), and the OPS content in the fermentation supernatant was measured by HPLC. The OPS content was 5.6 g / L.

[0192] CSma homogenate: The cell homogenate of Escherichia coli (E. coli) JM105×pCSma-pKKj strain was prepared from 2 × 50 mL of shake flask cultures as described in Example 3. This homogenate was supplemented with 15 μL of 500 mg / L PLP. The total volume of the homogenate was 4 mL.

[0193] In vivo conversion batch: A 50 mL thermostatable double-walled downward-tapering reaction vessel (an accessory of Titrator TitroLine alpha (Schott)) was connected to a thermostat (Lauda) by a hose connection, and the temperature was adjusted to 30 °C.

[0194] The reaction batch contained 6 mL of OPS-containing fermentation supernatant with an OPS content of 5.6 g / L, 4 mL of CSma homogenate, 0.2 mL of 3 M KCl, 0.1 mL of 0.1 M DTE (dithioerythritol, Sigma-Aldrich), and 0.3 mL of a 1 M solution of Na2SO3 in KPi 7.0 buffer. The batch volume was 10.6 mL. The OPS concentration in the batch was 17.1 mM (0.18 mmol of OPS in a batch volume of 10.6 mL). The batch was stirred with a magnetic stirrer. The batch was also equipped with a pH electrode (Mettler Toledo), which was connected to a pH control unit (TitroLine alpha titrator, Schott) and operated in pH-stat mode according to the manufacturer's instructions. Under pH-stat conditions, the pH in the reaction vessel was kept constant at the set pH 7.0 throughout the reaction by metering the addition of 0.5 M NaOH from a burette connected to the control unit.

[0195] The reaction time was 4 hours. Since 0.5 M NaOH was supplied from the burette to maintain the pH at 7.0 in the batch, the batch volume after 4 hours of reaction was 13.0 mL. At 2 hours and 4 hours after the start of the reaction, 50 μL aliquots of the batch were taken out in each case, and the contents of OPS and L-cysteic acid were analyzed by HPLC. The progress of the reaction over time is summarized in Table 5. After 4 hours of reaction time, the L-cysteic acid content in the batch was 2399.6 mg / L (14.2 mM). This corresponded to an absolute molar yield of 0.18 mmol of L-cysteic acid for a batch volume of 13.0 mL. The OPS used was completely consumed. Based on the 0.18 mmol of OPS used, this corresponded to a yield of 100%.

[0196]

Table 5

[0197] Example 7: Recombinant production of CSADcc derived from Cyprinus carpio (carp) in Escherichia coli (E. coli) CSADcc gene: The cDNA sequence derived from the mRNA of cysteine sulfinic acid decarboxylase (CSAD) from Cyprinus carpio (carp) is disclosed in Genbank sequence ID: AB220585.1 (coding region: nt82 - 1584) of the NCBI database (National Center for Biotechnology Information). Using the corresponding amino acid sequence, a DNA sequence codon-optimized for expression in Escherichia coli (E. coli) was derived (using the generally available Eurofins Genomics GENEius software), and this was synthetically produced (Eurofins Genomics). The synthetically produced DNA had the sequence disclosed in SEQ ID NO: 5. This contained the coding region of the gene (hereinafter referred to as the CSADcc coding region (SEQ ID NO: 5, nt31 - 1530)), which had the amino acid sequence disclosed in SEQ ID NO: 6 and encoded a protein designated CSADcc. For cloning purposes, the synthetically produced DNA contained an EcoRI cleavage site (SEQ ID NO: 5, nt25 - 30) at the 5' end and a HindIII cleavage site (SEQ ID NO: 5, nt1532 - 1537) at the 3' end.

[0198] Vector pCSADcc-pKKj: The vector pCSADcc-pKKj suitable for recombinant expression of the CSADcc coding region (Figure 4) was produced by cleaving synthetically produced DNA using EcoRI and HindIII and cloning it as an EcoRI / HindIII fragment into the vector pKKj cleaved with EcoRI and HindIII by a known method (see Example 3). Thereby, the vector pCSADcc-pKKj was obtained. By transforming the vector pCSADcc-pKKj into Escherichia coli (E. coli) K12 JM105 strain in a known manner, the CSADcc coding region was expressed in Escherichia coli (E. coli). The clones obtained by transformation were selected on an LB ampicillin plate. The clones were selected and cultured in a shaking flask. The CSADcc-producing strain was designated as Escherichia coli (E. coli) JM105×pCSADcc-pKKj. The CSADcc coding region was expressed by growth in a shaking flask in Escherichia coli (E. coli) JM105×pCSADcc-pKKj in the same manner as described in Example 3 for Escherichia coli (E. coli) JM105×pCSma-pKKj.

[0199] Culture in a shaking flask: 10 mL of preculture (OD of 3.1 / mL) 600 ) was used as the inoculum for the main culture (1 L Erlenmeyer flask) of 100 mL of 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 mg / L pyridoxal phosphate, and 100 mg / L ampicillin. The main culture was grown and induced as described in Example 3. Cells from 100 mL of shaking flask culture (OD of 7.1 / mL) 600 ) were isolated by centrifugation and the cell pellet was resuspended in 4 mL of KPi 7.0 buffer. This cell suspension was used directly in the in vivo conversion experiment.

[0200] Example 8: Production of taurine from L-cysteic acid by in vivo conversion First, 9 mL of the batch of Example 5 with an L-cysteic acid content of 84.5 mg / L (Table 4) was introduced into a 100 mL Erlenmeyer flask, and 1 mL of the cell suspension of the CSADcc enzyme of Example 7 was added. The batch volume was 10 mL. The batch was incubated in a chest shaker (Infors) at 37 °C and 140 rpm. After 2 hours, 1 mL of the batch was incubated at 80 °C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The L-cysteic acid used was completely consumed. The amount of taurine formed was 66.4 mg / L.

[0201] Example 9: Production of taurine from OPS by in vivo conversion First, 9 mL of the batch from the shake flask culture of Escherichia coli (E. coli) W3110-ΔserB strain (Example 2) with an OPS content of 94.7 mg / L was introduced into a 100 mL Erlenmeyer flask, and 0.5 mL of a 0.2 M solution of Na2SO3 in KPi 7.0 buffer, 1 mL of the CSma cell homogenate from the shake flask culture (Example 3), and 1 mL of the cell suspension of the CSADcc enzyme (Example 7) were added. The batch volume was 11.5 mL. The batch was incubated in a chest shaker (Infors) at 30 °C and 140 rpm. After 4 hours, 1 mL of the batch was incubated at 80 °C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The OPS used was completely consumed. At the same time, 34.4 mg / L of L-cysteic acid and 40.4 mg / L of taurine were formed.

[0202] Abbreviations used in the figures: bla: Ampicillin resistance gene (β-lactamase) kanR: Kanamycin resistance gene ORI: Replication origin pr-1: Primer binding site 1 pr-2: Primer binding site 2 FRT1: FLP recombinase recognition sequence 1 FRT2: FLP recombinase recognition sequence 2 araC: araC gene (repressor gene) P araC: Promoter of the araC gene ParaB: Promoter of the araB gene Gam: Recombinant gene of lambda phage Gam Bet: Recombinant gene of lambda phage Bet Exo: Recombinant gene of lambda phage Exo ORI101: Temperature-sensitive replication origin RepA: Gene of plasmid replication protein A Ptac: tac promoter EcoRI: Cleavage site of restriction enzyme EcoRI HindIII: Cleavage site of restriction enzyme HindIII CSma: Coding region of the cysteine synthase gene from Methanosarcina acetivorans CSADcc: Coding region of the cysteine sulfinate decarboxylase gene from Cyprinus carpio

Claims

1. A method for producing L-cysteic acid, comprising: a reaction of O-phospho-L-serine (OPS) with a sulfite salt (sulfite) and a cysteic acid synthase (CS enzyme) belonging to enzyme class EC 2.5.1.76 in in vivo conversion.

2. The method according to claim 1, wherein the CS enzyme is produced by the growth of a microbial strain of the family Enterobacteriaceae that heterologously expresses the CS enzyme in an enzymatically active form.

3. The method according to one or both of claims 1 and 2, wherein the CS enzyme is used in the reaction without a prior regeneration step.

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

5. The method according to one or more of claims 1 to 4, wherein the OPS used in the reaction is produced by biotechnology.

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

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

8. The method according to one or more of claims 1 to 7, wherein the molar yield of L-cysteic acid relative to the molar amount of OPS used is at least 60%.

9. A method for producing taurine, wherein the L-cysteic acid is produced by the method according to one or more of claims 1 to 8 and decarboxylated.

10. The method according to claim 9, wherein the decarboxylation is carried out by cysteine sulfinic acid decarboxylase (CSAD enzyme) belonging to enzyme class EC 4.1.1.

29.

11. The method according to claim 10, wherein the amino acid sequence of the CSAD enzyme is SEQ ID NO:

6.

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

13. The method according to one or more of claims 9 to 12, wherein all method steps are carried out in one reaction batch (one-pot method).

14. Use of L-cysteine acid produced by the method according to one or more of claims 1 to 8 for producing taurine.

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