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, comprising the reaction of O-phospho-L-serine (OPS), a sulfite salt, and a cysteate synthase (CS enzyme) belonging to enzyme class EC2.5.1.76 in biotransformation. The L-cysteic acid produced by the present invention can be decarboxylated to taurine. The present invention further relates to the use of the produced L-cysteic acid for taurine production. [Background technology]

[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 proteins. It is found in nature, for example in wool, as an oxidation product of L-cysteine, an amino acid that does constitute proteins. Cysteic acid is also an intermediate in the coenzyme M biosynthesis (CoM, 2-mercaptoethanesulfonic acid, CAS 3375-50-6) by methanogenic archaea. The biosynthetic pathway of L-cysteic acid in methanogenic bacteria follows formula (1), starting with O-phospho-L-serine (OPS, L-serine-O-phosphate, L-2-amino-3-hydroxypropanoic acid 3-phosphate, CAS 407-41-0), which reacts with sulfite in a reaction catalyzed by cysteate synthase (CS enzyme) to form L-cysteic acid. (1) OPS+SO3 2- -> L-cysteic acid + HPO4 2-

[0003] Graham et al., Biochem. J. (2009) 424: 467-478, disclosed that in the case of a gene product derived from Methanosarcina acetivorans, which is related to threonine synthase, recombinantly produced in Escherichia coli (E. coli), and concentrated as an enzymatically inactive protein in so-called inclusion bodies, detectable enzymatic activity is only measurable after complex regeneration of the inclusion bodies. After regeneration, analytical assays (HPLC and mass spectrometry) using the regenerated protein demonstrated the production of L-cysteic acid by reacting commercially available (chemically synthesized) OPS with sulfite according to formula (1). These studies established that the regenerated protein from Methanosarcina acetivorans is associated with cysteic acid synthase activity and that its genetic relationship with threonine synthase is 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 HCl in DMSO, or by oxidative cleavage of cystine. Furthermore, L-cysteic acid can also be produced by oxidation of L-cysteinesulfinic acid. Known methods for the chemical production of L-cysteic acid are not considered sustainable, use environmentally harmful chemicals, and are unacceptable to consumers, particularly 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, such as as an ingredient in Regu®-Slim (DSM) for skincare. 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, see above) showed that when the 44 kDa CS enzyme is heterologously expressed in E. coli, it is not produced in an active form, but is only concentrated in an inactive form within the inclusion bodies. Solubilization of the inclusion bodies and refolding to form the active enzyme yielded only a very low yield of 3 mg of refolded protein per liter of E. coli culture, and was therefore unsuitable as a preparative in vivo conversion process for the industrial-scale production of L-cysteic acid.

[0007] According to Graham et al. (2009, see above), enzyme activity without refolding was detectable only 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 conventional technique indicates that active CS enzyme can only be produced in E. coli in low yields through a laborious and costly process.

[0008] In a metabolic engineering approach, Joo et al. (2018), J. Agric. Food Chem. 66: 13454 - 13463 describes genetically modified strains of Corynebacterium glutamicum, a bacterium for taurine production using the CS gene derived from Methanosalquina acetylborans, as well as strains optimized for sulfur utilization. CS activity was analytically detected by enzyme assays for the gene expressed in C. glutamicum. The activity of the CS enzyme in the taurine-producing strains was indirectly inferred. As shown in Figure S2 of the "Supporting Information" in Joo et al. (2018, see above), CS-expressing C. glutamicum cells accumulate OPS, the substrate of the CS reaction, intracellularly, and further accumulate serine, cysteine, and taurine. Details of L-cysteic acid production remain unclear. Joo et al. (2018, see above) showed that even when intracellular production of cysteine ​​and other sulfur-containing compounds such as taurine occurs in measurable yields in the same strain, the simultaneous presence of OPS and CS enzymes in cells is insufficient for effective L-cysteic acid production.

[0009] Tevatia et al., Algal Research (2015) 9: 21-26 describes the spontaneous production of taurine in microalgae, with its detection as an intermediate of L-cysteic acid. As shown in Figure 1 of Tevatia et al. (2015, see above), the biosynthetic pathway in algae yields L-cysteic acid ("cysteic acid" in Figure 1) from L-serine. However, this synthetic pathway does not involve the enzymatic reaction of the CS enzyme of formula (1). None of the described biosynthetic pathways yield L-cysteic acid via OPS. The intracellular content of L-cysteic acid is extremely low, and it is accompanied by several by-products that make post-treatment difficult, such as methionine, cysteine, cysteinesulfinic acid, hypotaurine, and taurine, making the growth of microalgae unsuitable for L-cysteic acid production.

[0010] In a metabolic engineering approach, U.S. Patent Application Publication 2019 / 0062757 (KnipBio) describes heterologous production strains for the production of taurine or its precursors. The described strains express CS enzymes and other biosynthetic genes derived from taurine metabolism in various configurations. Yields of hypotaurine and taurine were extremely low, at a maximum of 419 ng / mL. The yield of L-cysteic acid production is not mentioned. While this prior art uses CS gene constructs, no enzyme assays are performed to detect CS enzyme activity. Furthermore, this metabolic engineering approach does not demonstrate that L-cysteic acid can be produced preparatively by biotechnology. Similarly, it is not disclosed whether the OPS or CS enzyme production strains described in this document are suitable for use in in vivo conversion to produce L-cysteic acid according to formula (1).

[0011] Steinfeld et al., ACS Chem. Biol. (2014) 9: 1104-1112 describes increased intracellular production of OPS in E. coli strains lacking the serB gene. Increased extracellular production of OPS is not described (see Figure 5 in Steinfeld et al.).

[0012] European Patent No. 2444481 of CJ CheilJedang Corporation (KR) describes a method for producing L-cysteine, comprising producing OPS using a strain with reduced SerB activity, and then reacting it with a sulfide or thiosulfate in an enzyme-catalyzed reaction to form L-cysteine. It is also unknown whether the enzyme class of O-phosphoserine sulfhydrase used here (OPSS, EC2.5.1.65) can utilize sulfites as a substrate with respect to the CS enzyme. In fact, Steiner et al. J. Bacteriol. (2014), 196: 3410-3420 investigated the reaction mechanism of the OPSS enzyme derived from Mycobacterium tuberculosis and found that sulfites such as Na2S2O5 and Na2SO3 are not suitable as S donors for the reaction with the enzyme-bound aminoacrylate intermediate produced when OPS binds to the OPSS enzyme and phosphate is removed, in contrast to Na2S (sulfide) and Na2S2O3 (thiosulfate) (Steiner et al., 2014, Figure 2, see above). Accordingly, European Patent No. 2444481 discloses the production of a strain that produces OPS by inactivating the serB gene, as also known from Steinfeld et al. (2014, see above). OPS was then used in biotransformation 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 does not provide a method for producing CS enzymes with sufficient activity for previously unknown industrially applicable methods, such as the preparative production of L-cysteic acid for further use, or the in vivo conversion of OPS with sulfite sulfites.

[0014] Therefore, there is a need for an environmentally friendly and sustainable method for producing L-cysteic acid on a preparative scale, for which biotechnology is well suited. As consumer-driven trends are moving away from chemically produced components, the objective of this invention is a biotechnology method for producing L-cysteic acid by biotransformation that is suitable for industrial-scale use. [Overview of the project]

[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 bioconversion 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 objective is achieved by a method for producing L-cysteic acid, which involves the reaction of O-phospho-L-serine (OPS) with a sulfite salt (sulfite) and a cysteate synthase (CS enzyme) belonging to enzyme class EC2.5.1.76 in biotransformation. [Modes for carrying out the invention]

[0017] In the context of the present invention, the manufacturing methods are distinguished as follows: 1.Chemical method 2. Biotechnological methods (a) Biotechnological methods using metabolic engineering Metabolic engineering (also known as "pathway design") is a biotechnological method that alters the metabolic pathways of an organism by optimizing or modifying genes and regulatory processes, as opposed to biotransformation. Enzymes can be introduced into an organism by supplementing the genome with genes for novel or modified enzymes, or by expressing genes for endogenous enzymes at enhanced or attenuated levels, thereby establishing new metabolic pathways in the organism or enhancing or attenuating existing ones. The goal of metabolic engineering is to enable the organism to produce new or intracellular metabolites in high yield. Metabolic engineering methods do not use enzyme substrates, such as OPS in this invention, which are characteristic starting materials for the metabolite; instead, they use only the nutrient medium necessary for the growth of the target organism, consisting of a carbon source (e.g., glucose), a nitrogen source (e.g., ammonium salts, or complex amino acid mixtures such as peptone or yeast extracts), and other salts necessary for growth. Such nutrient media are known to those skilled in the art from microbiological practice. (b) Biotechnological methods by biotransformation In vivo conversion is defined as the conversion of one or more reactants into a product under enzymatic catalysis, and the enzyme substrate is added to the reaction batch along with the enzyme. In the reaction batch, the added enzyme substrate, such as OPS in this invention, is enzymatically converted in the presence of a sulfite salt by an enzyme selected from the class of cysteate synthase (CS enzyme, EC2.5.1.76). The reactants may be derived from chemical or biotechnological production. The OPS used in the method of this invention may be obtained, for example, by chemical synthesis or by biotechnological production by propagation of a production strain. The enzyme used for enzymatic catalysis is preferably derived from biotechnological production by fermentation of a production strain of 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, for example, does not require 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 permeabilized chemically (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® Protein Assay Kit" and a Thermo Fisher Scientific Qubit 3.0 Fluorometer, in accordance with the manufacturer's instructions. (ii) In a solution buffered to pH 7 with potassium phosphate, first add OPS (final concentration 10 mM) and sodium sulfite (final concentration 20 mM), then add CS enzyme to start the reaction. The assay volume is 10 mL. The assay temperature 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, use at least 0.1 mg of the enzyme fraction prepared in (i). If using purified CS enzyme, use at least 10 μg of the purified enzyme fraction. After 1 hour, 2 hours, and 4 hours from the start of the reaction, take out 1 mL of the assay solution in each case, centrifuge for 10 minutes, and determine the content of OPS and L-cysteic acid by calibrating HPLC (see Example 4). A reference substance used for calibration is commercially available (Sigma-Aldrich).

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

[0022] Heterogenetic expression is understood to mean expressing a gene's cds (Cryptid-derived gene samples) or a cds of a portion of a gene in a host organism that does not originally possess that gene or gene fragment. Introducing heterogeneous gene cds into a host organism involves the use of recombinant DNA technology. Heterogeneous gene cds can be introduced into a host organism by integration into the host organism's genome or by extrachromosomal integration in the form of autonomously replicating gene constructs (plasmids, vectors).

[0023] It is preferable to introduce heterologous gene cds into a host organism in the form of autonomously replicating gene constructs (plasmids, vectors).

[0024] Depending on the genetic element (promoter) used to control the expression of heterologous cds, constitutive expression and inductive expression are distinguished. In constitutive expression, gene expression is activated at all stages of cell culture (unregulated). In inductive expression (regulated), gene expression is stimulated by adding an inducer molecule to the cell culture, for example, by adding the inducer molecule IPTG to induce the tac promoter in Examples 3 and 7 of the present invention. Inductive 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 in a host cell from the genome from which the gene originally originates.

[0026] If the heterologously expressed protein is, for example, an enzyme, it may be, for example, in an enzymatically active form, or it may be concentrated in an enzymatically inactive form within an inclusion body.

[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 to be introduced into the host strain are not encoded in the genome of the host strain, (ii) at least the cds of the gene encoding the CS enzyme are incorporated into the genome of the host organism on the chromosome by recombinant DNA technology, or preferably introduced into the host organism extrachromosome by an autonomous replication vector, and (iii) the CS enzyme is expressed in an enzymatically active form by the 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, following protein biosynthesis and any post-translational modifications, such as the incorporation of cofactors (e.g., pyridoxal phosphate in the case of the CS enzyme; see entry EC2.5.1.76 in the KEGG enzyme database), express the CS enzyme in an enzymatically active form within the microorganism. "Expressed in an enzymatically active form" does not mean that the protein is initially produced as an inactive protein in the inclusion body and only becomes enzymatically active after regeneration.

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

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

[0031] Within the scope of the present invention, reaction yield is defined as the amount of reactants used that are converted into a product under reaction conditions. Yield can be expressed as absolute yield of product (mmol or g), as volume yield (mM or g / L) in absolute amounts of product per unit volume, or as relative yield of product in proportion to the reactants used (taking into account the molecular weights of the reactants and products). Relative yield is also referred to as percentage yield.

[0032] In the context of this invention, the terms “growth” or synonymously “culture” of microbial cells encompass both shaking flask culture and fermentation. The medium used for the growth or culture of microorganisms is called a growth medium or culture medium, and in the case of fermentation, it is also called a fermentation medium. Growth / culture / fermentation of a production strain in a growth medium, culture medium, or fermentation medium yields a culture broth / fermentation broth. The culture broth / fermentation broth consists of the cellular biomass of the production strain and a biomass-free culture supernatant / fermentation supernatant formed during growth from the growth medium and from metabolites secreted by the cells.

[0033] Fermentation is a method and process for the production (cultivation) of cell cultures on an industrial scale (manufacturing scale), preferably involving the growth of a microbial production strain under specified conditions of culture medium, temperature, pH, oxygen supply, and medium mixture. Depending on the composition (genetic composition) of the production strain, the purpose of fermentation is to produce proteins / enzymes or metabolites in the highest possible yield for further use. 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 production strain cells (fermentation cells) and a biomass-free fermentation supernatant formed from the growth medium and metabolites secreted by the fermentation cells during fermentation. The target product of fermentation may be present in the fermentation cells or in the fermentation supernatant. For example, OPS is present in the fermentation supernatant, and CS enzymes are present in the fermentation cells.

[0034] Shaking flask culture is used to cultivate microorganisms on a laboratory scale, in contrast to production-scale fermentation. Shaking flask culture requires specifying a particular culture medium and pH, and culturing in the presence of oxygen under constant movement (shaking), although more precise conditions regarding the medium, temperature, pH, oxygen supply, and medium mixing can be established and controlled within a fermenter. Smaller-scale cultures, such as those in shaking flasks, can also be used as pre-cultures for larger-scale cultures, such as inoculation into a fermenter.

[0035] A production strain is defined as a microbial strain suitable for producing a product, for example, through fermentation. Production strains are distinguished by the fact that they enable (improved) production of the product as a result of genetic modification. Genetic modification may result from genome modification (chromosome modification), from the introduction of autonomously replicating extrachromosomal genetic elements such as plasmids, or from a combination of chromosomal and extrachromosomal modifications. An example of a chromosomally modified production strain is the E. coli W3110-ΔserB strain described in Example 1 for the production of OPS. An example of a production strain produced by plasmid introduction is the E. coli JM105×pCSma-pKKj strain described in Example 3 for the production of CS enzyme. A microbial strain possessing extrachromosomal genetic elements is called a host strain or host organism, and the extrachromosomal genetic elements are called gene constructs, plasmids, vectors, or expression vectors.

[0036] An open reading frame (ORF, also known as a cds or coding sequence) is a region of DNA or RNA that codes for the amino acid sequence of a protein, beginning with a start codon and ending with a stop codon. ORFs are also called coding regions or structural genes.

[0037] A gene is a portion of DNA that contains all the basic information necessary to produce biologically active RNA. A gene includes the portion of DNA from which a single-stranded RNA copy is produced through transcription, and expression signals involved in regulating this copying process. Expression signals include, for example, at least one promoter, transcription start site, translation start site, 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 instructions for assembling specific proteins within the cell. mRNA molecules transmit the message necessary for protein synthesis from genetic information (DNA) to ribosomes, which are responsible for protein synthesis. Within the cell, mRNA molecules are formed as transcripts of DNA portions corresponding to genes. The genetic information stored in DNA does not change through this process.

[0039] Eukaryotic genes, primarily known as mosaic genes, differ from prokaryotic genes in that they also contain non-coding regions called introns (intra-gene regions). Exons (expression regions), which are coding sequences, are parts of the DNA of eukaryotic genes. After being transcribed into RNA, they are translated into the amino acid sequence of a protein by ribosomes. After transcription from DNA to RNA, introns are spliced ​​from the primary transcript. Protein-coding RNA that does not contain 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, the prokaryote does not process the exon / intron structure. Therefore, the protein sequence or the coding region of mature mRNA must be back-translated into intron-free DNA. In the context of this invention, when referring to a gene sequence derived from a protein sequence or a gene sequence derived from mRNA, this back-translation process is precisely what is meant. Sequence optimization, i.e., adaptation to the codon utilization of the corresponding prokaryote (codon optimization), is preferably performed simultaneously with the back translation of the protein sequence or mRNA sequence into the DNA sequence.

[0040] A gene construct refers to a DNA molecule in which a gene is linked to other genetic elements (e.g., promoters, terminators, selection markers, origins of replication). In the context of this invention, a gene construct is a circular DNA molecule, also known as a plasmid, vector, or expression vector. The genetic elements of a gene construct induce extrachromosomal inheritance during cell growth, producing the protein encoded by the gene.

[0041] The abbreviation WT (Wt) refers to the wild type. A wild-type gene is a gene that arises naturally during the process of evolution and is present in the wild-type genome. The DNA sequences of Wt genes are publicly available in databases such as the NCBI (National Center for Biotechnology Information) database. Microbial strains that possess a Wt genome are called Wt strains.

[0042] L-cysteic acid obtained from the bioconversion of OPS using sulfites according to the present invention can be used directly without further post-processing 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, and crystallization.

[0043] Preferably, the method of the present invention is characterized by further use of the reaction batch containing L-cysteic acid without further workup, purification, or isolation steps.

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

[0045] In the context of this invention, denaturation refers to a structural change in molecules such as proteins, and specifically refers to a structural change that results in the loss of biological function in the molecule, even though its primary structure remains unchanged.

[0046] Denatured proteins are characterized by being enzymatically inactive, that is, in the context of the present invention, the CS enzyme, by lacking the ability to catalyze the synthesis of L-cysteic acid from OPS and sulfites. Denaturation can result from physical or chemical influences. Enzymatically inactive proteins that are not properly folded or are incompletely folded may accumulate in cells as protein aggregates (known as inclusions), which can be considered spontaneously denatured proteins. Inclusions are observed particularly at high expression levels, and the resulting high concentrations of newly synthesized protein chains imply that aggregation is preferred over folding into an enzymatically active three-dimensional form. It is unpredictable whether heterologously expressed proteins will occur in the form of insoluble inclusions or in an active form, and depends not only on the primary structure of the protein chain (sequence of amino acid sequences) but also on the expression system and parameters used (e.g., growth temperature, induction intensity of inductive promoters) that can control the rate of protein biosynthesis. However, in the case of the present invention, it is surprising that cysteate synthase derived from Metanosalkina acetylborans was produced in an enzymatically active form in Escherichia coli, because, according to prior art (Graham et al., 2009, see above), the protein was only produced in an inactive form in inclusion bodies heterologously expressed in Escherichia coli.

[0047] Regeneration refers to the reverse transformation of denatured proteins into biologically active spatial structures. In protein biochemistry, regeneration involves using chaotropic compounds to return denatured proteins in inclusion bodies to solution, and then removing the chaotropic compounds to regenerate the proteins. Urea and guanidine hydrochloride are particularly 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 regeneration step.

[0049] Regeneration processThe method includes the following steps: dissolving the denatured protein in a culture medium containing a chaotropic compound, and then removing the chaotropic compound. The extent to which the chaotropic compound is removed depends on the specific protein. The chaotropic compound can be removed, for example, by dialysis, by selective binding of the chaotropic compound to a supporting material, by selective binding of the regenerated protein to a supporting material and subsequent elution under regeneration conditions, or by diluting the chaotropic compound to a critical concentration below which it does not cause denaturation (see, e.g., Graham et al., 2009, above). Methods for protein regeneration are described in the prior art. Regeneration conditions include, for example, dissolving the denatured protein in a 6M aqueous urea solution or a 6M aqueous guanidine hydrochloride solution.

[0050] Diluting or removing chaotropic compounds below critical concentrations depends on the specific protein and means reducing the concentration of the chaotropic compound, such as urea or guanidine, to a level below which the target protein can revert to its three-dimensional structure of its active form.

[0051] Chaotropic compounds are chemical substances that disrupt the regular hydrogen bonding in water. Examples of chaotropic compounds include barium salts such as barium chloride or barium acetate, thiocyanates such as guanidine hydrochloride and guanidine thiocyanate, perchlorates, iodides, butanol, phenols, thiourea, urea, and / or surfactants.

[0052] Surfactants (also called detergents or soaps) are organic compounds that act as surfactants; that is, due to their structure, surfactants are positioned between two phase interfaces to reduce surface tension, and as a result, allow wetting, for example. By reducing surface tension, surfactants promote the mixing of two phases, sometimes leading to the formation of an emulsion. Surfactants are, Surfactants are distinguished by the polarity of their molecular structure; part of the molecule is hydrophilic, mediating solubility in water, while the other part is hydrophobic. Surfactants solubilize hydrophobic compounds, contributing to their solubilization in water. The surfactants used include 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, sulfoacetates), cationic surfactants based on quaternary ammonium compounds (distearyldimethylammonium chloride, "Esterquat"), and / or zwitterionic (amphoteric) surfactants based on betaine (e.g., "cocoamidopropyl betaine") or sulfobetaine (e.g., cocamidopropyl hydroxysultaine).

[0053] In preferred embodiments, the method of the present invention is characterized in that the CS enzyme is used in the reaction without a prior regeneration step. Working without a regeneration step is extremely economically advantageous because it eliminates the need for the complex, costly, and environmentally harmful regeneration steps described above. In the case of CS enzyme production by proliferation of production strains, particularly when the CS enzyme is produced by fermentation, the host cells expressing the CS enzyme do not need to be mechanically or chemically destroyed first in a complex, costly, and environmentally harmful method, for example, due to waste. Furthermore, in the case of inclusion bodies where the enzyme is concentrated in the inactive form described in Graham et al. (2009), there is no need to isolate and regenerate the protein from the cell lysate so that it can be used in the enzymatic reaction.

[0054] The present invention's method for producing L-cysteic acid by biotransformation requires the availability of OPS. OPS can be produced chemically or biotechnically, for example, by fermentation of an OPS-producing strain. Possible methods for the chemical production of OPS include, for example, phosphorylation of L-serine, or production of a directly usable racemic O-phospho-D / L-serine, or OPS may be obtained in advance from a racemic mixture, for example, by degradation.

[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 proliferation of an OPS-producing strain. Particularly preferred is the biotechnological production of OPS by the proliferation of an OPS-producing strain, in which OPS accumulates in the cell culture supernatant (extracellularly).

[0056] Those skilled in the art can determine, using isotope analysis, whether a substance such as OPS intended for use as a reactant in the method of the present invention originates from chemical production or from biotechnological production, such as fermentation. Distinguishing isotope analysis methods are described, for example, in Sieper et al., Rapid Commun. Mass Spectrom. (2006) 20: 2521-2527, and are based on determining different isotopic ratios, such as carbon or nitrogen isotope ratios, depending on whether the product originates from chemical production (petroleum-based) or from biotechnological production, such as fermentation (plant-based). A method for producing OPS is, for example, a biotechnological production method, such as fermentation (plant-based), if the glucose used for propagating the production strain originates from plant-based production, and this is also applicable to the method described in Example 2.

[0057] For example, in the cysteine ​​metabolism of E. coli, OPS acts as a precursor for the biosynthesis of L-serine. The latter is formed by the dephosphorylation of OPS. This reaction is enzymatically catalyzed by O-phospho-L-serine phosphatase (SerB, EC3.1.3.3). From conventional technology, it is known that E. coli strains with suppressed SerB activity can accumulate OPS. Therefore, microbial strains with suppressed SerB activity cannot produce L-serine by the 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. In this case, the microbial strain with suppressed SerB activity is used as the OPS production strain, and this suppression of SerB activity includes genetic modification of the microbial strain. The SerB activity of an unmodified microbial strain (Wt strain) is set to 100%, and the microbial strain with suppressed SerB activity is defined as having lower SerB activity compared to the 100% activity of the Wt strain, preferably 20% or less, particularly preferably 10% or less, and particularly preferably 0% (inactivation of the SerB gene) of the wild-type strain, with the wild-type strain set to 100% activity. The SerB activity that is still measurable in the modified microbial strain and expressed as a percentage based on the activity in the Wt strain is called residual activity.

[0059] Microbial strains with suppressed SerB activity are characterized by one or more of the following genetic modifications: • Deletion of the chromosomal gene encoding the enzyme SerB. • Introduction of mutations into the chromosomal gene encoding the enzyme SerB to reduce the activity of the endogenous gene. • Replacement of the chromosomal gene encoding the enzyme SerB with a mutated gene to reduce the activity of the endogenous gene. • Introduction of mutations into the regulatory region of the gene encoding the enzyme SerB to reduce endogenous enzyme activity. • Introduction of an antisense oligonucleotide complementary to the transcript of the gene encoding the enzyme SerB to inhibit mRNA translation.

[0060] As described in the prior art, 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 photoquantitatively determined at 340 nm as a molybdate complex. The SerB activity determined for the WT strain using this enzyme assay method is defined as 100% activity, and the residual activity of microbial strains with suppressed SerB activity is measured under the same conditions.

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

[0062] Microbial strains having suppressed SerB activity are preferably selected from the families Corynebacteriaceae and Enterobacteriaceae, particularly preferably from the genera Corynebacterium, Pantoea, and Escherichia, and especially preferably from the species Pantoea ananatis and 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 characterized by serine nutritional requirements, i.e., the strain itself is unable to form the amino acid L-serine for growth. Nutritional requirements can be overcome by adding serine or glycine to the culture medium (growth medium), which in each case can be added as a pure substance, as a component of a complex medium such as yeast extract, peptone, or tryptone, or as a mixture of a pure substance and a complex medium component. A mixture of a pure substance selected from glycine and L-serine and a complex medium component is preferred, and a mixture of glycine and a complex medium component is particularly preferred. Adding glycine to the growth medium is particularly preferred.

[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 especially preferably 0.3 g / L to 2 g / L.

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

[0066] Therefore, the regions in the serB gene that are susceptible to homologous recombination may include not only the region encoding O-phospho-L-serine phosphatase but also regions that are not. These susceptible regions may also include DNA sequences adjacent to the serB gene, namely the 5' region before the start of the coding region (the gene transcription promoter) and the 3' region after the end of the coding region (the gene transcription terminator). Modifications to these regions by homologous recombination can lead to inactivation of the serB gene, similar to modifications of the coding region.

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

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

[0069] Therefore, a DNA construct for inactivating the serB gene may consist of a sequence homologous to the serB gene, starting at the 5' end, followed by an expression cassette of a selection marker chosen from, for example, a class of antibiotic resistance genes, followed by further sequences homologous to the serB gene.

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

[0071] A selection marker gene is generally a gene whose gene product enables the propagation of a parent plant under selective conditions where the original parent plant cannot propagate.

[0072] Preferred selection marker genes are selected from a group of antibiotic resistance genes, such as ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, chloramphenicol resistance genes, or neomycin resistance genes. Other preferred selection marker genes enable parental strains with metabolic deficiencies (e.g., amino acid nutritional requirements) to grow under selective conditions as a result of the correction of these deficiencies through the expression of the selection marker gene. Finally, another possibility is a selection marker gene for a gene product that chemically alters a compound that is inherently toxic to the parental strain, thereby inactivating the compound (e.g., a gene for the acetamidase enzyme that breaks down acetamide, a compound toxic to many microorganisms, into the harmless products acetic acid and ammonia).

[0073] Among the selected marker genes, ampicillin resistance genes, tetracycline resistance genes, kanamycin resistance genes, and chloramphenicol resistance genes are particularly preferred. Tetracycline resistance genes and kanamycin resistance genes are especially preferred.

[0074] Homologous recombination-based systems also exist, offering the option of removing selection markers from the genome, in addition to inactivating target genes, thereby enabling the production of double and multiple mutants. One such system is the so-called Lambda Red technology, commercially available as the "Quick and Easy E. coli Gene Deletion Kit" based on Gene Bridges GmbH's Red® / ET® technology (see "Technical Protocol, Quick & Easy E. coli Gene Deletion Kit, by Red® / ET® Recombination, Cat. No. K006, Version 2.3, June 2012" and the cited literature, e.g., Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645).

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

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

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

[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 strain of Enterobacteriaceae microorganism that heterologously expresses the CS enzyme.

[0080] This microbial strain, also known as the 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 species Escherichia, and especially preferably Escherichia coli K12 JM105, which heterologously expresses the CS enzyme, and very preferably in an enzymatically active form.

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

[0083] Cysteic acid synthase is known, for example, as coenzyme M biosynthesis by the class Methanobacteria. Preferably, a method for producing L-cysteic acid is characterized in that the CS enzyme is derived from Methanosarkina acetylborans or has a sequence homologous thereto, and particularly preferably is the CS enzyme derived from Methanosarkina acetylborans. Particularly preferably, the coding DNA sequence is Sequence ID No. 3, which codes for a protein having the amino acid sequence Sequence 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 gene sequences derived from Methanocella paludicola (NCBI Gene ID: 8682885), Methanosarcina barkeri (NCBI Gene ID: 24822660), Methanoculleus marisnigri (NCBI Gene ID: 4845938), or nucleotide sequences homologous to these.

[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 aligning two or more nucleotide sequences are 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, the method of the present invention is characterized in that the CS enzyme has the amino acid sequence defined in SEQ ID NO: 4 or an amino acid sequence homologous thereto, wherein the amino acid sequence homologous to SEQ ID NO: 4 has at least 50%, preferably at least 70%, and particularly preferably at least 80% sequence identity with respect to SEQ ID NO: 4, and simultaneously has cysteate synthase activity. Cysteate synthase activity can be detected in a CS activity assay as defined above. The amino acid sequence of a homologous CS enzyme can be found in the NCBI database (National Center for Biotechnology Information) using the search term "cysteate synthase," or by entering the amino acid sequence SEQ ID NO: 4 into the auxiliary program "Protein BLAST." Enzymes homologous to the CS enzyme derived from Metanosarquina acetylborans are preferably selected from Metanosarquina paldicola (NCBI number: WP_012900738.1), Metanolinea mesophila (NCBI number: WP_245249687.1), Metanosarcinia barkeri (NCBI number: WP_011308449.1), and Metanocreus marisnigri (NCBI number: WP_011842967.1).

[0087] Searching the genes of CS enzymes using "Protein BLAST" reveals homologous protein sequences in numerous bacteria originating from the archaeal domain, including thermophilic organisms (growing at temperatures above 50°C to 110°C). These enzymes also typically exhibit optimal activity above 50°C. This invention also encompasses CS enzymes derived from the archaeal domain that exhibit optimal activity above 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 aligning two or more protein sequences are 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 part of a protein is intraframe fused in the laboratory with one or more DNA sequences encoding a further protein or part of a further protein, resulting in the encoding of a modified (extended) protein that does not exist in nature. The fusion DNA sequence can be joined at the 5' end, the 3' end, or at both ends of the 5' and 3' ends. It is also conceivable to insert the fusion DNA sequence within a protein-coding sequence (e.g., as a link between two domains of a protein). Fusion proteins are also called hybrids or hybrid enzymes. This means that the protein of the present invention having cysteate synthase activity is encoded only by the cds of the gene in question, and the CS cds are not extended by any further sequences added to the CS cds. Proteins having a coding region fused with a nucleotide sequence that encodes a protein sequence that is cleaved again during protein biosynthesis or post-translational modification, such as an export signal sequence that mediates protein secretion, are not included in the term “fusion protein.” In the context of the present invention, the term “fusion protein” always refers to a mature protein.

[0090] In the context of the present invention, "production of CS enzyme by proliferation of a CS enzyme-producing strain" refers to the production of CS enzyme as an enzymatically active protein without refolding, and preferably without relying on expression as a fusion protein. Particularly preferred is the production of CS enzyme from Metanosalkina acetylborans by proliferation of a CS enzyme-producing strain that is 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 Escherichia coli. Particularly preferred is the production of CS enzyme from Metanosalkina acetylborans by proliferation of the Escherichia coli production strain JM105×pCSma-pKKj described in Example 3.

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

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

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

[0094] In a preferred embodiment, the CS enzyme is produced as a cell homogenate following the proliferation of the CS enzyme-producing strain, and this 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, all conceivable sulfites, including the known salts Na2SO3, K2SO3, (NH4)2SO3, NaHSO3 (or its Na2S2O5 anhydride), or KHSO3, are suitable for the reaction in the method for producing L-cysteic acid. It is also conceivable to use gaseous sulfur dioxide, the anhydride of sulfurous acid, which can be introduced into the reaction batch, where it is hydrated with H2SO3 of sulfurous acid and, depending on the pH, deprotonated HSO3. - and SO3 2- And it reaches a state of equilibrium.

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

[0097] According to equation (1), the reaction of OPS to form L-cysteic acid releases a stoichiometric amount of phosphate, which can lower the pH in the batch as the reaction progresses. Since excessively low pH affects the activity of the CS enzyme, it is necessary to prevent the pH from dropping too low. This can be done passively with an appropriate high-concentration buffer in the batch, or actively by a measurement and control unit.

[0098] If the pH deviates from the target value, it is preferable to restore the desired pH by quantitatively adding an alkaline solution or acid (the so-called pH stat method), or to use 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 preferred, a reaction temperature of 15°C to 50°C is particularly preferred, and a reaction temperature of 20°C to 40°C is especially preferred.

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

[0101] The solvent used in the production method of 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 or continuous operation. In a discontinuous operation (batch operation), all reactants are added to the batch during the reaction, and the batch is post-processed after the reaction is complete. In a continuous operation, the CS enzyme is introduced as the stationary phase (for example, immobilized on a membrane reactor or support), and the substrate OPS and sulfite are metered and supplied as the mobile phase. The contact time between the mobile phase and the stationary phase is set so that the substrate OPS reacts completely 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 to be at least equimolar relative to the OPS, preferably at least 1.5 times molar excess, particularly preferably at least 2 times molar excess, and especially preferably at least 5 times molar excess.

[0104] The OPS concentration 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 method for producing 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 especially preferably at least 90%.

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

[0107] Surprisingly, contrary to conventional techniques, the CS enzyme heterologously recombinantly produced in the CS enzyme-producing strain was found to be enzymatically active in cell homogenates without refolding (regeneration), preferably without relying on a fusion partner, and suitable for the efficient production of L-cysteic acid in previously unknown in vivo conversions. For this purpose, OPS produced from the cell culture supernatant of an OPS-producing strain in which SerB enzyme activity is preferably suppressed, or commercially available OPS, can be reacted with sulfite according to formula (1).

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

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

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

[0111] The decarboxylation of L-cysteic acid to taurine can be carried out chemically or under enzymatic catalysts in biotransformation. Thermal decarboxylation at high temperatures under metal catalysts is known, but it is not considered sustainable and has the disadvantages of consuming a large amount of energy and producing a high proportion of byproducts. Preferably, the decarboxylation reaction is performed by enzymatic decarboxylation of L-cysteic acid to taurine in biotransformation.

[0112] To produce taurine by enzymatic decarboxylation, 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-processing steps. However, prior to enzymatic decarboxylation, it is also possible to remove particulate biomass from the reaction batch, for example by centrifugation, or to isolate the L-cysteic acid from the reaction batch beforehand.

[0113] It is preferable to use the L-cysteic acid-containing reaction batch directly, or to use the L-cysteic acid-containing reaction batch after removing particulate biomass.

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

[0115] For the enzymatic catalysis of L-cysteic acid to taurine according to formula (2), enzymes from the classes of L-cysteine ​​sulfinate decarboxylase (CSAD, EC 4.1.1.29), aspartate-1-decarboxylase (EC 4.1.1.11), or glutamate decarboxylase (EC 4.1.1.15) are preferred.

[0116] Preferably, the method for producing taurine is characterized in that decarboxylation is carried out by cysteinesulfinate decarboxylase (CSAD enzyme), which belongs to enzyme class EC4.1.1.29. CSAD enzyme is known to decarboxylate L-cysteinesulfinate to hypotaurine according to formula (3). (3) L-cysteine ​​sulfate -> hypotaurine + CO2

[0117] To varying degrees, these enzymes can also decarboxylate L-cysteic acid to taurine as a substrate. In particular, as shown in Examples 8 and 9, the CSADcc enzyme derived from Cyprinus carpio, for example, is suitable for decarboxylating L-cysteic acid to taurine according to formula (2).

[0118] CSAD enzymes are mainly found in metazoans (multicellular animals), including mammals such as humans (Homo sapiens), cattle (Bos taurus), rats (Rattus norvegicus), and mice (Mus musculus), but are also found in fish such as Cyprinus carpio. Enzymes with CSAD activity are also found in algae, such as single-celled organisms of the genus Synechococcus, as well as bacteria and fungi.

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

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

[0121] Particularly 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 from the NCBI database under GenBank sequence ID: AB220585.1 (code region: nt82~1584). From the corresponding amino acid sequence, it is preferable to derive a codon-optimized CSADcc coding region DNA sequence for expression in a particularly selected microorganism (e.g., E. coli) (for example, in E. coli, it is defined as nt31~1530 of SEQ ID NO: 5, which codes for a protein having the amino acid sequence of SEQ ID NO: 6). For codon optimization, commonly available software programs such as the Eurofins Genomics GENEius software used in Example 7 can be used.

[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, and more preferably CSADcc, is recombinantly produced by a microbial production strain. Recombinant production of the CSAD enzyme in an E. coli production strain is disclosed, for example, in Example 7. CSAD cds are cloned into an expression vector, e.g., vector pKKj (see Example 3), in a well-known manner, and a gene construct, e.g., pCSADcc-pKKj (Figure 4), is produced. The production strain is produced, similarly in a well-known manner, by transforming a gene construct containing CSAD cds, e.g., pCSADcc-pKKj, into a microbial host strain, e.g., E. coli strain JM105, and using the resulting production strain, e.g., E. coli JM105 × pCSADcc-pKKj, to produce the CSAD enzyme in a similarly well-known manner. The CSAD enzyme can be produced on a shaking flask scale for laboratory purposes (e.g., as described in Example 7) or by fermentation in a well-known manner.

[0124] The CSAD enzyme contains pyridoxal phosphate (PLP, CAS number 54-47-7) as a cofactor. Therefore, supplementing growth media or in vivo conversion batches for 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), is a suitable alternative for method improvement.

[0125] CSAD enzyme, preferably CSADcc, obtained by growth or fermentation in a shaking flask can be used as a culture broth without further post-processing, or as a cell suspension after re-isolation of cells, for example, by centrifugation. Furthermore, CSAD enzyme, preferably CSADcc, can be used in the form of cell homogenates after mechanical disruption of the cell suspension, or in the form of chemically permeable cells (e.g., by chloroform), or as a cell extract after removing particulate components from the cell homogenates, or as an enzyme purified, for example, by chromatography. It is preferable to use CSAD enzyme as a cell suspension after re-isolation of cells from the culture broth, as described, for example, in Examples 7 and 8.

[0126] The bioconversion of L-cysteic acid to taurine by the CSAD enzyme is carried out under pH and temperature conditions that allow for efficient decarboxylation from L-cysteic acid to taurine. The pH range for bioconversion is preferably 5.0 to 9.0, and the temperature range is preferably 20°C to 70°C.

[0127] The bioconversion of L-cysteic acid to produce taurine can be carried out in a discontinuous or continuous operation. In a discontinuous operation (batch operation), all reactants are added to the batch during the reaction, and the batch is post-processed after the reaction is complete. In a continuous operation, the CSAD enzyme is introduced as the stationary phase (e.g., immobilized on a membrane reactor or support), and the substrate L-cysteic acid is metered and supplied as the mobile phase. The contact time between the mobile phase and the stationary phase is set so that the substrate L-cysteic acid reacts completely to form the product taurine. The discontinuous (batch) operation is preferred.

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

[0129] Preferably, a 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 especially preferably at least 95%.

[0130] Preferably, the method steps for L-cysteic acid production (in vivo conversion 1) and taurine production proceed sequentially, i.e., one after the other. 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 steps of the method are performed in a single reaction batch, this method is also called the one-pot method or one-pot reaction.

[0132] Example 9 of the present invention discloses a method for carrying out such a one-pot reaction, in which the bioconversion of OPS to L-cysteic acid according to formula (1) and the bioconversion of L-cysteic acid to taurine according to formula (2) are carried out simultaneously, i.e., in a single reaction batch, in which OPS reacts with sulfite (salt of sulfite) in the presence of CS enzyme and CSAD enzyme. L-cysteic acid is formed in the first reaction and decarboxylated to taurine "in situ" 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 adding a sufficient amount of CSAD enzyme, L-cysteic acid can be quantitatively converted to taurine. It is preferable that the OPS used is converted to L-cysteic acid and taurine, and that the total molar yield of L-cysteic acid and taurine is 60% or more, particularly preferably 70% or more, and especially preferably 80% or more.

[0133] The ability to produce L-cysteic acid and perform the decarboxylation process to taurine in a single reaction batch is particularly interesting from an economic feasibility standpoint.

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

[0135] For taurine production, in vivo conversion is preferred, in which the constituent enzymes OPS, CS enzyme, and CSAD enzyme are produced individually.

[0136] Taurine can be used directly without further post-processing 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 a method for producing taurine.

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

[0139] The present invention will be further illustrated by the following embodiments, but will not be limited thereto. [Examples]

[0140] Example 1: Production of serB deletion mutants in Escherichia coli The strain used was Escherichia coli K12 W3110 (commercially available from DSMZ: Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures] under strain number DSM 5911). The target of gene inactivation was the coding sequence of the serB gene derived from E. coli. The DNA sequence of the coding region of the serB gene derived from E. coli K12 (sequence number 1, nt67~nt1032), which encodes a protein with the amino acid sequence of sequence number 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 Gene Bridges GmbH's Red® / ET® technology, as detailed below (see the user manual for 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 cited literature, e.g., Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97 (2000): 6640-6645). Plasmids pKD13, pKD46, and pCP20 were used for this purpose: The 3.4kb plasmid pKD13 (Figure 1) is disclosed in the gene database "GenBank" under accession number AY048744.1. The 6.3kb plasmid pKD46 (Figure 2) is disclosed in the gene database "GenBank" under accession number AY048746.1. The 9.4kb plasmid pCP20 is disclosed in Cherepanov and Wackernagel, Gene 158 (1995): 9-14.

[0142] To inactivate the serB gene of *E. coli* W3110 by homologous recombination using the Lambda Red system, the following steps were performed:

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

[0144] 2. PCR reactions using plasmid pKD13 (Figure 1) DNA and primers serb-1f (SEQ ID NO: 7) and serb-2r (SEQ ID NO: 8) ("Phusion® High-Fidelity" DNA polymerase, Thermo Scientific®) produced serB-specific DNA fragments suitable for inactivation. The PCR reaction formed a 1.4kb PCR product containing 30nt DNA segments specific to the serB gene derived from Escherichia coli (E. coli) W3110 at both the 5' and 3' ends. Furthermore, this PCR product contained an expression cassette of the kanamycin resistance gene contained in pKD13, and short segments of DNA adjacent to the 5' and 3' ends of the kanamycin expression cassette (so-called "FRT direct repeats" (referred to as "FRT1" and "FRT2" in Figure 1)) that are used as recognition sequences for "FLP recombinase" (contained in plasmid pCP20) in a later post-processing step to remove the antibiotic marker kanamycin.

[0145] The 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] The 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] 3. To remove residual pKD13 plasmid DNA, a 1.4 kb PCR product was isolated and treated with Dpn I, a restriction endonuclease familiar to those skilled in the art that cleaves only methylated DNA. Unmethylated DNA from the PCR reaction is not degraded.

[0148] 4. A 1.4kb PCR product containing an expression cassette specific to the serB gene and containing a kanamycin resistance 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 GIBCO® tryptone, 5 g / L BD Biosciences yeast extract, 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 on an LB kanamycin-glycine plate (i.e., isolated by singularization), and it was determined in a PCR reaction whether the kanamycin-resistant cassette had been correctly incorporated into the serB gene.

[0150] The genomic DNA used in the PCR reaction ("Phusion® High-Fidelity" DNA Polymerase, Thermo Scientific®) was isolated from cells of kanamycin-resistant Escherichia coli (E. coli) W3110×pKD46 grown 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 from wild-type E. coli (E. coli) W3110 was used as a control. The primers used in the PCR reaction were serb-3f (SEQ ID NO: 9, 5' flanking the serB gene, nt1~22 of SEQ ID NO: 1) and serb-4r (SEQ ID NO: 10, 3' flanking the serB gene, nt1066~1085 of SEQ ID NO: 1, reverse complementary morphology).

[0151] Wild-type E. coli (W3110) DNA yielded a 1.1 kb DNA fragment via PCR, as expected for an intact gene. On the other hand, the four kanamycin-resistant clones studied yielded approximately 1.6 kb DNA fragments via PCR, as expected if the 1.4 kb PCR product had been integrated into the serB gene at the sites defined by primers serb-1f and serb-2r. This result indicated that the kanamycin resistance gene had been successfully integrated into the serB gene locus, resulting in the inactivation of the serB gene. Clones with the inactivated serB gene were selected and treated at 42°C to remove the temperature-sensitive plasmid pKD46, thereby restoring the strain to ampicillin sensitivity. This strain was designated W3110-ΔserB::kan.

[0152] 6. To remove the kanamycin selection marker, W3110-ΔserB::kan was transformed with plasmid pCP20, and transformants were selected at 30°C. The 9.4kb vector pCP20 is disclosed in Cherepanov and Wackernagel (1995), Gene 158: 9-14. The FLP recombinase gene is present on vector pCP20. FLP recombinase recognizes the FRT sequence adjacent to the kanamycin resistance gene expression cassette, causing the removal of the kanamycin expression cassette. For this reason, clones obtained at 30°C were incubated at 37°C. Under these conditions, FLP recombinase expression was induced, and replication of the pCP20 vector was suppressed.

[0153] As a result of this process, clones were obtained in which the serB gene was inactivated and sensitivity to kanamycin was restored (so-called "hardening" 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 LB-glycine and LB-kanamycin-glycine on plates: Growth was positive on the LB-glycine plate, but no growth was observed on the LB-kanamycin-glycine plate. This indicates that the kanamycin cassette was successfully removed from the genome.

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

[0157] The strain isolated in this process was named *E. coli* W3110-ΔserB. This strain is distinguished by the presence of an inactivated serB gene and by its re-establishment of sensitivity to the antibiotic kanamycin.

[0158] Example 2: Production of OPS Production in a shaking flask: OPS was produced by growing E. coli (E. coli) strain W3110-ΔserB in a shaking flask. For comparison, OPS production in the wild-type strain E. coli (E. coli) W3110 was analyzed. As a pre-culture for shaking flask culture, E. coli (E. coli) strain W3110 and E. coli (E. coli) W3110-ΔserB strain were 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), respectively, and incubated at 30°C and 135 rpm in a shaker for 16 hours.

[0159] Main culture: Subsequently, a portion of each pre-culture solution was transferred to a 300 mL Erlenmeyer flask (with baffles) containing 30 mL of SM1 medium, which contained 15 g / L glucose, 5 mg / L vitamin B1 (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 Sigma-Aldrich).

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

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

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

[0163] Samples were collected after 24 hours, and the cell density (OD) was measured in both the culture supernatant and the cell pellet. 600The OPS content was measured at a rate of 6,000 rpm for 3 × 20 seconds with 30-second intervals in each case. For this purpose, 2 mL of cell culture medium was centrifuged at 13,000 rpm for 5 minutes in each case (Heraeus® Fresco® 21 centrifuge). The cell culture supernatant was directly analyzed for OPS content by HPLC. Cell extracts were prepared by resuspending the cell pellet in 2 mL of H2O in each case. This was achieved using an MP Biomedicals FastPrep-24® 5G cell homogenizer. In each case, a 2 × 1 mL 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 with 30-second intervals in each case). In each case, the resulting cell homogenates were combined and centrifuged at 13,000 rpm for 5 minutes to prepare the cell extract. The OPS content of this cell extract was analyzed by PLC. The results are summarized in Table 1.

[0164] [Table 1]

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

[0166] Eluent A: 25 mM sodium phosphate, pH 6.0. Eluent B: Methanol. Separation was performed in gradient mode: 0-10 minutes with 1%-15% eluent B, followed by 15 minutes with 15% eluent B, at a flow rate of 1.0 mL / min. Retention time of L-cysteic acid: 6.95 minutes. OPS retention time: 7.65 minutes. Taurine retention time: 21.9 minutes.

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

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

[0169] Preculture 2: Subsequently, 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 was shaken at 150 rpm for 17 hours in a 1 L Erlenmeyer flask at 32°C (Infors incubator shaker). After this culture, the cell density OD was measured. 600 The concentration 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 capacity 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 NaC1, 0.225 g / L CaCl2×2H2O, 1.2 g / L MgSO4×7H2O, 0.075 g / L FeSO4×7H2O, 1 g / L sodium 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 shaking flask culture).

[0171] The pH of the fermenter was adjusted to 7.0 at the start by supplying a 25% NH4OH solution by pump. During fermentation, the pH was maintained at 7.0 by automatic correction using 25% NH4OH or 4M H3PO4. Foam control was achieved by automatic quantitative addition of 4% (v / v) Struktol J673 (Schill & Seilacher) in H2O. For inoculation, pre-culture 2 (60 mL) was supplied to the fermenter vessel by pump. Therefore, the initial volume was approximately 660 mL. The culture was first stirred at 400 rpm and aerated with compressed air sterilized by a sterile filter at an aeration rate of 2 vvm (air volume per minute of culture volume). 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 fell below the target value, an adjustment cascade was initiated to restore the O2 saturation to the target value. This involved 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 the initial 10 g / L to approximately 2 g / L, a 56% (w / w) glucose solution was continuously added. The supply rate was then 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 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 at 23, 30, 47, and 53 hours after the start of fermentation, and the cell density (OD) was measured from one aliquot. 600 The concentration was measured at / mL. In each case, additional aliquots were incubated at 80°C for 5 minutes, centrifuged, and the OPS content was measured by HPLC from the cell culture supernatant. The cell density and OPS content are summarized in Table 2.

[0175] [Table 2]

[0176] Example 3: Production of CSma enzyme Cysteate synthase (CSma) derived from Metanosalkina acetivorans was used. The amino acid sequence of the CSma enzyme is accessible from the NCBI database under accession ID WP_048066469. Using this amino acid sequence, a codon-optimized DNA sequence for expression in Escherichia coli (E. coli) was induced (using the commonly available Eurofins Genomics GENEius software), and this was synthetically produced (Eurofins Genomics). The synthetically produced DNA had the sequence disclosed in Sequence ID No. 3, which contained the coding region of the gene (hereinafter referred to as the CSma coding region (Sequence ID No. 3)) that had the amino acid sequence disclosed in Sequence ID No. 4 and encoded a protein called CSma. 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 DNA synthetically produced using EcoRI and HindIII, and then cloning it as an EcoRI / HindIII fragment into the pKKj vector, which was also cleaved using EcoRI and HindIII, using 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 under accession number M77749.1. Approximately 1.7kb (bp262~1947 of the DNA sequence disclosed in M77749.1) was removed from a 4.6kb plasmid to obtain a 2.9kb expression vector pKKj.

[0178] The vector pCSma-pKKj was transformed into Escherichia coli (E. coli) K12 JM105 strain in a known manner to express the CSma coding region in Escherichia coli (E. coli). The Escherichia coli (E. 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 (E. coli) JM105×pCSma - pKKj. The CSma coding region was expressed in Escherichia coli (E. 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 (E. 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;600 The OD reached 2.0. Subsequently, the inducing agent IPTG (Sigma-Aldrich, final concentration 0.4 mg) was added, and cell proliferation was continued for another 20 hours at 30°C and 140 rpm in a chest shaker (Infers). At the end of proliferation, the cell density OD was 600 The value was 3.1 / mL.

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

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

[0184] Example 4: In vivo conversion of commercial OPS and Na using CSma enzyme 2 SO 3 Production of L-cysteic acid from Two batches were run in parallel:

[0185] Batch 1: In a 100 mL Erlenmeyer flask, 8.15 mL of KPi7.0 buffer was first added, followed by 1 mL of 0.2 M Na2SO3 solution in KPi7.0 buffer, 0.5 mL of CSma cell homogenate from shaking flask culture (Example 3), and 350 μL of 0.2 M OPS (Sigma-Aldrich) solution in KPi7.0 buffer. The batch volume was 10 mL.

[0186] Batch 2: Batch 2 (a comparison batch without Na2SO3) contained the same composition as Batch 1. Instead of the Na2SO3 solution, 1 mL of KPi7.0 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 batches were 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: In vivo conversion of OPS-containing culture supernatant and Na from shaking flask culture using CSma enzyme 2 SO 3 Production of L-cysteic acid from First, 9 mL of cell culture supernatant from a shaking flask culture of Escherichia coli (E. coli) strain W3110-ΔserB (Example 2) with an OPS content of 113.6 mg / L was added to a 100 mL Erlenmeyer flask. Then, 0.3 mL of 3 M KC, 0.2 M Na2SO3 in 0.5 mL of KPi7.0 buffer, and 1 mL of CSma cell homogenate from a shaking flask culture (Example 3) were added. 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 in each case, centrifuged, and the supernatant was analyzed by HPLC for OPS and L-cysteic acid content. The reaction course 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 bioconversion of OPS at a constant pH. OPS substrate: 10 mL of fermentation broth derived from the fermentation of Escherichia coli (E. coli) strain W3110-ΔserB (Example 2) was centrifuged (15,000 rpm for 10 minutes, Sorvall RC5C centrifuge equipped with 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: A cell homogenate of Escherichia coli (E. coli) strain JM105×pCSma-pKKj was prepared from a 2×50 mL shaking flask culture as described in Example 3. 15 μL of 500 mg / L PLP was added to this homogenate. The total volume of the homogenate was 4 mL.

[0193] In vivo conversion batch: A 50 mL thermostatic double-walled, downward-tapered reaction vessel (an accessory for the Titrator TitroLine alpha (Schott)) was connected to a thermostat (Lauda) via 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 1 M Na2SO3 solution in KPi7.0 buffer. The batch volume was 10.6 mL. The OPS concentration in the batch was 17.1 mM (0.18 mmol of OPS in 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 of 7.0 throughout the entire reaction period by adding 0.5 M NaOH by metering from a burette connected to the control unit.

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

[0196] [Table 5]

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

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

[0199] Culture in a shaking flask: 10 mL of pre-culture solution (3.1 / mL OD) 600 The ) was used as inoculum for the main culture (1 L Erlenmeyer flask) in 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. 100 mL shaking flask culture (7.1 / mL OD 600 Cells were isolated from the sample by centrifugation, and the cell pellet was resuspended in 4 mL of KPi7.0 buffer. This cell suspension was used directly in in vivo conversion experiments.

[0200] Example 8: Production of taurine from L-cysteic acid by biotransformation First, 9 mL of batch 5 containing 84.5 mg / L of L-cysteic acid (Table 4) was added to a 100 mL Erlenmeyer flask, and then 1 mL of the CSADcc enzyme cell suspension from 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, a 1 mL 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 bioconversion First, a 9 mL batch from a shaking flask culture of Escherichia coli (E. coli) strain W3110-ΔserB (Example 2) with an OPS content of 94.7 mg / L was added to a 100 mL Erlenmeyer flask. Then, 0.5 mL of 0.2 M Na2SO3 solution in KPi7.0 buffer, 1 mL of CSma cell homogenate from the shaking flask culture (Example 3), and 1 mL of CSADcc enzyme cell suspension (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, a 1 mL batch was incubated at 80°C for 5 minutes, centrifuged, and the supernatant was analyzed by HPLC. The OPS used was completely consumed. Simultaneously, 34.4 mg / L of L-cysteic acid and 40.4 mg / L of taurine were formed.

[0202] Abbreviations used in the diagram: bla: Ampicillin resistance gene (β-lactamase) kanR: Kanamycin resistance gene ORI: origin of replication 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 P araB: Promoter of the araB gene Gam: Lambda phage Gam recombinant gene Bet: Lambda phage Bet recombinant gene Exo: Lambda phage Exo recombinant gene ORI101: Temperature-sensitive origin of replication RepA: The gene for plasmid replication protein A Ptac:tac Promotor EcoRI: Cleavage site of restriction enzyme EcoRI HindIII: Cleavage site of restriction enzyme HindIII CSma: The coding region of the cysteate synthase gene from Metanosalkina acetivorans (M. acetivorans). CSADcc: The coding region of the cysteine ​​sulfinate decarboxylase gene from Cyprinus carpio (C. carpio).

Claims

1. A method for producing L-cysteic acid, This includes the reaction of O-phospho-L-serine (OPS) with a sulfite salt (sulfite) and cysteate synthase (CS enzyme) belonging to enzyme class EC2.5.1.

76. The concentration of the OPS in the batch is at least 1 g / L, The aforementioned CS enzyme is produced in an enzymatically active form by the proliferation of the microbial strain E. coli K12 JM105. The above reaction is carried out under active pH control, The method wherein the CS enzyme has the amino acid sequence defined in SEQ ID NO: 4 or an amino acid sequence homologous thereto, the amino acid sequence homologous to SEQ ID NO: 4 has at least 80% sequence identity with respect to SEQ ID NO: 4, and has cysteate synthase activity.

2. The CS enzyme having at least 80% sequence identity with the amino acid sequence defined in Sequence ID No. 4 and having cysteate synthase activity is a CS enzyme derived from Metanoscarcia acetylans (Sequence ID No. 4), a CS enzyme derived from Metanoscarcia paldicola (NCBI number: WP_012900738.1), and Metanolinea mesophylla. The method according to claim 1, wherein the enzyme is at least one selected from the group consisting of a CS enzyme derived from Mesophylla (NCBI number: WP_245249687.1), a CS enzyme derived from Metanosarkina barkeri (NCBI number: WP_011308449.1), and a CS enzyme derived from Metanocreus marisnigri (NCBI number: WP_011842967.1).

3. The method according to claim 1, wherein the CS enzyme is used in the reaction without undergoing a prior regeneration step.

4. The method according to claim 1, wherein the OPS used in the reaction is produced by biotechnology.

5. The method according to claim 1, wherein the OPS used in the reaction is produced using a microbial strain in which the activity of the O-phospho-L-serine phosphatase (SerB enzyme) belonging to enzyme class EC3.1.3.3 is suppressed.

6. The method according to claim 1, wherein the molar yield of L-cysteic acid with respect to the molar amount of OPS used is at least 60%.

7. A method for producing taurine, wherein the L-cysteic acid is produced and decarboxylated by the method described in claim 1.

8. The method according to claim 7, wherein the decarboxylation is carried out by a cysteine ​​sulfinate decarboxylase (CSAD enzyme) belonging to enzyme class EC4.1.1.

29.

9. The method according to claim 8, wherein the amino acid sequence of the CSAD enzyme is sequence number 6.

10. The method according to claim 7, wherein the molar yield of taurine based on the molar amount of L-cysteic acid used is at least 60%.

11. The method according to claim 7, wherein all method steps are carried out in a single reaction batch (one-pot method).

12. Use of L-cysteic acid produced by the method of claim 1 for the production of taurine.

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