WO2023280382A1 - Verfahren zur enzymatischen oxidation von sulfinsäuren zu sulfonsäuren - Google Patents
Verfahren zur enzymatischen oxidation von sulfinsäuren zu sulfonsäuren Download PDFInfo
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- WO2023280382A1 WO2023280382A1 PCT/EP2021/068556 EP2021068556W WO2023280382A1 WO 2023280382 A1 WO2023280382 A1 WO 2023280382A1 EP 2021068556 W EP2021068556 W EP 2021068556W WO 2023280382 A1 WO2023280382 A1 WO 2023280382A1
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- WIPO (PCT)
- Prior art keywords
- acid
- hypotaurine
- taurine
- cysteine
- oxidase
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C303/00—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides
- C07C303/02—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of sulfonic acids or halides thereof
- C07C303/16—Preparation of esters or amides of sulfuric acids; Preparation of sulfonic acids or of their esters, halides, anhydrides or amides of sulfonic acids or halides thereof by oxidation of thiols, sulfides, hydropolysulfides, or polysulfides with formation of sulfo or halosulfonyl groups
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P11/00—Preparation of sulfur-containing organic compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03004—Glucose oxidase (1.1.3.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
- C12Y101/03013—Alcohol oxidase (1.1.3.13)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Definitions
- the invention relates to a method for the enzymatic oxidation of sulfinic acids of the formula H2N-CH(R)-CH2-SO2H to sulfonic acids of the formula H2N-CH(R)-CH2-SO3H using an enzyme selected from the class of H2O2-generating oxidases in the presence its substrate, in particular for the enzymatic oxidation of L-cysteine sulfinic acid to L-cysteic acid and of hypotaurine to taurine.
- the salts of the sulfinic acids are the sulfinates.
- Sulfonic acids are organic sulfur compounds with the general structure R-SO2-OH, where R is an organic radical.
- R is an organic radical.
- Their salts and esters with the general structure R-S020 or R1-SO2-O-R2 (Ri, R2 are each organic residues) are called sulfonates.
- the naturally occurring sulfinic acids include, for example, L-cysteine sulfinic acid and hypotaurine.
- Occurring sulfonic acids include, for example, L-cysteic acid and taurine.
- Taurine (2-aminoethanesulfonic acid, CAS number 107-35-7) is an aminosulfonic acid that occurs naturally in nature as a breakdown product of the amino acids cysteine and methionine. Taurine is of economic importance, is e.g. a component of energy drinks and is also used in pet food, e.g. for cats or in fish farming (Salze and Davis, Aquaculture (2015) 437: 215-229). However, health-promoting effects are also attributed to taurine (Ripps and Shen, Molecular Vision (2012) 18: 2673-2686).
- taurine is currently produced chemically.
- a process from Changshu Yudong Chemical Factory is known, which starts with ethylene and leads to taurine via ethyleneimine.
- biotechnological processes for the production of taurine are increasingly being investigated.
- taurine occurs almost exclusively in the animal kingdom, with only a few examples of its occurrence in plants, algae or bacteria.
- There are various biosynthetic pathways to taurine see e.g. the KEGG Pathway database: "Taurine and hypotaurine metabolism", including starting from L-cysteine.
- the most important synthetic steps leading from L-cysteine to taurine are given in equations (1) to (5) shown.
- L-cysteic acid -> taurine + CO2 In a first step, L-cysteine is oxidized to L-cysteine sulfinic acid (3-sulfinoalanine, CAS number 207121-48-0) by the enzyme cysteine dioxygenase (CDO, EC 1.13.11.20).
- Cysteine sulfinate decarboxylase (CSAD, EC 4.1.1.29) decarboxylates L-cysteine sulfinic acid to hypotaurine (2-aminoethanesulphinic acid, CAS number 300-84-5) and can decarboxylate L-cysteic acid to taurine in a similar reaction.
- hypotaurine is generally the main product. If taurine is to be the product, chemical processes for the oxidation of hypotaurine must be used.
- hypotaurine could be chemically oxidized to taurine in cell extracts for analytical purposes by treatment with H2O2. No method has been described how hypotaurine can be converted to taurine in a non-chemical step for further use.
- WO 17/213142 A1 (Ajinomoto) describes a taurine-producing strain which was obtained by heterologous expression of a cysteine dioxygenase and an L-cysteinesulfinic acid decarboxylase in an originally cysteine-producing strain.
- the main product was hypotaurine with a maximum of 450 mM yield, which could only be converted to taurine by subsequent chemical-alkaline treatment and only in low yield.
- EM01 uses the cofactors NADH or NADPH in stoichiometric amounts.
- NADH or NADPH used in stoichiometric amounts.
- the use of these commercially expensive cofactors makes technical use uneconomical.
- the object of the present invention was therefore to provide an economically favorable biotechnological process for oxidizing sulfinic acids to sulfonic acids, in particular L-cysteine sulfinic acid to L-cysteic acid and hypotaurine to taurine.
- the problem was solved by a process for the enzymatic oxidation of sulfinic acids of the formula H2N-CH(R)-CH2-SO2H to sulfonic acids of the formula H2N-CH(R)-CH2-SO3H using an enzyme selected from the class of H2O2-generating oxidases in the presence of their substrate.
- the process is preferably characterized in that the sulfinic acid is aminoalkylsulfinic acid, particularly preferably 2-aminoalkylsulfinic acid.
- the process is preferably characterized in that the sulfonic acid is aminoalkylsulfonic acid, particularly preferably 2-
- Aminoalkylsulfonic acid is.
- the sulfinic acid is particularly preferably aminoalkylsulfinic acid and the sulphonic acid is aminoalkylsulphonic acid.
- the radical R can be any and is preferably hydrogen, an organic, linear, branched, cyclic, saturated or unsaturated, aromatic or heteroaromatic radical, with or without substituents. This means that the radicals R can be substituted or unsubstituted.
- Saturated or unsaturated radicals with C 1 -C 4 -, particularly preferably C 1 -C 4 -alkyl, vinyl, in particular methyl or ethyl, in particular methyl, are preferably used deployed.
- the invention thus also relates to the enzymatic oxidation of L-cysteine sulfinic acid to L-cysteic acid and of hypotaurine to taurine as a process step in a biotechnological production of taurine.
- the substrate of the enzyme selected from the class of H 2 O 2 -generating oxidases means the substance that can be oxidized by the oxidase.
- the oxidase reacts with its oxidizable substrate according to formula (7).
- Suitable H 2 O 2 -generating oxidases can be found in the "KEGG Enzyme” database under the search term "oxidase” as a subset of the enzymes listed there. Of the large number of H2O2-generating oxidases, however, only those oxidases that use an inexpensive substrate to generate H2O2 by oxidizing it according to Equation (7) are of interest for process engineering use.
- H 2 0 2 -generating oxidases examples include glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase ( EC 1.1.3.10), L-lactate oxidase (EC 1.1.3.2), aryl alcohol oxidase (EC 1.1.3.7), galactose oxidase (EC 1.1.3.4), glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase ( EC 1.1.3.10), L-lactate oxidase (EC 1.1.3.2), aryl alcohol oxidase (EC 1.1.3.7), galactose oxidas
- H 2 0 2 - generating oxidases are glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase (EC 1.1.3.10), L-lactate oxidase (EC 1.1.3.4), glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), alcohol oxidase (EC 1.1.3.13), secondary alcohol oxidase (EC 1.1.3.18), pyranose oxidase (EC 1.1.3.10), L-lactate oxidase (EC
- the method according to the invention is preferably characterized in that the combination of H2O2-generating oxidase and its oxidizable substrate is selected from glucose oxidase/glucose,
- alcohol oxidase/methanol1 alcohol oxidase/ethanol
- secondary alcohol oxidase/isopropanol secondary alcohol oxidase/isopropanol
- L-lactate oxidase/lactate L-lactate oxidase/lactate
- the method is characterized in that the H 2 O 2 -generating oxidase is alcohol oxidase and a primary alcohol, particularly preferably methanol, is present as the substrate.
- a primary alcohol particularly preferably methanol
- the H 2 O 2 -generating oxidase is an enzyme of the alcohol oxidase class EC 1.1.3.13 of the KEGG database.
- Alcohols are compounds of the general formula R-OH with one or more hydroxy groups that do not have a functional group with a higher priority. A distinction is made between alcohols according to the number of C and H atoms on the C atom of the functional group to which the hydroxyl group is also attached. In the case of primary alcohols, two H atoms are bonded to this C atom in addition to one C atom, so that the general Formula RCH2OH results. In addition, a primary alcohol becomes an aldehyde through oxidation. For example, ethanol becomes the aldehyde ethanal by splitting off 2 H atoms.
- Preferred primary alcohols include methanol and ethanol, most preferably the primary alcohol is methanol.
- R is an alkyl, alkenyl or alkynyl radical, but not an aryl radical, an acyl radical or a heteroatom.
- the method is characterized in that the H2O2-generating oxidase is glucose oxidase and glucose is present as the substrate.
- the H 2 O 2 -generating oxidase is an enzyme of the class EC 1.1.3.4 of the KEGG database, referred to as glucose oxidase.
- a prerequisite for the technical implementation of the process is the availability of the H 2 O 2 -generating oxidase and an inexpensive oxidizable substrate.
- H 2 O 2 -generating oxidases can be produced, for example, by cultivating a suitable production strain which naturally produces the relevant oxidase (homologous production) or as a result of expression of a suitable recombinant gene construct in a host organism (heterologous production).
- suitable production strain which naturally produces the relevant oxidase
- heterologous production or as a result of expression of a suitable recombinant gene construct in a host organism (heterologous production).
- various H2O2-generating oxidases are commercially available, which can have a positive effect on the economics of the process.
- oxidases examples include alcohol oxidase AOX from the yeast Pichia pastoris (available, for example, from Sigma-Aldrich) or glucose oxidase GOX from the fungus Aspergillus niger (available, for example, from Sigma-Aldrich from homologous and heterologous production).
- glucose oxidase is the enzyme sold under the trade name Gluzyme® (Novozymes) for Applications eg in the baking industry. It is preferred that the tbC ⁇ -generating oxidase is produced by fermentation.
- the substrate of the H2O2-generating oxidase is decisive for the economics of the process.
- the substrate is a primary alcohol, preferably selected from methanol or ethanol.
- the AOX enzyme generates H2O2 with methanol after reaction (9):
- the substrate is D-glucose.
- the GOX enzyme generates H2O2 with D-glucose after reaction (10):
- a process within the meaning of the invention is defined as a multi-stage sequence of work steps where, in one or more consecutive reaction batches, a starting material is converted into the product via the intermediate products determined by the reaction conditions in a predefined order.
- a biotechnological process within the meaning of the invention is defined as the use of enzymes, cells or whole organisms in technical applications for the production of chemical compounds, such as the production of taurine from hypotaurine using a glucose oxidase.
- chemical processes there are processes that are characterized by chemical process steps.
- a batch or reaction batch is defined as a mixture of educt (starting material), enzyme and possibly other reactants, in which the educt is converted into a product under defined conditions.
- An approach or reaction approach of The present invention comprises at least one sulfinic acid of the formula H 2 N—CH(R)—CH 2 —SO 2 H, at least one enzyme selected from the class of fhCh-generating oxidases, the substrate that can be oxidized by the oxidase, and atmospheric oxygen O 2 .
- Biotransformation is defined as the conversion of an educt into the product under enzymatic catalysis.
- the method according to the invention is a biotransformation.
- the yield of the reaction can be given as a volume yield in absolute volume-related amount of product (mM or g/L) or as a relative yield of product in percent of the starting material used (taking into account the molecular weights of the starting material and the product), also called as percentage yield.
- the molar yield denotes the total molar amount of sulfonic acid of the formula H 2 N—CH(R)—CH 2 —SO 3 H after enzymatic oxidation by an H 2 O 2 -generating oxidase in the presence their substrate in relation to the sum of the molar amounts of sulfinic acid of the formula H 2 N-CH (R)-CH 2 -SO 2 H and sulfonic acid of the formula H 2 N-CH (R)-CH 2 -S0 3 used in this reaction H.
- Fermentation is a process step for the production of cell cultures in which a microbial production strain is made to grow under defined conditions of culture medium, temperature, pH, oxygen supply and medium mixing.
- the aim of fermentation is to produce a protein/enzyme and/or a metabolite, each with the highest possible yield, for further use in a process.
- the enzyme activity is given in U/ml, 1 U/ml being defined as the conversion of 1 pmol substrate/min in 1 ml test mixture under test conditions.
- the area of the DNA or RNA that begins with a start codon and ends with a stop codon and codes for the amino acid sequence of a protein is called the open reading frame (ORF, synonymous with cds, coding sequence).
- the ORF is also referred to as the coding region or structural gene.
- the DNA section that contains all the basic information for the production of a biologically active RNA is referred to as a gene or expression unit.
- a gene contains the DNA section from which a single-stranded RNA copy is produced by transcription and the expression signals that are involved in the regulation of this copying process.
- the expression signals include, for example, at least one promoter, a transcription start, a translation start and a ribosome binding site. Furthermore, a terminator and one or more operators are possible as expression signals.
- mRNA also known as messenger RNA or messenger RNA
- messenger RNA is a single-stranded ribonucleic acid (RNA) that carries the genetic information for the construction of a protein.
- RNA ribonucleic acid
- An mRNA provides the building instructions for a specific protein in a cell.
- the mRNA molecule carries the message from the genetic information (DNA) necessary for protein construction to the protein-constructing ribosomes.
- DNA genetic information
- a cell it is formed as a transcript of a section of DNA belonging to a gene. The genetic information stored in the DNA is not changed in the process.
- mRNA messenger RNA
- the coding region of the mature mRNA is then translated into the protein sequence
- Eukaryotic gene with exon/intron structure are expressed in prokaryotic organisms, since the processing of the exon/intron structure does not take place in prokaryotes, it is necessary to retranslate the protein sequence or the coding region of the mature mRNA into intron-free DNA from the protein sequence or the mRNA derived gene sequences, exactly this process of reverse translation is meant.It is preferred that simultaneously with the reverse translation of the mRNA sequence into DNA sequence, a sequence optimization, ie adaptation to the codon usage of the corresponding prokaryote takes place (codon optimization).
- An operon is defined as a superordinate expression unit in which several genes are transcribed under the control of only one promoter, but each is translated from its own ribosome binding site.
- a gene construct is a circular DNA molecule (plasmid, vector) in which at least one gene is linked to other genetic elements (eg promoter, ribosome binding site (RBS), terminator, selection marker, origin of replication).
- the genetic elements of the gene construct cause its extrachromosomal inheritance during cell growth and the production of the protein encoded by the gene.
- tbC ⁇ -generating oxidases in combination with their oxidizable substrate are suitable for oxidizing sulfinic acids such as L-cysteine sulfinic acid and hypotaurine to the corresponding sulfonic acids L-cysteic acid and taurine, respectively. This observation was new and surprising.
- H2O2 is basically suitable for the oxidation of sulfinic acids to sulfonic acids.
- H2O2 must be used in at least stoichiometric amounts according to equation (8), so that correspondingly large amounts of H2O2 are required to produce larger amounts of a sulfonic acid.
- H 2 O 2 -generating oxidases have been known for a long time, it was therefore not possible for the person skilled in the art to foresee whether the amount of H2O2 formed from the alcohol oxidase reaction or the glucose oxidase reaction is sufficient to oxidize larger amounts of sulfinic acids.
- the method according to the invention therefore not only has the advantage that it is a biotechnological method, but also that it can be implemented on an industrial scale and it is an economically favorable method since it does not require expensive cofactors.
- hypotaurine in a concentration of 20 g/l relevant for chemical synthesis was surprisingly oxidized almost quantitatively to taurine by glucose oxidase/glucose.
- the invention thus provides a biotechnological process which is suitable for the ever increasing demand for the sustainable production of sulfonic acids such as taurine, avoiding chemical synthesis steps, for use in the food, feed or pharmaceutical sectors.
- the method is preferably characterized in that the sulfinic acid is 2-aminoethanesulphinic acid (hypotaurine) and the sulphonic acid formed is 2-aminoethanesulphonic acid (taurine).
- the enzymatic process according to the invention for the oxidation of a sulfinic acid to the sulfonic acid is preferably carried out at a temperature of 15°C to 80°C, particularly preferably from 20°C to 60°C and particularly preferably from 25°C to 50°C.
- the pH at which the method according to the invention can be carried out depends on the enzymatic properties of the H 2 O 2 -generating oxidase. As described in the examples, the reaction of the alcohol oxidase was carried out at pH 7.5 and that of the glucose oxidase at pH 5.5.
- the pH range at which the reaction is preferably carried out is pH 3.0 to pH 8.5, more preferably pH 4.0 to pH 8.0 and most preferably pH 4.5 to pH 7.5.
- the process according to the invention is carried out with the supply of atmospheric oxygen, either by passive input, as occurs by mixing the reaction mixture, e.g. on an incubation shaker, or by active input, as occurs by gassing with compressed air.
- sulfinic acid:oxidizable substrate preferably at least 1:1).
- a molar ratio of sulfinic acid:oxidizable substrate of at least 1:2 and particularly preferably of at least 1:5 is particularly preferred.
- the reaction time depends on the dosage of the H2O2-generating oxidase and is preferably a maximum of 72 hours, particularly preferably a maximum of 48 hours and particularly preferably a maximum of 24 hours.
- Water is preferably used as the solvent for the reaction.
- the concentration of the sulfinic acid in the batch is preferably at least 1 g/l, particularly preferably at least 10 g/l and particularly preferably at least 20 g/l.
- the molar conversion of sulfinic acid to sulfonic acid in the biotransformation according to the invention is preferably at least 60%, particularly preferably at least 80% and particularly preferably at least 90%.
- the process according to the invention for the oxidation of sulfinic acids can be operated in a batch or continuous manner.
- discontinuous operation batch operation
- all reactants are added to the batch in the course of the reaction and the batch is worked up after the reaction has ended.
- the oxidase enzyme is presented as a stationary phase, for example in a membrane reactor or immobilized on a carrier and brought the substrates comprising a sulfinic acid and a substrate oxidizable by the ThCh-generating oxidase as mobile phase with the stationary phase in contact.
- the contact time of the mobile phase with the stationary phase is preferably set so that the sulfinic acid can react to form the sulfonic acid product.
- the sulfonic acid from the enzymatic oxidation according to the invention can either be used further directly without further work-up steps or can be enriched or purified by means of known methods. The degree of enrichment depends on further use.
- the process is characterized in that the sulfonic acid, particularly preferably taurine, is isolated from the reaction mixture.
- Methods for isolating sulfonic acids, in particular taurine are known to those skilled in the art from methods for isolating, for example, amino acids. They include, for example, filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation, crystallization.
- the process according to the invention reveals a new and unexpected route for the enzymatic oxidation of sulfinic acids to the corresponding sulfonic acids and is particularly suitable for the biotechnological production of L-cysteic acid and taurine.
- the content of sulfinic acid (educt) and sulfonic acid (product) is determined at the beginning, at various times during the reaction and at the end of the reaction in order to monitor the progress of the reaction.
- the content of a solution or cell culture of sulfinic acid of the formula H 2 N-CH (R)-CH 2 - SO2H such as the hypotaurine content or the content of sulfonic acids of the formula H2N-CH (R)-CH2-SO3H such as the taurine content can be determined as follows:
- the content can be quantified directly from an aliquot of the batch, provided the concentration of the sulfinic acid starting material at the beginning of the reaction is at least 0.1 g/L, corresponding to an equivalent concentration of the sulphonic acid product at the end of the reaction with complete conversion, determined e.g. by HPLC.
- concentration of the sulfinic acid starting material at the beginning of the reaction is at least 0.1 g/L, corresponding to an equivalent concentration of the sulphonic acid product at the end of the reaction with complete conversion, determined e.g. by HPLC.
- an aliquot of 1 ml is taken from the mixture, incubated for 5 min at 80 °C, then all solid components are separated off, for example by centrifuging for 5 minutes at maximum speed in a table centrifuge, and the supernatant is quantified by HPLC calibrated for the sulfinic acid or sulfonic acid in question, as e.g. described in example 1 for hypotaurine and taurine.
- the sample can be concentrated beforehand to increase the measurement accuracy for both the sulfinic acid educt and the sulfonic acid product, e.g. by evaporating the sample and redissolving it in an appropriate volume of H2O (e.g. 10% of the sample volume, corresponding to a 10-fold concentration).
- an appropriate volume of H2O e.g. 10% of the sample volume, corresponding to a 10-fold concentration
- a culture broth containing sulfinic acid is used in the method according to the invention, it can be used directly in the quantification. Alternatively, it is also possible first to remove the cells from the culture broth, for example by centrifugation or filtration, and to use the resulting sulfinic acid-containing cell culture supernatant in the method according to the invention. It is also possible to isolate the sulfinic acid from the cell culture supernatant by means of methods known per se and to use the purified sulfinic acid in the process according to the invention. The following biotransformation assay can be used to demonstrate the method according to the invention and to determine the conversion:
- the solution or culture containing sulfinic acid such as culture broth containing hypotaurine, preferably fermenter broth, or the corresponding cell culture supernatant, the purified or commercially available sulfinic acid, such as hypotaurine, is used to produce a biotransformation approach.
- the biotransformation batch on a laboratory scale has a batch volume of 10 ml and contains at least 0.1 g/L sulfinic acid (as described above from the culture/culture broth/fermenter broth, the centrifuged culture supernatant or as a purified substance and quantified accordingly or as a commercial product dosed accordingly).
- the batch volume can also be higher depending on the production scale and is e.g. at least 1 L on the preparative scale.
- the H2O2-generating oxidase preferably AOX or GOX
- the substrate that can be oxidized by this oxidase are added so that the dosage of the enzyme activity is at least 1 U/ ml.
- AOX at least 1% (v/v) methanol, i.e. at least 0.1 ml per 10 ml reaction mixture, is added as the substrate of the AOX enzyme.
- a substrate of the GOX enzyme glucose is added to the reaction mixture in a dosage of at least 10 g/L.
- the biotransformation batch is supplied with atmospheric oxygen, either passively by mixing, e.g. on an incubation shaker or with a stirrer, in each case with access to air, or actively by introducing compressed air or pure oxygen with and without mixing.
- the pH of the reaction is 7.5 if AOX is used.
- the pH of the reaction is 5.5 if GOX is used.
- the pH of the batch can be kept constant by, for example, equipping it with a pH electrode which is connected to a pH control unit which doses a correction agent (lye or acid) from a burette (the so-called pH stat method).
- the reaction temperature is 25°C (AOX) or 30°C (GOX).
- the progress of the reaction can be increased by consuming the Starting material hypotaurine and formation of the product taurine can be monitored, with the quantitative determination of hypotaurine and taurine being able to be carried out, for example, by HPLC.
- the time at which the reaction ends is chosen according to the progress of the reaction.
- the end of the reaction is preferably selected when the molar yield of sulfonic acid of the formula H2N-CH(R)-CH2-SO3H, such as taurine, is at least 60%, particularly preferably at least 80% and particularly preferably at least 90%.
- the sulfonic acid of the formula H2N-CH(R)-CH2-SO3H formed in the reaction mixture is then available for further use.
- Sulfinic acids can originate from chemical or fermentative production, it being preferred that the sulfinic acid used in the process according to the invention originates from fermentative production.
- the method is particularly preferably characterized in that the sulfinic acid is hypotaurine and this originates from fermentative production.
- the sulfinic acids of the formula H2N-CH(R)-CH2-SO2H such as hypotaurine come from fermentative production and a sulfonic acid of the formula H2N-CH(R)-CH2-SO3H such as taurine is formed from this in the process according to the invention, the great advantage that by means of the present invention, a biotechnological process for the production of sulfonic acids of the formula H2N-CH (R) -CH2-SO3H such as taurine is made available. This method does not require any chemical reaction steps.
- Bacterial strains eg E. coli, Corynebacterium glutamicum, Pantoea ananatis, Bacillus subtillis), algae (eg Chlamydomonas reinhardtii), yeasts are suitable for the production of sulfinic acids of the formula H 2 N-CH(R)-CH 2 -SO 2 H such as hypotaurine (e.g. Saccharomyces cerevisiae, Yarrowia llpolytlca) or fungi (e.g. Aspergillus niger).
- hypotaurine e.g. Saccharomyces cerevisiae, Yarrowia llpolytlca
- fungi e.g. Aspergillus niger
- a microorganism strain suitable for hypotaurine production is used.
- a microorganism strain suitable for hypotaurine production is characterized in that it contains a metabolic pathway that leads to hypotaurine according to one of the metabolic pathways specified in the KEGG Pathway database "Taurine and hypotaurine metabolism". Since the biosynthesis of hypotaurine according to equations (1) and (3 ) starts from L-cysteine, a microorganism strain is preferred which is also suitable for cysteine production.As described, for example, by Wada and Takagi, Appl.Microbiol.Biotechnol.(2006) 73: 48-54, cysteine production is in a wild-type microorganism strictly regulated, so that a microorganism strain with a regulated cysteine biosynthetic pathway, as documented in the prior art, is not suitable for the production of economically usable amounts of hypotaurine.
- a microorganism strain with a deregulated cysteine biosynthetic pathway and thus suitable for cysteine production is characterized in that it has at least one of the following changes: a) The microorganism strain is characterized by a modified serA gene, coding for a 3-phosphoglycerate dehydrogenase (SerA ) with a feedback inhibition by L-serine that is reduced by a factor of at least two compared to the corresponding wild-type enzyme (as described, for example, in EP 1950 287 B1), it being possible for the SerA enzyme activity to be determined photometrically, for example, by the SerA substrate 3- Phosphohydroxypyruvate dependent oxidation of NADH as described by McKitrick and Pizer, J. Bacteriol. (1980) 141:235-245.
- SerA enzyme activity to be determined photometrically, for example, by the SerA substrate 3- Phosphohydroxypyruvate dependent oxidation of NADH as described by McKitrick and Pizer, J. Bacteriol.
- 3-phosphoglycerate dehydrogenase have a factor of at least 5 compared to the corresponding wild-type enzyme, in particular feedback inhibition by L-serine is preferably reduced by a factor of at least 10 and in an additionally preferred embodiment by a factor of at least 50.
- the microorganism strain contains an altered cysE gene, coding for a serine-O-acetyl-transferase (CysE), which has feedback inhibition by cysteine that is reduced by a factor of at least two compared to the corresponding wild-type enzyme (such as described for example in EP 0858 510 B1 or Nakamori et al., Appl Env Microbiol (1998) 64: 1607-1611), where the CysE enzyme activity can be determined photometrically, for example, by the consumption of the CysE substrate acetyl-CoA as a result the reaction with L-serine to O-acetyl-L-serine as described, for example, by Nakamori et al., Appl.
- CysE serine-O-acetyl-transferase
- cysE serine-O-acetyltransferase
- the microorganism strain has a cysteine export from the cell which is increased by a factor of at least two by overexpression of an efflux gene compared to the corresponding wild-type cell, it being possible for the cysteine export to be determined by photometric measurement of the extracellular cysteine content Gaitonde, Biochem. J.
- an efflux gene preferably leads to a cysteine export from the cell that is increased by a factor of at least 5, particularly preferably by a factor of at least 10, particularly preferably by a factor of at least 20, compared to a wild-type cell.
- the efflux gene preferably comes from the group ydeD (see EP 0885 962 B1), yfiK (see EP 1382 684 B1), cydDC (see WO 2004/113373 A1), bcr (see US 2005-221453 AA) and emrAB (see US 2005 -221453 AA) from E. coli or the corresponding homologous gene from another microorganism.
- the microorganism strain suitable for hypotaurine production is used according to the prior art, preferably by fermentation, for the production of hypotaurine.
- a hypotaurine-containing culture broth is produced, preferably fermenter broth.
- the content of hypotaurine and possibly taurine as a by-product can be quantified from the culture broth.
- an aliquot of 1 ml is taken from the culture broth with a cell density OD 6 oo/nil of at least 1.0/ml, incubated for 5 min at 80° C., then all solid components are separated off, for example by centrifuging for 5 minutes at maximum speed in a table centrifuge and the supernatant quantitated by HPLC calibrated for hypotaurine and taurine, eg as described in example 1 for hypotaurine and taurine.
- isotope analysis to determine whether a substance such as hypotaurine, which he wants to use as sulfinic acid in the process, comes from chemical or fermentative production.
- a method of isotope analysis suitable for differentiation is, for example, in Sieper et al., Rapid Commun. mass spectrum. (2006) 20: 2521-2527 and is based on the determination of the isotope ratios for eg C or N, which are different depending on whether a product comes from chemical (petroleum-based) or natural (plant-based) production.
- the method described in Example 5 for the production of hypotaurine is one of the methods of natural (plant-based) production, since the Cultivation of the production strain used glucose came from plant production according to the prior art.
- homologous genes, proteins or homologous sequences mean that the DNA or amino acid sequences of these genes, DNA sections or proteins are at least 70%, preferably at least 80% and particularly preferably at least 90% identical.
- the degree of DNA identity is determined by the "nucleotide blast” program found at http://blast.ncbi.nlm.nih.gov/, which is based on the blastn algorithm.
- algorithm parameters for an alignment of two or more nucleotide sequences the default parameters were used.
- the method is characterized in that the sulfinic acid is hypotaurine is and this hypotaurine comes from bacterial production, ie was produced with the help of a bacterial production strain.
- the bacterial production strain is preferably a strain of the species Escherichia coli.
- the preferred bacterial production strain is also characterized in that it is suitable for cysteine production.
- Strains suitable and preferred for cysteine production are the E. coli K12 W3110 x pCys and E. coli K12 W3110-ppsA-MHI x pCys strains disclosed in the examples.
- Example 5 of the present invention heterologous expression of a CDO gene encoding a cysteine dioxygenase and a CSAD gene encoding a cysteine sulfinic acid decarboxylase in the W3110 x pCys-derived strain W3110 x pCys-CDOrn- CSADhs as well as in the strain W3110-ppsA-MHI x pCys derived from W3110-ppsA-MHI x pCys for the production of hypotaurine and only little taurine is formed.
- a microorganism strain designated ppsA-MHI is characterized in that it lacks the coding sequence of the wt-ppsA gene and has replaced this with the ppsA-MHI cds, SEQ ID NO: 9, encoding a protein with the amino acid sequence SEQ ID NO: 10.
- ppsA designates the gene encoding a protein with the enzyme activity of the enzyme class designated in the KEGG database with the number EC 2.7.9.2.
- the corresponding protein is also referred to as PpsA or as phosphoenolpyruvate synthase (PEP synthase) or synonymously as phosphoenolpyruvate-H 2 O-dikinase.
- the enzyme class is defined as being able to produce pyruvate from phosphoenolpyruvate in a reversible reaction according to the formula:
- H2O2-generating oxidases in the hypotaurine production strain.
- Genes for suitable H2O2-generating oxidases can be identified in databases such as NCBI (National Center for Biotechnology Information).
- glucose oxidase heterologously produced in E. coli is the GOX gene from Penicillium amagasakiense (Witt et al., App. Environ. Microbiol (1998) 64: 1405-1411).
- a biotechnological process for the production of taurine is preferred in which hypotaurine is produced in a first step by growing a production strain and in a second step the hypotaurine formed is oxidized enzymatically to taurine without further processing, the enzymatic oxidation of hypotaurine to taurine by glucose oxidase /D-glucose is particularly preferred.
- the examples of the present invention disclose the biotechnological production of hypotaurine (example 5) and its enzymatic oxidation to taurine (example 6).
- the method is characterized in that the sulfinic acid is hypotaurine and this hypotaurine originates from bacterial production, ie was produced using a bacterial production strain which has a deregulated cysteine biosynthetic pathway.
- the definition given above applies for the microorganism strain with deregulated cysteine biosynthetic pathway.
- Cysteine is particularly preferably produced first, which according to equation (1) is further converted by the CDO enzyme contained in the production strain to cysteine sulfinic acid (CDO reaction) and according to equation (3) by the CSAD enzyme also contained in the production strain to hypotaurine (CSAD -Reaction) reacts.
- the method is particularly preferably characterized in that the sulfinic acid is hypotaurine produced by means of a bacterial production strain and the production strain is one of the strains E. coli K12 W3110 x pCYS-CDOrn-CSADhs or E. coli K12 W3110-ppsA-MHI x pCYS-CDOrn-CSADhs, particularly preferably the strain E. coli K12 W3110-ppsA-MHI x pCYS-CDOrn-CSADhs.
- the invention thus also shows production strains for the biotechnological production of hypotaurine.
- a particularly preferred starting strain is one of the E. coli K 12 W3110 x pCys E. coli K 12 W3110-ppsA-MHI x pCys strains suitable for cysteine production.
- the vector pCys (Fig. 1) was modified to incorporate the genes for cysteine dioxygenase from Rattus norvegicus (CDOrn),
- SEQ ID NO: 1 coding for a protein with the amino acid sequence SEQ ID NO: 2 and for L-cysteinesulfinic acid decarboxylase from Homo sapiens (CSADhs), SEQ ID NO: 3, nt 1 to nt 1509, coding for a protein with the amino acid sequence SEQ ID NO: 4 expanded.
- the vector pCys-CDOrn-CSADhs suitable for hypotaurine production was created (FIG. 2).
- the CDO gene used is not limited to the Rattus norvegicus species. Any CDO gene is suitable, its gene product (protein) derived from the mRNA according to Equation (1) capable of oxidizing L-cysteine to L-cysteine sulfinic acid. Such proteins can be found, for example, in the NCBI database if you search for CDO proteins in the "protein" sub-database using the search term "cysteine dioxygenase".
- CDO proteins known from the prior art and not limited to these are preferably the CDO protein from Homo sapiens (human), Cyprinus carpio (carp) or Synechococcus (alga) or a protein with a 70% homologous amino acid sequence, especially preferably a protein with an 80% homologous amino acid sequence and particularly preferably the amino acid sequence of the CDO protein has the sequence given in SEQ ID NO: 2 and is encoded by the DNA sequence given in SEQ ID NO: 1.
- the CSAD gene used is not limited to the species Homo sapiens. Any CSAD gene is suitable whose gene product (protein) derived from the mRNA according to equation (3) is suitable for decarboxylating L-cysteine sulfinic acid to hypotaurine. Such proteins can be found, for example, in the NCBI database by searching for CSAD proteins in the "protein" sub-database using the search term "cysteine sulfinic acid decarboxylase". Further CSAD proteins known from the prior art and not limited to these are preferably the CSAD protein from Rattus norvegicus (rat), Cyprinus carpio (carp), Synechococcus (algae) or E.
- Amino acid sequence, particularly preferably a protein with an 80% homologous amino acid sequence thereto and particularly preferably the amino acid sequence of the CSAD protein has the sequence given in SEQ ID NO: 4 and is encoded by the DNA given in SEQ ID NO: 3 of nt 1-1509 -Sequence.
- a gene construct containing the CDO and CSAD gene such as particularly preferably the construct pCys-CDOrn-CSADhs can according to the prior art using recombinant DNA techniques known to those skilled in the art, as described in detail in Example 4, for example.
- a CDO gene which codes for cysteine dioxygenase
- a CSAD gene which codes for a cysteine sulfinic acid decarboxylase
- the CDO and CSAD gene can each be cloned into the vector with its own promoter and possibly terminator as independent expression units or else, in any order, as an operon under the control of only one promoter will. Also possible is an operon structure in which the CDO and CSAD genes, each with their own ribosome binding site, are cloned behind one of the genes already present on the vector and are expressed under the control of the promoter of the relevant gene. In addition, all other conceivable combinations of expression under its own promoter and as an operon are possible for the CDO and the CSAD gene.
- CDO and CSAD gene as separate expression units independently of the vector ensuring deregulated cysteine biosynthesis, such as pCys, into a separate vector, or to integrate them into the genome of a host organism such as E. coli.
- the vector is particularly preferably pCys-CDOrn-CSADhs, in which the CDO and the CSAD gene are behind the in pCys contained serA317 gene are cloned so that their expression takes place under the control of the serA317 promoter according to a sequence of expression units: serA promoter->(serA317-cds)->RBS-(CDOrn-cds)->RBS-(CSADhs- cds)-rrnB terminator .
- a configuration of the expression units in which the CSAD cds is arranged in front of the CDO cds is also possible.
- pCys-CDOrn-CSADhs is also characterized by the fact that it ensures deregulated cysteine biosynthesis by expressing expression units for the feedback-resistant variant serA317 of 3-phosphoglycerate dehydrogenase (serA gene), the feedback-resistant variant CysEX of serine -O- acetyl transferase (cysE gene) and the ydeD gene of a cysteine efflux protein.
- the invention relates to vectors which contain at least one, preferably two and particularly preferably three of the genes serA317, cysEX or ydeD, it being possible for the genes concerned to be contained in any selection and sequence on the vector.
- CDO and CSAD have been cloned into a vector, this is then transformed into a suitable host strain in a known manner.
- a suitable host strain Any microorganism that is accessible to recombinant DNA techniques and that is suitable for the fermentative production of recombinant proteins is suitable as a host strain.
- the preferred host strain is a strain of the species Escherichia coli, particularly preferably the strain E. coli K12 W3110 or E. coli K12 W3110-ppsA-MHI.
- strains obtained from the transformation are suitable for the production of hypotaurine, as described in Example 5.
- the production of hypotaurine using particularly preferably one of the strains E. coli K12 W3110 x pCys-CDOrn-CSADhs or E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs can be done by cultivation in shake flasks (as described in example 5) or on a production scale by fermentation of the strain.
- the hypotaurine formed is converted to taurine by enzymatic oxidation.
- the hypotaurine-containing cell culture is incubated with an H 2 O 2 -generating oxidase in the presence of its substrate, such as, for example, with a glucose oxidase in the presence of glucose (see Example 6).
- the method is characterized in that the sulfinic acid is cysteine sulfinic acid and the resulting sulphonic acid is cysteic acid.
- the process is preferably characterized in that the molar yield of sulphonic acids of the formula H 2 N-CH(R)-CH 2 -SO 3 H based on the additive molar concentration of sulfinic acids of the formula H 2 N-CH(R)- used CH 2 -SO 2 H and sulfonic acids of the formula H 2 N-CH(R)-CH 2 -SO 3 H is at least 60%, more preferably at least 80% and most preferably at least 90%.
- the amount of sulfinic acid used is preferably at least 1 mM (109.2 mg/L), particularly preferably at least 10 mM (1.1 g/L) and particularly preferably at least 100 mM (10.9 g/L).
- the molar content of the sulfinic and sulfonic acids used in the enzymatic oxidation is determined at the beginning and at the end of the reaction by means of HPLC, as is known in the prior art. For example, the determination can be carried out as described in Example 1, taking into account the molecular weights 109.2 g/mol for hypotaurine and 125.2 g/mol for taurine.
- the molar content of sulfonic acid in the batch at the end of the reaction in relation to the total molar content of sulfinic acid and sulfonic acid at the beginning of the reaction gives the molar yield of the sulfonic acid expressed as a percentage.
- Figure 1 pCys.
- bla gene conferring resistance to ampicillin
- kanR gene conferring resistance to kanamycin araC: araC gene (repressor gene)
- P araC promoter of the araC gene
- P araB promoter of the araB gene
- Bet Lambda Phage Bet recombination gene
- RepA plasmid replication protein
- gene sacB levansucrase gene
- pr-f primer binding site f
- pr-r primer binding site r
- OriC origin of replication C
- TetR gene conferring resistance to tetracycline
- serA317 serA (3-phosphoglycerate dehydrogenase gene coding for amino acids 1 to 317)
- cds cysE X cysE (serine O-acetyltransferase gene, feedback resistant) eds
- ORF306 ydeD (cysteine efflux gene) eds Seal: restriction enzyme site Seal PpuMI: restriction enzyme site PpuMI CDOrn: CDO (cysteine dioxygenase) R. norvegicus eds CSADhs: CSAD (cysteine sulfinic acid decarboxylase) H. sapiens eds
- RBS ribosome binding site
- Example 1 Oxidation of hypotaurine and cysteine sulfinic acid with alcohol oxidase (AOX)
- the second batch without AOX received 30 mM 100 mM Na phosphate, pH 7.5.
- the batches were shaken at 25° C. and 140 rpm (Infors chest shaker).
- 1 ml of each batch was removed, incubated for 5 min at 80° C., centrifuged for 5 min at 13000 rpm (HeraeusTM FrescoTM 21 centrifuge) and the supernatants analyzed by HPLC.
- the content of L-cysteinesulfinic acid and L-cysteic acid in the batch with AOX and in the batch without AOX at the beginning (0 h) and after a reaction time of 5 h is summarized in Table 1.
- the reaction was investigated in two parallel runs, i.e. with and without AOX.
- 12 mg of hypotaurine (Sigma-Aldrich) were each weighed into two 100 ml Erlenmeyer flasks, dissolved in 9.9 ml of 100 mM Na phosphate, pH 7.5, and 0.1 ml of methanol (final concentration 1%, v/v) were added.
- 30 ml of commercially available AOX from Pichia pastoris (Sigma-Aldrich) dissolved in 100 mM Na phosphate, pH 7.5, were added in one batch. According to the manufacturer's information on the enzyme activity, the AOX activity in the batch was 5 U/ml.
- the batch without AOX received 30 mM 100 mM Na phosphate, pH 7.5.
- the batches were shaken at 25° C. and 140 rpm (Infors chest shaker).
- 1 ml of each batch was removed, incubated for 5 min at 80° C., centrifuged for 5 min at 13000 rpm (HeraeusTM FrescoTM 21 centrifuge) and the supernatants analyzed by HPLC.
- the content of hypotaurine and taurine in the mixture with alcohol oxidase (AOX) and in the mixture without alcohol oxidase at the beginning (0 h) and after a reaction time of 5 h is summarized in Table 2.
- An HPLC method calibrated for L-cysteinesulfinic acid, L-cysteic acid, hypotaurine and taurine was used for the quantitative determination of the compounds quantitatively analyzed in the examples, with all reference substances used for calibration being commercially available (Sigma-Aldrich).
- An HPLC device from Agilent, model 1260 Infinity II, equipped with a pre-column derivatization known from the analysis of amino acids with o-phthaldialdehyde (OPA derivatization) from the same manufacturer was used.
- the HPLC device was equipped with a fluorescence detector to detect the OPA-derivatized products of L-cysteine sulfinic acid, L-cysteic acid, hypotaurine and taurine. The detector was set to an excitation wavelength of 330 nm and an emission wavelength of 450 nm.
- An AccucoreTM aQ column from Thermo ScientificTM was also used.
- Eluent A 25 mM Na phosphate, pH 6.0
- Eluent B methanol
- the reaction was investigated in two parallel approaches, ie with and without GOX.
- 100 ml 12 mg of L-cysteine sulfinic acid ⁇ H2O (Sigma-Aldrich) were each weighed into Erlenmeyer flasks, dissolved in 9.5 ml of 100 mM Na acetate, pH 5.5, and 0.5 ml of a 200 g/L glucose solution in the same buffer was added.
- 50 ml of commercially available GOX from Aspergillus niger (Sigma-Aldrich), dissolved in 100 mM Na acetate, pH 5.5, were added in one batch. According to the manufacturer's information, the GOX activity in the batch was 5 U/ml.
- the batch without GOX received 50 mM 100 mM Na acetate, pH 5.5.
- the batches were shaken at 30° C. and 140 rpm (Infors chest shaker).
- 1 ml of each batch was removed, incubated for 5 min at 80° C., centrifuged for 5 min at 13000 rpm (HeraeusTM FrescoTM 21 centrifuge) and the supernatants analyzed by HPLC as described above.
- the reaction was investigated in two parallel approaches, ie with and without GOX.
- 12 mg of hypotaurine (Sigma-Aldrich) were each weighed into two 100 ml Erlenmeyer flasks, dissolved in 9.5 ml of 100 mM Na acetate, pH 5.5, and 0.5 ml of a 200 g/L glucose solution in the same added buffer.
- 50 ml of commercially available GOX from Aspergillus niger (Sigma-Aldrich) dissolved in 100 mM Na acetate, pH 5.5, were added in one batch. According to the manufacturer's information, the GOX activity in the batch was 5 U/ml.
- the batch without GOX received 50 mM 100 mM Na acetate, pH 5.5.
- the batches were shaken at 30° C. and 140 rpm (Infors chest shaker).
- 1 ml of each batch was removed, incubated for 5 min at 80° C., centrifuged for 5 min at 13000 rpm (HeraeusTM FrescoTM 21 centrifuge) and the supernatants analyzed by HPLC as described above.
- the reaction was investigated in two parallel approaches, i.e. with different doses of the enzyme GOX, with the concentration of the substrate to be oxidized, hypotaurine, being 20 g/L in each case.
- Example 4 Preparation of the hypotaurine production strains E. coli K12 W3110 x pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs Cysteine dioxygenase CDOrn:
- CDOrn The amino acid sequence of the cysteine dioxygenase from Rattus norvegicus is disclosed in the NCBI (National Center for Biotechnology Information) database under the sequence ID: AAH70509.1.
- a DNA sequence codon-optimized for expression in E. coli publicly available Eurofins Genomics GENEius software was derived from the amino acid sequence and produced synthetically (Eurofins Genomics).
- This DNA sequence designated CDOrn is disclosed in SEQ ID NO: 1 encoding a protein having the amino acid sequence of SEQ ID NO: 2.
- CSADhs The amino acid sequence of the cysteine sulfinic acid decarboxylase (CSADhs) from Homo sapiens is disclosed in the NCBI (National Center for Biotechnology Information) database under the sequence ID: XP_016861786.1.
- a DNA sequence codon-optimized for expression in E. coli publicly accessible Eurofins Genomics GENEius software was derived from the amino acid sequence.
- This DNA sequence designated CSADhs is disclosed in SEQ ID NO: 3, nt 1 to 1509, encoding a protein having the amino acid sequence of SEQ ID NO: 4.
- the DNA sequence of the E. coli rrnB terminator (SEQ ID NO :3, nt 1510 to 1842) was linked to nt 1509.
- the DNA sequence of the rrnB terminator is disclosed in Orosz et al., Eur. J. Biochem. (1991) 201:653-659.
- Fig. 1 denotes the plasmid pACYC184-cysEX-GAPDH-ORF306-serA317, a derivative of the plasmid disclosed in EP 0885 962 B1 pACYC184-cysEX-GAPDH-ORF306.
- the plasmid pACYCl84-cysEX-GAPDH-ORF3 06 contains in addition to the Origin of replication and a tetracycline resistance gene (starting vector pACYC184) the cysEX allele, which codes for a serine-O-acetyl-transferase with reduced feedback inhibition by cysteine, and the efflux gene ydeD (ORF306), whose expression is controlled by the constitutive GAPDH promoter will.
- serA317 In order to obtain pACYC184-cysEX-GAPDH-ORF306-serA317, the serA317 gene fragment coding for the N-terminal 317 amino acids of the SerA protein from E. coli and disclosed in Bell et al., Eur. J. Biochem. (2002) 269: 4176-4184 (referred to therein as "NSD:317") behind the ydeD (ORF306) efflux gene.
- SerA317 encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase.
- the expression of serA317 is regulated by the serA promoter.
- E. coli cells transformed with pCys produce cysteine, the starting product for the subsequent products cysteine sulfinic acid and hypotaurine.
- pCys was cut with Seal and PpuMI.
- the 6.1 kb vector fragment released in the process was isolated after preparative agarose gel electrophoresis ( QIAquick® Gel Extraction Kit, Qiagen).
- the CDOrn DNA was derived from the synthetic DNA SEQ ID NO: 1 (CDOrn) by PCR (“PhusionTM High-Fidelity" DNA Polymerase, Thermo ScientificTM) with the primers cdorn-lf (SEQ ID NO: 5) and csadhs- 2r (SEQ ID NO:6) and isolated as a 0.6 kb fragment.
- Primer cdorn-lf comprised, starting from the 5' end, 28 nt overlapping with the 3' end of the 6.1 kb pCys Scal/PpuMI vector fragment (nt 1 to 28 in SEQ ID NO: 5) obtained by Scal digestion, a ribosome binding site (RBS) (nt 31 to 36 in SEQ ID NO: 5) and the first 22 nt of the CDOrn eds (SEQ ID NO 5, nt 44 to 65).
- RBS ribosome binding site
- Primer csadhs-2r comprised, in reverse complement form, starting from the 5' end, 22 nt overlapping the start of the CSADhs cds (SEQ ID NO: 6, nt 1 to 22), followed by a ribosome binding site (SEQ ID NO: 6, nt 30 to 35) and the last 20 nt of the CDOrn cds (SEQ ID NO: 6, nt 38 to 57).
- Primer csadhs-3f comprised, starting from the 5' end, 20 nt overlapping with the 3' end of the CDOrn cds (nt 1 to 20 in SEQ ID NO: 7), a ribosome binding site (nt 23 to 28 in SEQ ID NO: 7) and the first 22 nt of the CSADhs cds (SEQ ID NO 7, nt 36 to 57).
- Primer glf-2r comprised, in reverse complementary form, starting from the 5' end, 33 nt overlapping with the 5' end obtained by PpuMI digestion of the 6.1 kb pCys Scal/PpuMI vector fragment (nt 1 to 33 in SEQ ID NO: 8) followed by the last 22 nt of the rrnB terminator (SEQ ID NO: 8, nt 34 to 55).
- E. coli NEB® 10-beta cells were then transformed with the ligation mixture. Clones from the transformation were selected on LBtet. LBtet contained 10 g/L tryptone (GIBCOTM), 5 g/L yeast extract (BD Biosciences), 5 g/L NaCl, 15 g/L agar and 15 mg/L tetracycline (Sigma-Aldrich). A single clone from the transformation was analyzed by culturing in LBtet liquid medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl and 15 mg/L tetracycline) and isolating the vector from the cell pellet of cultivation. The correct 8.5 kb vector was named pCys-CDOrn-CSADhs ( Figure 2).
- the strain Escherichia coli K12 W3110 (commercially available under the strain number DSM 5911 from the DSMZ German Collection of Microorganisms and Cell Cultures GmbH) was used.
- E. coli K12 W3110-ppsA-MHI The strain E. coli K12 W3110-ppsA-MHI was used.
- E. coli K12 W3110-ppsA-MHI is characterized by the mutated ppsA gene ppsA-MHI (SEQ ID NO: 9), coding for a protein with the protein sequence from SEQ ID NO: 10 and the enzyme activity of a PEP synthase (in enzyme class designated with the number EC 2.7.9.2 in the KEGG database).
- E. coli K12 W3110-ppsA-MHI was produced using the combination of Lambda-Red recombination and counter-selection screening for genetic modification known to those skilled in the art (see e.g. Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245).
- E. coli K12 W3110 was transformed with the plasmid pKD46 (FIG. 3, disclosed in the “GenBank” gene database under the accession number AY048746.1) and the E. coli W3110 ⁇ pKD46 strain was isolated (ampicillin selection).
- the 3.2 kb Kan-sacB cassette was isolated by PCR with the primers pps-9f (SEQ ID NO: 11, binds to the site designated "pr-f" in Fig. 4) and pps-10r (SEQ ID NO: 12, binds at the site designated "pr-r” in Fig. 4) isolated.
- the plasmid pKansacB contains expression cassettes both for the kanamycin (Kan) resistance gene and for the sacB gene, coding for the enzyme levansucrase.
- coli kanamycin resistance gene (Kan), encoding an aminoglycoside Phosphotransferase is disclosed in the NCBI database under accession number SH02_03400.
- the B. subtilis sacB gene is disclosed in the NCBI database under accession number 936413.
- Primer pps-9f contains the first 30 nt of the ppsA WT gene (identical to SEQ ID NO: 9, nt 1 to 30) followed by 20 nt of the site designated "pr-f" in Figure 4.
- Primer pps -lOr contains the last 30 nt of the ppsA WT gene, in reverse complementary form (identical to SEQ ID NO: 9, nt 2350 - 2379) and connected thereto 20 nt of the site designated "pr-r” in Fig. 4 .
- E. coli W3110 x pKD46 was transformed with the 3.2 kb Kan-sacB cassette and kanamycin-resistant clones isolated.
- LBSC plates (10 g/L tryptone, 5 g/L yeast extract, 7% sucrose, 1.5% agar and 15 mg/L kanamycin). Clones with an integrated sacB gene produced toxic levan from sucrose, which led to growth inhibition (sucrose-sensitive). A kanamycin resistant and sucrose sensitive clone was selected and designated W3110-ppsA::Kan-sacB x pKD46.
- W3110-ppsA::Kan-sacB x pKD46 was transformed with DNA of the ppsA-MHI gene (SEQ ID NO: 9, produced synthetically, Eurofins Genomics) and clones on LBS plates (10 g/L tryptone, 5 g/L yeast extract, 7% sucrose, 1.5% agar) without kanamycin. Only clones that no longer contained an active sacB gene could grow on LBS plates.
- clones were transferred to LBkan plates (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 1.5% agar, 15 mg/L kanamycin) in order to select those clones that also had no active Kan gene and whose growth was inhibited in the presence of kanamycin.
- E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI strains were transformed into E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI strains.
- E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI were transformed with the vector pCys.
- Transformants were selected on LBtet. One clone each was isolated.
- the strains were named E. coli K12 W3110 x pCys-CDOrn-CSADhs and E. coli K12 W3110 x pCys or, analogously, E.
- E. coli K12 W3110 x pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs were used as production strains to produce hypotaurine.
- E. coli K12 W3110 x pCys-CDOrn-CSADhs From E. coli K12 W3110 x pCys-CDOrn-CSADhs, E. coli K12 W3110 x pCys, E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI x pCys strains a preculture was produced in LBtet liquid medium (cultivation at 37° C. and 120 rpm overnight).
- Main culture 0.5 ml of the preculture was placed in a 300 ml Erlenmeyer flask (with baffle) with 30 ml SM1-Ac medium, also containing 15 g/L glucose, 2 g/L Na2S2O3 x 5 H2O, 0.1 g/L L L-isoleucine, 0.1 g/L D,L-methionine, 0.1 g/L L-threonine, 5 mg/L vitamin B1 and 15 mg/L tetracycline.
- SM1-Ac medium also containing 15 g/L glucose, 2 g/L Na2S2O3 x 5 H2O, 0.1 g/L L L-isoleucine, 0.1 g/L D,L-methionine, 0.1 g/L L-threonine, 5 mg/L vitamin B1 and 15 mg/L tetracycline.
- Composition of the SM1-Ac medium 12 g/LK 2 HPO 4 , 3 g/L KH 2 PO 4 , 5 g/L NH 4 acetate, 0.3 g/L MgSO 4 x 7 H 2 0, 0.015 g /L CaCl2 x 2 H 2 0.002 g/L FeSO 4 x 7 H 2 0.1 g/L Na 3 citrate x 2 H 2 0.0.1 g/L NaCl; 1 ml/L trace element solution.
- composition of the trace element solution 0.15 g/L Na 2 Mo0 4 x 2 H 2 0, 2.5 g/L H3BO3, 0.7 g/L CoCl 2 x 6 H 2 0, 0.25 g/L CuS0 4 x 5 H 2 0. 1.6 g/L MnCl 2 x 4 H 2 0. 0.3 g/L ZnS0 4 x 7 H 2 0.
- the main culture was incubated for 24 h at 30° C. and 140 rpm in a chest shaker (Infors). After 24 h, 1 ml samples were taken and the cell density OD ö oo/nil (optical density of the main culture, measured photometrically at 600 nm) measured with a GenesysTM 10S UV-Vis spectrophotometer from Thermo-ScientificTM, as well as the content of Hypotaurine and taurine determined by HPLC. The content of hypotaurine in the culture supernatant determined by HPLC was for E. coli K12 W3110 x pCys-CDOrn-CSADhs (cell density OD ö oo/nil of the culture: 5.3/ml)
- the hypotaurine contained in the shake flask culture (Example 5) of 157.3 mg/L (strain W3110 x pCys-CDOrn-CSADhs) and 1059.2 mg/L (strain W3110-ppsA-MHI x pCys -CDOrn-CSADhs) was completely consumed and taurine was formed in a concentration of 212.8 mg/L or 1355.6 mg/L.
- the molar yield was determined taking into account the different molecular weights (109.2 g/mol for hypotaurine, 125.2 g/mol for taurine).
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US18/035,610 US20230406817A1 (en) | 2021-07-05 | 2021-07-05 | Method for enzymatic oxidation of sulfinic acids to sulfonic acids |
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