METHOD FOR ENZYMATIC OXIDATION OF SULFINIC ACIDS TO SULFONIC ACIDS

Abstract

A process for the enzymatic oxidation of sulfinic acids includes sulfinic acids of formula H.sub.2NCH(R)CH.sub.2SO.sub.2H to sulfonic acids of formula H.sub.2NCH(R)CH.sub.2SO.sub.3H and an enzyme selected from the class of H.sub.2O.sub.2-generating oxidases in the presence of the substrate of said enzyme.

Claims

1-14. (canceled)

15. A process for the enzymatic oxidation of sulfinic acids of formula H.sub.2NCH(R)CH.sub.2SO.sub.2H to sulfonic acids of formula H.sub.2NCH(R)CH.sub.2SO.sub.3H with an enzyme selected from the class of H.sub.2O.sub.2-generating oxidases in the presence of the substrate of said enzyme.

16. The process as claimed in claim 15, wherein the H.sub.2O.sub.2-generating oxidase is alcohol oxidase and that the substrate present is a primary alcohol.

17. The process as claimed in claim 15, wherein the H.sub.2O.sub.2-generating oxidase is glucose oxidase and that the substrate present is glucose.

18. The process as claimed in one or more of claims 15, wherein the sulfinic acid is aminoalkyl sulfinic acid and that the sulfonic acid is aminoalkyl sulfonic acid.

19. The process as claimed in claim 15, wherein the concentration of the sulfinic acid in the batch is at least 1 g/l.

20. The process as claimed in claim 15, wherein the sulfinic acid is 2-aminoethanesulfinic acid (hypotaurine) and that the sulfonic acid formed is 2-aminoethanesulfonic acid (taurine).

21. The process as claimed in claim 15, wherein the sulfonic acid is isolated from the reaction batch.

22. The process as claimed in claim 20, wherein the hypotaurine is hypotaurine from fermentative production.

23. The process as claimed in claim 22, wherein the hypotaurine originates from bacterial production by way of using a bacterial production strain.

24. The process as claimed in claim 23, wherein the bacterial production strain is a strain of the species Escherichia coli.

25. The process as claimed in claim 23, wherein the production strain is a strain having a deregulated cysteine biosynthetic pathway.

26. The process as claimed in claim 23, wherein the production strain is one of the strains E. coli W3110 x pCys-CDOrn-CSADhs or W3110-ppsA-MHI x pCys-CDOrn-CSADhs.

27. The process as claimed in claim 15, wherein the sulfinic acid is cysteine sulfinic acid and that the sulfonic acid formed is cysteic acid.

28. The process as claimed in claim 15, wherein the molar yield of sulfonic acid of formula H.sub.2NCH(R)CH.sub.2SO.sub.3H based on the total molar concentration of sulfinic acid of formula H.sub.2NCH(R)CH.sub.2SO.sub.2H and sulfonic acid of formula H.sub.2NCH(R)CH.sub.2SO.sub.3H used in this reaction is at least 60%.

Description

[0111] The figures show the plasmids used in the examples.

[0112] FIG. 1: pCys.

[0113] FIG. 2: pCys-CDOrn-CSADhs.

[0114] FIG. 3: pKD46.

[0115] FIG. 4: pKan-SacB.

ABBREVIATIONS USED IN THE FIGURES

[0116] bla: Gene conferring resistance to ampicillin (-lactamase)

[0117] kanR: Gene conferring resistance to kanamycin

[0118] araC: araC gene (repressor gene)

[0119] P araC: Promoter of the araC gene

[0120] P araB: Promoter of the araB gene

[0121] Gam: Lambda phage Gam recombination gene

[0122] Bet: Lambda phage Bet recombination gene

[0123] Exo: Lambda phage Exo recombination gene

[0124] ORI101: Temperature-sensitive origin of replication

[0125] RepA: Gene for plasmid replication protein A

[0126] sacB: Levansucrase gene

[0127] pr-f: Binding site f for primer (forward)

[0128] pr-r: Binding site r for primer (reverse)

[0129] OriC: Origin of replication C

[0130] TetR: Gene conferring resistance to tetracycline

[0131] P15A ORI: Origin of replication

[0132] serA317: serA (3-phosphoglycerate dehydrogenase gene encoding amino acids 1 to 317) cds

[0133] cysE X: cysE (serine O-acetyltransferase gene, feedback resistant) cds

[0134] ORF306: ydeD (cysteine efflux gene) cds

[0135] ScaI: Cleavage site for the restriction enzyme ScaI

[0136] PpuMI: Cleavage site for the restriction enzyme PpuMI

[0137] CDOrn: CDO (cysteine dioxygenase) R. norvegicus cds

[0138] CSADhs: CSAD (cysteine sulfinic acid decarboxylase) H. sapiens cds

[0139] RBS: Ribosome binding site

[0140] The following examples serve to further elucidate the invention further without restricting it thereto.

EXAMPLES

Example 1

Oxidation of Hypotaurine and Cysteine Sulfinic Acid with Alcohol Oxidase (AOX)

[0141] A) Oxidation of Cysteine Sulfinic Acid to Cysteic Acid with AOX: The reaction was investigated in two parallel batches, i.e. with and without AOX. 12 mg of L-cysteine sulfinic acid monohydrate (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.9 ml of 100 mM Na phosphate pH 7.5 and 0.1 ml of methanol was added (end concentration 1% v/v). To start the reaction, 30 l of a commercially available solution of AOX from Pichia pastoris (Sigma-Aldrich) in 100 mM Na phosphate pH 7.5 was added to one batch. In accordance with the manufacturer's information on the enzyme activity, the AOX activity in the batch was 5 U/ml. The second batch without AOX (comparison batch) was treated with 30 l of 100 mM Na phosphate pH 7.5. The batches were shaken at 25 C. and 140 rpm (Infors incubator shaker). At the start and 5 h after the start of the reaction, 1 ml from each batch was taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatants analyzed by HPLC. The content of L-cysteine sulfinic acid and L-cysteic acid in the batch with AOX and in the batch without AOX at the start (0 h) and after a reaction time of 5 h is summarized in Table 1.

TABLE-US-00001 TABLE 1 Time course of the oxidation of L-cysteine sulfinic acid to L- cysteic acid by AOX in the presence of methanol +5 U/ml AOX without AOX L-Cysteine L-Cysteine sulfinic L-Cysteic sulfinic L-Cysteic acid acid acid acid Time [mg/L] [mg/L] [mg/L] [mg/L] 0 h 1171.5 0.0 1239.3 0.0 5 h 13.4 1023.3 1244.3 0.0 [0142] B) Oxidation of Hypotaurine to Taurine with AOX: The reaction was investigated in two parallel batches, i.e. with and without AOX. 12 mg of hypotaurine (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was dissolved in 9.9 ml of 100 mM Na phosphate pH 7.5 and 0.1 ml of methanol was added (end concentration 1% v/v). To start the reaction, 30 l of a commercially available solution of AOX from Pichia pastoris (Sigma-Aldrich) in 100 mM Na phosphate pH 7.5 was added to one batch. In accordance with the manufacturer's information on the enzyme activity, the AOX activity in the batch was 5 U/ml. The batch without AOX (comparison batch) was treated with 30 l of 100 mM Na phosphate pH 7.5. The batches were shaken at 25 C. and 140 rpm (Infors incubator shaker). At the start and 5 h after the start of the reaction, 1 ml from each batch was taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatants analyzed by HPLC. The content of hypotaurine and taurine in the batch with alcohol oxidase (AOX) and in the batch without alcohol oxidase at the start (0 h) and after a reaction time of 5 h is summarized in Table 2.

TABLE-US-00002 TABLE 2 Time course of the oxidation of hypotaurine to taurine by AOX in the presence of methanol +5 U/ml AOX without AOX Hypotaurine Taurine Hypotaurine Taurine Time [mg/L] [mg/L] [mg/L] [mg/L] 0 h 1030.2 0.0 1112.3 0.0 5 h 0.0 1124.3 1169.3 0.0

HPLC Analysis of L-Cysteine Sulfinic Acid, L-Cysteic Acid, Hypotaurine, and Taurine:

[0143] For quantitative determination of the compounds quantitatively analyzed in the examples, an HPLC method calibrated respectively for L-cysteine sulfinic acid, L-cysteic acid, hypotaurine, and taurine was employed; all reference substances used for calibration were commercially available (Sigma-Aldrich). An Agilent 1260 Infinity II HPLC system was used, which was equipped with a unit from the same manufacturer for pre-column derivatization with o-phthaldialdehyde (OPA derivatization) as is known from the analysis of amino acids. For detection of the OPA-derivatized products of L-cysteine sulfinic acid, L-cysteic acid, hypotaurine, 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. Also used were an Accucore aQ column from Thermo Scientific, length 100 mm, internal diameter 4.6 mm, particle size 2.6 m, thermally equilibrated at 40 C. in a column oven.

Eluent A: 25 mM Na phosphate pH 6.0

Eluent B: Methanol

[0144] The separation was carried out in gradient mode: 10% eluent B to 60% eluent B over min, followed by 60% eluent B to 100% eluent B over 2 min, followed by 100% eluent B for a further 2 min, at a flow rate of 0.5 ml/min. Retention time of L-cysteic acid: 3.2 min. Retention time of L-cysteine sulfinic acid: 4.1 min. Retention time of taurine: 14.8 min. Retention time of hypotaurine: 15.7 min.

Example 2

Oxidation of Hypotaurine and Cysteine Sulfinic Acid with Glucose Oxidase (GOX)

[0145] A) Oxidation of Cysteine Sulfinic Acid to Cysteic Acid with GOX: The reaction was investigated in two parallel batches, i.e. with and without GOX. 12 mg of L-cysteine sulfinic acid monohydrate (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was 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. To start the reaction, 50 l of a commercially available solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM Na acetate pH 5.5 was added to one batch. In accordance with the manufacturer's information, the GOX activity in the batch was 5 U/ml. The batch without GOX (comparison batch) was treated with 50 l of 100 mM Na acetate pH 5.5. The batches were shaken at 30 C. and 140 rpm (Infors incubator shaker). At the start and 5 h after the start of the reaction, 1 ml from each batch was taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatants analyzed by HPLC as described above. The content of L-cysteine sulfinic acid and L-cysteic acid in the batch with GOX and in the batch without GOX at the start (0 h) and after a reaction time of 5 h is summarized in Table 3.

TABLE-US-00003 TABLE 3 Time course of the oxidation of L-cysteine sulfinic acid to L- cysteic acid by GOX in the presence of glucose without GOX +5 U/ml GOX L-Cysteine L-Cysteine sulfinic L-Cysteic sulfinic L-Cysteic acid acid Time acid [mg/L] acid [mg/L] [mg/L] [mg/L] 0 h 1322.7 0.0 1146.3 0.0 5 h 3.0 1279.3 1171.3 0.0 [0146] B) Oxidation of Hypotaurine to Taurine with GOX: The reaction was investigated in two parallel batches, i.e. with and without GOX. 12 mg of hypotaurine (Sigma-Aldrich) was weighed into each of two 100 ml conical flasks; this was 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. To start the reaction, 50 l of a commercially available solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM Na acetate pH 5.5 was added to one batch. In accordance with the manufacturer's information, the GOX activity in the batch was 5 U/ml. The batch without GOX (comparison batch) was treated with 50 l of 100 mM Na acetate pH 5.5. The batches were shaken at 30 C. and 140 rpm (Infors incubator shaker). At the start and 5 h after the start of the reaction, 1 ml from each batch was taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatants analyzed by HPLC as described above. The content of hypotaurine and taurine in the batch with GOX and in the batch without GOX at the start (0 h) and after a reaction time of 5 h is summarized in Table 4.

TABLE-US-00004 TABLE 4 Time course of the oxidation of hypotaurine to taurine by GOX in the presence of glucose +5 U/ml GOX without GOX Hypotaurine Taurine Hypotaurine Taurine Time [mg/L] [mg/L] [mg/L] [mg/L] 0 h 1238.3 0.0 1037.4 0.0 5 h 0.0 1519.7 1103.3 0.0

Example 3

Preparative Oxidation of Hypotaurine to Taurine with GOX

[0147] The reaction was investigated in two parallel batches, i.e. with different dosing of the enzyme GOX, the concentration of the hypotaurine substrate undergoing oxidation being 20 g/L in each case.

[0148] In batch 1, 200 mg of hypotaurine (Sigma-Aldrich) was weighed into a 100 ml conical flask, dissolved in 6.95 ml of 100 mM Na acetate pH 5.5, and 3 ml of a 200 g/L glucose solution in the same buffer was added. To start the reaction, 50 l of a commercially available solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM Na acetate pH 5.5 was added. In accordance with the manufacturer's information on the enzyme activity, the GOX activity in the batch was 5 U/ml. In batch 2, 200 mg of hypotaurine (Sigma-Aldrich) was weighed into a 100 ml conical flask, dissolved in 6.5 ml of 100 mM Na acetate pH 5.5, and 3 ml of a 200 g/L glucose solution in the same buffer was added. To start the reaction, 500 l of a commercially available solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM Na acetate pH 5.5 was added. In accordance with the manufacturer's information on the enzyme activity, the GOX activity in the batch was 50 U/ml.

[0149] Batches 1 and 2 were shaken at 30 C. and 140 rpm (Infors incubator shaker). 3 h, 6 h, and 24 h after the start of the reaction, 1 ml aliquots of each batch were taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatants analyzed by HPLC as described above. The course of the reactions over time is summarized in Table 5.

TABLE-US-00005 TABLE 5 Time course of the oxidation of hypotaurine to taurine by GOX in the presence of glucose +5 U/ml GOX +50 U/ml GOX Hypotaurine Taurine Hypotaurine Taurine Time [g/L] [g/L] [g/L] [g/L] 0 h 20.0 0.0 20.0 0.0 3 h 12.3 6.8 8.1 10.9 6 h 7.6 12.2 3.0 18.7 24 h 0.5 22.3 0.6 23.4

Example 4

Production 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:

[0150] CDOrn: The amino acid sequence of cysteine dioxygenase from Rattus norvegicus is disclosed in the NCBI (National Center for Biotechnology Information) database under the sequence ID: AAH70509.1. The amino acid sequence was used to derive a DNA sequence codon-optimized for expression in E. coli (publicly available Eurofins Genomics GENEius software), which was synthetically produced (Eurofins Genomics). This DNA sequence, designated CDOrn, is disclosed in SEQ ID NO: 1 and encodes a protein having the amino acid sequence from SEQ ID NO: 2.

Cysteine Sulfinic Acid Decarboxylase CSADhs:

[0151] CSADhs: The amino acid sequence of cysteine sulfinic acid decarboxylase (CSADhs) from homo sapiens is disclosed in the NCBI (National Center for Biotechnology Information) database under the sequence ID: XP_016861786.1. The amino acid sequence was used to derive a DNA sequence codon-optimized for expression in E. coli (publicly available Eurofins Genomics GENEius software). This DNA sequence, designated CSADhs, is disclosed in SEQ ID NO: 3, nt 1 to 1509 and encodes a protein having the amino acid sequence from SEQ ID NO: 4. The DNA sequence of the E. coli rrnB terminator (SEQ ID NO: 3, nt 1510 to 1842) was coupled to nt 1509. The DNA sequence of the rrnB terminator is disclosed in Orosz et al., Eur. J. Biochem. (1991) 201: 653-659. The DNA disclosed in SEQ ID NO: 3, consisting of the CSADhs cds and the rrnB terminator, was produced synthetically (Eurofins Genomics) and given the designation CSADhs-rrnB.

Production of the Vector pCys-CDOrn-CSADhs: [0152] Vector pCys (FIG. 1) refers to the plasmid pACYC184-cysEX-GAPDH-ORF306-serA317, a derivative of the plasmid pACYC184-cysEX-GAPDH-ORF306 disclosed in EP 0 885 962 B1. The plasmid pACYC184-cysEX-GAPDH-ORF306 contains not only the origin of replication and a tetracycline resistance gene (parent vector pACYC184), but also the cysEX allele, which encodes a serine O-acetyltransferase having reduced feedback inhibition by cysteine, and also the efflux gene ydeD (ORF306), the expression of which is controlled by the constitutive GAPDH promoter. [0153] To obtain pACYC184-cysEX-GAPDH-ORF306-serA317, the serA317 gene fragment encoding 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) was cloned in pACYC184-cysEX-GAPDH-ORF306 after the ydeD (ORF306) efflux gene. SerA317 encodes a serine feedback-resistant variant of 3-phosphoglycerate dehydrogenase. The expression of serA317 is controlled by the serA promoter. [0154] As a consequence of deregulated biosynthesis, E. coli cells transformed with pCys produce cysteine, the starting product for the derived products cysteine sulfinic acid and hypotaurine. [0155] Vector pCys-CDOrn-CSADhs (FIG. 2): [0156] pCys was cut with Scal and PpuMI. The 6.1 kb vector fragment thereby released was isolated by preparative agarose gel electrophoresis (QIAquick Gel Extraction Kit, Qiagen). [0157] The CDOrn DNA was amplified from the synthetic DNA SEQ ID NO: 1 (CDOrn) by PCR (Phusion High Fidelity DNA polymerase, Thermo Scientific) using the primers cdorn-1f (SEQ ID NO: 5) and csadhs-2r (SEQ ID NO: 6) and isolated as a kb fragment. [0158] Primer cdorn-1f 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 cds (SEQ ID NO 5, nt 44 to 65). [0159] Primer csadhs-2r comprised, in reverse complement form, starting from the 5 end, 22 nt overlapping with 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). [0160] CSADhs-rrnB DNA: The DNA fragment was amplified from the synthetic DNA SEQ ID NO: 3 (CSADhs-rrnB) by PCR (Phusion High Fidelity DNA polymerase, Thermo Scientific) using the primers csadhs-3f (SEQ ID NO: 7) and glf-2r (SEQ ID NO: 8) and isolated as a 1.8 kb fragment. [0161] 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). [0162] Primer glf-2r comprised, in reverse complement form, starting from the 5 end, 33 nt overlapping with the 5 end of the 6.1 kb pCys Scal/PpuMI vector fragment (nt 1 to 33 in SEQ ID NO: 8) obtained by PpuMI digestion, followed by the last 22 nt of the rrnB terminator (SEQ ID NO: 8, nt 34 to 55). [0163] Vector pCys-CDOrn-CSADhs: The 6.1 kb pCys Scal/PpuMI vector fragment, the 0.6 kb CDOrn PCR product (CDOrn DNA), and the 1.8 kb CSADhs-rrnB PCR product (CSADhs-rrnB DNA) were ligated using the NEBuilder cloning kit (NEB New England Biolabs) according to the manufacturer's instructions. [0164] E. coli NEB 10-beta cells (NEB New England Biolabs) were then transformed with the ligation mixture. Clones from the transformation were selected on LBtet. LBtet contained 10 g/L of tryptone (Gibco), 5 g/L of yeast extract (BD Biosciences), 5 g/L of NaCl, 15 g/L of agar, and 15 mg/L of tetracycline (Sigma-Aldrich). A single clone from the transformation was analyzed by culturing in LBtet liquid medium (10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of NaCl, and 15 mg/L of tetracycline) and isolating the vector from the cell pellet from culturing. The correct 8.5 kb vector was designated pCys-CDOrn-CSADhs (FIG. 2). [0165] Strain E. coli W3110: [0166] The strain used was Escherichia coli K12 W3110 (commercially available under the strain number DSM 5911 from the DSMZ: Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH [German Collection of Microorganisms and Cell Cultures]). [0167] Strain E. coli W3110-ppsA-MHI: [0168] The strain used was E. coli K12 W3110-ppsA-MHI. E. coli K12 W3110-ppsA-MHI is characterized by the mutated ppsA gene ppsA-MHI (SEQ ID NO: 9) encoding a protein having the protein sequence from SEQ ID NO: 10 and the enzymatic activity of a PEP synthase (enzyme class designated EC 2.7.9.2 in the KEGG database). E. coli K12 W3110-ppsA-MHI was produced by using the combination known to those skilled in the art of Lambda-Red recombination and counter-selection screening for genetic modification (see for example Sun et al., Appl. Env. Microbiol. (2008) 74: 4241-4245). To replace the ppsA WT gene of E. coli K12 W3110 with ppsA-MHI, the following steps were carried out: [0169] 1) E. coli K12 W3110 was transformed with the plasmid pKD46 (FIG. 3, disclosed in the GenBank gene database under access number AY048746.1) and the strain E. coli W3110 x pKD46 was isolated (ampicillin selection). [0170] 2) From the plasmid pKan-sacB (FIG. 4), the 3.2 kb Kan-sacB cassette was isolated by PCR using 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 to the site designated pr-r in FIG. 4). The plasmid pKan-sacB contains expression cassettes both for the kanamycin (Kan) resistance gene and for the sacB gene encoding the enzyme levansucrase. The E. coli kanamycin resistance gene (Kan) encoding an aminoglycoside phosphotransferase is disclosed in the NCBI database under access number SH02_03400. The B. subtilis sacB gene is disclosed in the NCBI database under access 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) and, connected thereto, 20 nt of the site designated pr-f in FIG. 4. Primer pps-10r contains the last 30 nt of the ppsA WT gene, in reverse complement form (identical to SEQ ID NO: 9, nt 2350 to 2379) and, connected thereto, 20 nt of the site designated pr-r in FIG. 4. [0171] E. coli W3110 x pKD46 was transformed with the 3.2 kb Kan-sacB cassette and kanamycin-resistant clones were isolated. [0172] 4) The clones were seeded onto LBSC plates (10 g/L of tryptone, 5 g/L of yeast extract, 7% sucrose, 1.5% agar, and 15 mg/L of kanamycin). Clones having 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. [0173] 5) W3110-ppsA::Kan-sacB x pKD46 was transformed with DNA of the ppsA-MHI gene (SEQ ID NO: 9, produced synthetically, from Eurofins Genomics) and clones selected on LBS plates (10 g/L of tryptone, 5 g/L of yeast extract, 7% sucrose, 1.5% agar) without kanamycin. Only clones that no longer contained an active sacB gene were able to grow on LBS plates. These clones were seeded onto LBkan plates (10 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of NaCl, 1.5% agar, 15 mg/L of kanamycin) in order to select those clones that also no longer contained an active Kan gene and the growth of which was inhibited in the presence of kanamycin. [0174] 6) A clone exhibiting positive growth in the presence of sucrose and negative growth in the presence of kanamycin was selected, the ppsA MHI gene was isolated by PCR from genomic DNA of the strain, and DNA sequencing (Eurofins Genomics) confirmed that the ppsA MHI gene having the DNA sequence disclosed in SEQ ID NO: 9 had been integrated, encoding a protein corresponding to the sequence from SEQ ID NO: 10. The strain, after removal of the plasmid pKD46 by incubation at 42 C., was given the designation E. coli W3110-ppsA-MHI. [0175] Production strains: Plasmid DNA of the vector pCys-CDOrn-CSADhs was transformed into the strains E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI. For comparison, E. coli K12 W3110 and E. coli K12 W3110-ppsA-MHI were transformed with the vector pCys. Transformants were selected on LBtet. One clone was in each case isolated. The strains were given the designation E. coli K12 W3110 x pCys-CDOrn-CSADhs and E. coli K12 W3110 x pCys and, by analogy, E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI x pCys respectively. E. coli K12 W3110 x pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs were used as production strains for the production of hypotaurine.

Example 5

Production of Hypotaurine in Shake Flasks

[0176] A preculture in LBtet liquid medium was produced from each of the strains 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 (cultured overnight at 37 C. and 120 rpm).

[0177] Main culture: 0.5 ml of the preculture was transferred to a 300 ml conical flask (baffled) with 30 ml of SM1-Ac medium, also containing 15 g/L of glucose, 2 g/L of Na.sub.2S.sub.2O.sub.3.Math.5H.sub.2O, 0.1 g/L of L-isoleucine, 0.1 g/L of D,L-methionine, 0.1 g/L of L-threonine, 5 mg/L of vitamin B1, and 15 mg/L of tetracycline.

[0178] Composition of the SM1-Ac medium: 12 g/L of K.sub.2HPO.sub.4, 3 g/L of KH.sub.2PO.sub.4, 5 g/L of NH.sub.4 acetate, 0.3 g/L of MgSO.sub.4.Math.7H.sub.2O, 0.015 g/L of CaCl.sub.2.Math.2H.sub.2O, 0.002 g/L of FeSO.sub.4.Math.7H.sub.2O, 1 g/L of trisodium citrate dihydrate, 0.1 g/L of NaCl; 1 ml/L of trace element solution.

[0179] Composition of the trace element solution: 0.15 g/L of Na.sub.2MoO.sub.4.Math.2H.sub.2O, 2.5 g/L of H.sub.3BO.sub.3, 0.7 g/L of CoCl.sub.2.Math..6H.sub.2O, 0.25 g/L of CuSO.sub.4.Math.5H.sub.2O, 1.6 g/L of MnCl.sub.2.Math..4H.sub.2O, 0.3 g/L of ZnSO.sub.4.Math..7H.sub.2O.

[0180] The main culture was incubated at 30 C. and 140 rpm for 24 h in an incubator shaker (Infors). After 24 h, 1 ml samples were taken and the cell density OD.sub.600/ml (optical density of the main culture, measured photometrically at 600 nm), measured using a Genesys 10S UV/visible spectrophotometer from Thermo Scientific, and the content of hypotaurine and taurine determined by HPLC. The content determined by HPLC of hypotaurine in the culture supernatant was 157.3 mg/L for E. coli K12 W3110 x pCys-CDOrn-CSADhs (cell density OD.sub.600/ml of culture: 5.3/ml). The taurine content in the culture supernatant was 52.8 mg/L.

[0181] For E. coli K12 W3110-ppsA-MHI x pCys-CDOrn-CSADhs (cell density OD.sub.600/ml of culture: 7.1/ml), the content determined by HPLC of hypotaurine in the culture supernatant was 1059.2 mg/L and the content of taurine 304.1 mg/L.

[0182] For the two comparison strains E. coli K12 W3110 x pCys (cell density OD.sub.600/ml of culture: 6.1/mL) and E. coli K12 W3110-ppsA-MHI x pCys (cell density OD.sub.600/ml of culture: 7.5/ml) neither hypotaurine nor taurine could be detected.

Example 6

Oxidation of Hypotaurine FROM the Shake-Flask Culture to Taurine

[0183] 10 ml each of the batches from the shake-flask culture of E. coli W3110 x pCys-CDOrn-CSADhs and E. coli W3110-ppsA-MHI x pCys-CDOrn-CSADhs (example 5) was centrifuged at 4000 rpm for 10 min (Heraeus Megafuge 1.0 R) and 9.4 ml of the respective supernatant transferred to a 100 ml conical flask and adjusted to pH 5.5 with 0.7 M NaOH. To this were added 0.5 ml of a 200 g/L solution of glucose in H.sub.2O (end concentration in the mixture 10 g/L) and 100 l of a 1 U/l stock solution of GOX from Aspergillus niger (Sigma-Aldrich) in 100 mM Na acetate pH 5.5 (end concentration 10 U/ml) and the batches (volume 10 ml) were incubated at 30 C. and 140 rpm in an incubator shaker (Infors). At the start and 2 h after the start of the incubation, 1 ml aliquots of each batch were taken, incubated at 80 C. for 5 min, and centrifuged at 13 000 rpm for 5 min (Heraeus Fresco 21 centrifuge) and the supernatant analyzed by HPLC.

[0184] As summarized in Table 6, the hypotaurine present in the shake-flask culture (example 5)157.3 mg/L for strain W3110 x pCys-CDOrn-CSADhs and 1059.2 mg/L for strain W3110-ppsA-MHI x pCys-CDOrn-CSADhswas completely consumed, with the formation of taurine in a concentration of 212.8 mg/L and 1355.6 mg/L respectively. The molar yield was determined taking into account the different molecular weights (109.2 g/mol for hypotaurine, 125.2 g/mol for taurine). As indicated in Table 6, for the strain W3110 x pCys-CDOrn-CSADhs, the combined content of hypotaurine (1.4 mM) and taurine (0.4 mM) at the start of the reaction was 1.8 mM. 2 h after the start of the reaction, the taurine content was 1.7 mM and hypotaurine was no longer detectable. The molar yield of taurine from the enzymatic oxidation of a hypotaurine/taurine product mixture, i.e. based on the total input of hypotaurine and taurine (1.4+0.4=1.8 mM) from the shake-flask culture was 94.4%. For the strain W3110-ppsA-MHI x pCys-CDOrn-CSADhs, the combined content of hypotaurine (9.7 mM) and taurine (2.4 mM) at the start of the reaction was 12.1 mM. 2 h after the start of the reaction, the taurine content was 10.8 mM and hypotaurine was no longer detectable. The molar yield of taurine from the enzymatic oxidation of a hypotaurine/taurine product mixture, i.e. based on the total input of hypotaurine and taurine (9.7+2.4=12.1 mM) from the shake-flask culture was 89.3%.

TABLE-US-00006 TABLE 6 Oxidation of hypotaurine from the shake-flask culture of the strains E. coli K12 W3110 pCys-CDOrn-CSADhs and E. coli K12 W3110-ppsA-MHI pCys-CDOrn-CSADhs to taurine after incubating with GOX for 2 h in the presence of glucose Hypotaurine Taurine mg/L mM mg/L mM W3110 pCys-CDOrn-CSADhs: 0 h 157.3 1.4 52.8 0.4 2 h 0.0 0.0 212.8 1.7 W3110-ppsA-MHI pCys-CDOrn-CSADhs: 0 h 1059.2 9.7 304.1 2.4 2 h 0.0 0.0 1355.6 10.8