METHOD FOR PRODUCING A HYDROXYTYROSOL

Abstract

A process for producing hydroxytyrosol (HTS) by enzymatic conversion of tyrosol to HTS is provided. The process includes a reaction mixture comprising tyrosol, a compound selected from the group consisting of erythorbic acid and erythorbate, and an oxidase with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and an amino acid sequence homologous with SEQ ID NO: 2. HTS is isolated from the reaction mixture.

Claims

1. A process for producing hydroxytyrosol (HTS) by enzymatic conversion of tyrosol to HTS, characterized in that the reaction mixture comprises i) tyrosol and ii) a compound selected from the group consisting of erythorbic acid and erythorbate and iii) an oxidase with an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and an amino acid sequence homologous with SEQ ID NO: 2 and HTS is isolated from the reaction mixture.

2. The process as claimed in claim 1, characterized in that the amino acid sequence homologous with SEQ ID NO: 2 is SEQ ID NO: 3.

3. The process as claimed in either of claims 1 and 2, characterized in that the oxidase is produced by recombinant means by fermentation in E. coli.

4. The process as claimed in claim 3, characterized in that the fermentation for production of the oxidase is effected in the presence of a concentration of at least 0.02 mM Cu(II) ions.

5. The process as claimed in either of claims 3 and 4, characterized in that the fermenter broth from the fermentation for production of the oxidase is used directly without further workup in the process for producing HTS.

6. The process as claimed in any of claims 1 to 5, characterized in that tyrosol is used in a concentration of more than 200 mM.

7. The process as claimed in any of claims 1 to 6, characterized in that the reaction mixture contains erythorbic acid or erythorbate in a concentration of at least 0.4 M.

8. The process as claimed in any of claims 1 to 7, characterized in that the molar ratio of tyrosol to erythorbic acid or erythorbate is not more than 1:1.50.

9. The process as claimed in any of claims 1 to 8, characterized in that the proportion by volume of the fermenter broth from the fermentation for production of the oxidase in the reaction mixture is up to 90%.

10. The process as claimed in any of claims 1 to 9, characterized in that the reaction mixture is reacted for a period of time until at least 90% of the tyrosol used has been converted to HTS.

11. The process as claimed in any of claims 1 to 10, characterized in that HTS is produced from tyrosol with a molar yield of at least 70%.

12. The process as claimed in any of claims 1 to 11, characterized in that HTS is isolated from the reaction mixture by extraction with ethyl acetate.

13. The process as claimed in any of claims 1 to 12, characterized in that HTS is isolated from the reaction mixture with a purity of at least 80%.

Description

BRIEF ELUCIDATION OF THE FIGURES

[0110] FIG. 1 shows the 4.5 kb vector pRscK60 produced in example 1.

[0111] FIG. 2 shows the 4.4 kb vector pRscK60-del produced in example 1.

ABBREVIATIONS USED IN THE APPLICATON

[0112] cds coding DNA sequence (see above for definition) [0113] nt nucleotide(s) [0114] HTS hydroxytyrosol [0115] HPLC high-performance liquid chromatogrphy

[0116] The invention is elucidated by the examples that follow without being restricted thereby:

EXAMPLE 1

Production of the Expression Vectors pRscK60 and pRscK60-Del

[0117] For the isolation of the cds of the RscK60 gene, genomic DNA from the Ralstonia solanacearum K60 strain (commercially available under strain number DSM 9544 at the DSMZ German Collection of Microorganisms and Cell Cultures GmbH) was used.

[0118] The coding sequence of RscK60 to be isolated (referred to hereinafter as rsck60-cds, SEQ ID NO: 1), encoding a putative polyphenol oxidase/catechol oxidase, is disclosed in the NCBI (National Center for Biotechnology Information) nucleotide database under Locus Tag CAGT01000120.1, nt 3460-5091 (SEQ ID NO: 1), encoding a protein with Genbank accession number CCF97399.1 (SEQ ID NO: 2), referred to hereinafter as RscK60 oxidase.

[0119] The vectors pRscK60 and pRscK60-del were produced using the following DNA fractions from the putative cds of the polyphenol oxidase/catechol oxidase: [0120] rsck60-cds: SEQ ID NO: 1 nt 1-nt 1632, encoding a protein with amino acid sequence SEQ ID NO: 2, referred to hereinafter as RscK60 oxidase. [0121] rscK60-del-cds: SEQ ID NO: 1 nt 142-nt 1632, encoding a protein with amino acid sequence SEQ ID NO: 3, referred to hereinafter as RscK60-del oxidase. RscK60-del oxidase is a protein with N-terminal truncation by 47 amino acids compared to RscK60 oxidase.

[0122] The DNA fragment rscK60-cds was isolated in a PCR reaction (Phusion? High-Fidelity DNA polymerase, Thermo Scientific?) as a 1.6 kb fragment. For this purpose, genomic DNA from the R. solanacearum K60 strain and the primers rsck60-If (SEQ ID NO: 4) and rsck60-2r (SEQ ID NO: 5) were used.

[0123] The DNA fragment rscK60-del-cds was isolated in a PCR reaction (Phusion? High-Fidelity DNA polymerase, Thermo Scientific?) as a 1.5 kb fragment. For this purpose, genomic DNA from the R. solanacearum K60 strain and the primers rsck60-3f (SEQ ID NO: 6) and rsck60-2r (SEQ ID NO: 5) were used.

[0124] Primer rsck60-1f contains an EcoRI cleavage site adjoined by 23 nucleotides (nt) beginning with the start of the cds of RscK60 oxidase (nt 1-23 in SEQ ID NO: 1).

[0125] Primer rsck60-2r contains a HindIII cleavage site adjoined by 24 nucleotides (nt) from the 3 region of the cds of RscK60 oxidase (nt 1609-1632 in SEQ ID NO: 1, in reversed complementary form).

[0126] Primer rsck60-3f contains an EcoRI cleavage site adjoined by 24 nucleotides (nt) from the 5? region of the cds of RscK60 oxidase (nt 142-165 in SEQ ID NO: 1).

[0127] The PCR products were cleaved with EcoRI (present in the primers rsck60-If and rsck60-3f) and HindIII (present in primer rsck60-2r) and cloned into the pKKj expression vector that had been cleaved beforehand with EcoRI and HindIII. This gave rise to the 4.5 kb expression vector pRscK60 (FIG. 1) and the 4.4 kb expression vector pRscK60-del (FIG. 2).

[0128] The expression vector pKKj, disclosed in EP 2 670 837 A1, is a derivative of the expression vector pKK223-3. The DNA sequence of pKK223-3 is disclosed in the GenBank gene database under accession number M77749.1. About 1.7 kb were removed from the 4.6 kb plasmid (bp 262-1947 of the DNA sequence disclosed in M77749.1), which gave rise to the 2.9 kb expression vector pKKj.

EXAMPLE 2

Analytical Tests

Cell Suspensions and Isolated Cells:

[0129] Cells cultured in a shaken flask (example 3) or in a fermenter (example 4) were either used directly as cell suspensions (culture broth, fermenter broth) without further isolation for analytical tests or the cells were isolated.

[0130] The cells were isolated from a suspension (culture broth, fermenter broth) by centrifugation of the suspension (10 min 15 000 rpm, Sorvall RC5C centrifuge, equipped with an SS34 rotor). The resultant cell pellet was washed once with 0.9% (w/v) NaCl. For further use as isolated cells, the cell pellet from a 100 mL culture was suspended in 20 mL of KPi buffer (50 mM potassium phosphate, 1 mM EDTA, pH 6.5).

Production of a Cell Homogenate:

[0131] For production of a cell homogenate, the FastPrep-24? 5G cell homogenizer from MP Biomedicals was used. 2?1 mL of cell suspension was digested in 1.5 mL tubes containing glass beads (Lysing Matrix B) that had been prefabricated by the manufacturer (3?20 sec at a shaken frequency of 6000 rpm with breaks of 30 sec in each case between the intervals).

Production of an Enzyme Extract:

[0132] A cell-free enzyme extract was produced from the cell homogenate by centrifugation (10 min 15 000 rpm, Sorvall RC5C centrifuge, equipped with an SS34 rotor) and isolation of the resulting supernatant.

Determination of the Protein Content of a Sample:

[0133] The protein content of cell suspensions, isolated cells, cell homogenates or enzyme extracts was determined with a Qubit 3.0 fluorometer from Thermo Fisher Scientific using the Qubit? Protein Assay Kit according to the manufacturer's instructions.

Determination of the Oxidase Enzyme Activity (L-DOPA Test):

[0134] The oxidase enzyme activity was determined using a photometric test in which the oxidation of the L-DOPA enzyme substrate (3,4-dihydroxy-L-phenylalanine, CAS number 59-92-7) to the dopachrome chromophore (CAS number 3571-34-4) at a wavelength of 475 nm is monitored (Behbahani et al. (1993), Microchemical J. 47: 251-260). The enzyme test was conducted with cell suspensions (e.g. monitoring of the progression of production in the fermenter), isolated cells, cell homogenate or cell-free enzyme extract.

[0135] A test batch contained, in a 100 mL Erlenmeyer flask, in a batch volume of 8 mL: 4 mL of KPi buffer, 10 mM L-DOPA (Sigma-Aldrich) and 4 mL of the sample to be tested (cell suspension, isolated cells, cell homogenate or cell-free enzyme extract).

[0136] If samples from the shaken flask culture were to be tested, the cells were first concentrated by centrifuging 25 mL of shaken flask mixture for isolation of a cell pellet (10 min 15 000 rpm, Sorvall RC5C centrifuge, equipped with an SS34 rotor), the resultant cell pellet was used as described above for production of isolated cells, cell homogenate or cell-free enzyme extract, and, for further use, the volume of the sample to be tested was adjusted to 4 mL with KPi buffer.

[0137] If samples from fermentation were to be tested, 1.6 mL of fermentation batch was used directly as cell suspension or as described above for production of isolated cells, cell homogenate or cell-free enzyme extract, and, for further use, the volume of the sample to be tested was adjusted to 4 mL with KPi buffer.

[0138] The reaction was started by adding the respective sample to be tested. The test batches were incubated in a shaker (Infors) at 37? C. and 140 rpm. 1 mL aliquots were taken at the time points 0 min, 30 min, 60 min and 120 min, and centrifuged immediately at 13 000 rpm for 5 min (Heraeus? Fresco? 21 centrifuge, Thermo Scientific?), and the absorbance of the supernatant was determined at 475 nm (Genesys? 10S UV-VIS spectrophotometer, Thermo Scientific?).

[0139] 1 unit (U) of oxidase activity is defined as the amount of enzyme that produces 1 ?mol dopachrome/min from L-DOPA under test conditions (extinction coefficient of dopachrome ?.sub.475nm=0.37 x 10.sup.4 L x Mol.sup.?1 x cm.sup.?1).

[0140] The specific oxidase activity was calculated by basing the oxidase enzyme activity on 1 mg of total protein in the measured sample (cell extract, homogenate or cell suspension) (U/mg of protein).

[0141] The (specific) oxidase activity determined by L-DOPA test is referred to hereinafter as (specific) oxidase activity in the L-DOPA test.

Determination of Tyrosol and HTS by HPLC (HPLC Test):

[0142] A test biotransformation of tyrosol to HTS was conducted on an analytical scale. Tyrosol solution: 7 mg of tyrosol (Sigma-Aldrich, final concentration 5.1 mM in the test) was weighed into a 100 mL Erlenmeyer flask, dissolved in 4.9 mL of KPiE buffer (50 mM potassium phosphate, 10 mM EDTA, pH 6.5), and supplemented with 0. 1 mL of 1 M sodium D-erythorbate x H.sub.2O (Sigma-Aldrich, final concentration 10 mM in the test). For comparative purposes, in individual tests, 7 mg of tyrosol was dissolved in 5 mL of KPiE buffer without addition of sodium D-ery thorbate.

[0143] Test batches: 50 mL of suspension of the cells cultured in a shaken flask, or 10 mL of the cells cultured in the fermenter, was suspended in 5 mL of KPiE buffer and added to the tyrosol solution at the start of the reaction. The test batches (volume 10 mL) were incubated on a shaker (Infors) at 30? C. and 140 rpm. Samples each of 1 mL were taken at the time points 0 h, 1 h, 2 h and 4 h, and, in order to stop the reaction, admixed immediately with 0.1 mL in each case of conc. H.sub.3PO.sub.4. After centrifugation (5 min at 13 000 rpm, Heraeus? Fresco? 21 centrifuge, Thermo Scientific?M), 1 mL in each case of the supernatant was dispensed for the determination of tyrosol and HTS by HPLC.

HPLC Analysis of Tyrosol and HTS:

[0144] For quantitative determination of tyrosol and HTS, an HPLC method respectively calibrated for tyrosol and HTS was used. The reference substances tyrosol and HTS for calibration came from Sigma-Aldrich. An Agilent Infinity II HPLC instrument was used, equipped with a diode array detector. The detector was set to the wavelength of 274 nm. In addition, a Luna C18(2) column from Phenomenex, length 250 mm, internal diameter 4.6 mm, particle size 5 ?m, was adjusted to a temperature of 30? C. in the column oven. Eluent A: 5 mL of H.sub.3PO.sub.4 in 1 L of H.sub.2O. Eluent B: acetonitrile. The separation was effected in gradient mode from 5% to 10% eluent B within 5 min, followed by 10% to 16% eluent B within 15 min at a flow rate of 1 mL/min. Retention time of tyrosol: 13.9 min: retention time of HTS: 10 min.

[0145] The yield of the reaction in the context of the invention is defined as the amount of tyrosol used (reactant) which is converted under reaction conditions to HTS (product). The yield may be reported as volume yield in the absolute amount of product based on volume (mM or g/L) or as relative yield of the product in percent (also referred to as percentage yield), i.e. the absolute yield is based on the tyrosol used (reactant) (taking account of the molecular weights of 138.2 g/mol for tyrosol (reactant) and 154.2 g/mol for HTS (product)).

EXAMPLE 3

Expression of RscK60 and of RscK60-Del Oxidase in E. coli by Shaken Flask Culture

[0146] For isolation of plasmid DNA, the expression vectors pRscK60 and pRscK60-del from example I were each transformed into a commercially available E. coli strain NEBR 10-beta (New England Biolabs) which is used for cloning purposes. A cell culture was produced (37? C., 120 rpm, Infors tray shaker) for each clone from the transformation by culturing in LBamp medium (10 g/L tryptone, GIBCOTM, 5 g/L yeast extract from BD Biosciences, 5 g/L NaCl, 100 mg/L ampicillin), and plasmid DNA was isolated from the cells with a plasmid DNA isolation kit in accordance with the manufacturer's instructions (QIAprep? Spin Miniprep Kit, Qiagen).

[0147] Plasmid DNA of the expression vectors pRscK60 and pRscK60-del was transformed by known methods into the E. coli K12 strain JM105. The E. coli JM105 strain is commercially available under strain number DSM 3949 from DSMZ German Collection of Microorganisms and Cell Cultures GmbH.

[0148] Clones for the transformation were selected on Lbamp plates (10 g/L tryptone, GIBCO?, 5 g/L yeast extract from BD Biosciences, 5 g/L NaCl, 15 g/L agar, 100 mg/mL ampicillin) and respectively identified as JM105 x pRscK60 and JM105 x pRscK60-del. The control used was E. coli JM105, transformed with the pKKj vector (E. coli JM105 x pKKj), from which transformants were produced in the same way.

[0149] A pre-culture was produced (culture at 37? C. and 120 rpm overnight, Infors tray shaker) for each clone of the E. coli strains JM105 x pKKj, JM105 x pRscK60 and JM105 x pRscK60-del in 30 mL of Lbamp medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 100 mg/mL ampicillin).

[0150] 2 mL of each preculture were used as inoculum for a main culture of 100 mL of SM3 medium (1 L Erlenmeyer flask), supplemented with 15 g/L glucose, 0.5 mM CuSO.sub.4 x 5 H.sub.2O and 100 mg/L ampicillin. The main culture was shaken at 30? C. and 140 rpm until a cell density OD.sub.600 of 2.0 was attained (OD.sub.600: photometric determination of cell density by determination of absorbance at 600 nm). Then the IPTG inductor (isopropyl-?-thiogalactoside, final concentration 0.4 mM, Sigma-Aldrich) was added and the mixture was shaken at 30? C. and 140 rpm overnight.

[0151] Composition of the SM3 medium: 12 g/L K.sub.2HPO.sub.4, 3 g/L KH.sub.2PO.sub.4, 5 g/L (NH.sub.4).sub.2SO.sub.4, 0.3 g/L MgSO.sub.4 x 7 H.sub.2O, 0.015 g/L CaCl.sub.2 x 2 H.sub.2O, 0.002 g/L FeSO.sub.4 x 7 H.sub.2O, 1 g/L Na.sub.3 citrate x 2 H.sub.2O, 0.1 g/L NaCl: 5 g/L peptone (Oxoid): 2.5 g/L yeast extract (BD Biosciences): 0.005 g/L vitamin B1 (Sigma-Aldrich): 1 mL/L trace element solution.

[0152] Composition of the trace element solution: 0.15 g/L Na.sub.2MoO.sub.4 x 2 H.sub.2O, 2.5 g/L H.sub.3BO.sub.3, 0.7 g/L CoCl.sub.2 x 6 H.sub.2O, 0.25 g/L CuSO.sub.4 x 5 H.sub.2O, 1.6 g/L MnCl.sub.2 x 4 H.sub.2O, 0.3 g/L ZnSO.sub.4 x 7 H.sub.2O.

[0153] Subsequently, the cells from the shaken flask culturing were used to verify enzyme activity by the HPLC test and the photometric L-DOPA test.

Comparison of Activity of RscK60 Oxidase with the Activity of RscK60-Del Oxidase:

[0154] For HPLC tests, the cells from 50 mL of a shaken flask culture of the E. coli JM105x pRscK60 (cell density OD.sub.600 of 6.4/mL) and JM105 x pRscK60-del (cell density OD.sub.600 of 5.9/mL) strains were isolated by centrifugation and in each case suspended in 5 mL of KPiE buffer. 5 mL in each case of the isolated and resuspended cells was used in the HPLC test described in example 2.

[0155] Two HPLC tests were conducted. One batch contained, in a batch volume of 10 mL: 5 mL of the isolated and resuspended JM105 x pRscK60-del cells from the above-described shaken flask culture, 7 mg of tyrosol (final concentration in the batch 5.1 mM), 4.9 mL KPiE buffer and 0.1 mL of 1 M sodium D-erythorbate x H.sub.2O, dissolved in KPiE buffer (see HPLC test in example 2). The second batch contained, likewise in a batch volume of 10 mL: 5 mL of the isolated and resuspended JM105 x pRscK60 cells from the above-described shaken flask culture, 7 mg of tyrosol, 4.9 mL of KPiE buffer and 0.1 mL of 1 M sodium D-erythorbate x H.sub.2O, dissolved in KPiE buffer. The batches were incubated on a shaker (Infors) at 30? C. and 140 rpm. Samples from the test were taken after 0 h, 2 h, 4 h and 6 h, and analyzed by HPLC. The results are reported in table 1.

TABLE-US-00001 TABLE 1 Activity of isolated E. coli K12 JM105 ? pRscK60-del cells that express RscK60-del oxidase and of isolated E. coli K12 JM105 ? pRscK60 cells that express RscK60 oxidase from the shaken flask culture with regard to the biotransformation of tyrosol to HTS RscK60-del oxidase RscK60 oxidase Time [h] Tyrosol [mM] HTS [mM] Tyrosol [mM] HTS [mM] 0 5.1 0.1 4.1 0.1 2 2.1 3.7 2.0 2.3 4 0.0 5.1 1.0 3.5 6 0.0 5.1 0.4 4.1

Photometric L-DOPA Test:

[0156] The enzyme activity of the JM105 x pRscK60-del strain was determined by comparison with the comparative strain JM105 x pKKj by the photometric L-DOPA test. For cells of the two strains, a cell homogenate was produced (digestion of the cells as described in example 2) and used in the L-DOPA test with L-DOPA as enzyme substrate. The specific enzyme activity, determined in the L-DOPA test with L-DOPA as enzyme substrate, was 1.5 mU/mg for the cell homogenate of the E. coli JM105 x pRscK60-del strain and 0 mU/mg for that of the JM105 x pKKj control strain.

Variation of the Cu(II) Concentration in the Culture Medium:

[0157] In preliminary experiments for optimization of the RscK60-del enzyme activity in the shaken flask culture, one parameter varied was the concentration of the Cu(II) concentration in the culture medium. The supplementation of the SM3 culture medium with Cu(II) ions was found to be advantageous. The SM3 culture medium, from the addition of the trace element solution with 0.25 mg/L CuSO.sub.4 x 5 H.sub.2O (final concentration in the culture medium 1 ?M), intrinsically contained only a low content of Cu(II) ions.

[0158] The effect of Cu(II) ions on the enzyme activity of RscK60-del oxidase was tested, as described above, by shaken flask culturing of the E. coli JM105 x pRscK60-del strain in SM3 medium, 15 g/L glucose, 100 mg/L ampicillin, to which CuSO.sub.4 x 5 H.sub.2O had additionally been added in concentrations of 0 mM to 0.5 mM. The cells from the batches were used as cell homogenate for the determination of activity by L-DOPA test (table 2).

TABLE-US-00002 TABLE 2 Dependence of the oxidase activity determined in the L-DOPA test on the Cu(II) ion concentration in the culture medium CuSO.sub.4 ? 5 H.sub.2O Oxidase activity in the L-DOPA test [mM]* [mU/mL] 0 0.432 0.02 0.649 0.05 0.892 0.1 1.198 0.2 2.631 0.5 5.802 *All values are based on the concentration of the Cu(II) ions that have been added to the SM3 medium. The concentration of Cu(II) ions present in the SM3 medium of 1 ?m was not taken into account.

Effect of Sodium D-Erythorbate on HTS Stability:

[0159] For HPLC tests, the combined cells from 2 x 50 mL shaken flask cultures of the E. coli JM105 x pRscK60-del strain (cell density OD.sub.600 of 4.7/mL) were isolated by centrifugation and resuspended in 10 mL of KPiE buffer. 5 mL in each case of the isolated and resuspended cells were used in the HPLC test described in example 2.

[0160] Two comparative test biotransformations were conducted. One batch contained, in a batch volume of 10 mL: 5 mL of the isolated and resuspended JM105 x pRscK60-del cells from the above-described shaken flask culture, 7 mg of tyrosol (final concentration in the batch 5.1 mM) and 5 mL of KPiE buffer. The second batch contained, likewise in a batch volume of 10 mL: 5 mL of the isolated and resuspended JM105 x pRscK60-del cells, 7 mg of tyrosol, 4.9 mL of KPiE buffer and 0.1 mL of 1 M sodium D-erythorbate x H.sub.2O, dissolved in KPiE buffer (see HPLC test in example 2). The batches were incubated on a shaker (Infors) at 30? C. and 140 rpm. Samples from the test were taken after 0 h, 1 h, and 4 h, and analyzed by HPLC. The results are reported in table 3.

TABLE-US-00003 TABLE 3 Activity of isolated E. coli K12 JM105 ? pRscK60-del cells that express RscK60-del oxidase from the shaken flask culture with regard to the biotransformation of tyrosol to HTS without or in the presence of sodium D-erythorbate Tyrosol [mM] HTS [mM] Tyrosol [mM] HTS [mM] without without in the in the addition addition presence of presence of Time of sodium of sodium 10 mM sodium 10 mM sodium [h] erythorbate erythorbate erythorbate erythorbate 0 5.1 0.0 5.1 0.0 1 1.6 1.7 0.8 4.5 4 0.0 2.1 0.0 5.2

EXAMPLE 4

Expression of RscK60-Del Oxidase in E. coli by Fermentation

[0161] For the fermentation, the E. coli JM105 x pRscK60-del strain was used. The fermentations were conducted in Biostat B fermenters (working volume 2 1) from Sartorius BBI Systems GmbH.

Shaken Flask Preculture:

[0162] 2 x 100 mL of LBamp medium in 1 1 baffled Erlenmeyer flasks were inoculated from an agar plate with the JM105 x pRscK60-del strain and incubated on an incubation shaker (Infors) at 30? C. and a speed of 120 rpm for 7-8 h up to a cell density OD.sub.600 of 2/mL-4/mL.

Prefermenter:

[0163] 1.5 L of FM2 medium, supplemented with 40 g/L glucose and 100 mg/L ampicillin, was inoculated with 7.5 mL of the shaken flask preculture. The fermentation conditions were: temperature 30? C.: pH constant at 7.0 (automatic correction with 25% NH.sub.4OH and 6.8 N HPO.sub.4 as described below); foam control by automatic metering of 4% v/v Struktol J673 in H.sub.2O (Schill & Seilacher): stirrer speed 450-1300 rpm: constant ventilation with compressed air sterilized by means of a sterile filter at 1.7 vvm (vvm: input of compressed air into the fermentation batch reported in liters of compressed air per liter of fermentation volume per minute): pO.sub.2?50%. The partial oxygen pressure pO.sub.2 was controlled via the stirrer speed. After a fermentation time of 16 h, a cell density OD.sub.600 of 45/mL-60/mL was achieved.

Production Fermenter:

[0164] 1.35 L of FM2 medium, pH 7.0, supplemented with 20 g/L glucose, 0.5 mM CuSO.sub.4 x 5 H.sub.2O and 100 mg/L ampicillin, was inoculated with 150 mL of prefermenter culture. The fermentation conditions were: temperature 30? C.: pH constant at 7.0 (automatic correction with 25% NH.sub.4OH and 6.8 N H.sub.3PO.sub.4 as described below); foam control by automatic dosage of 4% v/v Struktol J673 in H.sub.2O (Schill & Seilacher): stirrer speed 450-1300 rpm: constant ventilation at 1.7 vvm: pO.sub.2?50%. The partial oxygen pressure pO.sub.2 was controlled via the stirrer speed. The fermentation time was 30-32 h.

[0165] FM2 medium: (NH.sub.4).sub.2SO.sub.4, 5 g/L: NaCl, 0.50 g/L: FeSO.sub.4 x 7 H.sub.2O, 0.075 g/L: Na.sub.3 citrate, 1 g/L, MgSO.sub.4 x 7 H.sub.2O, 0.30 g/L, CaCl.sub.2 x 2 H.sub.2O, 0.015 g/L, KH.sub.2PO.sub.4, 1.50 g/L, vitamin B1 (Sigma-Aldrich), 0.005 g/L: peptone (Oxoid), 5.00 g/L: yeast extract (BD Biosciences), 2.50 g/L: trace element solution, 10 mL/L (corresponding to that used in example 2).

[0166] The pH in the fermenter was adjusted to 7.0 at the start by pumping in a 25% NH.sub.4OH solution. During the fermentation, the pH was kept at a value of 7.0 by automatic correction with 25% NH.sub.4OH, or 6.8 N H.sub.3PO.sub.4. For inoculation, 150 mL of prefermenter culture was pumped into the fermenter vessel. The starting volume was thus 1.5 L. At the start, the cultures were stirred at 350 rpm and sparged at a ventilation rate of 1.7 vvm. Under these starting conditions, the oxygen probe was calibrated to 100% saturation before the inoculation.

[0167] The target value for the O.sub.2 saturation (pO.sub.2) during the fermentation was adjusted to 50%. After the O.sub.2 saturation had dropped below the target value, a closed-loop control cascade was started, in order to bring the O.sub.2 saturation back to the target value. The stirrer speed was increased continuously (up to max. 1500 rpm).

[0168] The fermentation was conducted at a temperature of 30? C. Once the glucose content in the fermenter, from initially 20 g/L, had dropped to about 5 g/L, a 60% (w/w) glucose solution was fed in continuously. The feed rate was adjusted such that the glucose concentration in the fermenter never exceeded 2 g/L again thereafter. Glucose was determined with a glucose analyzer from YSI (Yellow Springs, Ohio, USA).

[0169] Once the target density in the fermenter had reached an OD.sub.600 of 50/mL-60/mL (fermentation time 7.5 h), the expression of the oxidase RscK60-del was started by a single addition of the IPTG inductor (final concentration 0.4 mM). 22.5 h after induction, corresponding to a total fermentation time of 30 h, the fermentation was stopped and the enzyme activity (L-DOPA test) and the conversion of tyrosol to HTS on an analytical scale (HPLC test) were quantified in a sample of the fermentation batch as described in example 2. In both tests, the fermenter broth was used directly without further workup. The remaining fermenter broth was dispensed in 50 mL aliquots, frozen and stored at ?20? C. for further experiments.

[0170] The specific enzyme activity of the fermenter broth without further workup with L-DOPA as enzyme substrate (L-DOPA test) was 28.4 mU/mg protein, using 1.6 mL of fermenter broth with a protein concentration of 5 mg/mL in the L-DOPA test.

[0171] In the HPLC test for examination of the enzyme activity in the presence of sodium D-erythorbate (example 2), 100 ?l of fermenter broth (OD.sub.600 63.8/mL) was used in the HPLC test after a fermentation time of 30 h, such that the actual cell density in the 10 mL test batch was 0.64/mL. Samples from the test were taken after 0 h, 1 h, 2 h and 4 h, the content of tyrosol and HTS was analyzed by HPLC, and the percentage yield, i.e. the proportion of tyrosol used that was converted to HTS, was determined (percentage of HTS). After an incubation time of 4 h, 99.4% of the tyrosol used had been converted to HTS (see table 4).

TABLE-US-00004 TABLE 4 Percentage of HTS over the course of a biotransformation of tyrosol to HTS with E. coli K12 JM105 ? pRscK60-del fermenter cells that express RscK60-del oxidase Time [h] Proportion of HTS [%] 0 0.7 1 82.6 2 95.0 4 99.4

[0172] In a further batch, the suitability of the fermenter cells of E. coli strain JM105 x pRscK60-del, which is important for economic viability of the process, for the biotransformation of tyrosol to HTS was examined with regard to the conversion of tyrosol in a maximum concentration in a minimum period of time (space-time yield).

[0173] An initial charge of 249 mg of tyrosol (final concentration 180 mM) and 389 mg of sodium D-erythorbate x H.sub.2O (180 mM) in a 100 mL Erlenmeyer flask was dissolved in 1 mL of KPi buffer, and 9 mL fermenter broth of the RscK60-del oxidase-expressing JM105 x pRscK60-del cells was added without further workup, in order to start the reaction. The batch volume was 10 mL. Molar ratio of tyrosol to sodium D-erythorbate x H.sub.2O was 1:1. The pH of the reaction actually measured in the batch was 6.7. The batch was incubated on a shaker (Infors) at 37? C. and 140 rpm. Sampling and analysis by HPLC (as described in example 2) were effected after 0 h, 2 h and 4 h. The progression of the reaction is shown in table 5.

TABLE-US-00005 TABLE 5 Progression against time of the biotransformation of tyrosol to hydroxytyrosol (HTS) with RscK60-del fermenter cells Time [h] Tyrosol [g/L] HTS [g/L] 0 24.9 0.0 2 10.2 14.0 4 0.0 25.5

EXAMPLE 5

Production of HTS

[0174] Experiment 1: Biotransformation of 30 g/L tyrosol with JM105 x pRscK60-del fermenter cells that express RscK60-del oxidase in the presence of sodium D-erythorbate x H.sub.2O in a molar ratio of tyrosol to sodium D-erythorbate of 1:1

[0175] An initial charge of 300 mg of tyrosol (final concentration 220 mM) and 475.4 mg of sodium D-erythorbate x H.sub.2O (220 mM) in a 100 mL Erlenmeyer flask was dissolved in 5 mL of KPIE buffer, and 5 mL of fermenter broth of the E. coli K12 JM105 x pRscK60-del strain from example 4 was added without further workup in order to start the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x H.sub.2O was 1:1. The pH of the reaction actually measured in the batch was 6.7. The batch was incubated on a shaker (Infors) at 30? C. and 140 rpm. Sampling and analysis by HPLC (example 2) were effected after 0 h, 3 h and 24 h. The progression of the reaction is shown in table 6.

TABLE-US-00006 TABLE 6 Progression against time of the biotransformation of tyrosol to HTS with E. coli K12 JM105 ? pRscK60-del fermenter cells Tyrosol Tyrosol HTS HTS Time [h] [g/L] [mM] [g/L] [mM] 0 28.36 205.20 0.10 0.63 3 22.30 161.34 7.98 51.78 24 0.00 0.00 30.53 197.99

[0176] Experiment 2: Biotransformation of 60 g/L tyrosol with RscK60-del fermenter cells in the presence of sodium D-erythorbate x H.sub.2O (molar ratio of tyrosol to sodium D-erythorbate 1:1.2)

[0177] An initial charge of 600 mg of tyrosol (final concentration 434 mM) and 1126 mg of sodium D-erythorbate x H.sub.2O (521 mM) in a 100 mL Erlenmeyer flask was suspended in 5 mL of KPiE buffer. While sodium D-erythorbate x H.sub.2O had good solubility under these conditions, tyrosol could be reacted only in suspension. 5 mL of fermenter broth of the E. coli K12 JM105 x pRscK60-del strain from example 4 was added without further workup in order to start the reaction. The batch volume was 10 mL. The molar ratio of tyrosol to sodium D-erythorbate x H.sub.2O was 1:1.2. The batch was incubated on a shaker (Infors) at 30? C. and 140 rpm. Sampling and analysis by HPLC (example 2) were effected after 0 h, 3 h and 24 h. The progression of the reaction is shown in table 7.

TABLE-US-00007 TABLE 7 Progression against time of the biotransformation of tyrosol to HTS with E. coli K12 JM105 ? pRscK60-del fermenter cells Tyrosol Tyrosol HTS HTS Time [h] [g/L] [mM] [g/L] [mM] 0 60.0 434 0.1 1 3 50.6 366 8.6 56 24 0.0 0 66.7 433

[0178] Experiment 3: Biotransformation of 100 g/L tyrosol with RscK60-del fermenter cells in the presence of sodium D-erythorbate x H.sub.2O (molar ratio of tyrosol to sodium D-erythorbate 1:1.2)

[0179] To an initial charge of 1000 mg of tyrosol (final concentration 724 mM) and 1885 mg of sodium D-erythorbate x H.sub.2O (872 mM) in a 100 mL Erlenmeyer flask were added 1 mL of KP.sub.2 buffer (500 mM potassium phosphate, 10 mM EDTA, pH 6.5) and 9 mL of fermenter broth of the cells JM105 x pRscK60 that express RscK60-del oxidase from example 4 without further workup. While sodium D-erythorbate x H.sub.2O had good solubility under these conditions, tyrosol could be reacted only in suspension. The batch volume was 10 mL. The molar ratio of tyrosol and sodium D-erythorbate x H.sub.2O was 1:1.2. The batch was incubated on a shaker (Infors) at 37? C. and 140 rpm. Sampling and analysis by HPLC (example 2) were effected after 3 h, 6 h, 24 h and 29 h. The progression of the biotransformation against time is shown in table 8.

TABLE-US-00008 TABLE 8 Progression against time of the biotransformation of tyrosol to HTS with E. coli K12 JM105 ? pRscK60-del fermenter cells Tyrosol Tyrosol HTS HTS Time [h] [g/L] [mM] [g/L] [mM] 3 104.2 750 14.6 90 6 108.3 780 34.5 220 24 17.5 130 85.9 560 29 6.1 40 109.0 707

EXAMPLE 6

Extraction of HTS

[0180] 6.5 mL of the biotransformation from example 5 experiment 2 was extracted four times with 10 mL of ethyl acetate each time (ethyl ethanoate, CAS number 141-78-6) and, after phase separation, the upper ethyl acetate phases were removed and combined. The combined ethyl acetate phases (volume 40 mL) were analyzed by HPLC for the presence of tyrosol and HTS as described in example 2, but only HTS could be detected. On the basis of the HPLC analysis, the purity of the HTS was 97%.

[0181] The molar yield of HTS was calculated. The amount of the tyrosol reactant used was 390 mg (6.5 mL of the 60 g/L tyrosol batch), corresponding to 2.8 mmol of tyrosol (molecular weight of tyrosol: 138.2 g/mol). Given a 100% yield, the result was 2.8 mmol HTS, corresponding to 431.8 mg (molecular weight of HTS: 154.2 g/mol). Determination by HPLC found 404.8 mg of HTS, which corresponded to 93.8% of the theoretical yield.

[0182] The solvent of the extract was separated off in a rotary evaporator (B?chi Rotavapor R-205)(bath temperature 62? C., reduced pressure of 500 mbar) and residues of the solvent were removed by reducing the pressure. The remaining brownish-yellow oil was weighed. The yield was 400 mg, which was in good agreement with the yield determined by HPLC.

EXAMPLE 7

Production of HTS on a Preparative Scale

[0183] A jacketed 1 L thermostatable glass vessel (Diehm) was connected via a hose connection to a thermostat (Lauda) and adjusted to a temperature of 37? C. To an initial charge of 35 g of tyrosol (final concentration 507 mM) and 65.6 g of sodium D-erythorbate x H.sub.2O (607 mM) in the glass vessel were added 250 mL of KP.sub.3 buffer (50 mM potassium phosphate, 5 mM EDTA, pH 6.5) and 250 mL of fermenter broth of the E. coli K12 JM105 x pRscK60-del strain (example 4) without further workup. The molar ratio of tyrosol to sodium D-erythorbate x H.sub.2O was 1:1.2. The batch volume was 0.5 L. The mixing was effected by means of a magnetic stirrer. For oxygen supply, compressed air was introduced into the batch by a glass tube. The reaction was started by starting the magnetic stirrer and the sparging. Sampling and analysis by HPLC (example 2) were effected after 0 h, 3 h, 6 h, and 24 h. At that time, the tyrosol had been 100% converted. The progression of the biotransformation against time is summarized in table 9.

TABLE-US-00009 TABLE 9 Biotransformation of tyrosol to HTS with RscK60- del fermenter cells on a preparative scale Tyrosol Tyrosol HTS HTS Time [h] [g/L] [mM] [g/L] [mM] 0 70.0 506.5 0.0 0.0 3 46.9 339.2 17.1 110.6 6 26.5 191.8 41.0 266.1 24 0.0 0.0 70.6 458.0

[0184] The molar yield of 458 mM HTS was 90.4%, based on the amount of 506.5 mM tyrosol used at the start of the reaction.

[0185] For isolation of HTS, 0.5 L of the biotransformation was first incubated at 80? C. while mixing with a magnetic stirrer for 30 min and then centrifuged at 4000 rpm for 30 min (Heraeus Megafuge 1.0 R) in order to separate off particulate material. The supernatant was extracted three times with ethyl acetate. The first extraction was with 1 L of ethyl acetate, and the second and third extractions each with 0.5 L of ethyl acetate. The ethyl acetate phases were combined and gave 2 L of extract.

[0186] The ethyl acetate was distilled off in a rotary evaporator (B?chi Rotavapor R-205), first at a reduced pressure of 270 mbar and a temperature of 60? C. and then at a reduced pressure of 20 mbar and a temperature of 85? C., in order to remove residual ethyl acetate. Removal of the ethyl acetate left a residue of 34.1 g, which corresponded to the HTS product of the process. To determine the purity, 32 mg of the HTS product was weighed out, dissolved in 1 mL of H.sub.2O (concentration 32 mg/mL) and analyzed by HPLC. The HPLC analysis gave an HTS content of 30 mg/mL), which corresponded to a purity of 93.8% based on the amount of 32 mg of the HTS product weighed out.

[0187] The yield of HTS was calculated. The amount of the tyrosol reactant used was 35 g. Taking account of the differences in molecular weight (138.2 g/mol for tyrosol, 154.2 g/mol for HTS), a maximum yield of 39 g of HTS was to be expected. Taking account of the purity of 93.8%, 34.1 g of the HTS product contained 31.9 g of HTS. For the overall process, this corresponded to a yield of 81.7%, based on the maximum achievable yield of 39 g of HTS.