POLYNUCLEOTIDE ENCODING AN AMINO ACID SEQUENCE, ENCODING AN OXIDOREDUCTASE

Abstract

A polynucleotide, encoding an amino acid sequence, encoding an oxidoreductase, that is ≥50% identical to an amino acid sequence of SEQ ID NO:1 (Geobacillus sp. PA9), SEQ ID NO:3 (Thermus thermophilus), SEQ ID NO:4 (Streptomyces globisporus), SEQ ID NO:5 (Clostridium aminobutyricum), SEQ ID:6 (Burkholderai cepacia), SEQ ID NO:8 (Oscillatoria sp. PCC 6506), or SEQ ID NO:9 (Paraburkholderia phymatum). The polynucleotide has an amino acid exchange in one or more of positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 of SEQ ID NO:1, or at a corresponding position of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9.

Claims

1. A polynucleotide, encoding an amino acid sequence, encoding an oxidoreductase, that is ≥50% identical to an amino acid sequence of SEQ ID NO:1 (Geobacillus sp. PA9), SEQ ID NO:3 (Thermus thermophilus), SEQ ID NO:4 (Streptomyces globisporus), SEQ ID NO:5 (Clostridium aminobutyricum), SEQ ID:6 (Burkholderai cepacia), SEQ ID NO:8 (Oscillatoria sp. PCC 6506), or SEQ ID NO:9 (Paraburkholderia phymatum), wherein the polynucleotide has an amino acid exchange in one or more of positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 of SEQ ID NO:1, or at a corresponding position of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9, wherein the amino acid exchange is not A210S or S212A.

2. The polynucleotide, encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, that is ≥65%, identical to the amino acid sequence of SEQ ID NO:1 (Geobacillus sp. PA9), SEQ ID NO:3 (Thermus thermophilus), SEQ ID NO:4 (Streptomyces globisporus), SEQ ID NO:5 (Clostridium aminobutyricum), SEQ ID:6 (Burkholderai cepacia), SEQ ID NO:7 (Cupriavidus necator), SEQ ID NO:8 (Oscillatoria sp. PCC 6506), or SEQ ID NO:9 (Paraburkholderia phymatum), wherein the polynucleotide has an amino acid exchange in one or more of positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 of SEQ ID NO:1, or at a corresponding position of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

3. The polynucleotide, encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, that is ≥90%, identical to the amino acid sequence of SEQ ID NO:1 (Geobacillus sp. PA9), SEQ ID NO:3 (Thermus thermophilus), SEQ ID NO:4 (Streptomyces globisporus), SEQ ID NO:5 (Clostridium aminobutyricum), SEQ ID:6 (Burkholderai cepacia), SEQ ID NO:7 (Cupriavidus necator), SEQ ID NO:8 (Oscillatoria sp. PCC 6506), SEQ ID NO:9 (Paraburkholderia phymatum), or SEQ ID NO:10 (Ralstonia pickettii), wherein the polynucleotide has an amino acid exchange in one or more of positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 of SEQ ID NO:1, or at a corresponding position of the amino acid sequence of SEQ ID NO:3, SEQ ID NO:4, SEQ ID:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

4. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, selected from SEQ ID NO:1 (Geobacillus sp. PA9) with an amino acid exchange at one or more of the positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214; SEQ ID NO:3 (Thermus thermophilus) with an amino acid exchange at one or more of the positions 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205; or SEQ ID NO:21; SEQ ID NO:4 (Streptomyces globisporus) with an amino acid exchange at one or more of the positions 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222; or SEQ ID NO:22; SEQ ID NO:5 (Clostridium aminobutyricum) with an amino acid exchange at one or more of the positions 202, 203, 204, 205, 206, 207, 208, 209, 210; or SEQ ID NO:23; SEQ ID:6 (Burkholderai cepacia) with an amino acid exchange at one or more of the positions 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212; or SEQ ID NO:24; SEQ ID NO:7 (Cupriavidus necator) with an amino acid exchange at one or more of the positions 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213; or SEQ ID NO:25; SEQ ID NO:8 (Oscillatoria sp. PCC 6506) with an amino acid exchange at one or more of the positions 209, 210, 211, 212, 213, 214, 215, 216, 217; or SEQ ID NO:26; SEQ ID NO:9 (Paraburkholderia phymatum) with an amino acid exchange at one or more of the positions 192, 193, 194, 195, 196, 197, 198, 199, 200, 201; or SEQ ID NO:27; or SEQ ID NO:10 (Ralstonia pickettii) with an amino acid exchange at one or more of the positions 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 or SEQ ID NO:28.

5. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, that is ≥92% identical to the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14 wherein SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, at position 207, or at a corresponding position of the amino acid sequence, has a proteinogenic amino acid other than L-valine.

6. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, wherein the polynucleotide is a replicable nucleotide sequence encoding an enzyme 4-hydroxyphenylacetate 3-monooxygenase from microorganisms of a genus Geobacillus, wherein protein sequences encoded thereby contain a proteinogenic amino acid other than L-valine at a position corresponding to position 207 of SEQ ID NO:1.

7. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, wherein the amino acid sequence encoded thereby has, at the position 207 or a corresponding position, an amino acid which is selected from the group consisting of threonine, leucine, glutamine and glycine.

8. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, wherein the amino acid sequence encoded thereby contain a proteinogenic amino acid other than L-threonine at position 206, or at a corresponding position of the amino acid sequence; or contain a proteinogenic amino acid other than L-lysine at position 208, or at a corresponding position of the amino acid sequence.

9. The polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, according to claim 1, that is ≥90%, identical to the amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32.

10. A vector comprising the polynucleotide according to claim 1.

11. The vector according to claim 10, which is suitable for replication in microorganisms of a genera Escherichia, Pseudomonas or Corynebacterium.

12. A polypeptide comprising an amino acid sequence encoded by the polynucleotide according to claim 1.

13. A microorganism of a genera Escherichia, Pseudomonas or Corynebacterium comprising the polynucleotide according to claim 1.

14. The microorganism according to claim 13, in which the polynucleotide is present in an overexpressed form.

15. The microorganism according to claim 13 having a capability of producing a fine chemical.

16. A fermentative process for producing a fine chemical comprising: a) fermenting a microorganism comprising a polynucleotide encoding an amino acid sequence, encoding an oxidoreductase, that is ≥90%, identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3 to SEQ ID NO:32 in a medium, and b) accumulating the fine chemical in the medium, wherein a fermentation broth is obtained.

17. The fermentative process according to claim 16, wherein the fine chemical or a liquid or solid fine chemical-containing product is obtained from the fermentation broth and the fine chemical is L-DOPA.

18. A method of making L-DOPA comprising fermenting the microorganism according to claim 13.

Description

EXAMPLES

[0142] The present invention will be described in more detail hereinafter with reference to exemplary embodiments.

Example 1: Production of E. coli Strains with Different HpaB (Geobacillus) Mutants

[0143] A pOM17c-plasmid, which has been described previously in DE102004043748A1, where the complete sequence of the plasmid (containing the sequence of the cyanidase gene from Pseudomonas stutzeri AK61) is disclosed, was used as a starting point. Sequences of origin of replication and ampicillin resistance gene on this plasmid were maintained and the wildtype Geobacillus sp. PA-9 hpaB gene, the wildtype E. coli hpaC gene, the Pseudomonas oleovorans PalkB promoter and the E. coli rrnB terminator sequences, as well as the alkS transcriptional regulator gene (Yuste et al., J Bacteriol. 1998 October; 180(19):5218-26) were added. This plasmid was digested using the Geobacillus sp. wildtype single cutter restriction enzymes AgeI and PmlI [New England Biolabs GmbH, Brüningstraße 50, Geb. B852, 65926 Frankfurt am Main]. Synthesized DNA-fragments [Eurofins Genomics Germany GmbH Anzinger Str. 7a DE-85560 Ebersberg] were cloned with the plasmid backbone using the NEBuilder® HiFi DNA Assembly Master Mix [New England Biolabs GmbH Brüningstrasse 50, Geb. B852 65926 Frankfurt am Main] following the manufacturer's instructions. Thereby, the plasmid contained both E. coli hpaC and the different hpaB variants. After transformation of NEB® 5-alpha Electrocompetent E. coli [New England Biolabs GmbH Brüningstrasse 50, Geb. B852 65926 Frankfurt am Main], cells were plated on LB-Agar containing 100 μg/ml ampicillin. Restriction analysis and sequencing were done to select correctly cloned plasmids. If mentioned, the plasmid stabilizing toxin-antitoxin sequences hok/sok (Thisted, EMBO J. 1994 Apr. 15; 13(8):1950-9) and the cer determinant (Summers & Sherratt, 1984, DOI: 10.1016/0092-8674(84)90060-6) were added to the plasmids to increase plasmid stability.

[0144] L-tyrosine producing strain DPD6021-S was used for the transformation of the selected plasmids. DPD6021-S is an S-phase-variant of DPD4145, which is described in U.S. Pat. No. 7,700,328 B2 and where additionally the pMT100 plasmid was eliminated. For plasmid transformation, the strain was inoculated to an OD.sub.600 of 0.05 from an LB-overnight culture and grown to an OD.sub.600 of 0.7. After 30 min incubation on ice, the cells were harvested (10 min, 5500 g; 4° C.) and washed twice with 50 ml H.sub.2O.sub.demin. After an additional wash step with 1 ml pre-chilled 10% (v/v) Glycerol, cells were resuspended in 200 μl pre-chilled 10% (v/v) glycerol and aliquots of 40 μl were used for transformation. Therefore, 100 ng of plasmid were transferred to an electroporation cuvette, mixed with 40 μl cell solution and pulsed in a Gene Pulser Xcell Electorporation System [Bio-Rad Laboratories GmbH, Kapellenstraße 12, D-85622 Feldkirchen] at 2500 V, 200Ω & 25 μF. After the addition of 1 ml SOC media and regeneration for 45 min at 37° C., 100 μl of the suspension were plated to LB-Agar containing 100 μg/ml of Ampicillin. The plasmid was isolated from resistant colonies and authenticity of the plasmids was confirmed by restriction analysis.

[0145] The following strains were generated: [0146] hpaBC_Ec: E. coli hpaB (wildtype; reference); [0147] hpaB_Gs: [0148] hpaB_V207L_Gs: [0149] hpaB_V207T_Gs: [0150] hpaB_V207Q_Gs: [0151] hpaB_V207G_Gs: [0152] hpaB_V207T_K208R_Gs: [0153] hpaB_T206M_V207T_Gs: [0154] hpaB_T206A_V207T_Gs: [0155] Geobacillus sp. PA-9 hpaB (wildtype); [0156] Geobacillus sp. PA-9 hpaB (Mutation V207L); [0157] Geobacillus sp. PA-9 hpaB (Mutation V207T); [0158] Geobacillus sp. PA-9 hpaB (Mutation V207Q); [0159] Geobacillus sp. PA-9 hpaB (Mutation V207G); [0160] Geobacillus sp. PA-9 hpaB (Mutation V207T_K208R); [0161] Geobacillus sp. PA-9 hpaB (Mutation T206M_V207T); [0162] Geobacillus sp. PA-9 hpaB (Mutation T206A_V207T). [0163] hpaB_V207T_A210S_T211N_S212T: Geobacillus sp. PA-9 hpaB (Mutation V207T_A210S_T211N_S212T) [0164] hpaB_V207T_A210V_T211M_S212N: Geobacillus sp. PA-9 hpaB (Mutation V207T_A210V_T211M_S212N) [0165] hpaB_V207T_G213A_E214Q_D215N: Geobacillus sp. PA-9 hpaB (Mutation V207T_G213A_E214Q_D215N) [0166] hpaB_1152 L_V207T: Geobacillus sp. PA-9 hpaB (Mutation 1152L_V207T)

Example 2: Production of L-DOPA and L-Tyrosine (Using BioLector Screening)

[0167] DPD6021-S strains with plasmids expressing wildtype or mutated variants of the Geobacillus sp. PA-9 gene as well as a reference strain expressing the wildtype E. coli gene were tested in a 20 BioLector [m2p-labs; Arnold-Sommerfeld-Ring 2, 52499 Baesweiler] small scale test system.

[0168] Therefore, the strains were cultivated in 10 ml LB-media (100 μg/ml ampicillin) in baffled shake flasks at 37° C., 200 rpm for 18 h. Cultures were seeded into BioLector Flowerplates containing 1 ml LB-media, pH 5.5 (supplemented with 7.5 mM L-tyrosine; 100 μg/ml ampicillin; 0.25% (v/v) DCPK) to yield a starting OD.sub.600 of 0.1. Cultivation was done at 37° C., 1200 rpm and relative humidity of 85% for 24 h, until the process was stopped, and L-DOPA and L-tyrosine concentrations were measured using High performance liquid chromatography (HPLC).

[0169] HPLC was performed on an Agilent 1200 (Agilent Technologies, Palo Alto, Calif.). An Inertsil ODS-3, 5 μm, 4.6×150 mm column (Agilent Technologies) was used. The method used required a column flow rate of 1.00 ml/min with a stop time of 18 minutes. The mobile phase was composed of ratios of Solvent A (2.72 g/L KH2PO4, 2.5 ml/L concentrated phosphoric acid, 40 ml/L acetonitrile) and Solvent B (acetonitrile) as described in table 1.

TABLE-US-00001 TABLE 1 Composition of mobile phase for HPLC analysis Time [min] Solvent A [%] Solvent B [%] 0 100 0 6.5 100 0 10 75 25 13 75 25 13.01 100 0 18 100 0

[0170] The spectrum was scanned from 100 nm to 380 nm, with signal for L-DOPA being recorded at 290 nm and a retention time of 2.8 min, signal for L-tyrosine being recorded at 278 nm and a retention time of 3.9 minutes.

TABLE-US-00002 TABLE 2 Production of L-DOPA and L-tyrosine with single mutants using BioLector screening L-DOPA [mM] L-Tyrosin [mM] LD-EC-12 [hpaBC_Ec] 2.7 5.4 hpaB_Gs 3.6 4.5 hpaB_V207Q_Gs 4.0 4.0 hpaB_V207T_Gs 4.3 3.6 hpaB_V207L_Gs 4.4 3.8

TABLE-US-00003 TABLE 3 Production of L-DOPA and L-tyrosine with single and multiple mutants using BioLector screening (plasmid containing plasmid stabilizing elements) L-DOPA L-Tyrosin [mM] [mM] hpaB_V207T_Gs 5.2 3.5 hpaB_T206M_V207T_Gs 6.4 2.2 hpaB_V207T_K208R_Gs 5.4 3.6 hpaB_V207G_Gs 4.4 4.4 hpaB_T206A_V207T_Gs 4.7 3.8 hpaB_V207T_A210S_T211N_S212T 4.0 4.5 hpaB_V207T_A210V_T211M_S212N 4.0 4.6 hpaB_V207T_G213A_E214Q_D215N 3.8 4.7 hpaB_I152L_V207T 4.0 5.0

[0171] The results of the screening are summarized in table 2 and table 3 and visualized in FIG. 1 and FIG. 2. FIG. 1 shows the production of L-DOPA and L-tyrosine with single mutants using BioLector screening and FIG. 2 shows the production of L-DOPA and L-tyrosine with single and multiple mutants using BioLector screening (plasmid contain a plasmid stabilizing element).

Example 3: Production of L-DOPA and L-Tyrosine (Fermentative Process)

[0172] Fermentation was carried out as described in Example 8 of U.S. Pat. No. 7,700,328 B2 for strain DPD4145 and the strains were evaluated for production of L-tyrosine and L-DOPA. Unlike in Example 8 of U.S. Pat. No. 7,700,328 B2, fermentation was not induced with IPTG, but with Dicyclopropyl ketone (DCPK) and fermentation was performed in the presence of ampicillin (100 mg/L) at pH 6.8. Samples were drawn from the fermenter periodically and analyzed for L-tyrosine, L-DOPA and biomass concentration. The results are summarized in tables 4-6, table 4 showing the production of L-DOPA over 48 h, table 5 showing the production of L-tyrosine over 48 h for the same fermentation process and table 6 showing the ratio of L-DOPA/L-tyrosine. The results are visualized in corresponding FIGS. 3-4.

[0173] FIG. 3 shows the production of L-DOPA and L-tyrosine over 48 h with E. coli HpaB (top) and Geobacillus spec. HpaB (bottom) using a fermentative process and FIG. 4 shows the production of L-DOPA and L-tyrosine over 48 h with Geobacillus spec. HpaB V207T mutant (top) and Geobacillus spec. HpaB V207L mutant (bottom) using a fermentative process.

TABLE-US-00004 TABLE 4 Production of L-DOPA with fermentative process L-DOPA [g/L] t [h] hpaB_Ec hpaB_Gs hpaB_Gs_V207T hpaB_Gs_V207L 0 0.0 0.0 0.0 0.0 18 8.2 4.3 7.2 7.3 25 11.4 10.6 13.9 13.1 48 15.3 13.7 21.4 20.3

TABLE-US-00005 TABLE 5 Production of L-Tyrosine with fermentative process L-Tyrosine [g/L] t [h] hpaB_Ec hpaB_Gs hpaB_Gs_V207T hpaB_Gs_V207L 0 0.0 0.0 0.0 0.0 18 2.0 3.6 2.5 2.7 25 1.4 3.0 1.1 1.3 48 3.4 1.9 below detection limit 0.5

TABLE-US-00006 TABLE 6 Ratio of L-DOPA/L-Tyrosine with fermentative process Ratio L-D/L-T t [h] hpaB_Ec hpaB_Gs hpaB_Gs_V207T hpaB_Gs_V207L 0 18 4.0 1.2 2.9 2.7 25 8.2 3.5 12.9 9.7 48 4.5 7.1 L-tyrosine below 42.5 detection limit

[0174] It could be clearly shown that by using HpaB from Geobacillus as a wildtype enzyme and with different mutations for the fermentative production of L-DOPA for 48 h, the ratio of L-DOPA/L-tyrosine was higher as when using the E. coli wildtype enzyme (table 6). The mutants of the Geobacillus HpaB enzyme even have higher values for the ratio of L-DOPA/L-tyrosine.

[0175] Further mutants Gs_V207T-T206M and Gs_V207T-K208R were also tested in a fermentative process and similar results were obtained with high values for the ratio of L-DOPA/L-tyrosine.

Example 5: Analysis of Structural Homologies

[0176] Regarding the oxidoreductases of the present invention, the Geobacillus sp. HpaB sequence was used to search the Protein Data Bank (PDB) for structural homologues. The structural alignment was constructed using MATT (Menke, M., Berger, B. & Cowen, L. Matt: Local Flexibility Aids Protein Multiple Structure Alignment. PLoS Comput. Biol. 4, el 0 (2008)).

[0177] Such an alignment for the oxidoreductases of the present invention is shown in FIG. 5, which shows an alignment of Geobacillus sp. HpaB and 2yyj (oxidoreductase from Thermus thermophilus).

[0178] FIG. 6: Alignment of Geobacillus sp. HpaB and 4002 (oxidoreductase from Streptomyces globisporus)

[0179] FIG. 7: Alignment of Geobacillus sp. HpaB and 1 uv8 (oxidoreductase from Clostridium aminobutyricum)

[0180] FIG. 8: Alignment of Geobacillus sp. HpaB and 3hwc (oxidoreductase from Burkholderia cepacia)

[0181] FIG. 9: Alignment of Geobacillus sp. HpaB and 4g5e (oxidoreductase from Cupriavidus necator)

[0182] FIG. 10: Alignment of Geobacillus sp. HpaB and 4irn (oxidoreductase from Oscillatoria sp. PCC 6506)

[0183] FIG. 11: Alignment of Geobacillus sp. HpaB and 5idu (oxidoreductase from Paraburkholderia phymatum)

[0184] FIG. 12: Alignment of Geobacillus sp. HpaB and 6jhm (oxidoreductase from Ralstonia pickettii)

[0185] The superposition of the modeled HpaB structures shows that the proteins are well aligned.

Example 6: Production of E. coli Strains with Different Oxidoreductase Mutants

[0186] Restriction of the pOM17c plasmid described in example 1 bearing the wildtype Geobacillus sp. PA-9 hpaB gene and the wildtype E. coli hpaC gene with the Enyzme Earl [New England Biolabs GmbH Brüningstrasse 50, Geb. B852 65926 Frankfurt am Main] is done to remove the wildtype Geobacillus sp. PA-9 hpaB gene, as well as the PalkB, alkS sequences and a part of the E. coli hpaC sequence. The resulting plasmid backbone is cloned with synthesized DNA fragments containing the previously removed PalkB and alkS sequences and a part of the E. coli hpaC sequence as well as the wildtype sequences of genes coding for the enzymes with amino acid sequences of SEQ ID NO:3 (Thermus thermophilus), SEQ ID NO:4 (Streptomyces globisporus), SEQ ID NO:5 (Clostridium aminobutyricum), SEQ ID:6 (Burkholderai cepacian), SEQ ID NO:7 (Cupriavidus necator), SEQ ID NO:8 (Oscillatoria sp. PCC 6506), SEQ ID NO:9 (Paraburkholderia phymatum), SEQ ID NO:10 (Ralstonia pickettii).

[0187] Furthermore, the mentioned plasmid backbone is cloned with synthesized DNA fragments containing the previously removed PalkB and alkS sequences and a part of the E. coli hpaC sequence as well as mutant sequences of genes resulting in enzyme variants with improved activity with the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28.

[0188] The resulting plasmids are used for transformation of the L-tyrosine producing strain DPD6021-S as described in Example 1.

[0189] The resulting strains are cultivated in small scale screening system as described in Example 2 or fermentation as described in Example 3 to evaluate the conversion rate of the strains expressing mutant enzyme variants. L-DOPA formation is quantified as described in Example 1. L-DOPA production was detected for all variants.

TABLE-US-00007 Protein sequences SEQ ID NO: 1 Geobacillus sp. PA9 HpaB SEQ ID NO: 2 E. coli (HpaB) 4-hydroxyphenylacetate 3-monooxygenase oxygenase component SEQ ID NO: 3 Thermus thermophilus SEQ ID NO: 4 Streptomyces globisporus SEQ ID NO: 5 Clostridium aminobutyricum SEQ ID NO: 6 Burkholderia cepacia SEQ ID NO: 7 Cupriavidus necator SEQ ID NO: 8 Oscillatoria sp. PCC 6506 SEQ ID NO: 9 Paraburkholderia phymatum SEQ ID NO: 10 Ralstonia pickettii SEQ ID NO: 11 Geobacillus sp. PA9 HpaB V207L SEQ ID NO: 12 Geobacillus sp. PA9 HpaB V207T SEQ ID NO: 13 Geobacillus sp. PA9 HpaB V207Q SEQ ID NO: 14 Geobacillus sp. PA9 HpaB V207G SEQ ID NO: 15 Geobacillus sp. PA9 HpaB T206M SEQ ID NO: 16 Geobacillus sp. PA9 HpaB T206A SEQ ID NO: 17 Geobacillus sp. PA9 HpaB K208R SEQ ID NO: 18 Geobacillus sp. PA9 HpaB V207T_K208R SEQ ID NO: 19 Geobacillus sp. PA9 HpaB T206M_V207T SEQ ID NO: 20 Geobacillus sp. PA9 HpaB T206A_V207T SEQ ID NO: 21 Thermus thermophilus (mutated) SEQ ID NO: 22 Streptomyces globisporus (mutated) SEQ ID NO: 23 Clostridium aminobutyricum (mutated) SEQ ID NO: 24 Burkholderia cepacia (mutated) SEQ ID NO: 25 Cupriavidus necator (mutated) SEQ ID NO: 26 Oscillatoria sp. PCC 6506 (mutated) SEQ ID NO: 27 Paraburkholderia phymatum (mutated) SEQ ID NO: 28 Ralstonia pickettii (mutated) SEQ ID NO: 29 Geobacillus sp. PA9 HpaB V207T_A210S_T211N_S212T SEQ ID NO: 30 Geobacillus sp. PA9 HpaB V207T_A210V_T211M_S212N SEQ ID NO: 31 Geobacillus sp. PA9 HpaB V207T_G213A_E214Q_D215N SEQ ID NO: 32 Geobacillus sp. PA9 HpaB I152L_V207T