Bacterial unspecific peroxygenases (BUPO's) and methods and uses thereof

Abstract

Novel polypeptides having peroxygenase activity, and methods and uses related thereto. A method for the production of melanin or a melanin-like pigment, comprising the use of a polypeptide having pigment producing activity, wherein said polypeptide is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 50% pairwise sequence identity when aligned to at least 200 consecutive amino acid residues of Seq. no. 16 and comprising at least two of the following motifs: i) RXFWXRWXXGHQ; ii) LXXLXXCXD; iii) PRXXYH; iv) RXR[ML]ALQH; v) CXXL; vi) HXXIAXH; vii) DLXHXG; and viii) VDGXHHPV; (b) a polypeptide comprising an amino acid sequence having at least 30% pairwise sequence identity when aligned to at least 150 amino acid residues of Seq. no. 12, and comprising the motif HXXXC; and c) a fragment of the polypeptide of (a) or (b) that has peroxygenase activity.

Claims

1. A method for the production of melanin and/or melanin-like pigment(s), comprising the use of a polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 50% pairwise sequence identity when aligned to at least 200 consecutive amino acid residues of Seq. no. 16 of FIG. 1, and comprising at least two of the following motifs: i) RXFWXRWXXGHQ, preferably R[LV]FWYRWIAGHQ; ii) LXXLXXCXD, preferably L[DE][ALV]L[ACST][TAS]C[IV]D; iii) PRXXYH, preferably PR[AD][HQ]YH; iv) RXR[ML]ALQH, preferably R[APT]R[ML]ALQH; v) CXXL, preferably C[EAR][AE]L; vi) HXXIAXH, preferably H[DS][HF]IA[ND]H; vii) DLXHXG, preferably DL[AS]H[NH]G; and viii) VDGXHHPV, preferably VDG[AR]HHPV; wherein X is any amino acid; (b) a polypeptide comprising an amino acid sequence having at least 30% pairwise sequence identity when aligned to at least 150 consecutive amino acid residues of Seq. no. 12 of FIG. 2, and comprising the motif HXXXC, wherein X is any amino acid, preferably H[IRKAQVG][GNELSYRHM][VI]C, more preferably HARVC; (c) a fragment of the polypeptide of (a) or (b) that has pigment producing activity, wherein the method comprises the fermentative production of melanin and/or a melanin-like pigment, the method comprising the steps of: i) providing a microbial host cell expressing the heterologous polypeptide having piment producing activity; ii) culturing the host cell in a culture medium and allowing for the production of melanin and/or melanin-like pigment; and iii) isolating melanin and/or melanin-like pigment.

2. The method of claim 1, comprising the hydroxylation of L-tyrosine to L-DOPA and subsequent oxidation to dopachrome and the formation of melanin and/or a melanin-like pigment.

3. (canceled)

4. A method according to claim 3, wherein the host cell is a bacterial, yeast or fungal host cell, preferably a bacterial host cell, preferably E. coli.

5. The method according to claim 3, wherein the culture medium comprises at least one melanin(-like) pigment precursor, preferably L-Tyrosine, optionally in combination with L-cysteine.

6. A method for the hydroxylation or oxidation of a substituted or unsubstituted, linear or branched, aliphatic or aromatic substrate, comprising contacting the substrate with a polypeptide having peroxygenase activity and a source of hydrogen peroxide, wherein said polypeptide is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 50% pairwise sequence identity when aligned to at least 200 consecutive amino acid residues of Seq. no. 16 of FIG. 1, and comprising at least two of the following motifs: i) RXFWXRWXXGHQ, preferably R[LV]FWYRWIAGHQ; ii) LXXLXXCXD, preferably L[DE][ALV]L[ACST][TAS]C[IV]D; iii) PRXXYH, preferably PR[AD][HQ]YH; iv) RXR[ML]ALQH, preferably R[APT]R[ML]ALQH; v) CXXL, preferably C[EAR][AE]L; vi) HXXIAXH, preferably H[DS][HF]IA[ND]H; vii) DLXHXG, preferably DL[AS]H[NH]G; and viii) VDGXHHPV, preferably VDG[AR]HHPV; wherein X is any amino acid; (b) a polypeptide comprising an amino acid sequence having at least 30% pairwise sequence identity when aligned to at least 150 consecutive amino acid residues of Seq. no. 12 of FIG. 2, and comprising the motif HXXXC, wherein X is any amino acid, preferably H[IRKAQVG][GNELSYRHM][VI]C, more preferably HARVC; (c) a fragment of the polypeptide of (a) or (b) that has peroxygenase activity.

7. The method according to claim 6, comprising one or more of the following reactions: the enantioselective sulfoxidation of an optionally substituted alkyl sulfide, aryl sulfide or aryl alkyl sulfide substrate, preferably the enantioselective sulfoxidation of a substrate selected from the group consisting of methyl phenyl sulfide, benzyl phenyl sulfide, allyl phenyl sulfide, benzyl methyl sulfide, N-butyl methyl sulfide, ethyl phenyl sulphide and isopropyl phenyl sulphide; the manufacture of a substituted or unsubstituted indigo dye by contacting a substituted or unsubstituted indole substrate with a source of hydrogen peroxide and the polypeptide; the oxidation of a primary alcohol, preferably the oxidation of veratryl alcohol to veratryl aldehyde.

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. The method according to claim 1, wherein the polypeptide is comprised in whole cells or a cell-free extract, or wherein the polypeptide is used as purified enzyme.

13. The method according to claim 1, wherein the polypeptide under (a) furthermore comprises one or both of the following motifs: a. LWRAM b. EDL[YF]DN[FY][FY], preferably EDLYDNFF. and/or contains one or more of the residues corresponding to Arg115, Met118, Phe125, Ser126, Leu180, Tyr200 and Phe204 of Seq. no. 16.

14. (canceled)

15. The method according to claim 1, wherein the polypeptide comprises a sequence that has at least 60%, at least 70%, at least 80%, or at least 90% pairwise sequence identity with any one of Seq. no. 15, 16, 17 and 18 of Table 1, or a fragment thereof that has peroxygenase activity.

16. The method according to claim 1, wherein the polypeptide under (b) furthermore comprises one or more of the following residues/motifs: i) DXXFXXXR; preferably D[LEAFDSRHT][AGFHY]F[GNCLR][IAV][VELKIRSD]R, more preferably DSYFLVER; ii) R[WR]XX[GQ]HXXF; preferably R[WR][VIRMKH][RYCLVQK][GQ]-H[HLYQR][VLIAS]F, more preferably RWKQQHQLF; iii) YXXXXR[PV]; preferably Y[N/T/Q/V/E/A/H/D/R/Q][E/T/D/S/Q/A]-[Q/R/E/S/A/I/G/F/T/V/M/L/N][IV]RP, more preferably YESRIRP; iv) H232; v) [LM]281; preferably L281 wherein X is any amino acid and wherein the numbering corresponds to the amino acid sequence of Seq. no. 12, and preferably, wherein in addition, comprises a sequence that has at least 40%, at least 50%, at least 60%, or at least 70% pairwise sequence identity with any one of Seq. no. 2-13 and 19-29 of Table 2, or a fragment thereof that has peroxygenase activity.

17. (canceled)

18. The method of claim 15, wherein the polypeptide is any one of Seq. no. 2, 3, 4, 7, 8, 11, 12, 13, 20, 21 and 28, or a fragment thereof that has peroxygenase activity.

19. The method according to claim 1, wherein the polypeptide is any of Seq. no. 2, 8, 11, 12, 13, 15, 16, 17, 18 and 20, preferably Seq. no. 15, 16 or 17, or a fragment thereof having pigment producing activity.

20. The method according to claim 1, wherein the polypeptide further comprises an N- and/or C-terminal protein tag allowing for enhanced expression, solubilization, purification and/or immobilization.

21. (canceled)

22. (canceled)

23. The method according to claim 6, wherein the polypeptide is comprised in whole cells or a cell-free extract, or wherein the polypeptide is used as purified enzyme.

24. The method according to claim 6, wherein the polypeptide under (a) furthermore comprises one or both of the following motifs: a. LWRAM b. EDL[YF]DN[FY][FY], preferably EDLYDNFF, and/or contains one or more of the residues corresponding to Arg115, Met118, Phe125, Ser126, Leu180, Tyr200 and Phe204 of Seq. no. 16.

25. The method according to claim 6, wherein the polypeptide comprises a sequence that has at least 60%, at least 70%, at least 80%, or at least 90% pairwise sequence identity with any one of Seq. no. 15, 16, 17 and 18 of Table 1, or a fragment thereof that has peroxygenase activity.

26. The method according to claim 6, wherein the polypeptide under (b) furthermore comprises one or more of the following residues/motifs: i) DXXFXXXR; preferably D[LEAFDSRHT][AGFHY]F[GNCLR][IAV][VELKIRSD]R, more preferably DSYFLVER; ii) R[WR]XX[GQ]HXXF; preferably R[WR][VIRMKH][RYCLVQK][GQ]-H[HLYQR][VLIAS]F, more preferably RWKQQHQLF; iii) YXXXXR[PV]; preferably Y[N/T/Q/V/E/A/H/D/R/Q][E/T/D/S/Q/A]-[Q/R/E/S/A/I/G/F/T/V/M/L/N][IV]RP, more preferably YESRIRP; iv) H232; v) [LM]281; preferably L281 wherein X is any amino acid and wherein the numbering corresponds to the amino acid sequence of Seq. no. 12, and preferably, wherein in addition, the polypeptide comprises a sequence that has at least 40%, at least 50%, at least 60%, or at least 70% pairwise sequence identity with any one of Seq. no. 2-13 and 19-29 of Table 2, or a fragment thereof that has peroxygenase activity.

27. The method of claim 25, wherein the polypeptide is any one of Seq. no. 2, 3, 4, 7, 8, 11, 12, 13, 20, 21 and 28, or a fragment thereof that has peroxygenase activity.

28. The method according to claim 6, wherein the polypeptide further comprises an N- and/or C-terminal protein tag allowing for enhanced expression, solubilization, purification and/or immobilization.

Description

LEGEND TO THE FIGURES

[0167] FIG. 1: Amino acid sequence alignment of a number of novel Type II BUPO polypeptides. Conserved sequence motifs (i) through (viii), and (a) and (b) are indicated on top.

[0168] FIG. 2: Amino acid sequence alignment of a number of novel Type I BUPO polypeptides. Conserved sequence motifs (i) through (v) and HXXC are indicated on top.

[0169] FIG. 3: Phylogenetic tree showing the evolutionary relationships among novel BUPOs of the invention. Known enzymes SfmD (Seq. no. 1) and lmbB2 (Seq. no. 14) are also included.

[0170] FIG. 4: UV-Vis spectra of exemplary purified BUPOs. Presence of the Soret band at 405 nm shows heme incorporation. Panel A: Seq. no. 12; panel B: Seq. no. 7; panel C: Seq. no. 11; panel D: Seq. no. 18; panel E: Seq. no. 17; panel F: Seq. no. 16.

[0171] FIG. 5: HPLC chromatogram of L-tyrosine conversion to L-DOPA by enzyme having Seq.no.12, Seq.no.17 or Seq.no.16. Standard for L-tyrosine has a retention time of 2.50 min, while L-DOPA standard (*) elutes at retention time of 2.41 min. Most of the L-tyrosine was converted to L-DOPA by Seq. no. 12 and 16, while for Seq. no. 17 a partial conversion was observed.

[0172] FIG. 6: Screening of novel BUPO enzymes for peroxygenase activity against different substrates. Rows A, B: melanin production from tyrosine; rows C, D: Indigo production from indole. Rows E, F: veratryl alcohol oxidation; rows G, H: p-cresol oxidation. In all cases, the reaction mixture contained 5 mM substrate in 50 mM K-phosphate buffer pH 7.5 and 1-20 M enzyme solution. Reactions were started by addition of hydrogen peroxide (2 mM final concentration). Picture was taken after 1h. Enzymes tested: Seq.1 (SfmD)-A1, C1, E1, G1; Seq.2A2, C2, E2, G2; Seq.5A3, C3, E3, G3; Seq.3A4, C4. E4, G4; Seq.13A5, C5, E5, G5; Seq.4A6, C6, E6, G6; Seq.12A7, C7, E7, G7; Seq.7B1, D1, F1, H1; Seq.11B2, D2, F2, H2; Seq.15B3, D3, F3, H3; Seq.18B4, D4, F4, H4; Seq.17B5, D5, F5, H5; Seq.16B6, D6, F6, H6; Control (no enzyme)B7, D7, F7, H7. Colored products were observed in wells A6, A7, B5 and B6 corresponding to melanin production; D3, D4, D5 and D6 correspond to indigo production; H5 and H6 correspond to p-cresol oxidation and polymerization.

[0173] FIG. 7: Representative HPLC chromatograms of reaction mixtures obtained by reacting exemplary BUPO enzymes with various substrates. [0174] Panel (A) reaction of p-cresol with enzyme Seq.no.7, Seq.no.16 or Seq.no.17. p-Cresol standard has a retention time of 9.38 min, while observed hydroxylation/oxidation products (*) eluted at retention times of 2.85 min (for no. 7) and at 8.05 min and 8.55 min (for no. 16 and no. 17). Enzyme having Seq. no. 16 converted most of the substrate under given conditions. [0175] Panel (B) reaction of m-cresol with enzyme Seq.no. 16 or Seq.no. 17. Standard for m-cresol has a retention time of 9.40 min, while observed products (*) eluted at retention times 7.6 min, 8.05 min and 8.55 min. [0176] Panel (C) reaction of p-nitrophenol with enzymes Seq.no. 16 or Seq.no. 17. Standard for p-nitrophenol has a retention time of 3.5 min, while observed products (*) eluted at retention times 1.25 min, 5.4 min and 5.75 min. [0177] Panel (D) reaction of protocatechuic acid with enzyme Seq.no. 15, Seq.no. 16, Seq.no. 17 or Seq.no. 18. Standard for protocatechuic acid has a retention time of 1.2 min, while observed products (*) eluted at retention times 5.1 min, 5.4 min and 7.0 min.

[0178] FIG. 8: Representative GCMS chromatograms of reaction mixtures obtained by reacting methyl phenyl sulfide (thioanisole) with exemplary BUPO enzymes. Thioanisole has a retention time of 12.42 min, while the observed oxidation product methyl phenyl sulfoxide eluted at retention time 17.50 min. Panel A: control (no enzyme); panel B: Seq. no. 4; panel C: Seq. no. 15; panel D: Seq. no. 16; panel E: Seq. no. 17; panel F: Seq. no. 18.

[0179] FIG. 9: Supernatants of the cultures of microbial cells expressing representative pigment-producing BUPOs cultured in defined media supplemented with tyrosine.

[0180] FIG. 10: Effect of mutations in polypeptide of Seq. no. 16 on the melanin producing activity. Dark color correlates with the level of melanin-type pigment.

EXPERIMENTAL SECTION

Example 1: Cloning, Expression and Purification of Novel BUPOs

[0181] Using either SfmD or LmbB2 as query sequences, an extensive BLAST search was performed against entire NCBI database of non-redundant sequences. Then, a phylogenetic tree (FIG. 3) was constructed for SfmD-like (Type I) and Orf13/LmbB2-like (Type II) enzymes. Representatives of distinct branches were selected and ordered as synthetic genes.

[0182] Synthetic genes coding for the selected putative BUPOs were cloned either in pET28a, pBAD-His or pBAD-His-SUMO vectors. Following the confirmation of the sequence by Sanger sequencing, the final constructs were transformed into chemically competent E. coli BL21 strains (C43 or BL21AI) for pET28-based constructs or into a E. coli NEB10B for pBAD-based constructs. Single colonies were picked and grown overnight in 5 mL Luria Bertani (LB) media supplemented with corresponding antibiotic.

[0183] On the following day, the over-night culture was diluted 1:100 in terrific broth (TB) with corresponding antibiotic. Cultures were incubated at 37 C. until OD600=0.6. Expression was induced at this point by addition of 1 mM IPTG (for pET28 constructs) or 0.02 w/v % arabinose (for pBAD-constructs), together with 0.5 mM 5-aminolevulinic acid (a heme precursor); all concentrations are given as final concentrations. Expression was carried out using baffled Erlenmeyer flasks at 17 C. for 46-72 hours in orbital shakers at 150-200 rpm with 2.5-5 cm orbital.

[0184] Cells were harvested using a cooling centrifuge at 4000g at 4 C. Cell pellets were resuspended in K-phosphate (KPi) buffer 50 mM pH 7.8 with 150 mM NaCl and with addition of 0.1 mM PMSF and 0.1 mg/ml lysozyme. Cells were disrupted using VibraCell sonicator (5 sec on, 10 sec off, 5 min total time, 70% amplitude). Clarified cell-free extract (CFE) was obtained by using cooling centrifuge at 19000g at 4 C.

[0185] CFE was loaded on a preequilibrated Ni-Sepharose column (KPi buffer 50 mM pH 7.8 with 150 mM NaCl). Unbound proteins were washed with 3 CV of starting buffer, followed by 3 CV of starting buffer containing 30 mM imidazole, and eluted in starting buffer containing 0.5 M imidazole. Eluted fractions were pooled and buffer was exchanged using the EconoPac desalting columns (BioRad), into a KPi buffer 50 mM pH 7.8; 150 mM NaCl. UV-Vis spectra were collected to estimate the concentration of the purified protein. Proteins were then flash-frozen in liquid nitrogen and stored at 70 C. It was possible to purify most of BUPOs with a yield of 60 mg/L of terrific broth (TB) media. These enzymes were red and show the presence of Soret band in their UV-Vise spectra which is a proof of heme incorporation. See FIG. 4 for the spectra of some exemplary enzymes.

Example 2: Qualitative Analysis of Peroxygenase Activity

[0186] The first batch of purified enzymes was tested for peroxygenase activity using the established Russig's blue assay (Yamada et al. (2017) PLoS ONE 12(4):e0175846). The standard assay mixture comprised 15% (v/v) ethanol, 100 mM KPi (pH 7.5), an excess (5-10 mM) of H.sub.2O.sub.2, 1-methoxynaphthalene (1-MN), and 10 L purified enzyme in a total volume of 100 l. The reaction was started by the addition of the enzyme and was carried out for 5 min at room temperature. The production of the reaction product Russig's blue was determined from the increase in the absorbance at 610 nm [(c) 1.45104 M1 cm1]. The assay was performed in a plate reader.

[0187] It was found that at least the Type II enzymes of Seq. no. 15, 17 and 18 can utilize 1-MN as a substrate, while the SfmD enzyme known in the art did not accept 1-MN as a substrate. It was furthermore interesting to note that, whereas enzymes SphingoUPO nor SpinosaUPO (Seq. no. 11 and 13) were not active against 1-MN, their expression in bacterial host cells did give rise to a dark/black coloured culture medium. This points to the different substrate scopes between the selected enzymes.

Example 3: Hydroxylation of L-Tyr to L-DOPA and Melanin

[0188] Reaction mixtures (0.5 mL) consisting of 5 mM L-Tyr in 50 mM KPi pH 7.5, 5-20 M of the purified enzyme (seqs 12, 16 and 17) and 1 mM H.sub.2O.sub.2 were incubated for 1 h at 25 C. and stopped by heating at 95 C. for 5 min. Reaction mixtures were centrifuged for 5 min at 15000g and the resulting supernatant was analyzed by reverse-phase HPLC-DAD using Waters Xselect CSH Fluoro-phenyl 5 m 4.6250 mm column with a linear gradient (10-50%) of water with 0.8% formic acid (A) and acetonitrile (B) over 18 min with a flow rate of 1.2 mL/min. L-Tyr and commercially available L-DOPA were used as standards. All enzymes tested were found to give L-DOPA (see FIG. 5) and melanin (see FIG. 6, rows A, B) as products, which means that these enzymes possess monophenolase and diphenolase activity.

[0189] Some of the enzyme-expressing bacterial cultures were colored dark, indicating the ability of the expressed proteins to produce dark (brown/black) pigment, melanin-like, that either can diffuse through the cell membrane or it is formed in the media from the reaction products which can diffuse through the cell. The building block is most likely tyrosine taken from the metabolic pathways.

[0190] Appearance of a dark color was not observed for enzymes known in the art (SfmD, Seq.no.1 and LmbB2, Seq.no.14), but clearly for various representatives of the newly discovered enzymes (Seq. numbers 2, 8, 11, 13, 16, 17 and 20). The observed pigment could pass through an ultrafiltration membrane with a molecular weight cut-off (MWCO) of 30 kDa. Experiments were carried out with individual purified enzymes to test whether similar result can be achieved in vitro (FIG. 6).

[0191] Reaction scheme for conversion of tyrosine to L-DOPA and dopachrome:

##STR00001##

Example 4: Hydroxylation of Substituted Phenols and Phenolic Acids

[0192] Using the same procedure as described above, hydroxylation and further oxidation to oligomers and insoluble polymers was shown for p-cresol (FIG. 6, rows G,H; and FIG. 7A) and m-cresol (FIG. 7B) with enzymes 12, 15, 16, 17 and 18, using reverse-phase HPLC. Insoluble polymers were removed by centrifugation at 15000g for 5 min prior to HPLC analysis. Hydroxylation of p-nitrophenol (FIG. 7C) and protocatechuic acid (FIG. 7D) was confirmed by HPLC as well. For p-nitrophenol, activity was observed using enzymes with sequence numbers 12, 16 and 17, while for protocatechuic acid the activity was confirmed for enzymes with sequence numbers 12, 15, 16, 17 and 18.

Example 5: Fermentative Production of Melanin-Type Pigment(s)

[0193] Methods

[0194] Production of melanin was tested using E. coli cells overexpressing different BUPOs in either pET or pBAD-based vectors. For pET28-based constructs E. coli BL21 cells were used in autoinduction media supplemented with kanamycin and 5-aminolevulinic acid. For the pBAD-based constructs E. coli NEB10 beta cells were used in TB media supplemented with ampicillin, 5-aminolevulininc acid and 0.02% arabinose. Expression was done by mixing 100 L of pre-inoculum (overnight culture) with 900 L of abovementioned media and expression was carried out in deep-well microtiter plates (DWMTP) for 20h at 30 C. Furthermore, the same experiment was carried out with addition of L-tyrosine (1 g/L) to the media. Autoinduction TB composition (g/L): Tryptone (12), Yeast Extract (24), (NH.sub.4).sub.2SO.sub.4 (3.3), MgSO.sub.4 (0.15), Glucose (0.5), Lactose (2.0) with addition of K-phosphate buffer (14.85, same composition as standard TB-buffer).

[0195] Results

[0196] In the first set of experiments (without addition of tyrosine to the media), dark melanin-type pigment formation was observed only in the culture using pET28 constructs. The pigment formation was observed in cultures of host cells expressing enzymes corresponding to Seq. no.: 7, 8, 12 or 20 (lower intensity), or Seq. no. 11, 18, 17, 16 and 15 (strong intensity).

[0197] When the medium was supplemented with tyrosine, some of the cultures with pBAD-based constructs also showed the melanin production. These were cultures corresponding to the following seq.no.: 13, 12 and 2 (strong intensity). The cultures of host cells expressing BUPOs from a pET construct gave same result with increase in the melanin production for all the cultures, most notably for cultures of enzymes 12, 8 and 20.

[0198] Following the experiment of fermentative melanin production in complex media (TB and autoinduction media), the melanin production was evaluated in minimal media (M9+trace elements, a recipe known in the art) with glucose as a sole carbon source and supplemented with 0.5 g/L tyrosine. The expression was performed using E. coli BL21 cells expressing the heterologous BUPO polypeptides from pET28 constructs. Induction was performed using 1 mM IPTG with addition of 0.5 mM 5-aminolevulinic acid. The enzymes tested in this experiment included the following: Seq. no. 20, 7, 4, 16 (two double mutants: R115A/L180A and R115A/L180S), 12, 8, 13, 18, 11, 16, 17 and 15.

[0199] Resulting cultures showed a range of melanin production, which could be visually observed based on the colored pigment (darker cultures correspond to higher amounts of melanin). Unexpectedly, some of the cultures were able to produce larger amounts of melanin than others. For example, as is shown in FIG. 9, the cultures of enzymes with Seq. no. 20, 7 or 4, and two mutants of enzyme 16 produced melanin only relatively small amounts; cultures of enzymes 12, 8, 13, 18 and 11 produced moderate amounts of melanin, while cultures of enzymes 16, 17 and 15 produced melanin in large amounts.

Example 6: Mutagenesis Study

[0200] Next, the polypeptide of Seq.no. 16 was used as representative pigment-producing enzyme in a mutagenesis study designed to identify residue(s) that are relevant for the manufacture of melanin-type pigments.

[0201] Changes to alanine and to other similar residues or residues present in the phylogenetic study were assessed. These included Trp50, Trp53, Tyr110, Arg115, Met118, Phe125, Ser126, Leu180, Tyr200 and Phe204. Some mutations were found to significantly affect the expression level of the polypeptide. For example, no protein could be produced from constructs encoding one of the following single point mutants: Tyr110Ala, Trp53Ala, Phe125Ala, Tyr200His, Tyr200Leu, Tyr200Val, Tyr200Ala, Phe204His and Phe204Ala. The remaining mutants could be expressed and purified, although for some the expression yield was reduced (data not shown).

[0202] Furthermore, the melanin production activity was severely affected by some of the point mutations. These are Leu180Ser, Leu180Ala, Arg115Ala and Phe204Leu. See FIG. 10. These data demonstrate that at least residues Leu180 and Arg115 in the Type II BUPO's are essential for melanin-producing activity.

Example 7: Hydroxylation of Indole

[0203] Representative enzymes were tested for their ability to form the blue pigment indigo by catalyzing the hydroxylation of indole.

[0204] Reactions were performed using 5 mM indole as substrate in a K-phosphate buffer pH 8 at room temperature. Reactions were started by the addition of 2 mM hydrogen peroxide. The formation of indigo as a strong blue color was observed for enzymes NobraUPO (Seq. no. 17) and NotUPO (Seq. no. 16), and a light blue color was observed for ActUPO (seq. no. 18), see FIG. 6, rows C, D. These findings indicate that these enzymes can be used for conversion of indole and substituted indoles for production of indigoid dyes, using either whole cells, cell-free extract, or purified enzymes in a soluble or an immobilized form.

Example 8: Oxidation of Primary Alcohols

[0205] Oxidation of veratryl alcohol (VA) to veratryl aldehyde is usually taken as a measure of lignin-peroxidase activity. It can be performed by measuring the absorbance of the formed aldehyde at 340 nm (c340=93 mM-lcm-1).

[0206] All enzymes (10 L) were mixed with 170 L of 5 mM VA in 50 mM KPi pH 8.0 and the reaction was started with addition of 20 L 50 mM H.sub.2O.sub.2 (5 mM f.c.). Control reactions were set up without addition of H.sub.2O.sub.2. A change in absorbance was measured in the plate reader.

[0207] A clear confirmation of catalysis of VA oxidation was observed for Nobra (seq 17), Not (seq 16) and StrpetUPO (seq 15), Ppac (seq 12) and Act (seq 18). See FIG. 6, rows E, F.

Example 9: Oxyfunctionalization of Indene, Thionaphthene and Sulfoxidation Reactions

[0208] All substrates were dissolved in 1 mL of methanol and diluted with 7 mL of KPi pH 7.5 to give 5 mM substrate solutions. Reaction setup consisted of mixing 400 L of 50 mM KPi pH 7.5 containing 5 mM substrate, 50 L of enzyme stock solution and 25-50 L of 20 mM H.sub.2O.sub.2 (final concentration 1-2 mM). Reactions were incubated in Eppendorf tubes with orbital shaking (1-inch orbital, 150 rpm, 28 C.) for 1-24h, after which the reaction was stopped by extracting with ethyl-acetate (for GCMS or chiral GC analysis). Ethyl-acetate extract was dried over anhydrous sodium-sulphate and 1 L was injected on HP5 column for GCMS or on Chiraldex G-TA column for chiral GC-FID analysis (both performed using Shimadzu instruments). Alternatively HPLC-UV/Vis with the chiral OD-H column can be used for quantification of enantiomeric excess.

[0209] Conversion of indene to 1,2-epoxyindane and 2-inden-2-one, as well as conversion of thionaphtene to hydroxy-benzothiophene and benzothiophen-2-one, was confirmed using GC-MS for enzymes with sequence numbers 16 and 17.

[0210] Sulfoxidation of various sulfide substrates (methyl phenyl sulfide, benzyl phenyl sulfide, allyl phenyl sulfide, benzyl methyl sulfide, N-butyl methyl sulfide, ethyl phenyl sulfide and isopropyl phenyl sulfide) was confirmed for enzymes with sequence numbers 4, 15, 16, 17 and 18 using GCMS analysis.

[0211] Moreover, using chiral GC and chiral HPLC analysis it was found that BUPOs are able to catalyze the enantioselective sulfoxidation of thioanisole (data not shown). This is another proof of true peroxygenase activity as it shows that oxygen is transferred from the heme iron. The enzymes tested were found to mainly produce the S-enantiomer (enzyme 18: 42% ee, enzyme 17: 23% ee, enzyme 16: 33% ee).

TABLE-US-00001 TABLE1 TypeIIenzymeshavingperoxygenase/ pigment-producingactivity NCBI Seq. accession Aminoacid No. number source sequence 15 NEA50176.1 Streptomyces MQPQLLFMPQVGHPY sp. QRPAEPPHTPGTAER SID10815 ELPEYDLLGARPVDA QRLFWYRWIAGHQIS FVLWRAMGDILWRHP DDPPGDRELDALATC VDGYSAMLLYSATVP RAHYHAHTRARMALQ HPSFSGAWAPDYRPV RRIFRGRFPWQGGRS CEALDQAIARNGVTH DHIANHLVPDGRSLL QQSAGAPGVSVSREK EDLYDNFFLTVRRPV GHTELVAQLDTRVAE VATDLAHNGLYPDVD GRHHPVVTWQSAGVM EPLLTGVLRVLDRAT RLMAHLRLEEVRS 16 WP_ Nocardia MQPHVLFMPPLGDSF 040743483.1 tenerifensis DGSAESDHSTEVTDR DWPRYNVFGNEPVEP ERLFWYRWIAGHQVS FLLWRAMCDVVWHHP DEDAPSERELDLLCA CIDGYSAVLLYSSTV PRDQYHADIRPRMAL QHPAFSGTWAPDYRP VRRLFRGKMPWQEDA SCAALGEAVARNGIT HSHIADHLVPEGRSL LQESAGAPGVSVSRE KEDLYDNFFLTVRRP VSQTEFVAQLDSRLA DLAADLAHNGLYPNV DGAHHPVVTEQADDE MRPFVTGVLDVLDRA ARLVSEMRLEEAHR 17 WP_ Nocardia MQPRLLFMPSLGEPF 029903812.1 brasiliensis DESAPDEDVVLAARD APEYPLFGTEPVEAE RLFWYRWIAGHQISF LLWRAMCDVVCQYPD EVPGERELEVLSACI DGYSAMLLYSSTVPR DHYHADIRPRMALQH PAFSGTWAPDYRPVR RLFHGRLPWQDDPSC RALDAAVDRNELTHS HIADHLVPDGRSLLQ QSVGALGVSVSREKE DLYDNFFLTVRRPVS HAEFVAQLDARITEL TVDLSHHGMYPMVDG RHHPVVGDRSEAVMQ PLITYAVQVVDRAAR LVAKMRLEGVRR 18 WP_ Actinokineospora MTHDRRRPQASTDPA 121392645.1 cianjurensis LQPRLLFLPATDGDY ADSDDTAHGADLDHL PDYTLFGTRPVEARR VFWYRWLAGHQISFA LWRAMSDVVTRRGDD LPSERELEVLTSCVD GYSAMLLYSATVPRD HYHANIRTRMALQHP SFSGAWAPDHKPIRL LFRGRFDWQDDPSCR ELDEAVARNRRTHDF IADHLVPDGRSLLQK SAGTVRQGVSRDKED LYDNFFLTIRRPVGH AELVTQLDDRVAELA EDLAHHGLYPEVDGR HHPVVTGRPDPALRP LIAEMPATLRRAAHL VAGMHYAGARS

TABLE-US-00002 TypeIenzymeshavingperoxygenase/pigment-producingactivity NCBI Seq. accession No. number source Aminoacidsequence 2 WP_078759802.1 Marinactinospora MDWSPSQSADIPFHLGIPEELTALPPIGPVPDLVA thermotolerans PITERTGAIDVRGDLSLFAEAMRAARKTLPLPAPE PVEADPRESQANRDNDEAFGIVRVAGPPLLLMLDG LLKAAAGVVEAAADYGTRIPAEEWADLVHGFDTVL GWLGGDDRPSSPPPLAFPPEEGPESNADDGVLVRW VRGHHLFMAICQACALALAHARQAQQRGALASART ALGVAAVMMGSGRATLRFAADASPTDYTTRIRPTL MPPVAPPRMSGLHWRDHEELIRQIRAVSRSWAQFA RHAPGQVEEFESVLATTYDAHRNVCAHFVGEEPSL LASSGSRGPASLSGPSVLGRLRRQRLSLLSLPPRE 3 WP_033325725.1 Streptomyces MTDYLEHVATVPFRIARPADLPTTIPDLAVVGGAA yerevanensis AARVSELLLAPAARAGLPALCDAVRRAGDELGPVP STLVTDDPDESRPNRDNDAAFGIERHSGDPAQLLR LALLHALEEVLDLTAASGTGLDEETWRELVDGFDV LLHWLAHPDRVLPADAAARVPAAPAQRVSTPMEGL KRWVRGHHVFMPFSQGCGLALASMSDAAQRGDEDA AVVGARVAIRTMRAARAALCFAGDATATQYQDEIR PTLMPPVAPPQMSGLRWRDHEYLVERLTAAGPAWH WLAARGFEALLRDFLAALDGAYEAHKEVCAHFVGT ASPSLLATARSHRPAVGVIEQFRQIRRSAVSAPGE QPAAKEKP 4 AAL33759.1 Pseudomonas MESIAFPIAHKPFILGCPENLPATERALAPSAAMA fluorescens RQVLEYLEACPQAKNLEQYLGTLREVLAHLPCAST GLMTDDPRENQENRDNDFAFGIERHQGDTVTLMVK ATLDAAIQTGELVQRSGTSLDHSEWSDMMSVAQVI LQTIADPRVMPESRLTFQAPKSKVEEDDQDPLRRW VRGHLLFMVLCQGMSLCTNLLISAAHDKDLELACA QANRLIQLMNISRITLEFATDLNSQQYVSQIRPTL MPPIAPPKMSGINWRDHVVMIRWMRQSTDAWNFIE QAYPQLAERMRTTLAQVYSAHRGVCEKFVGEENTS LLAKENATNTAGQVLENLKKSRLKYLKTKGCAGAG 5 WP_157246732.1 Nonomuraeasp. MQDYLQGVVTEPFTLCRPDDLPGTTGELSSAAGLV p1410 RPGARTVTGPGARRDLAGLLAALGEARSALDRGPV TPVGEDPTESDLNKANDLGFGILRVRGEPVRVLTD AALRSIAAVLGLAARNGTGFEPATWRRLVGGFDAL LLWLADPGRAPASHPVPAPGERQPLEPGGALRRWI YGHHVFMVFSQGCTLSLACLRERAAAGDGSGAAAA AATAVRLMWASQGALSFAGDVAMEDYATEIRPTLM PPIAPPRMTGLHWRDHEALVRELAAARDAWQWLAG SRPELLEEFRDAIDSAYSAHRGVCASFVGDRSPSL LATSRSSRPAVAVLEQFHRRRLDMLPPSARDDGDG R 6 QCC21379.1 Candidatus MREISPIFYLLQPENLPISRDRLRCGDIVQEVLSH Endohaliclona ILNLTRNENTILDGIHQAFSLLAPSFEGISVVKED renieramycinifaciens PLESLENRANDEAFNIKRVNGNPSYVLTYSTLQSY AGMLEDALDIGTSLSEVEWTKLNLAYISIFDMLRG GITPINSFKFSTIMPKKSRTYNNENERDGMRRWVC GHYVFMSIVQAMIVALNFFSSEVANDDIQASQLAI DRAILLMKGSESALKFTGDFSRRSYEQSVRPSLSP PKAPRGMSGVNWRDHEYLIKKVFRKLQPIFLNPPL EVKAKLYDFLDALKDAYDAHKLVCSFFVGNDKPSL LSKKSAMNMLESYKQNRVAVLKKPQKGLRV 7 WP_152646293.1 Streptacidiphilus MIDVETQARHPDVPALHLLKAEQLPASMSAIIGMM albus NQRTADALRTVIDTNPQNSDRHALAAVDAALACLT APAATTKYTDAERNSLPEDLYEEHDLFFGAIRVTG NPVETFAHTMLQALRSQLNDALSAGTSLCSGERGQ LREAFWSVFHELWRIGVAGTPFEYAARARPLDTGL GLDDAEADPLIRWRLGHQVFFALIQALIVSVGCLE ECLRDDPDDIESACRLLEDATVLMIGSGASMRYAG DFTRTHYTDAVRPAMMPPHINAKFSGLQLRDHRIL LKLLNRVKPLLASPAPAVDKSYRQLLDAMSTAYDA HISVCSRFGGDRESSLRTPGSSLPAVGVLERFRSR RIGAATPDRPAE 10 WP_093605566.1 Lentzea MTLILLHPTSLPRREDQLSLAGDAPAPLRLARRLA waywayandensis AGVSGADLLDRLRTLPARSGAVALAERVDSGVPYT YDHRLREFDDHFGVERVDVDTDGVFARSLLVAYRS LLEEGLASGTRMSWARWSVLVSVLQNMIVRCAGEE ADLPVAVRPPLLRWHLDPERRWRVGHHVFFVVTQC LIVALQSFATALGEDDLPAARRALRLATRLLDASS AAFVFTAEFGANQYHQAVRPTMEPPFVSAGFSGLL SPDHHYLVRLFAEVRPMLRQLPADLVADHAAFVRA LGAVYESHKYVCARFGGDTGVSLRTSALPAVEVLH ALKLARTKIVGRP 11 WP_156139503.1 Sphingomonassp. MVALEGLLARSDERTDTSSLDAPAAPPRRGSQIAR 35-24ZXX KGKVRKGGVKQVQAKLARVFLRGVEPVVVRSAPTS GSRLPRADLRGLTTALTANPKSHQHVVHRVRSRLV HIHALPATHVPTPSQVQAALGHNRLQQYAELMPER RTAHVLAALSTQLARPAQSLSAVVDSPNVRDNELH DAYFCVVRVDPRNLASHVEGIILAAQALRREMRSG SDRLGIADFGEIADGIELAFTLATGIRSPRIAMLR TVHRAALSETMIARRWMRGHQLFLVITQAMIASLN NLEVIAGHGSDDSEMAGAIEQLVRLLRAAAGSMRL TGDFPPDIYDTIIRPSMAPPELPDGFSGIFSSDHR FLVTRLREITPKFSGLKERLHDAHALLVKAFADLY DDHINVCANFVSSEKNSLLMADGARCTALEQLGKF KKSRLRLLGKP 12 WP_092059473.1 Poseidonocella MQVESLTLPLPHDWFAAETTLPKHRFRMSQLMASG pacifica NAMTGLAARVTQAVANEEFAPSESTLDGAQRLFDS YFLVERASGAPVEMLRAAMAKALPLCAADIETGSA LSDVQLQTLARGLHRLADWALIPTAAPAAASPHVT ADPLFRWKQQHQLFFLIIHGMLYLLHVLEETLDRE HAPVTQAVLSDFADLMEASTVAFHLAADFSPEDYE SRIRPDMTAHDPHFSGLFYADHKELVTSLRVLKRV PDDFEEELDRISRAISETYDAHARVCLRFVGETAS LASKDDSRIAAESIRGKYVKRTKVIAGLAKPRA 13 PKW18123.1 Saccharopolyspora MSCEKWNTMPDEQKADSFLRLLDPNHLPVERESLA spinosa RAAIRLVGDHRDDQVLRVTEPERHRARRVLTDELL GHEPIPPARSGRRALDQDEHDRHFGVVRVHGDSGA IFLTSLLVSYLDMLDDVERGGTTLGQAQWTDLLRV PTLVFDFALRRQASAFPGSDFPIPAGRCELLPHRR WHVGHRLFFTLIQTTTQAVTSLSQAVRAGDEAAEA DVRGCVDFVRLTTLGSTAALRLTADFSPVDYEQRV RPTMLPPHVRPGFSGLQTRDHRHLLNAMRKLREDM ATVAELGEPHAELIAAIRSLHDAHAYVCTRFGGDT APSLRMAALGQQDGKGVEVVRGLARHRESLLEPSR ANAHDQPRAYQRSDEDGHE 19 WP149740153.1 Rhizobiumsp.RU20A MPEDLPGRTAFCAAIPATLRADCAADIAASHMQFR RTLPALKALMPSAAAIETGRIDFMIDTGQACDIDS HDHFFRIRRSRTGAAPALLASQIQTLQILKTEVTA GPVTVPAERIVAMADAIRAAIAFLVGTQATTPTEL TFRSVTEGDPPAERAMQRWVRGHQIFAGLCQSLVV ALGALESAVQDQDDATVDSAGTLVASLLSASATAL ELTGDFPEARYNAGIRVAMDAPYFPTGFSGVLSRD HRQLVSRMKMMRPAIESLGRRHPALHASIGAALSS VYASHKHVCARFVEPRSTSLLMAATAERPATEQID RFRAMRLRAWDVSAEHPGVSGSSSE 20 WP_015586201.1 Rhodococcus MTPSPTSIDRVVLPTDSTIPVARSAEYDTYFRISR SNDPHAWAVSALTVVTAITTDLPQSTLDNDSRLRL LDALAVVVGDASAAAAVSARERDPSQLAAPTDHSA DNRTPQAPNDSARQAITRWKKGHHLFHLFVISMNS RLAAVLECLANKQWPTLAHQLTELGTLYAGATESM RYASDFSPEIYRDFVRPSMEPPWLEPGFSAVFNRD HDVMLTRAKIVRTTLKTAELPGVAADSARRLWKSQ AENRRAHQLICQRFVPGGDSLLQEHFDSTGQSN 21 WP_019808821.1 Saccharomonospora MNTENKTDLIETIALRDDARAPTSETAADSRHYDD halophila YFRIDRTDDSATWWAGAQRVARLVVDDLPATGLDE DLRADLLAGVSRLLLASASAPAHSGTTMPVRTPTH GEDSVAARRWKLGHHLFHHMLSLMNSHADGIASAL DEPDPVTVGLLVEELTVLYDSATATMHYASSFPIS AYAETVRPSMEPPWMPPGFSGVFNREHETLLSSLT GLKPRLRGRTAERSLPADVKTASRALWKAQSRNRR EHKMICERFVPGGGSLLQDYFDETRKG 23 WP_091674400.1 Amycolatopsismarina MTDADAPESARIAMRRWRLGHHAFHLQLVVMNTLL TRADQACAEGRWADLTSDLVRLRTLYNGATATMAL AAAFDSDMYNQVVRPSMEPPFLAPGFSAAFGREHT IMLRRIERLHHRIRAGSSTKEGRPDQVLRAHRELQ RAQARNRREHVLICRRFVQDGKSLLRESEATH 24 WP_098424542.1 Bacillus MIKQITEAHQQAMVRRKQGHQVFHVILVCMNTLLE thuringiensis ESKSALEQNNFLKLQSILEQLTVLYDSATVSMKYT ADFSPKYYEEMIRPSMMPPFLSPGFSGTQNKDHQQ MIEGLGQLQKQMLNKLGDKNKWPDNVLQAWNLLSK SQIRNRKHHGLVCQNHVPEGASLLKQFYQNNK 25 WP_162035492.1 Chengkuizengella MQSNSVESISTAYQQAVLRWKQGHQSFQIIIITMN sediminis TLLESSIQALDQNEWHLLTKSLDRLSNLMKASTAT MKYTSDFSPKSYEELIRPSMMPPFLSEGFSGVLNI DHKLMLIKFRKLRDLMVQKLGDKHQWPSMITRSWN HLMDAQTHNREHHGLVCQHFVDDGVSLMQNFYKEK REAKN 26 WP_156623984.1 Mycobacteriumsp. MEQSTAALLSPEQKTAIRRWKLGHHIFHIYLFAMN 852002- TNLHTAREQLHRESWDQLVDSLQSLVQLYDAATAS 40037_SCH5390672 MKYAADMDRAEYQSLIRPSMAPPFLKPGFSGQQNK DHTTMVQLIRKLRREIRERGHRDNAYLPQEVYAAA KALWEAQARNRRHHILVCDRLVPGEGSLLQTYFAS IRSRGISKEGN 27 WP_093637903.1 unclassified MHISAEQQTAVRRWKLGHHVFHLHLTVMNTYLASL Streptomyces EKSINEEDWRSVSPLLTKLSRLYGAATSCMRYASD FPETAYESLIRPSMEPPWLNPGFSGKFNSDHERML DLMRTIRTSLKRAIRSGEVPEEVERAATQLWRAQS HNRANHKLICEKFVPGGQSLLQDYFNANA 28 WP_093231630.1 Thermoflavimicrobium MSVTNPSIPASYQQAVLRWKQGHHVFHVILVTMNT dichotomicum CLEESLRALNQQDWSRLIQLLERLATLYDAATATM KYSSNFSRKYYEEVIRPSMMPPFLKPGFSGKLNRE HNVMLDLFQTLRAELKKKEELPLGVEEAWRKLVQS QKRNRKHHGLVCQQFVDDGVSLLQEFYRSQTK 29 ADQ55481.1 unculturedorganism MIIIQGLIISLQCFIFEYSKKKFLQSLLYLNNAIK LMKATEVALYYTGEFSSKSYNENVRPTLMPPISQP EMSGLNWRDHQFMVKNCMRSIGKLNFTSYPIIQKK YNIFIISLKKAYHAHKYVCGKFVGPASGSLRSNEY SAVQEIEKFKKLRLKILKG