Methods for producing halogenated anthraquinone pigments and dyes

Abstract

The present disclosure pertains to the provision of a novel halogenase enzyme, nucleic acid constructs and expression vectors comprising a nucleotide sequence coding for said halogenase enzyme, a host cell expressing said halogenase enzyme, and methods using said halogenase enzyme or host cell expressing said halogenase enzyme for producing halogenated anthraquinone compounds, in particular chloro-endocrocin, chloro-dermolutein, chloro-emodin.

Description

[0001] METHODS FOR PRODUCING HALOGENATED ANTHRAQUINONE PIGMENTS AND DYES

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure pertains to the provision of a novel halogen- ase enzyme, nucleic acid constructs and expression vectors comprising a nucleotide sequence coding for said halogenase enzyme, a host cell expressing said halogenase enzyme, and methods using said halogenase enzyme or host cell expressing said halogenase enzyme for producing halogenated anthraquinone compounds, in particular chloro-endocrocin, chloro-dermolutein, chloroemodin.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Colorants for dyeing textiles, plastics, food, feed, cosmetics etc. are produced by chemical synthesis from non-renewable fossil raw materials. These colorants are recalcitrant to biodegradation in the environment, and are often toxic to the environment, to the workers who prepare and use them, and sometimes even to the consumers. Their chemical synthesis uses toxic reagents and produces huge amounts of toxic wastes, especially wastewater.

[0006] Anthraquinone pigments such as emodin, endocrocin and their analogues and derivatives, including the chlorinated anthraquinones chloro-endo- crocin, chloro-dermolutein, chloro-emodin, are produced by Cortinarius spp. mushrooms in the Dermocybe clan. However, these mushrooms are not cultivated, and can be foraged from the environment seasonally in non-industrial quantities, and can only be collected and processed manually. This leads to a variable and only periodic availability of these pigments; amounts that are not sufficient for industrial manufacturing processes; relatively high prices; and uneven quality of the colorant product. Cortinarius anthraquinone products are also not pure, but a complex mixture of emodin and endocrocin derivatives with other unidentified metabolites. Thus, these products display different hues, physicochemical properties, bioactivities, and toxicity and allergenicity profiles. In particular, the chlorinated anthraquinones are a minor fraction of the various anthraquinones produced by Cortinarius. They are very difficult to purify from the other anthraquinones present in much larger amounts and can be isolated from Cortinarius mushrooms in very small amounts only. This limits the utilization of chlorinated anthraquinones as colorants, in spite of their known advantageous properties. For example, the colour intensity of chlorinated anthraquinones is higher than those of other anthraquinones.

[0007] The pigment industry for the textiles, plastics, paints and coatings, food colorants and cosmetics market segments demand anthraquinones that are available year-round at a reasonable price in large quantities, at constant quality, and with defined chemical composition and purity. Such a supply can only be created by the synthetic biological production of anthraquinones.

[0008] Cohen, P. A. et al. Phytochemistry 44(2): 271 -274 (1997) discloses biocatalytic chlorination of emodin and 7-choloroemodin in vitro using a commercially available fungal (Caldariomyces fumago) chloroperoxidase or a semipurified enzyme preparation from the lichen Nephroma laevigatum. The disclosed C. fumago chloroperoxidase belongs to the family of heme-dependent haloperoxides and the halogenase activity of the enzyme preparation semi-purified from N. laevigatum also appears dependent on hydrogen peroxide. Thus, the chlorination reaction mechanism of the enzymes disclosed in Cohen et al. is different from that of the FAD-dependent halogenases. Chloroperoxidases typically show low substrate and regio specificity as opposed to FAD-dependent halogenases.

[0009] WO 2019 / 171046 A2 discloses halogenases Dsg92 and Dsg96, which, however, have not been purified and their enzymatic activity and substrate specificity have not been experimentally characterized. Methods for chlorinating endocrocin, dermolutein or emodin are not disclosed.

[0010] Menon, B. R. K. et al. Catalysis Reviews, 2022, 64(3): 533-591 ; Menon, B. R. K. et al. Angewandte Chemie International Edition, 2017, 56(39): 11841 -11845; Cochereau, B. et al. Molecules, 2022, 27(10): 3157; EP 4 242 311 A1 ; and Luo et al. Antibiotics, 2022, 11 (10): 1304 disclose flavin-dependent halogenases and uses of these. Methods for chlorinating endocrocin, dermolutein or emodin are not disclosed. Chemical halogenation uses harsh reagents that are toxic to the environment and hazardous to workers. Chemical halogenation is not regiospecific and provides a mixture of regio-isomeric products.

[0011] WO 2022 / 254047 A1 discloses production of chlorinated emodin by treating emodin isolated from the rhizomes of Japanese knotweed with / V- chlorosuccinimide (NCS) in THF solvent, and the use of chlorinated emodin in dyeing textiles.

[0012] Lei Sun et al. Metabolic Engineering, 2019, 54: 212-221 , discloses metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin. Halogenases and chlorinated anthraquinones are not described.

[0013] Cummings M, et al. 2019 PLoS Biol 17(7): e3000347 discloses methods of biological engineering of aromatic polyketides in E. coli. Methods for chlorinating endocrocin, dermolutein or emodin are not disclosed.

[0014] BRIEF DESCRIPTION OF THE DISCLOSURE

[0015] To address the above challenges in the prior art, inventors have identified halogenase-encoding genes in two Cortinarius mushrooms, C. semisan- guineus and C. sp. KIS3. These halogenases show very little sequence similarity to other FAD-dependent halogenases that had previously been functionally characterized in the literature. The present inventors cloned these coding sequences (prepared by reverse transcription from mRNA or gene synthesis following codon optimization and removal of introns) with appropriate promoter and terminator sequences into the genome of a suitable host cell, here a Saccharomyces cerevisiae strain that had previously been engineered to produce emodin, endocrocin, and dermolutein. In addition, the yeast host carries the fre flavin reductase gene from E. co / / whose protein product supplies FADH for the halogenation reaction within the cell. The resulting recombinant strain produced chloro-endocrocin, chloro-dermolutein, and chloro-emodin as detected by LC- MS analysis of the cultures. The newly discovered halogenase enzymes can be isolated, purified and used in vitro to chlorinate anthraquinones of interest, such as endocrocin, emodin, and dermolutein. The halogenase expressing host cells can likewise be used to chlorinate anthraquinones of interest, such as emodin, endocrocin, and dermolutein, either by supplying such suitable substrates of the halogenase enzyme to the culture medium of the halogenase-expressing strains or by further engineering the host cell to produce these substrates. Alternatively, the host cell for expressing the halogenases may be selected among strains that are natively able to produce the substrates. The produced chlorinated anthraquinones such as chloro-endocrocin, chloro-dermolutein, chloro-emodin, as well as their structural analogues and derivatives can then be isolated from the fermentations and used as intense yellow colorants to dye textiles, plastics, foods, feeds, and cosmetics, for example.

[0016] The invention thus provides a sustainable, year-round, efficient, and scalable production system to obtain these chlorinated anthraquinones with much increased purity compared to natural sources, and without the use of toxic reagents and without producing large amounts of toxic waste.

[0017] The challenges in the prior art are thus overcome by the provision of a novel halogenase enzyme, nucleic acid constructs and expression vectors comprising a nucleotide sequence coding for said halogenase, a host cell expressing said halogenase, and methods using said halogenase enzyme or host cell expressing said halogenase enzyme for producing halogenated anthraquinone compounds (for example chloro-endocrocin, chloro-dermolutein, chloroemodin), which are characterized by what is stated in the independent claims. The preferred embodiments of the invention are disclosed in the dependent claims.

[0018] More specifically, the present disclosure provides an enzyme with halogenase activity comprising or consisting of an amino acid sequence with at least 82% sequence identity over the full length of SEQ ID NO: 1 (halogenase consensus), in particular wherein in SEQ ID NO: 1

[0019] X at position 7 is I, L or V, in particular wherein X is I or L; and / or

[0020] X at position 50 is V, I or L, in particular wherein X is V or I; and / or X at position 98 is I, V or L, in particular wherein X is I or V; and / or

[0021] X at position 105 is N, S, Q, A or T, more preferably wherein X is N or S; and / or

[0022] X at position 106 is A, S, G or T, more preferably wherein X is A or S; and / or X at position 303 is N, D, Q or E, preferably wherein X is N, D or Q, most preferably wherein X is N or D; and / or

[0023] X at position 320 is I, V or L, in particular wherein X is I or V; and / or

[0024] X at position 325 is T, S, A, G or N, most preferably wherein X is T or S; and / or X at position 331 is N, S, Q, A, T or G, most preferably wherein X is N or S; and / or

[0025] X at position 371 i particular wherein X is V or I; and / or X at position 514 i particular wherein X is I or L; and / or

[0026] X at position 518 i N, most preferably wherein X is E or D; and / or

[0027] X at position 555 i particular wherein X is I or V; and / or

[0028] X at position 570 i E, more preferably wherein X is D, N or Q, most preferably wherein X is D or N; and / or

[0029] X at position 572 is Y, F, W, H, L or M, preferably wherein X is Y, F, W or H, most preferably wherein X is Y or F.

[0030] Further provided is a nucleic acid construct, comprising a nucleotide sequence coding for the enzyme with halogenase activity of the present disclosure, and an expression vector, comprising a nucleotide sequence coding for the enzyme with halogenase activity of the present disclosure in functional linkage with a promoter and a terminator sequence. Moreover, the present disclosure provides a host cell recombinantly producing the enzyme with halogenase activity of the present disclosure. The enzyme with halogenase activity of the present disclosure or the host cell recombinantly producing said enzyme can be advantageously used in the production of halogenated anthraquinone compounds.

[0031] In this context, the present disclosure furthermore provides an in vitro method of producing halogenated anthraquinone compounds, comprising the step of providing an enzyme with halogenase activity of the present disclosure with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound; in particular wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof.

[0032] Furthermore, the present disclosure provides a method of producing halogenated anthraquinone compounds, comprising the steps of

[0033] (i) culturing a host cell of the present disclosure in a suitable culture medium, and

[0034] (ii) isolating the halogenated anthraquinone compound from the host cell culture.

[0035] In embodiments, the method further comprises the step of supplementing the culture medium of step (i) with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound; in particular wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin and derivatives thereof. In embodiments, the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye; in particular selected from the group consisting of chloroemodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

[0036] BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In the following the invention will be described in greater detail by means of preferred embodiments with reference to the drawings, in which

[0038] Figure 1 provides a sequence alignment of the two isolated halogen- ase enzymes.

[0039] Figure 2 shows the biosynthesis of halogenated anthraquinones in Cortinarius spp. CkPKS3 (in Cortinarius sp. KIS-3) or CsPKS2 (in C. semisan- guineus) are responsible for the iterative condensation of malonyl-CoA precursors to produce atrochrysone carboxylic acid (3) that is then converted to emodin (1) or endocrocin (2). CkOMT2 or CsOMT5 O-methylate endocrocin to afford dermolutein (5). CkHALI (in Cortinarius sp. KIS-3) or CsHAL2 (in C. semisan- guineus) described herein halogenate these anthraquinone scaffolds to produce their halogenated derivatives.

[0040] Figure 3 shows the de novo production of halogenated anthraquinones in S. cerevisiae H6825 and H6827 culture extracts. (A) LC-MS detection of halogenated anthraquinones (selected ion chromatograms with the extracted m / z values of anthraquinone compounds) in PKS3-OMT5 strain expressing the CkHALI (strain H6825) or the CsHAL2 halogenase (strain H6827) compared to a strain expressing only PKS3-OMT5 (strain H6698). The chromatographic trace at the bottom represents the overlay of extracted ion chromatograms of authentic standards. (B) MS spectrum of chloro-emodin, chloro-endocrocin, and chloro- dermolutein, detected in strain H6827 (PKS3-OMT5-CsHAL2) culture extracts. The circled peaks correspond to the two m / z values of the compounds due to the presence of both naturally occurring chlorine isotopes (Cl35and Cl37). (C) MS spectrum of chloro-emodin, chloro-endocrocin, and chloro-dermolutein standards. The circled peaks correspond to the two m / z values of the compounds due to the presence of both naturally occurring chlorine isotopes (Cl35and Cl37). Legend: Emodin (1 ), Endocrocin (2), Dermolutein (5), Chloro-emodin (6), Chloro-endocrocin (7), Chloro-dermolutein (8).

[0041] Figure 4 illustrates the LC-MS detection of halogenated anthraquinones in culture extracts of Aspergillus oryzae Ao_CkHAL1 (M7541 ) and Ao_CsHAL2 (M7542), compared to that of the control parental strain M6576. The trace at the bottom represents the overlay of extracted ion chromatograms of authentic standards. Legend: 1. Emodin, 2. Endocrocin, 5. Dermolutein, 6. Chloro-emodin, 7. Chloro-endocrocin, 8. Chloro-dermolutein.

[0042] Figure 5 illustrates the LC-MS detection of halogenated anthraquinones in culture extracts of Trichoderma reesei Tr_CkHAL1 (M7543) and Tr_CsHAL2 (M7544), compared to that of the control parental strain M6578. The trace at the bottom represents the overlay of extracted ion chromatograms of authentic standards. Legend: 1. Emodin, 2. Endocrocin, 5. Dermolutein, 6. Chloro-emodin, 7. Chloro-endocrocin, 8. Chloro-dermolutein.

[0043] Figure 6 shows product profiles (selected ion chromatograms with the extracted m / z values of anthraquinone compounds) from cultures of S. cere- visiae strain (A) expressing Cortinarius halogenases (CkHALI or CsHAL2) and other known FAD-dependent tryptophan halogenases from literature, together with the Fre flavin reductase. The trace at the bottom represents the overlay of extracted ion chromatograms of authentic standards. (B) MS spectrum of the peak (9) corresponding to the putative chloro-emodin regioisomer detected from the culture extracts of H6674 (RadH-Fre), and that from the peak corresponding to chloro-emodin (6) from strain H6827 (CsHAL2-Fre). Legend: Emodin (1 ), Endocrocin (2), Dermolutein (5), Chloro-emodin (6), Chloro-endocrocin (7), Chloro- dermolutein (8), Uncharacterized chloro-emodin (9). Figure 7 illustrates biocatalytic production of halogenated anthraquinones with recombinant S. cerevisiae strain expressing halogenase CkHALI (strain H6823) or CsHAL2 (strain H6824), compared to the control parental strain H4590. (A) Feeding with emodin (1) as substrate. (B) Feeding with endocrocin (2) as substrate (C) Feeding with dermolutein (5) as substrate. The trace at the bottom of each panel represents the overlay of extracted ion chromatograms of authentic standards. Legend: Emodin (1 ), Endocrocin (2), Dermolutein (5), Chloro-emodin (6), Chloro-endocrocin (7), Chloro-dermolutein (8).

[0044] Figure 8 illustrates the production of chloro-endocrocin with recombinant Aspergillus fumigatus. (A) LC-MS detection of chloro-endocrocin from culture extracts of recombinant Aspergillus fumigatus. (B) MS spectrum of chloro-endocrocin peak detected from the culture extracts of recombinant As- pergillus fumigatus. The circled peaks correspond to the two m / z values of chloro-endocrocin due to the presence of both naturally occurring chlorine isotopes (Cl35and Cl37).

[0045] Figure 9 illustrates the production of chloro-emodin with recombinant Aspergillus ochraceus. (A) LC-MS detection of chloro-emodin from culture extracts of recombinant Aspergillus ochraceus. (B) MS spectrum of chloro-emodin peak detected from the culture extracts of recombinant Aspergillus ochraceus. The two peaks correspond to the two m / z values of chloro-emodin due to the presence of both naturally occurring chlorine isotopes (Cl35and Cl37).

[0046] Figure 10 shows production of halogenated anthraquinones with isolated recombinant halogenase CkHALI or CsHAL2, compared to the control reaction without enzyme. (A) In vitro assay with emodin (1) as substrate and chloro-emodin (6) as the product. (B) In vitro assay with endocrocin (2) as substrate and chloro-endocrocin (7) as the product. (C) In vitro assay with dermolutein (8) as substrate and chloro-dermolutein (8) as the product. The trace at the bottom of each panel represents the overlay of extracted ion chromatograms of authentic standards. Legend: 1. Emodin, 2. Endocrocin, 5. Dermolutein, 6. Chloro-emodin, 7. Chloro-endocrocin, 8. Chloro-dermolutein.

[0047] Figure 11 illustrates a sequence alignment of SEQ ID NO: 2 (CkHALI ), SEQ ID NO: 3 (CsHAL2), and RadH. Residues forming the FAD binding site are boxed, and the catalytic residues are highlighted.

[0048] Figure 12 illustrates a superimposition of Alphafold-generated structures of SEQ ID NO: 2 (CkHALI ) or SEQ ID NO: 3 (CsHAL2) with the experimentally determined crystal structure of RadH (PDB id: 8GU0, Peh et al. 2023). A) Superimposition of RadH (in white) with CkHALI (in black); B) Superimposition of RadH (in white) with CsHAL2 (in black).

[0049] Figure 13 illustrates a superimposition of the active sites from the Alphafold-generated structures of SEQ ID NO: 2 (CkHALI ) or SEQ ID NO: 3 (CsHAL2) with that of the experimentally determined crystal structure of RadH (PDB id: 8GLI0, Peh et al. 2023). The FAD coproduct is shown as white sticks, while the chlorine ion is shown as a sphere. The catalytic residues are shown as sticks. A) Superimposition of the RadH active site (in white) with that of CkHALI (in black); B) Superimposition of the RadH active site (in white) with that of CsHAL2 (in black).

[0050] Figure 14 illustrates comparative production of chloro-dermolutein and chloro-emodin in the S. cerevisiae H6827 (CsHAL2-Fre) and H6954 (CsHAL2) strain. (A) Percentage conversion of chloro-dermolutein in H6827 and H6954 strain (B) Percentage conversion of chloro-emodin in H6827 and H6954 strain.

[0051] DETAILED DESCRIPTION

[0052] The present inventors have identified halogenase-encoding genes in two Cortinarius mushrooms, C. sp. KIS3 and C. semisanguineus. These halo- genases show very little sequence similarity to other FAD-dependent halogen- ases that had previously been functionally characterized in the literature. The amino acid sequences of the encoded gene products are provided herein as SEQ ID NO: 2 and SEQ ID NO: 3, respectively. An alignment of both sequences is shown in Figure 1 , which shows that the two sequences have 95% mutual sequence identity over their respective full length. The alignment shows which amino acid residues are conserved between these two halogenases, and which amino acid positions are apparently less restrictive. A consensus sequence of this alignment is provided herein as SEQ ID NO: 1.

[0053] Based on these findings, the present disclosure provides an enzyme with halogenase activity comprising, in particular consisting of, an amino acid sequence with at least 91 % sequence identity over the full length of SEQ ID NO: 1 (halogenase consensus sequence).

[0054] As used herein, an amino acid sequence is said to have X % sequence identity with a given source SEQ ID NO over the full length of said SEQ ID NO if the sequence is aligned with said source SEQ ID NO and the sequence identity between those two aligned sequences is at least X %. Such an alignment can be performed using for example publicly available computer sequence similarity programs such as the “BLAST” program, in particular “blastp” provided at the NCBI website at http: / / www.ncbi.nlm.nih.gov / blast / blast.cgi, using the default settings provided therein. Subsequently, identical residues are counted and calculated as the percentage identity by dividing the number of identities over the full length of the source SEQ ID NO. Further methods of calculating sequence identity percentages of two polypeptides are known in the art.

[0055] In embodiments, the enzyme with halogenase activity comprises, in particular consists of, an amino acid sequence, which has at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% sequence identity over the full length of SEQ ID NO: 1 .

[0056] The amino acids are herein represented by their one-letter or three- letter code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (lie); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan (Trp); and Y: tyrosine (Tyr).

[0057] The consensus sequence of SEQ ID NO: 1 contains several positions at which the amino acid may be any amino acid. These positions indicated in SEQ ID NO: 1 with “X” corresponding to those positions, in which SEQ ID NO: 2 and SEQ ID NO: 3 differ from each other (see Figure 1 ). In preferred embodiments, said amino acid “X” can be a semi-conservative substitution of the corresponding amino acid at the same position in SEQ ID NO: 2 and / or of the corresponding amino acid at the same position in SEQ ID NO: 3. In even more preferred embodiments, said amino acid “X” can be a conservative substitution of the corresponding amino acid at the same position in SEQ ID NO: 2 and / or of the corresponding amino acid at the same position in SEQ ID NO: 3. Semi-con- servative and conservative substitutions are those wherein one amino acid is replaced by another amino acid within a family of amino acids that share same characteristics in their side chains and / or have similar chemical properties. Examples of such families are amino acids with basic side chains, acidic side chains, non-polar aliphatic side chains, non-polar aromatic side chains, uncharged polar side chains, small side chains, large side chains, etc. Typical semi-conservative and conservative substitutions are: In light of the foregoing, in embodiments the enzyme with halogenase activity is an enzyme, wherein in SEQ ID NO: 1

[0058] X at position 7 is any amino acid, preferably wherein X is I, L, V, M, A, F, Y, W, G, H or C; more preferably wherein X is I, L, V, M, or A, most preferably wherein X is I, L or V, in particular wherein X is I or L; and / or X at position 50 is any amino acid, preferably wherein X is V, I, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is V, I, L, M, or A, most preferably wherein X is V, I or L, in particular wherein X is V or I; and / or

[0059] X at position 78 is any amino acid, preferably wherein X is selected from R, H, K, F, Y, N, Q, S, T, D, E, A, more preferably wherein X is selected from R, H, K, Y, F, more preferably selected from R, H, K, and most preferably selected from R and H; and / or

[0060] X at position 98 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V; and / or

[0061] X at position 105 is any amino acid, preferably wherein X is N, S, Q, A, T, G, D, E, K, R, more preferably wherein X is N, S, Q, A, T, more preferably wherein X is N or S; and / or

[0062] X at position 106 is any amino acid, preferably wherein X is A, S, G, T, N, V, D,

[0063] E, R or K, more preferably wherein X is A, S, G, T or N, more preferably wherein X is A, S, G or T, more preferably wherein X is A or S; and / or

[0064] X at position 174 is any amino acid, preferably wherein X is S, P, A, T, G, N, V, I, D, E, R, K, L, M, W, Y, C or F, more preferably wherein X is S, P, A, T, G, N, V, or I, more preferably wherein X is S or P; and / or

[0065] X at position 238 is any amino acid, preferably wherein X is C, G, A, V, L, M, I,

[0066] F, S, N, T, D, E or Q, more preferably wherein X is C, G, A, V or L, more preferably wherein X is C, G or A, most preferably wherein X is C or G; and / or

[0067] X at position 239 is any amino acid, preferably wherein X is P, S, A, T, G, N, V, I, D, E, R, K, L, M, W, Y, C or F, more preferably wherein X is P, S, A, T, G, N,

[0068] V, or I, more preferably wherein X is P or S; and / or

[0069] X at position 240 is any amino acid, preferably wherein X is G, R, A,K, H, S, N, T, D, E, Q, more preferably wherein X is G, R, A,K, or H, most preferably wherein X is G or R; and / or

[0070] X at position 244 is any amino acid, preferably wherein X is Y, Q, F, W, H, N, L,

[0071] M, I, V, C, D, E, A, S, T, K or R, more preferably wherein X is Y, Q, F, W, H or

[0072] N, most preferably wherein X is Y or Q; and / or

[0073] X at position 296 is any amino acid, preferably wherein X is Y, S, F, W, H, A, T,

[0074] G, N, L, M, I, V, C, D, E, R or K, more preferably wherein X is Y, S, F, W, H, A, T, G or N, most preferably wherein X is Y or S; and / or

[0075] X at position 303 is any amino acid, preferably wherein X is N, D, Q, E, S, T, A, G, K, R or H, more preferably wherein X is N, D, Q or E, more preferably wherein X is N, D or Q, most preferably wherein X is N or D

[0076] X at position 320 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y,

[0077] W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V; and / or

[0078] X at position 325 is any amino acid, preferably wherein X is T, S, A, G, N, V, D, E, R, K or I, more preferably wherein X is T, S, A, G, N or V, more preferably wherein X is T, S, A, G or N, most preferably wherein X is T or S; and / or

[0079] X at position 327 is any amino acid, preferably wherein X is V, A, L, I, G, S, T, C, N, M or V, more preferably wherein X is V, A, L, I, G, S, T, C or N, more preferably wherein X is V, A, L, I, G, S or T, more preferably wherein X is V, A or G, most preferably wherein X is V or A; and / or

[0080] X at position 331 is any amino acid, preferably wherein X is N, S, Q, A, T, G, D, E, K or R, preferably wherein X is N, S, Q, A, T or G, most preferably wherein X is N or S; and / or

[0081] X at position 351 is any amino acid, preferably wherein X is N, Y, Q, F, W, H, D, E, S, T, A, G, K, R, L, M, I, V or C, preferably wherein X is N, Y, Q, F, W or H, most preferably wherein X is N or Y; and / or

[0082] X at position 371 is any amino acid, preferably wherein X is V, I, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is V, I, L, M, or A, most preferably wherein X is V, I or L, in particular wherein X is V or I; and / or

[0083] X at position 396 is any amino acid, preferably wherein X is L, S, M, I, V, A, T,

[0084] G, N, F, Y, W, H, C, D, E, R or K, preferably wherein X is L, S, M, I, V, A, T, G or N, most preferably wherein X is L or S; and / or

[0085] X at position 491 is any amino acid, preferably wherein X is R, S, K, H, A, T, G, N, Q, D or E, more preferably wherein X is R, S, K, H, A, T, G or N, most preferably wherein X is R or S; and / or

[0086] X at position 493 is any amino acid, preferably wherein X is D, G, E, N, Q, A, S, T, K, R or H, more preferably wherein X is D, G, E, N, Q, A, S or T, more preferably wherein X is D, G, E, N, Q or A, most preferably wherein X is D or G.

[0087] X at position 495 is any amino aciod, preferably wherein X is T, A, S, G, N, V, C, D, E, R, K or I, preferably wherein X is T, A, S, G, N, V or C, more preferablt wherein X is T, A, S, G, N or V, more preferably wherein X is T, A, S or G, most prefertably wherein X is T or A; and / or

[0088] X at position 514 is any amino acid, preferably wherein X is I, L, V, M, A, F, Y, W, G, H or C; more preferably wherein X is I, L, V, M, or A, most preferably wherein X is I, L or V, in particular wherein X is I or L; and / or

[0089] X at position 518 is any amino acid, preferably wherein X is E, D, Q, N, A, S, T, K, R or H, more preferably wherein X is E, D, Q or N, most preferably wherein X is E or D; and / or

[0090] X at position 523 is any amino acid, preferably wherein X is F, S, A, W, Y, L, M,

[0091] H, T, G, N, I, V, D, E, R or K, preferably wherein X is F, S, A, W, Y, L, M, H, T, G or N, more preferably wherein X is F, S or A, most preferably wherein X is F or S; and / or

[0092] X at position 555 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V; and / or

[0093] X at position 568 is any amino acid, preferably wherein X is L, P, V, A, M, I, F, Y, W, C, S or T, preferably wherein X is L, P, V, A, M, I, F, Y, W or C, more preferably wherein X is L, P, V, A, M or I, more preferably wherein X is L, P, V or I, most preferably wherein X is L or P; and / or

[0094] X at position 570 is any amino acid, preferably wherein X is D, N, Q, E, S, T, A, G, K, R or H, more preferably wherein X is D, N, Q or E, more preferably wherein X is D, N or Q, most preferably wherein X is D or N; and / or

[0095] X at position 572 is any amino acid, preferably wherein X is Y, F, W, H, L, M, I, V, C or A, more preferably wherein X is Y, F, W, H, L, M, I or V, more preferably wherein X is Y, F, W, H, L or M, more preferably wherein X is Y, F, W or H, most preferably wherein X is Y or F; and / or

[0096] X at position 587 is any amino acid, preferably wherein X is C, S, A, G, V, L, T, N, M, I, F, D, E, R or K, preferably wherein X is C, S, A, G, V, L, T or N, more preferably wherein X is C, S, A or G, most preferably wherein X is C or S.

[0097] As a consequence, the enzyme with halogenase activity can comprise, in particular consist of, an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 2 (CkHALI ).

[0098] In addition, or alternatively, the enzyme with halogenase activity may comprise, in particular or consist of, an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 3 (CsHAL2).

[0099] The enzyme of the present disclosure has a flavin-dependent halogenase activity in accordance with NC-IUBMB class EC 1.14.19. Specifically, the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin, endocrocin to chloro-endocrocin, dermolutein to chloro-der- molutein, and / or dermorubin to chloro-dermorubin. In addition, it is known that most flavin-dependent halogenases also work to a small extent with Br, while only a few examples exist for F and I (see, for example, Chapter Eleven: “Structure, mechanisms and applications of flavindependent halogenases" by A. Phintha, K. Prakinee and P. Chaiyen, in: The Enzymes, Volume 47, 2020, ISSN 1874-6047; and S. Mori et al. Nature Communication (2019) 10: 1255; the content of which is incorporated herewith by reference). Hence, it is expected that the enzyme with halogenase activity is capable to a smaller extent of selectively converting emodin to bromo-emodin, endocrocin to bromo-endocrocin, dermolutein to bromo-dermolutein, and / or der- morubin to bromo-dermorubin.

[0100] Whether an enzyme has the required halogenase activity can be tested for as described in the examples section. For example, the enzyme can be produced in a S. cerevisiae H4590 host cell grown for 72 hours with shaking at 220 rpm at 23°C in TSB medium supplemented with either 5 mM purified endocrocin, emodin, or dermolutein. The cells are separated by centrifugation at 4,000 rpm for 5 min, and the supernatants are extracted twice with ethyl acetate. The organic fractions are collected, evaporated under reduced pressure, and the resulting extracts are dissolved in methanol. The methanol extracts are analyzed by LC-MS for the presence of chloro-endocrocin, chloro-dermolutein, and / or chloro-dermorubin; and / or for the presence of bromo-emodin, bromo- endocrocin, bromo-dermolutein, and / or bromo-dermorubin.

[0101] Preferred embodiments of the enzyme with halogenase activity are those, which have significant halogenase activity. Hence, in embodiments, the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2). In addition, or alternatively, the enzyme with halogenase activity is capable of selectively converting endocrocin to chloro-endocrocin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2); and / or the enzyme with halogenase activity is capable of selectively converting dermolutein to chloro- dermolutein with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2). In embodiments, the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ), and / or the enzyme with halogenase activity is capable of selectively converting endocrocin to chloro-endocrocin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ); and / or the enzyme with halogenase activity is capable of selectively converting dermolutein to chloro-dermolutein with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ).

[0102] Activity of the enzymes may be determined and / or compared by any suitable means. For example, one can conduct comparative in vitro tests using the candidate enzyme with halogenase activity and the halogenase of SEQ ID NO: 3 or SEQ ID NO: 2, respectively, in equimolar amounts and under identical starting conditions, and by comparing the amounts of halogenated products. Alternatively, one can determine and compare the Km, kcat, or kcat / Km for both enzymes under identical conditions. In case of conflict, the enzymes are compared in terms of their Km.

[0103] Preferred embodiments are wherein the enzyme comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3. In one preferred embodiment, the enzyme comprises, preferably consists of SEQ ID NO: 3 (CsHAL2). In a further preferred embodiment, the enzyme comprises, preferably consists of SEQ ID NO: 2 (CkHALI ).

[0104] Further contemplated is a nucleic acid construct, comprising a nucleotide sequence coding for the enzyme with halogenase activity as disclosed herein. The nucleic acid construct may, for example, comprise flanking regions for incorporation of the coding sequence into the genome of a host cell. The nucleic acid construct may or may not comprise further regulatory sequences. Such regulatory sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, ribosome binding site and transcription terminator. The regulatory sequences may facilitate expression of the enzyme with halogenase activity.

[0105] Further provided is an expression vector, comprising a nucleotide sequence coding for the enzyme with halogenase activity of the present disclosure in functional linkage with a promoter and a terminator sequence. The term "expression vector" means a DNA molecule that comprises an expression cassette. Preferably, the expression vector is a linear or circular double stranded DNA molecule. The term "in functional linkage” describes a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, in such a way that the control sequence directs expression of the coding sequence. Thus, further provided is a host cell recombinantly producing the enzyme with halogenase activity of the present disclosure. The term, "host cell" means any cell type that is susceptible to transformation, transfection, transduction, mating, crossing or the like with a nucleic acid construct or expression vector comprising a polynucleotide encoding for the enzyme with halogenase activity as disclosed herein. The term " host cell " also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication. The host cell is preferably a microbial host cell, wherein the term “microbial host cell” refers to a prokaryotic or eukaryotic microorganisms such as a bacterium, a filamentous fungus or a yeast. A host cell recombinantly producing the enzyme with halogenase activity means that said host cell has been genetically modified or altered to comprise an exogenous nucleic acid sequence coding for the enzyme with halogenase activity, which is not native to the genomic DNA in the host cell. Also contemplated are host cells comprising an additional expression cassette for the enzyme having halogenase activity in addition to their endogenous gene coding for such an enzyme, i.e. a host cell genetically modified to over-express the enzyme with halogenase activity. It is common to all these kinds of host cells recombinantly producing the enzyme with halogenase activity that they are not found in nature.

[0106] In embodiments, the host cell is a yeast host cell, preferably selected from a group of yeast host cells consisting of Saccharomyces, Yarrowia, Kluy- veromyces, Pichia, Candida, Hansenula, Schizosaccharomyces, Debaryomy- ces, and Phaffia; more preferably selected from group of yeast host cells consisting of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Yarrowia lipolytica, Kluyve- romyces lactis, Pichia pastoris, Debaryomyces hansenii, Hansenula polymor- pha (=Ogataea polymorpha), Pichia kudriavzevii, Saccharomyces bayanus, Phaffia rhodozyma (=Xanthophyllomyces dendrorhous), and Kluyveromyces marxianus, and most preferably wherein the host cell is a Saccharomyces cerevisiae yeast host cell.

[0107] In other embodiments, the host cell is a filamentous fungal host cell, preferably selected from group of fungal host cells consisting of Aspergillus, Trichoderma, Corti na us, Acremonium, Aureobasidium, Bjerkandera, Ceripori- opsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypo- cladium, Thermothelomyces, and Trametes', more preferably selected from the group of filamentous fungal host cells consisting of Aspergillus oryzae, Tricho- derma reesei, Cortinarius sanguineus, C. californicus, C. marylandensis, C. smithii, C. birkebakii, C. uligi nosus, C. persplendidus,, C. semisanguineus and C. sp. KIS3, Aspergillus niger, A. awamori, A. foetidus, A. fumigatus, A. japoni- cus, A. nidulans, Trichoderma harzianum, T. koningii, T. longibrachiatum, T. viride, Bjerkandera adusta, Ceriporiopsis aneirina, C. caregiea, C. gilvescens, C. pannocinta, C. rivulosa, C. subrufa, C. subvermispora, Chrysosporium inops, Ch. keratinophilum, Ch. lucknowense, Ch. merdarium, Ch. pannicola, Ch. queenslandicum, Ch. tropicum, Ch. zonatum, Coprinus cinereus, Coriolus hir- sutus, F. bactridioides, F. cerealis, F. crookwellense, F. culmorum, F. gramine- arum, F. graminum, F. heterosporum, F. negundi, F. oxysporum, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sporotrichioides, F. sulphureum, F. torulosum, F. trichothecioides, F. venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Penicillium chrysogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Paecilomyces variotii, Paecilomyces lilacin us, Thermothelomyces heterothallica, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, and Trametes versicolor; and most preferably wherein the host cell is a Aspergillus oryzae or Trichoderma reesei filamentous fungal host cell.

[0108] In further embodiments, the host cell is a bacterial host cell, preferably selected from group of bacterial host cells consisting of Escherichia, Bacillus, Campylobacter, Clostridium, Enterococcus, Flavobacterium, Fusobacterium, Geobacillus, Helicobacter, llyobacter, Lactobacillus, Lactococcus, Micromono- spora, Neisseria, Nocardia, Oceanobacillus, Pseudomonas, Staphylococcus, Streptococcus, Streptomyces, Salmonella, and Ureaplasma', more preferably the bacterial host cell is selected from the group consisting of Escherichia coli, Bacillus alkalophilus, B. amyloliquefaciens, B. brevis, B. circulans, B. clausii, B. coagulans, B. firm us, B.s lautus, B. lentus, B. licheniformis, B. megaterium, B. pumilus, B. stearothermophilus, B. subtilis, and B. thuringiensis cells. In an embodiment, the bacterial host cell is Bacillus amyloliquefaciens, B. licheniformis, B. subtilis, Nocardia corallina, Pseudomonas aeruginosa, Pseudomonas putida, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces clavuligerus, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces Hvidans; and most preferably the bacterial host cell is a E. coli bacterial host cell.

[0109] The host cell may be additionally genetically modified to further support production of halogenated anthraquinone compounds by the enzyme with halogenase activity as disclosed herein. For example, said enzyme with halo- genase activity disclosed herein is a flavin-dependent halogenase, i.e. it requires FADH as a co-factor. Hence, in order to avoid any bottlenecks in the provision of FADH, the host cell may be additionally genetically modified to further recom- binantly expresses a flavin reductase gene whose protein product supplies FADH within the cell. This strategy is demonstrated in the examples, in which the host cells were genetically modified with the fre flavin reductase gene from E. coli.

[0110] The host cell can advantageously natively produce one or more anthraquinone substrates of the enzyme with halogenase activity disclosed herein. In embodiments, the one or more anthraquinone substrates of the enzyme with halogenase activity of the present disclosure is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof.

[0111] The term “anthraquinone” refers to an aromatic polycyclic hydrocarbon wherein R1 , R2, R3, R4, R5, R6, R7 and R8 are independently selected, and each R group is a substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic, aliphatic or aromatic hydrocarbon group, optionally comprising one or more heteroatoms (such as 0, N, S), hydroxyl group, methoxy group or hydrogen (Formula I). The term “endocrocin” refers to an anthraquinone where R1 , R6 and R8 are hydroxyl groups, R2 is a carboxylic acid group, R3 is a methyl group and R4, R5 and R7 are hydrogen atoms (Formula II).

[0112] The term “derivative” refers to a chemical compound that can be obtained from another compound by substituting any of its constituent atoms with another atom or group of atoms using a chemical reaction.

[0113] The term “endocrocin derivative” or “endocrocin derivatives” refers to compounds structurally similar to endocrocin but with substitution of one or more atoms by an atom of a different element or a functional group.

[0114] The term “emodin” refers to an anthraquinone where R1 , R6 and R8 are hydroxyl groups, R3 is a methyl group and R2, R4, R5 and R7 are hydrogen atoms (Formula III).

[0115] The term “emodin derivative” or “emodin derivatives” refers to compounds structurally similar to emodin but with substitution of one or more atoms by an atom of a different element or a functional group.

[0116] The term “dermolutein” refers to an anthraquinone where R1 and R6 are hydroxyl groups, R2 is a carboxylic acid group, R8 is O-methyl, R3 is a methyl group and R4, R5 and R7 are hydrogen atoms (Formula IV).

[0117] to compounds structurally similar to dermolutein but with substitution of one or more atoms by an atom of a different element or a functional group.

[0118] The term “dermorubin” refers to an anthraquinone where R1 , R4 and R6 are hydroxyl groups, R2 is a carboxylic acid group, R3 is a methyl group, R8 is O-methyl, and R5 and R7 are hydrogen atoms (Formula V).

[0119] The term refers to compounds structurally similar to dermorubin but with substitution of one or more atoms by an atom of a different element or a functional group.

[0120] Alternatively, or in addition, the host cell further recombinantly produces one or more anthraquinone substrates of the disclosed enzyme with hal- ogenase activity. The latter may be achieved, for example, by recombinantly producing in said host cell a polyketide synthase, such as PKS2 and / or PKS3, which uses malonyl-CoA to synthesize atrochrysone carboxylic acid, a precursor compound of emodin and endocrocin (see Figure 2). The host cell may be further genetically modified to produce O-m ethyltransferase 2 (0MT2) or O-me- thyltransferase 5 (0MT5) to convert endocrocin to dermolutein (see Figure 2). For example, the use of Aspergillus is generally advantageous, since Aspergillus is able to produce emodin and endocrocin natively. An Aspergillus host cell, which has been genetically modified to produce O-m ethyltransferase 2 (0MT2) or O-m ethyltransferase 5 (0MT5) is also capable of producing dermolutein (see also Figure 2). For further details, reference is made to application FI20235675, the disclosure of which is incorporated herein by reference.

[0121] The enzyme or the host cell producing the enzyme can be advantageously used in a method for producing halogenated anthraquinone compounds. In case it is decided to use the disclosed enzyme with halogenase activity, it may be necessary to first produce, isolate, and / or purify the enzyme prior to its intended use. Therefore, the present disclosure also contemplates a method of producing an enzyme with halogenase activity, comprising the steps of

[0122] (i) culturing the disclosed host cell producing the disclosed enzyme with halogenase acitivity in a suitable culture medium, and

[0123] (ii) isolating the enzyme with halogenase activity from the host cell culture.

[0124] Having regard to the enzyme as such, provided is an in vitro method of producing halogenated anthraquinone compounds, comprising the step of (i) providing an enzyme with halogenase activity as disclosed herein, and (ii) contacting said enzyme with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound.

[0125] When using the host cell of the present disclosure, a distinction should be made whether the host cells is capable of producing the anthraquinone substrate, or not. In case the host cell is not capable of producing the required anthraquinone substrate, a method of producing halogenated anthraquinone compounds is provided, comprising the steps of

[0126] (i) culturing the disclosed host cell producing the disclosed enzyme with halogenase activity in a suitable culture medium,

[0127] (ii) supplementing the culture medium of step (i) with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound, and

[0128] (iii) optionally isolating the halogenated anthraquinone compound from the host cell culture.

[0129] In case the host cell is capable of producing the required anthraquinone substrate, a method of producing halogenated anthraquinone compounds is disclosed, comprising the steps of

[0130] (i) culturing the disclosed host cell producing the disclosed enzyme with halogenase activity in a suitable culture medium, and (ii) isolating the halogenated anthraquinone compound from the host cell culture.

[0131] Common to these methods is that the halogenated anthraquinone compound may be a halogenated anthraquinone pigment or dye, in particular a chlorinated or brominated anthraquinone pigment or dye. In some embodiments, the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof. Accordingly, in embodiments, the halogenated anthraquinone compound may be selected from the group consisting of chloro-emodin, chloro-en- docrocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-endo- crocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof. Preferably, the anthraquinone compound is a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro- endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

[0132] Further contemplated is the use of an enzyme with halogenase activity as disclosed herein for producing halogenated anthraquinone compounds; and the use of a host cell as disclosed herein for producing halogenated anthraquinone compounds. The halogenated anthraquinone compound may be a halogenated anthraquinone pigment or dye, in particular a chlorinated or brominated anthraquinone pigment or dye. In some embodiments, the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof. Accordingly, in embodiments, the halogenated anthraquinone compound may be selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro- dermolutein, chloro-dermorubin, bromo-emodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof. Preferably, the anthraquinone compound is a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro- dermolutein, chloro-dermorubin, and derivatives thereof.

[0133] In particular envisaged are the following embodiments:

[0134] 1 . An enzyme with flavin-dependent halogenase activity in accordance with NC-IUBMB class EC 1.14.19, comprising or consisting of an amino acid sequence with at least 91 % sequence identity over the full length of SEQ ID NO: 1 (halogenase consensus).

[0135] 2. The enzyme with halogenase activity of embodiment 1 , wherein in SEQ

[0136] ID NO: 1 X at position 7 is I, L or V, in particular wherein X is I or L;

[0137] X at position 50 is V, I or L, in particular wherein X is V or I;

[0138] X at position 98 is I, V or L, in particular wherein X is I or V;

[0139] X at position 105 is N, S, Q, A or T, more preferably wherein X is N or S;

[0140] X at position 106 is A, S, G or T, more preferably wherein X is A or S;

[0141] X at position 303 is N, D, Q or E, preferably wherein X is N, D or Q, most preferably wherein X is N or D;

[0142] X at position 320 is I, V or L, in particular wherein X is I or V;

[0143] X at position 325 is T, S, A, G or N, most preferably wherein X is T or S;

[0144] X at position 331 is N, S, Q, A, T or G, most preferably wherein X is N or S;

[0145] X at position 371 i particular wherein X is V or I;

[0146] X at position 514 i particular wherein X is I or L;

[0147] X at position 518 i N, most preferably wherein X is E or D;

[0148] X at position 555 i particular wherein X is I or V

[0149] X at position 570 i E, more preferably wherein X is D, N or Q, most preferably wherein X is D or N;

[0150] X at position 572 is Y, F, W, H, L or M, preferably wherein X is Y, F, W or H, most preferably wherein X is Y or F.

[0151] 3. The enzyme with halogenase activity of embodiment 1 or embodiment 2, wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence which has at least 92%, preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% sequence identity over the full length of SEQ ID NO: 1 .

[0152] 4. The enzyme with halogenase activity of any of embodiments 1 -3, wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 2 (CkHALI ).

[0153] 5. The enzyme with halogenase activity of any of embodiments 1 -4, wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 3 (CsHAL2).

[0154] 6. The enzyme with halogenase activity of any one of embodiments 1 -5, wherein the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin, endocrocin to chloro-endocrocin, dermolutein to chloro-dermolutein, and / or dermorubin to chloro-dermoru- bin; and / or wherein the enzyme with halogenase activity is capable of selectively converting emodin to bromo-emodin, endocrocin to bromo-endocrocin, dermolutein to bromo-dermolutein, and / or dermorubin to bromo-dermoru- bin.

[0155] 7. The enzyme with halogenase activity of any one of embodiments 1 -6, wherein the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2).

[0156] 8. The enzyme with halogenase activity of any one of embodiments 1 -7, wherein the enzyme with halogenase activity is capable of selectively converting endocrocin to chloro-endocrocin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2).

[0157] 9. The enzyme with halogenase activity of any one of embodiments 1 -8, wherein the enzyme with halogenase activity is capable of selectively converting dermolutein to chloro-dermolutein with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 3 (CsHAL2).

[0158] 10. The enzyme with halogenase activity of any one of embodiments 1 -6, wherein the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ).

[0159] 11 . The enzyme with halogenase activity of any one of embodiments 1 -6 or 10, wherein the enzyme with halogenase activity is capable of selectively converting endocrocin to chloro-endocrocin with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ).

[0160] 12. The enzyme with halogenase activity of any one of embodiments 1 -6 or 10-11 , wherein the enzyme with halogenase activity is capable of selectively converting dermolutein to chloro-dermolutein with at least 80% of the activity of the enzyme with halogenase activity consisting of SEQ ID NO: 2 (CkHALI ).

[0161] 13. The enzyme with halogenase activity of any one of embodiments 1 -12, wherein the enzyme comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3.

[0162] 14. The enzyme with halogenase activity of any one of embodiments 1 -13, wherein the enzyme comprises, preferably consists of SEQ ID NO: 3 (CsHAL2).

[0163] 15. The enzyme with halogenase activity of any one of embodiments 1 -13, wherein the enzyme comprises, preferably consists of SEQ ID NO: 2 (CkHALI ).

[0164] 16. A nucleic acid construct, comprising a nucleotide sequence coding for the enzyme with halogenase activity as defined in any one of embodiments 1-15.

[0165] 17. An expression vector, comprising a nucleotide sequence coding for the enzyme with halogenase activity as defined in any one of embodiments 1-15 in functional linkage with a promoter and a terminator sequence.

[0166] 18. A host cell recombinantly producing the enzyme with halogenase activity as defined in any one of embodiments 1-15.

[0167] 19. The host cell of embodiment 18, wherein the host cell is a yeast host cell, preferably selected from a group of yeast host cells consisting of Saccharomyces, Yarrowia, Kluyveromyces, Pichia, Candida, Hansenula, Schiz- osaccharomyces, Debaryomyces, and Phaffia; more preferably selected from group of yeast host cells consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Yarrowia lipolytica, Debaryomyces hansenii, Hansenula polymorpha (=Ogataea polymor- pha), Pichia pastoris, Pichia kudriavzevii, Phaffia rhodozyma (=Xantho- phyllomyces dendrorhous), Kluyveromyces lactis, and Kluyveromyces marxianus, and most preferably wherein the host cell is a Saccharomy- ces cerevisiae yeast host cell.

[0168] 20. The host cell of embodiment 18, wherein the host cell is a filamentous fungal host cell, preferably selected from group of fungal host cells consisting of Aspergillus, Trichoderma, Cortinarius, Acremonium, Aureo- basidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Corio- lus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mu- cor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Tal- aromyces, Thermoascus, Thielavia, Tolypocladium, Thermothelomyces, and Trametes', more preferably selected from the group of filamentous fungal host cells consisting of Aspergillus oryzae, Trichoderma reesei, Cortinarius sanguineus, C. californicus, C. marylandensis, C. smithii, C. birkebakii, C. uliginosus, C. persplendidus, C. semisanguineus, and C. sp. KIS3, Zts- pergillus niger, A. awamori, A. foetidus, A. fumigatus, A. japonicus, A. nidulans, Trichoderma harzianum, T. koningii, T. longibrachiatum, T. viride, Bjerkandera adusta, Ceriporiopsis aneirina, C. caregiea, C. gil- vescens, C. pan noci nta, C. rivulosa, C. subrufa, C. subvermispora, Chrysosporium inops, Ch. keratinophilum, Ch. lucknowense, Ch. merdarium, Ch. pannicola, Ch. queenslandicum, Ch. tropicum, Ch. zona- tum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, F. ce- realis, F. crookwellense, F. culmorum, F. graminearum, F. graminum, F. heterosporum, F. negundi, F. oxysporum, F. reticulatum, F. roseum, F. sambucinum, F. sarcochroum, F. sporotrichioides, F. sulphureum, F. torulosum, F. trichothecioides, F. venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Penicillium chrysogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Paecilomyces variotii, Paecilomyces lilacinus, Thermothelomyces heterothallica, Tala- romyces emersonii, Thielavia terrestris, Trametes villosa, and Trametes versicolor; and most preferably wherein the host cell is an Aspergillus oryzae or Trichoderma reesei filamentous fungal host cell.

[0169] 21 . The host cell of embodiment 18, wherein the host cell is a bacterial host cell, preferably selected from group of bacterial host cells consisting of Escherichia, Bacillus, Campylobacter, Clostridium, Enterococcus, Flavo- bacterium, Fusobacterium, Geobacillus, Helicobacter, llyobacter, Lactobacillus, Lactococcus, Micromonospora, Neisseria, Nocardia, Oceanoba- cillus, Pseudomonas, Staphylococcus, Streptococcus, Streptomyces, Salmonella, and Ureaplasma', more preferably wherein the bacterial host cell is selected from the group consisting of Escherichia coli, Bacillus alkalophilus, B. amyloliquefaciens, B. brevis, B. circulans, B. clausii, B. coagulans, B. firmus, B. lautus, B. lentus, B. licheniformis, B. megaterium, B. pumilus, B. stearothermophi- lus, B. subtilis, and B. thuringiensis cells. In an embodiment, the bacterial cell / s a Bacillus amyloliquefaciens, B. licheniformis, B. subtilis, Micromonospora purpurea, Nocardia corallina, Pseudomonas aeruginosa, Pseudomonas putida, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces clavuligerus, Streptomyces coeli color, Streptomyces griseus, and Streptomyces lividans; and most preferably wherein the bacterial host cell is a E. coli bacterial host cell.

[0170] 22. The host cell of any one of embodiments 18-21 , wherein the host cell further recombinantly expresses a flavin reductase gene whose protein product supplies FADH within the cell, preferably the fre flavin reductase gene from E. coli.

[0171] 23. The host cell of any one of embodiments 18-22, wherein the host cell natively produces one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of embodiments 1 -15.

[0172] 24. The host cell of any one of embodiments 18-23, wherein the host cell further recombinantly produces one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of embodiments 1 -15.

[0173] 25. The host cell of any one of embodiment 23 or embodiment 24, wherein the one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of embodiments 1 -15 is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof.

[0174] 26. An in vitro method of producing halogenated anthraquinone compounds, comprising the step of providing an enzyme with halogenase activity as defined in any one of embodiments 1 -15, and contacting said enzyme with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound. The method of embodiment 26, wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof. The method of embodiment 26 or embodiment 27, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye, more particularly selected from the group consisting of chloroemodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-endocrocin, bromo-dermolutein, bromo-dermoru- bin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof. A method of producing halogenated anthraquinone compounds, comprising the steps of

[0175] (i) culturing the host cell as defined in any one of embodiments 18- 22 in a suitable culture medium,

[0176] (ii) supplementing the culture medium of step (i) with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound, and

[0177] (iii) optionally isolating the halogenated anthraquinone compound from the host cell culture. The method of embodiment 29, wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin and derivatives thereof The method of embodiment 29 or embodiment 30, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromoemodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye; in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof. A method of producing halogenated anthraquinone compounds, comprising the steps of

[0178] (i) culturing the host cell as defined in any one of embodiments 23- 25 in a suitable culture medium, and

[0179] (ii) isolating the halogenated anthraquinone compound from the host cell culture. The method of embodiment 32, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular selected from the group consisting of chloro-emodin, chloro- endocrocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof. A method of producing an enzyme with halogenase activity, comprising the steps of

[0180] (iii) culturing the host cell as defined in any one of embodiments 18-22 expressing an enzyme with halogenase acitivity in a suitable culture medium, and

[0181] (iv) isolating the enzyme with halogenase activity from the host cell culture. Use of an enzyme with halogenase activity as defined in any one of embodiments 1-15 for producing halogenated anthraquinone compounds. The use of embodiment 35, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromoemodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

[0182] 37. Use of a host cell as defined in any one of embodiments 18-25 for producing halogenated anthraquinone compounds.

[0183] 38. The use of embodiment 37, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromoemodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

[0184] BRIEF DESCRIPTION OF THE SEQUENCES

[0185] SEQ ID NO: 1

[0186] MSLSSSXPPQ HTQVLI IGGG PAGSYAASVL AREGINVTLL EASKFPRYHX GESLIPSVRH 60

[0187] YLRFIGAEEK MMNYGFIXKP GSAIKFNQYK REGYTDFXAL GHNNXXWNVV RSEFDQLLLN 120 HARSTGASVY EQTKVDS ISF SSTIPGKPIS VSWTHEPPPC PPSPPASPKD SSFXGFFSSP 180 VSPSKNGLVH GNTTETHLID ATGRSGIMST RYLKNRHFNA SLKNIAVWGY WTNVGQYXXX 240 SSRXGAPWFE ALTDESGWAW FIPLHNGTTS VGVVMNEKLY RAKSQMPLPP SPFASXSAPY 300 PVXSTMVTCY LSNLSCAPGX VKLIXPXGAL XLGSVKSASD FSYSSPSYAG XGYRIVGDAG 360 AFIDPFFSSG XHLAMTSALS AAATICAS IR NHCHEXQAAD WHTRRVSTSY TRFQVVVLSA 420 YKQIQSQSVD ILSDIDEDNY DRAFSFLRPV IQGASDMGAR LSETELQKSL DFCVNLFNPT 480 SPDQHERLSK XSXFXKELLD VASPVVDPTS FENXLHVXYY DPXGNSNEDR GNSSDTEKAT 540 ETRMVLNKIN ARRVXHPEYA INNLEQEXLX GXVVKLERGK LGLVKMX 587

[0188] X at position 7 is any amino acid, preferably wherein X is I, L, V, M, A, F, Y, W, G, H or C; more preferably wherein X is I, L, V, M, or A, most preferably wherein X is I, L or V, in particular wherein X is I or L.

[0189] X at position 50 is any amino acid, preferably wherein X is V, I, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is V, I, L, M, or A, most preferably wherein X is V, I or L, in particular wherein X is V or I.

[0190] X at position 78 is any amino acid, preferably wherein X is selected from R, H, K, F, Y, N, Q, S, T, D, E, A, more preferably wherein X is selected from R, H, K, Y, F, more preferably selected from R, H, K, and most preferably selected from R and H.

[0191] X at position 98 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V.

[0192] X at position 105 is any amino acid, preferably wherein X is N, S, Q, A, T, G, D, E, K, R, more preferably wherein X is N, S, Q, A, T, more preferably wherein X is N or S.

[0193] X at position 106 is any amino acid, preferably wherein X is A, S, G, T, N, V, D,

[0194] E, R or K, more preferably wherein X is A, S, G, T or N, more preferably wherein X is A, S, G or T, more preferably wherein X is A or S.

[0195] X at position 174 is any amino acid, preferably wherein X is S, P, A, T, G, N, V, I, D, E, R, K, L, M, W, Y, C or F, more preferably wherein X is S, P, A, T, G, N, V, or I, more preferably wherein X is S or P.

[0196] X at position 238 is any amino acid, preferably wherein X is C, G, A, V, L, M, I,

[0197] F, S, N, T, D, E or Q, more preferably wherein X is C, G, A, V or L, more preferably wherein X is C, G or A, most preferably wherein X is C or G.

[0198] X at position 239 is any amino acid, preferably wherein X is P, S, A, T, G, N, V, I, D, E, R, K, L, M, W, Y, C or F, more preferably wherein X is P, S, A, T, G, N,

[0199] V, or I, more preferably wherein X is P or S.

[0200] X at position 240 is any amino acid, preferably wherein X is G, R, A,K, H, S, N, T, D, E, Q, more preferably wherein X is G, R, A,K, or H, most preferably wherein X is G or R.

[0201] X at position 244 is any amino acid, preferably wherein X is Y, Q, F, W, H, N, L,

[0202] M, I, V, C, D, E, A, S, T, K or R, more preferably wherein X is Y, Q, F, W, H or

[0203] N, most preferably wherein X is Y or Q.

[0204] X at position 296 is any amino acid, preferably wherein X is Y, S, F, W, H, A, T,

[0205] G, N, L, M, I, V, C, D, E, R or K, more preferably wherein X is Y, S, F, W, H, A, T, G or N, most preferably wherein X is Y or S.

[0206] X at position 303 is any amino acid, preferably wherein X is N, D, Q, E, S, T, A, G, K, R or H, more preferably wherein X is N, D, Q or E, more preferably wherein X is N, D or Q, most preferably wherein X is N or D.

[0207] X at position 320 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y,

[0208] W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V. X at position 325 is any amino acid, preferably wherein X is T, S, A, G, N, V, D, E, R, K or I, more preferably wherein X is T, S, A, G, N or V, more preferably wherein X is T, S, A, G or N, most preferably wherein X is T or S.

[0209] X at position 327 is any amino acid, preferably wherein X is V, A, L, I, G, S, T, C, N, M or V, more preferably wherein X is V, A, L, I, G, S, T, C or N, more preferably wherein X is V, A, L, I, G, S or T, more preferably wherein X is V, A or G, most preferably wherein X is V or A.

[0210] X at position 331 is any amino acid, preferably wherein X is N, S, Q, A, T, G, D, E, K or R, preferably wherein X is N, S, Q, A, T or G, most preferably wherein X is N or S.

[0211] X at position 351 is any amino acid, preferably wherein X is N, Y, Q, F, W, H, D, E, S, T, A, G, K, R, L, M, I, V or C, preferably wherein X is N, Y, Q, F, W or H, most preferably wherein X is N or Y.

[0212] X at position 371 is any amino acid, preferably wherein X is V, I, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is V, I, L, M, or A, most preferably wherein X is V, I or L, in particular wherein X is V or I.

[0213] X at position 396 is any amino acid, preferably wherein X is L, S, M, I, V, A, T, G, N, F, Y, W, H, C, D, E, R or K, preferably wherein X is L, S, M, I, V, A, T, G or N, most preferably wherein X is L or S.

[0214] X at position 491 is any amino acid, preferably wherein X is R, S, K, H, A, T, G, N, Q, D or E, more preferably wherein X is R, S, K, H, A, T, G or N, most preferably wherein X is R or S.

[0215] X at position 493 is any amino acid, preferably wherein X is D, G, E, N, Q, A, S, T, K, R or H, more preferably wherein X is D, G, E, N, Q, A, S or T, more preferably wherein X is D, G, E, N, Q or A, most preferably wherein X is D or G.

[0216] X at position 495 is any amino aciod, preferably wherein X is T, A, S, G, N, V, C, D, E, R, K or I, preferably wherein X is T, A, S, G, N, V or C, more preferablt wherein X is T, A, S, G, N or V, more preferably wherein X is T, A, S or G, most prefertably wherein X is T or A.

[0217] X at position 514 is any amino acid, preferably wherein X is I, L, V, M, A, F, Y, W, G, H or C; more preferably wherein X is I, L, V, M, or A, most preferably wherein X is I, L or V, in particular wherein X is I or L.

[0218] X at position 518 is any amino acid, preferably wherein X is E, D, Q, N, A, S, T, K, R or H, more preferably wherein X is E, D, Q or N, most preferably wherein X is E or D.

[0219] X at position 523 is any amino acid, preferably wherein X is F, S, A, W, Y, L, M, H, T, G, N, I, V, D, E, R or K, preferably wherein X is F, S, A, W, Y, L, M, H, T, G or N, more preferably wherein X is F, S or A, most preferably wherein X is F or S.

[0220] X at position 555 is any amino acid, preferably wherein X is I, V, L, A, M, F, Y, W, G, T, C or N; more preferably wherein X is I, V, L, M, or A, most preferably wherein X is I, V or L, in particular wherein X is I or V.

[0221] X at position 568 is any amino acid, preferably wherein X is L, P, V, A, M, I, F, Y, W, C, S or T, preferably wherein X is L, P, V, A, M, I, F, Y, W or C, more preferably wherein X is L, P, V, A, M or I, more preferably wherein X is L, P, V or I, most preferably wherein X is L or P.

[0222] X at position 570 is any amino acid, preferably wherein X is D, N, Q, E, S, T, A, G, K, R or H, more preferably wherein X is D, N, Q or E, more preferably wherein X is D, N or Q, most preferably wherein X is D or N.

[0223] X at position 572 is any amino acid, preferably wherein X is Y, F, W, H, L, M, I, V, C or A, more preferably wherein X is Y, F, W, H, L, M, I or V, more preferably wherein X is Y, F, W, H, L or M, more preferably wherein X is Y, F, W or H, most preferably wherein X is Y or F.

[0224] X at position 587 is any amino acid, preferably wherein X is C, S, A, G, V, L, T, N, M, I, F, D, E, R or K, preferably wherein X is C, S, A, G, V, L, T or N, more preferably wherein X is C, S, A or G, most preferably wherein X is C or S.

[0225] SEQ ID NO: 2 (CkHALI , Cortinarius sp. KIS3)

[0226] MSLSSS IPPQ HTQVLI IGGG PAGSYAASVL AREGINVTLL EASKFPRYHI GESLIPSVRH 60

[0227] YLRFIGAEEK MMNYGFIHKP GSAIKFNQYK REGYTDFVAL GHNNSSWNVV RSEFDQLLLN 120

[0228] HARSTGASVY EQTKVDS ISF SSTIPGKPIS VSWTHEPPPC PPSPPASPKD SSFPGFFSSP 180

[0229] VSPSKNGLVH GNTTETHLID ATGRSGIMST RYLKNRHFNA SLKNIAVWGY WTNVGQYGSR 240

[0230] SSRQGAPWFE ALTDESGWAW FIPLHNGTTS VGVVMNEKLY RAKSQMPLPP SPFASSSAPY 300

[0231] PVDSTMVTCY LSNLSCAPGV VKLISPAGAL SLGSVKSASD FSYSSPSYAG YGYRIVGDAG 360

[0232] AFIDPFFSSG IHLAMTSALS AAATICAS IR NHCHESQAAD WHTRRVSTSY TRFQVVVLSA 420

[0233] YKQIQSQSVD ILSDIDEDNY DRAFSFLRPV IQGASDMGAR LSETELQKSL DFCVNLFNPT 480

[0234] SPDQHERLSK SSGFAKELLD VASPVVDPTS FENLLHVDYY DPSGNSNEDR GNSSDTEKAT 540

[0235] ETRMVLNKIN ARRVVHPEYA INNLEQEPLN GFVVKLERGK LGLVKMS 587 SEQ ID NO: 3 (CsHAL2, Cortinarius semisanguineus)

[0236] MSLSSSLPPQ HTQVLI IGGG PAGSYAASVL AREGINVTLL EASKFPRYHV GESLIPSVRH 60

[0237] YLRFIGAEEK MMNYGFIRKP GSAIKFNQYK REGYTDFIAL GHNNNAWNVV RSEFDQLLLN 120

[0238] HARSTGASVY EQTKVDS ISF SSTIPGKPIS VSWTHEPPPC PPSPPASPKD SSFSGFFSSP 180

[0239] VSPSKNGLVH GNTTETHLID ATGRSGIMST RYLKNRHFNA SLKNIAVWGY WTNVGQYCPG 240

[0240] SSRYGAPWFE ALTDESGWAW FIPLHNGTTS VGVVMNEKLY RAKSQMPLPP SPFASYSAPY 300

[0241] PVNSTMVTCY LSNLSCAPGI VKLITPVGAL NLGSVKSASD FSYSSPSYAG NGYRIVGDAG 360

[0242] AFIDPFFSSG VHLAMTSALS AAATICAS IR NHCHELQAAD WHTRRVSTSY TRFQVVVLSA 420

[0243] YKQIQSQSVD ILSDIDEDNY DRAFSFLRPV IQGASDMGAR LSETELQKSL DFCVNLFNPT 480

[0244] SPDQHERLSK RSDFTKELLD VASPVVDPTS FENILHVEYY DPFGNSNEDR GNSSDTEKAT 540

[0245] ETRMVLNKIN ARRVIHPEYA INNLEQELLD GYVVKLERGK LGLVKMC 587

[0246] The sequence of RadH is publicly available from the Protein Data Bank (PDB) under accession number 8GU0 (https: / / www.rcsb.org / fasta / entry / 8GU0 / dis- Play).

[0247] EXAMPLES

[0248] EXAMPLE 1

[0249] BIOINFORMATIC IDENTIFICATION OF ANTHRAQUINONE HALOGENASES FROM CORTINARIUS SP. KIS-3 AND CORTINARIUS SEMISANGUINEUS

[0250] The Cortinarius PKS gene clusters do not encode halogenase enzymes that could perform the chlorination of the anthraquinones emodin, endocrocin, and dermolutein. In silico screening of putative FAD dependent tryptophan halogenase across the Cortinarius genome led to the identification of CkHALI (JGI MycoCosm 1247128) in Cortinarius sp. KIS-3 and CsHAL2 (VTT_DN1604_HAL2) in Cortinarius semisanguineus.

[0251] CONSTRUCTION OF SACCHAROMYCES CEREVISIAE PLASMIDS

[0252] The halogenase-encoding genes CkHALI (Cortinarius sp. KIS-3, JGI MycoCosm: 1247128), and CsHAL2 (Cortinarius semisanguineus, contig DN1604) were codon-optimized for Aspergillus niger and obtained as synthetic gene blocks (Integrated DNA Technologies, USA). The fre reductase gene was amplified by PCR using E. coli genomic DNA as the template. The fre reductase gene, SES bi-directional promoter, along with the halogenase gene (CkHALI or CsHAL2) were assembled into the Sa / I and BamHI-digested pB40 vector that included targeting arms to the S. cerevisiae X-1 locus.

[0253] All S. cerevisiae plasmids were constructed with Gibson assembly (NEB Gibson Assembly® Master Mix kit) and verified by sequencing.

[0254] Tablel. Plasmids used in this study.

[0255] CONSTRUCTION OF SACCHAROMYCES CEREVISIAE STRAINS

[0256] The halogenase + reductase expression cassettes CkHAL1 -Fre or CsHAL2-Fre were transformed separately to the PKS3-OMT5 strain (H6698; Table 2) using the CRISPR-Cas9 method. These expression cassettes were released from the above-described Saccharomyces cerevisiae plasmids, respectively, by / Wssl digestion. The pB158 Cas9-gRNA plasmid was co-transformed with the expression cassettes. The transformants were selected on YPD+NAT (nourseothricin, 200ug / mL) plates. The transformed colonies were verified by qPCR for the presence of the expression cassette and the deletion of the target loci. Table 2. Saccharomyces cerevisiae strains used in this study.

[0257] EXAMPLE 2

[0258] STRAIN CULTIVATION AND SAMPLE PREPARATION: S. CEREVISIAE Three validated transformants for each strain were cultivated in 4 mL

[0259] YP (20 g peptone, 10 g yeast extract per one liter water) + 4% dextrose and / or TSB (17 g Tryptone, 3 g Soytone, 5 g NaCI, 2.5 g K2HPO4 per one liter water, pH 7.3) + 2% glucose medium in 24-well plates at 28°C for 72 hours with shaking at 200rpm. The cultures were then centrifuged, and the supernatants were collected and analyzed by LC-MS. For extraction of compounds from the cell pellet, the cells were incubated in 1 mL MeOH at 37°C with shaking at 800 rpm for 1 hour. The mixtures were centrifuged at 4000 rpm for 5 min, the supernatants were collected, and these MeOH extracts were analysed by LC-MS.

[0260] LC-MS METHOD

[0261] LC-MS analysis was performed on an Acquity UHPLC system, Waters (Milford, MA, USA) and Synapt G2-S MS system, Waters (Milford, MA, USA). Chromatography was performed using an ACQUITY UPLC BEH HSS T3 column, 1.8 pm 2.1x100 mm, (Waters), kept at 45 °C. The experiment was carried out at a flow rate of 0.4 ml / min with mobile phase A (0.1 % formic acid in water) and B (0.1 % formic acid in acetonitrile). The gradient elution started at 5% B maintained for 0.2 min, then increased to 90% B within 14 min, then directly returned to 5% B and maintained for 3 min. Mass spectrometry was carried out by electrospray ionization (ESI) in negative polarity using the capillary voltage at 2.0 kV and cone voltage 40 kV. Source temperature was 150 °C and desolvation temperature was 500 °C. The desolvation gas flow was set at 800 L / h (nitrogen).

[0262] Functional identification of CkHALI and CsHAL2 as anthraquinone halo- genases

[0263] Expression of the CkHALI or CsHAL2 gene together with the fre reductase gene of E. coli in the anthraquinone-producing S. cerevisiae H6698 strain (Table 1 ) led to the production of chloro-emodin, chloro-endocrocin, and chloro-dermolutein (Figure 3A). This demonstrates that CkHALI from Cortinar- ius sp. KIS-3 and CsHAL2 from C. semisanguineus are the enzymes that carry out the chlorination of these anthraquinone compounds.

[0264] The identities of these compounds were confirmed using LC-MS / MS, by comparing the fragmentation patterns with the standards or data available from literature (Figure 3B and 3C), and the percentage ppm error was found to be within the permissible range of ±20ppm (Table 3). Table 3. Percentage error in mass measured using LC-MS / MS.

[0265] EXAMPLE 3

[0266] Because the functionality of the SES expression system utilized in the expression cassettes described in Example 1 is independent of the identity of the fungal host strains, the same expression cassettes were also introduced to various additional fungal hosts.

[0267] CONSTRUCTION OF ASPERGILLUS ORYZAE PLASMIDS

[0268] All the Aspergillus oryzae plasmids (Ao-CkHAL1 and Ao_CsHAL2) were constructed by classical ligation. S. cerevisiae constructs of Example 1 were digested with Pac\ and BamH\ and the CkHALI or the CsHAL2 expression cassettes, respectively, were extracted from the agarose gel using Gene JET Gel Extraction Kit. Similarly, vector B13678 (flanks to A. oryzae pepS locus) was digested with the same restriction enzymes and the vector backbone was then procured from the gel. Both the digested fragments were then ligated using T4 DNA ligase kit (NEB, England).

[0269] CONSTRUCTION OF TRICHODERMA REESEI PLASMIDS

[0270] All the Trichoderma reesei plasmids (Tr-CkHAL1 and Tr_CsHAL2) were constructed by classical ligation. The CkHALI or the CsHAL2 expression cassettes were obtained by digesting S. cerevisiae constructs of Example 1 with Pac\ and BamH\. Vector B13685 (flanks to Cel3c locus) was digested with the same restriction enzymes. Both the digested fragments were then extracted from the agarose gel and ligated as described previously.

[0271] CONSTRUCTION OF ASPERGILLUS ORYZAE AND TRICHODERMA REESEI STRAINS

[0272] A. oryzae M6576 and T. reesei M6578 (Table 3) were grown on potato dextrose agar plates supplemented with 1 M sorbitol and 5mM uridine for 4- 5 days at 28°C. Spores were collected into 2 mL glycerol stock solution (20% glycerol, 0.8% NaCI and 0.025% Tween-20) and were used to inoculate 300 mL YP+3% gelatin media supplemented with 10 mM uridine, and cultured at 28°C for 16 hours with shaking at 200 rpm. Mycelium was collected by filtering the culture through a layer of sterile Miracloth (Calbiochem, Germany). The collected mycelium was washed first with sterile water and then with cold KMC (1 M KCI, 25 mM CaCl2, 10 mM Tris-HCI, pH 5.8) buffer. The filtered mycelia were resuspended in the lytic solution (20 mg / mL Trichoderma harzianum lysing enzyme in 20mL KMC buffer for A. oryzae, and 10 mg / mL yatalase in 10 mL KMC for T. reesei) and incubated at 30°C with shaking at 80 rpm for 3 hours. The resulting protoplast solutions were filtered through a sterile cotton plug filter. The filtered protoplast solutions were centrifuged and washed with cold KMC buffer, followed by final washing with STC solution (1.33 M sorbitol, 10 mM Tris-HCI, 50 mM CaCl2, pH 8.0). All the centrifugation steps were done for 4 min at 1500 rpm at 4°C. After washing, protoplasts were resuspended in STC to a total volume of about 400 pL.

[0273] The CkHALI or the CsHAL2 expression cassettes were obtained by digesting the corresponding plasmids with / Wssl. The digested plasmids were mixed with 100 pL of protoplast solution and incubated with 100 pL of transformation solution (25% PEG 6000, 50 mM CaCl2, 10 mM Tris-HCI, pH 7.5) on ice for 20 min. Two mL of the transformation solution was then separately added to the protoplasts and the mixtures were incubated at room temperature for 5 min. The transformation mixtures were then diluted with STC, mixed with 7 mL of molten TOP agar and poured on the selection plates. The plates were incubated for 3 days, and the colonies were then replated on selection plates followed by screening using qPCR.

[0274] Table 4. Aspergillus oryzae and Trichoderma reesei strains used in this study.

[0275] Ten transformants for each strain were cultivated in 4 mL BMDY media (10 g Yeast extract, 20 g Bacto peptone, 0.4 mg Biotin, 20 g glucose, 31 g Ca3(PO4)2, 13.4 g Yeast Nitrogen base with ammonium sulphate without amino acids per one liter water) in 24-well plates at 28°C with shaking at 200 rpm for 5 days. Supernatant was collected by centrifugation and used directly for analysis.

[0276] For extraction of compounds from the cells, mycelia were collected and resuspended in 1 mL MeOH. 500 pL of glass beads were added and cell lysis was performed by vortexing twice at 6500 rpm for 30 seconds. Samples were then centrifuged, and the supernatant was then collected for LC-MS analysis, as described in Example 2. Introduction of the CkHALI (Cortinarius sp. KIS-3, JGI Mycocosm: 1247128) and CsHAL2 (C. semisanguineus DN1604) to A. oryzae M6576 led to the production of the halogenated anthraquinone compounds chloro-emodin, chloro-endocrocin, and chloro-dermolutein (Figure 4).

[0277] In a similar way, T. reesei was also used as a host to produce the same compounds (Figure 5).

[0278] EXAMPLE 4

[0279] COMPARISON OF THE SUBSTRATE SPECIFICITIES OF CKHAL1 AND CSHAL2 WITH THOSE OF FAD-DEPENDENT TRYPTOPHAN HALOGEN- ASES FROM THE LITERATURE

[0280] To compare the substrate selectivities of the discovered Cortinarius halogenases with known halogenases (Table 5) that may chlorinate similar anthraquinone scaffolds, we expressed the genes for RadH (Cummings M, Peters AD, Whitehead GFS, Menon BRK, Micklefield J, Webb SJ, and Takano E. (2019) Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli. PLoS Biol 17(7): e3000347. https: / / doi.org / 10.1371 / journal.pbio.3000347 ) or GedL (Nielsen, M. T., Nielsen, J. B., Anyaogu, D. C., Holm, D. K., Nielsen, K. F., Larsen, T. 0., & Mortensen, U. H. (2013). Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach. PloS one, 8(8), e72871. https: / / doi.org / 10.1371 / journal.pone.0072871 ), together with that for the Fre reductase, in the S. cerevisiae H6118 strain.

[0281] Table 5. Halogenase candidates tested in this study.

[0282] Expression of GedL produced no new metabolites and thus was proven not to be able to recognize emodin, endocrocin or dermolutein as its substrate. Expression of the RadH-encoding gene in S. cerevisiae H6118 strain led to the production of a chlorinated emodin derivative. However, this emodin derivative displays a different retention time as compared to the chlorinated emodin derivative produced by the strain expressing genes for CkHALI or CsHAL2 (Figure 6). This demonstrates that although both RadH and CkHALI / CsHAL2 chlorinate emodin, the products of these enzymes are regioi- somers whereby the chlorine functionality is attached to emodin at differing positions.

[0283] Further, although RadH could chlorinate emodin, this enzyme did not accept the carboxylic group-containing anthraquinone compounds endocrocin or dermolutein as substrates. Thus, the substrate selectivity of RadH and those of the Cortinarius halogenases CkHALI / CsHAL2 are different. Apart from CkHALI and CsHAL2, no other known halogenase can halogenate endocrocin and dermolutein. Thus, the substrate specificities of CkHALI and CsHAL2 are unique in nature to perform halogenation of these anthraquinone scaffolds.

[0284] EXAMPLE 5

[0285] BIOCATALYTIC PRODUCTION OF CHLORINATED ANTHRAQUINONES WITH RECOMBINANT S. CEREVISIAE STRAINS EXPRESSING CKHAL1 AND CSHAL2

[0286] The genes encoding CkHALI (Cortinarius sp. KIS-3, JGI Mycocosm: 1247128) and CsHAL2 (C. semisanguineus DN1604) along with the Fre reductase were inserted separately into the pB40 expression vector. These expression vectors were separately transformed into the S. cerevisiae H4590 host to integrate the expression cassettes into the X-1 genomic locus. The resulting recombinant S. cerevisiae strains were grown for 72 hours with shaking at 220 rpm at 23°C in TSB medium supplemented (5 mM, final concentration) with any of purified endocrocin, emodin, or dermolutein. The cells were separated by centrifugation at 4,000 rpm for 5 min, and the supernatants were extracted twice with ethyl acetate. The organic fractions were collected, evaporated under reduced pressure, and the resulting extracts were dissolved in methanol. The methanol extracts were analysed by LC-MS. The production of chloro-emodin, chloro-endocrocin, and chloro-dermolutein, respectively, were detected in these methanol extracts (Figure 7). The identities of these compounds were confirmed using LC-MS / MS (Figure 7), and the percentage ppm error was found to be within the permissible range of ±20ppm (Table 3).

[0287] EXAMPLE 6

[0288] PRODUCTION OF HALOGENATED ANTHRAQUINONES WITH ANTHRA- QUINONE-PRODUCING ASPERGILLUS STRAINS EXPRESSING HALO- GENASE FROM CORTINARIUS KISS AND CORTINARIUS SEMI SANGUINEUS

[0289] The genes encoding CkHALI (Cortinarius sp. KIS-3, JGI Mycocosm: 1247128) and CsHAL2 (C. semisanguineus DN1604) were inserted separately into an expression vector. These expression vectors were separately transformed into Aspergillus fumigatus, a natural producer of endocrocin (Lim FY, Hou Y, Chen Y, Oh J, Lee I, Bugni TS, Keller NP. Genome-Based Cluster Deletion Reveals an Endocrocin Biosynthetic Pathway in Aspergillus fumigatus. Appt Environ Microbiol, 2012; 78; 4117-4125) to integrate the expression cassettes into the genome of the host. The resulting recombinant A. fumigatus strains were grown at 29°C for 10 days on solid GMM medium. The cultures were homogenized and extracted twice with ethyl acetate. The organic fractions were collected, filtered through a filter paper, evaporated under reduced pressure, and the resulting extracts were dissolved in methanol. The methanol extracts were analyzed by LC-MS. Chloro-endocrocin was identified in these extracts by comparison of its chromatographic mobility, UV spectrum, and MS / MS spectra with those of an authentic standard (Figure 8).

[0290] In addition, the gene encoding CsHAL2 (C. semisanguineus DN1604) was inserted into an expression vector. This expression vector was transformed into Aspergillus ochraceus, a natural producer of emodin (Lu P., Zhao X., Cui T. Full Length Research Paper Production of emodin from Aspergillus ochraceus at preparative scale. Afr. J. Biotechnol. 2010;9:512-517), to integrate the expression cassette into the genome of the host. The resulting recombinant A ochraceus strain was grown at 32 °C for 120 hours in Czapek- Dox medium with shaking at 220 rpm. The mycelia were collected by centrifugation at 3,500 rpm for 10 min, the supernatant was discarded, and the mycelia were extracted twice with ethyl acetate. The organic fractions were collected, filtered through a filter paper, evaporated under reduced pressure, and the resulting extracts were dissolved in methanol. The methanol extracts were analyzed by LC-MS. Chloro-emodin was identified in these extracts by comparison of its chromatographic mobility, UV spectrum, and MS / MS spectra with those of an authentic standard (Figure 9).

[0291] EXAMPLE 7

[0292] PRODUCTION OF HALOGENATED ANTHRAQUINONES WITH ISOLATED RECOMBINANT HALOGENASE FROM CORTINARIUS KIS-3 AND CORTINARIUS SEMISANGUINEUS

[0293] The genes encoding CkHALI (Cortinarius sp. KIS-3, JGI Mycocosm: 1247128), CsHAL2 (C. semisanguineus DN1604), and Fre reductase (E. coli) were tagged with a Hise tag at their 5’ ends and inserted separately into the B13490 expression vector to produce expression vectors B13491 , B13492, and B13493, respectively. These expression vectors were separately transformed into S. cerevisiae H4590 host. The resulting recombinant S. cerevisiae strains were grown at 30°C in YPD+NAT (nourseothricin, 200ug / mL) medium for 24 hours with shaking at 220 rpm. The cells were collected by centrifugation at 4,000 rpm for 10 min, resuspended in 50 mL of ice-cold lysis buffer (Binding buffer: 20 mM sodium phosphate, 0.5 M NaCI, 20mM imidazole, pH 7.4), supplemented with protease inhibitors (1 x complete, EDTA-free Protease Inhibitor Cocktail, Roche). The cell suspensions were combined with 0.5 mm dia. glass beads (Biospec) and lysed using a BeadBeater (Biospec). The extent of the cell lysis was monitored using a microscope. The resulting cell lysates were centrifuged at 27,000 g for 45 min at 4 °C, and the resulting clarified lysates were loaded onto 5 mL HiTrap Chelating HP columns equilibrated with Binding buffer. The CkHALI , CsHAL2, and Fre reductase enzymes were eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCI, 500mM imidazole, pH 7.4), and fractions were analyzed by SDS-PAGE. The fractions containing purified CkHALI and separately, the fractions containing purified CsHAL2, and the fractions containing the purified Fre reductase enzymes were then used for the in vitro assays.

[0294] Reaction mixtures containing 10 pM halogenase enzyme (CkHALI or CsHAL2), 50mM NaCI, 100 pM purified substrates (emodin, endocrocin, or der- molutein), 2 mM NADH, 10 pM FAD, and 2.5 pM Fre reductase enzyme in 20 mM sodium phosphate buffer pH 7.0 were incubated for an hour at 30°C. The reactions were terminated by adding an equal amount of methanol and analyzed by LC-MS. The halogenated products chloro-emodin, chloro-endocrocin, and chloro-dermolutein were identified by comparison of their chromatographic mobilities, UV spectra, and MS / MS spectra with those of authentic standards (Figure 10).

[0295] EXAMPLE 8

[0296] STRUCTURAL FEATURES OF CORTINARIUS HALOGENASES CKHAL1 AND CSHAL2

[0297] A sequence alignment of SEQ ID NO: 2 (CkHALI ), SEQ ID NO: 3 (CsHAL2), and RadH (PDB accession number: 8GU0, Wang et al., 2008, Menon et al., 2017, Cummings et al., 2019, Peh et al. 2023), is presented in Figure 11 . The primary amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 show low level of identities (~40% identity, 99% coverage) to RadH (PDB id: 8GU0, Peh et al. 2023). Nevertheless, this alignment allows the initial assignment of residues for the flavin adenine dinucleotide (FAD) binding site, and the catalytic active site residues of these enzymes, as shown in Figure 11 .

[0298] The canonical GxGxxG and WxWxlP sequence fingerprints are conserved in both Cortinarius halogenases. These sequence motifs are involved in cofactor (FAD) binding and allowing the enzymes to perform a halogenation instead of an oxygenation reaction, respectively.

[0299] To gain further insight into the structure / function relationships of these proteins, we generated AlphaFold protein structure models for SEQ ID NO: 2 (CkHALI ) and SEQ ID NO: 3 (CsHAL2). These two models were practically identical, with key amino acids (see below) completely conserved, in agreement with that the primary amino acid sequences of the two proteins being 94% identical.

[0300] Our structure models for SEQ ID NO: 2 (CkHALI ) and SEQ ID NO: 3 (CsHAL2) displayed an excellent alignment with the recently published crystal structure of RadH, with RMSD (Root mean square deviation) of 0.722 A and 0.642 A to CkHALI and CsHAL2, respectively (Figure 12).

[0301] To understand the catalytic action of the Cotinarius halogenases, the active site cavity areas of these enzymes were superimposed with that of RadH (Figure 13). The active site residues of the Cortinarius halogenases (CkHALI and CsHAL2) are also conserved with those of RadH (Figure 11 and 13) and may carry out the halogenation reaction in a similar way as proposed for RadH (Menon et al., 2017). The phenylalanine residue F367 in CkHALI or CsHAL2 (F328 in RadH) may be responsible for the TT-TT stacking interactions with the substrates. The residue D364 in CkHALI or CsHAL2 (D325 in RadH) may serve as a potential base to abstract the proton from the phenol group ortho to the halogenation site. Possible rearrangements of the substrate would then generate a carbanion on the C5 position of the substrate, making it nucleophilic. The C5 carbanion may then attack the chloroamine moiety (generated by the reaction of the hypohalous acid with the amino group of lysine at K79 of CkHALI or CsHAL2, similar to K74 of RadH) to abstract the chlorine to perform chlorination of the substrate.

[0302] EXAMPLE 9

[0303] IMPROVEMENT IN HALOGENATION BY CO-EXPRESSION OF CORTINARIUS HALOGENASE (CSHAL2) TOGETHER WITH FRE REDUCTASE

[0304] To validate if the co-expression of fre flavin reductase gene from E. co / / with the CsHAL2 (C. semisanguineus DN1604) halogenase is beneficial for improving the chlorination of the anthraquinones, we constructed H6954 strain which only expresses only CsHAL2 halogenase and compared its metabolite profile with H6827 strain expressing both the fre reductase and CsHAL2 halogenase. Co-expression of the fre reductase together with CsHAL2 (H6827) led to improved production of chloro-dermolutein (Figure 14A) and chloroemodin (Figure 14B) compared to that of H6954 strain. This supports the conclusion that the expression of the CsHAL2 halogenase alone can produce the chlorinated anthraquinones; however, the conversion is more efficient by providing excess of the required flavin cofactor through expression of a flavin reductase.

[0305] It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described above but may vary within the scope of the claims.

[0306] REFERENCES

[0307] Cohen, P. A. et al. Chlorination of anthraquinones by lichen and fungal enzymes. Phytochemistry, 1997, Vol. 44, No. 2, pages 271 -274

[0308] WO 2019 / 171046 A2

[0309] Menon, B. R. K. et al. Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals. Catalysis Reviews, 2022, Vol. 64, No. 3, pages 533-591

[0310] Menon, B. R. K. et al. RadH: A Versatile Halogenase for Integration into Synthetic Pathways, Angewandte Chemie International Edition, 2017, Vol. 56, No. 39, pages 11841 -11845.

[0311] Cochereau, B. et al. Halogenation in Fungi: What Do We Know and What Remains to Be Discovered? Molecules, 2022, 27(10):3157

[0312] EP 4 242 311 A1

[0313] Luo et al. Halogenase-Targeted Genome Mining Leads to the Discovery of (±) Pestalachlorides A1a, A2a, and Their Atropisomers Antibiotics, 2022, 11 (10): 1304

[0314] WO 2022 / 254047 A1

[0315] Lei Sun et al. Metabolic engineering of Saccharomyces cerevisiae for efficient production of endocrocin and emodin. Metabolic Engineering, 2019, Vol. 54, pages 212-221

[0316] Cummings M, et al. (2019) Assembling a plug-and-play production line for combinatorial biosynthesis of aromatic polyketides in Escherichia coli. PLoS Biol 17(7): e3000347.

[0317] Chapter Eleven: “Structure, mechanisms and applications of flavin-dependent halogenases" by A. Phintha, K. Prakinee and P. Chaiyen, in: The Enzymes, Volume 47, 2020, ISSN 1874-6047.

[0318] S. Mori et al. Unusual substrate and halide versatility of phenolic halogenase PltM, Nature Communication (2019) 10: 1255.

[0319] Lu P., Zhao X., Cui T. Full Length Research Paper Production of emodin from Aspergillus ochraceus at preparative scale. Afr. J. Biotechnol. 2010;9:512- 517.

[0320] Nielsen, M. T., Nielsen, J. B., Anyaogu, D. C., Holm, D. K., Nielsen, K. F., Larsen, T. O., & Mortensen, U. H. (2013). Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach. PloS one, 8(8), e72871.

[0321] Wang, S., Xu, Y., Maine, E. A., Wijeratne, E. M. K., Espinosa-Artiles, P., Gun- atilaka, A. A. L., & Molnar, I. (2008). Functional Characterization of the Biosynthesis of Radicicol, an Hsp90 Inhibitor Resorcylic Acid Lactone from Chaeto- mium chiversii. Chemistry and Biology, 15(^2), 1328-1338. https: / / doi.Org / 10.1016 / j.chembiol.2008.10.006

[0322] Peh, G. R., Gunawan, G. A., Tay, T., Tiong, E., Tan, L. L., Jiang, S., Goh, Y. L., Ye, S., Wong, J., Brown, C. J., Zhao, H., Ang, E. L., Wong, F. T., & Lim, Y. H. (2023). Further Characterization of Fungal Halogenase RadH and Its Homologs. Biomolecules, 13(7). https: / / doi.org / 10.3390 / biom13071081

Claims

Claims1 . An enzyme with flavin-dependent halogenase activity in accordance with NC-IUBMB class EC 1.14.19, comprising or consisting of an amino acid sequence with at least 91 % sequence identity over the full length of SEQ ID NO: 1 (halogenase consensus).

2. The enzyme with halogenase activity of claim 1 , wherein in SEQ ID NO: 1 X at position 7 is I, L or V, in particular wherein X is I or L;X at position 50 is V, I or L, in particular wherein X is V or I;X at position 98 is I, V or L, in particular wherein X is I or V;X at position 105 is N, S, Q, A or T, more preferably wherein X is N or S; X at position 106 is A, S, G or T, more preferably wherein X is A or S;X at position 303 is N, D, Q or E, preferably wherein X is N, D or Q, most preferably wherein X is N or D;X at position 320 is I, V or L, in particular wherein X is I or V;X at position 325 is T, S, A, G or N, most preferably wherein X is T or S;X at position 331 is N, S, Q, A, T or G, most preferably wherein X is N orS;X at position 371 i particular wherein X is V or I;X at position 514 i particular wherein X is I or L;X at position 518 i N, most preferably wherein X is E or D;X at position 555 i particular wherein X is I or VX at position 570 iE, more preferably wherein X is D, N or Q, most preferably wherein X is D or N;X at position 572 is Y, F, W, H, L or M, preferably wherein X is Y, F, W or H, most preferably wherein X is Y or F.

3. The enzyme with halogenase activity of claim 1 or claim 2, wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence which has at least 92%, preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and most preferably 100% sequence identity over the full length of SEQ ID NO: 1 .

4. The enzyme with halogenase activity of any of claims 1 -3, wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 2 (CkHALI ); or wherein the enzyme with halogenase activity comprises or consists of an amino acid sequence with at least 91 %, preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably 99% sequence identity over the full length of SEQ ID NO: 3 (CsHAL2).

5. The enzyme with halogenase activity of any one of claims 1 -4, wherein the enzyme with halogenase activity is capable of selectively converting emodin to chloro-emodin, endocrocin to chloro-endocrocin, dermolutein to chloro-dermolutein, and / or dermorubin to chloro-dermorubin; and / or wherein the enzyme with halogenase activity is capable of selectively converting emodin to bromo-emodin, endocrocin to bromo-endocrocin, dermolutein to bromo-dermolutein, and / or dermorubin to bromo-dermoru- bin.

6. The enzyme with halogenase activity of any one of claims 1 -5, wherein the enzyme comprises, preferably consists of, an amino acid sequence selected from SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3; in particular wherein the enzyme comprises, preferably consists of SEQ ID NO: 3 (CsHAL2), or wherein the enzyme comprises, preferably consists of SEQ ID NO: 2 (CkHALI ).

7. A nucleic acid construct, comprising a nucleotide sequence coding for the enzyme with halogenase activity as defined in any one of claims 1 -6.

8. An expression vector, comprising a nucleotide sequence coding for the enzyme with halogenase activity as defined in any one of claims 1 -6 in functional linkage with a promoter and a terminator sequence.

9. A host cell recombinantly producing the enzyme with halogenase activity as defined in any one of claims 1 -6; optionally wherein the host cell further recombinantly expresses a flavin reductase gene whose protein product supplies FADH within the cell, preferably the fre flavin reductase gene from E. coli.

10. The host cell of claim 9, wherein the host cell natively produces one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of claims 1 -6, or wherein the host cell further recombinantly produces one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of claims 1 -6; in particular wherein the one or more anthraquinone substrates of the enzyme with halogenase activity as defined in any one of claims 1 -6 is selected from the group consisting of emodin, endocrocin, dermolutein, der- morubin, and derivatives thereof.

11. An in vitro method of producing halogenated anthraquinone compounds, comprising the step of providing an enzyme with halogenase activity as defined in any one of claims 1 -6 and contacting said enzyme with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound; in particular wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin, and derivatives thereof.

12. The method of claim 11 , wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye, more particularly selected from the group consisting of chloro-emodin, chloro-endo- crocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-en- docrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof;preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

13. A method of producing halogenated anthraquinone compounds, comprising the steps of(i) culturing the host cell as defined in any one of claims 9-10 in a suitable culture medium, and(ii) isolating the halogenated anthraquinone compound from the host cell culture.

14. The method of claim 13, further comprising the steps of supplementing the culture medium of step (i) with one or more anthraquinone substrate compound, thereby producing a halogenated anthraquinone compound; in particular wherein the one or more anthraquinone substrate compound is selected from the group consisting of emodin, endocrocin, dermolutein, dermorubin and derivatives thereof.

15. The method of claim 13 or claim 14, wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromoemodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye; in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

16. Use of an enzyme with halogenase activity as defined in any one of claims 1-6 for producing halogenated anthraquinone compounds; optionally wherein the halogenated anthraquinone compound is a halogenated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromo-emodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

17. Use of a host cell as defined in any one of claims 9-10 for producing halogenated anthraquinone compounds; optionally wherein the halogenated anthraquinone compound is a halo- genated anthraquinone pigment or dye; in particular a chlorinated or brominated anthraquinone pigment or dye; more particularly selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, bromoemodin, bromo-endocrocin, bromo-dermolutein, bromo-dermorubin, and derivatives thereof; preferably a chlorinated anthraquinone pigment or dye, in particular selected from the group consisting of chloro-emodin, chloro-endocrocin, chloro-dermolutein, chloro-dermorubin, and derivatives thereof.

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