A biocatalytic route to indole, and optionally indican and / or indigo

Abstract

The present invention concerns in vitro enzyme biosynthesis of indole, and optionally further production conversion of the indole to indican and / or indigo; preferably provided as a one-pot solution.

Description

[0001] TITLE: A biocatalytic route to indole, and optionally indican and / or indigo

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns in vitro enzymatic biosynthesis of indole, and optionally further conversion of the indole to indican and / or indigo; preferably provided as a simple one-pot reaction. The present invention is a greener alternative to current industrial processes for colored fabrics and other products.

[0004] BACKGROUND OF THE INVENTION

[0005] Indigo is well-known as a dyeing agent and is primarily applied in denim. Its intrinsic properties result in the distinctive washed-out look of denim. The chemical synthesis of indigo was a major breakthrough in the late 19th century that resulted in exponential growth of the dye's production, which had previously been extracted from plants. Although the chemical synthesis has been optimized, the petroleum-based synthetic route has remained similar for decades.

[0006] Blue denim is traditionally dyed with chemically synthesized indigo under harsh and environmentally challenging conditions. As a final step in the synthesis, indigo forms spontaneously from indoxyl by oxidation by air, but for use in dyeing, indigo further needs to be solubilized with a strong reducing agent (e.g. Na2S2O4), which is likewise environmentally challenging (Figure 1).

[0007] In the context of sustainability, there have been made several attempts at developing a scalable bio-based production method of indigo. However, the microbial approaches relying on whole-cell production of indigo are challenged by the poor solubility of indigo and economics.

[0008] Glycosyltransferase (GT) enzymes have been proposed as part of a green biotech alternative to current industrial processes for blue denim production (Figure 1). Specifically, the hydroxyl group of chemically synthesized indoxyl may be glycosylated by GT, thereby protecting the reactive functional group and generating the stable soluble (colorless) indican molecule. Indican may then later be hydrolyzed by beta-glucosidase (BGL) back to indoxyl which can then spontaneously oxidize to form blue indigo directly on the fabric (Figure 1). This is a "green" alternative to the traditional industrial process, providing an alternative solution to the final steps of the indigo dyeing process.

[0009] The present invention provides a complete bio-based solution, without need for chemical synthesis of substrates, and omitting the use of any harsh chemicals. SUMMARY OF THE INVENTION

[0010] The present invention provides a novel bio-based enzymatic method for indole synthesis, and optionally followed by further enzymatic step(s) for indican and / or indigo synthesis. By the sequential action of several enzymes, basic building blocks are converted to indole, and optionally to indican and / or indigo. These basic building blocks are readily available and / or can be produced from renewable sources.

[0011] In one aspect, the present invention provides an in vitro method of producing indole, and optionally producing indican and / or indigo, comprising the steps

[0012] (i) providing ribose-5-phosphate (R5P);

[0013] (ii) enzymatically converting the R5P to phosphoribosyl pyrophosphate (PRPP), preferably facilitated by a phosphoribosylpyrophosphate synthase (PRS);

[0014] (iii) enzymatically converting the PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a N-phosphoribosyl anthranilate synthase (trpD);

[0015] (iv) enzymatically converting the N-PRA to l-(2-carboxyphenylamino)-l-deoxy- D-ribulose 5-phosphate (CdRP), preferably facilitated by a N-(5'- phosphoribosyl)anthranilate isomerase (trpF);

[0016] (v) enzymatically converting the CdRP to indole-3-glycerol phosphate (IGP), preferably facilitated by an indole-3-glycerol phosphate synthase (trpC); and

[0017] (vi) enzymatically converting the IGP to indole, preferably facilitated by an indole- 3-glycerol-phosphate lyase (IGL); and optionally comprising the steps

[0018] (a) enzymatically converting the indole to indoxyl, and

[0019] (bl) enzymatically converting the indoxyl to indican, and / or (b2) converting the indoxyl to indigo by exposure to oxygen.

[0020] In one embodiment, the ribose-5-phosphate (R5P) in step (i) is provided by

[0021] (I) enzymatically converting ribose to R5P, or

[0022] (II) enzymatically converting glucose or starch to glucose-6-phosphate (G6P), enzymatically converting G6P to 6-phosphogluconate (6PG), enzymatically converting 6PG to ribulose 5-phosphate (Ru5P), and enzymatically converting Ru5P to R5P.

[0023] In one such embodiment (I), the conversion of ribose to R5P is facilitated by a ribokinase (RK). In one such embodiment (II), the conversion of glucose to G6P is facilitated by a hexokinase (HK); the conversion of starch to G6P is facilitated by an alphaamylase, a glucoamylase, and a hexokinase (HK), or by an alpha-glucan phosphorylase (aGP) and a phosphoglucomutase (PGM); the conversion of G6P to 6PG is facilitated by a glucose 6-phosphate dehydrogenase (G6PDH); the conversion of 6PG to Ru5P is facilitated by a 6-phosphogluconate dehydrogenase (6PGDH), and the conversion of Ru5P to R5P is facilitated by a ribose 5-phosphate isomerase (RPI). In one preferred embodiment, the in vitro method of the present invention is a method for producing indigo, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a) and (b2), wherein indole is an intermediate compound.

[0024] In one preferred embodiment, the in vitro method of the present invention is a method for producing indican, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a) and (bl), wherein indole is an intermediate compound.

[0025] Preferably, step (a) is facilitated by an oxidizing enzyme, such as an oxidizing enzyme selected from an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin-containing monooxygenase (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1), and an unspecific peroxygenase (EC 1.11.2.1).

[0026] In a further embodiment, the in vitro method further comprises co-factor regeneration systems, such as an ATP-regenerating enzyme, a NADPH-regenerating enzyme, NAD(P)+-regenerating enzyme, and / or UDP-GIc-regenerating enzyme.

[0027] Preferably, step (bl) is facilitated by a glycosyltransferase (GT) (EC 2.4.1), such as a UDP-glycosyltransferases, preferably a glycosyltransferase wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0028] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0029] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0030] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, or and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0031] In one aspect, the present invention concerns a one-pot method for producing indole, and optionally producing indican and / or indigo.

[0032] In one preferred embodiment, steps (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a one-pot reaction; optionally further comprising the regeneration systems as disclosed herein in the one-pot reaction.

[0033] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a one-pot reaction; optionally further comprising the regeneration systems as disclosed herein in the one-pot reaction.

[0034] In one embodiment, all enzymatic reactions take place in a one-pot reaction. In one embodiment, sequential one-pot synthesis is used, where enzymes, substrates and / or co-factors are added to a reactor one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0035] In another aspect, an in vitro method of producing indican is provided, comprising the steps

[0036] (t) enzymatically converting tryptophan to indole, preferably facilitated by a L- tryptophan indole lyase (TIL);

[0037] (a) enzymatically converting indole to indoxyl, preferably facilitated by an oxidizing enzyme, such as an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin-containing monooxygenase (FMO) (EC 1.14.13.8), an unspecific peroxygenase (EC 1.11.2.1), or P450 (EC 1.14.14.1); and

[0038] (bl) enzymatically converting indoxyl to indican, preferably facilitated by a glycosyltransferase (GT) (EC 2.4.1), such as a UDP-glycosyltransferases, preferably a glycosyltransferase wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0039] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0040] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0041] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0042] In one aspect, the present invention concerns a one-pot method for producing indican from tryptophan, wherein all enzymatic reactions take place in a one-pot reaction.

[0043] DESCRIPTION OF THE INVENTION

[0044] Brief description of the figures:

[0045] Figure 1: The 'traditional process' involves chemically synthesized indigo and addition of reducing agents (e.g. sodium dithionite, Na2S2O4) to the indigo vat for reduction to dye-competent, soluble leucoindigo. In the 'chemoenzymatic process', indoxyl is glucosylated giving indican as product. Indican is stable and can be stored. The glucosyl group is removed on the fabric, during the dyeing step, allowing the regenerated indoxyl to oxidize to indigo on the fabric. GT= glycosyltransferase, BGL=p-glycosyl hydrolase. Figure 2: Overview of biosynthetic enzymatic routes to indole, indican and indigo. R5P= ribose-5-phosphate, PRPP= phosphoribosyl pyrophosphate, / V-PRA= phosphoribosyl pyrophosphate, CdRP=l-(o-carboxy phenylamino)-l- deoxyribulose 5-phosphate, IGP= indole-3-glycerol-phosphate, G1P= glucose-l-phosphate, G6P= glucose-6-phosphate, 6PG=6-phosphogluconate, Ru5P= ribulose 5-phosphate.

[0046] Figure 3:

[0047] (A) Example of enzymatic conversion of ribose-5-phosphate (R5P) and anthranilate to indole. R5P is converted to phosphoribosyl pyrophosphate (PRPP) by phosphoribosylpyrophosphate synthase (PRS); PRPP is converted to phosphoribosyl pyrophosphate (N-PRA) by N-phosphoribosyl anthranilate synthase (trpD); N-PRA is converted to l-(o-carboxy phenylamino)-l- deoxyribulose 5-phosphate (CdRP) by N- phosphoribosyl anthranilate isomerase (trpF); CdRP is converted to indole-3-glycerol- phosphate (IGP) by indole-3-glycerol-phosphate synthase (trpC); and IGP is converted to indole by indole-3-glycerol-phosphate lyase (IGL). Enzymatic cofactor recycling system may be used to regenerate ATP from AMP. PPK2-II = AMP kinase. PolyP = Polyphosphate. Anth = anthranilate. PPi = Inorganic pyrophosphate. G3P = Glycerol-3- phosphate.

[0048] (B) Example of conversion of ribose to ribose-5-phosphate (R5P) facilitated by ribokinase (RK). Enzymatic cofactor recycling system may be used to regenerate ATP from ADP. PPK2-I = ADP kinase. PolyP = Polyphosphate.

[0049] (C) Example of conversion of glucose to ribose-5-phosphate (R5P). Glucose is converted to glucose-6-phosphate (G6P) by a hexokinase (HK), G6P is converted to 6- phosphogluconate (6PG) by a glucose 6-phosphate dehydrogenase (G6PDH), 6PG is converted to ribulose 5-phosphate (Ru5P) by a 6-phosphogluconate dehydrogenase (6PGDH), and Ru5P is converted to R5P by a ribose 5-phosphate isomerase (RPI). Enzymatic cofactor recycling system may be used to regenerate ATP from AMP, and NAD(P)+ from NAD(P)H. PPK2-I = ADP kinase. PolyP = Polyphosphate. GR = Glutathione-disulfide reductase. GSSG = Glutathione disulfide. GSH = Glutathione.

[0050] (D) Example of conversion of starch to ribose-5-phosphate (R5P). Starch is converted to glucose by an alpha-amylase and a glucoamylase, glucose is converted to glucose-6- phosphate (G6P) by a hexokinase (HK), G6P is converted to 6-phosphogluconate (6PG) by a glucose 6-phosphate dehydrogenase (G6PDH), 6PG is converted to ribulose 5- phosphate (Ru5P) by a 6-phosphogluconate dehydrogenase (6PGDH), and Ru5P is converted to R5P by a ribose 5-phosphate isomerase (RPI). Enzymatic cofactor recycling system may be used to regenerate ATP from AMP, and NAD(P)+from NAD(P)H. PPK2-I = ADP kinase. PolyP = Polyphosphate. GR = Glutathione-disulfide reductase. GSSG = Glutathione disulfide. GSH = Glutathione. (E) Example of conversion of starch to ribose-5-phosphate (R5P). Starch is converted to glucose-l-phosphate (G1P) by an alpha-glucan phosphorylase (aGP), G1P is converted to glucose-6-phosphate (G6P) by a phosphoglucomutase (PGM), G6P is converted to 6-phosphogluconate (6PG) by a glucose 6-phosphate dehydrogenase (G6PDH), 6PG is converted to ribulose 5-phosphate (Ru5P) by a 6-phosphogluconate dehydrogenase (6PGDH), and Ru5P is converted to R5P by a ribose 5-phosphate isomerase (RPI). Enzymatic cofactor recycling system may be used to regenerate NAD(P)+from NAD(P)H. GR = Glutathione-disulfide reductase. GSSG = Glutathione disulfide. GSH = Glutathione. Pi = Inorganic phosphate.

[0051] Figure 4: Examples of enzymatic conversion of indole to indican. (A) Indole is converted to indoxyl by an oxidoreductase; and indoxyl is converted to indican by UDP glycosyltransferase (UGT). Enzymatic cofactor recycling systems are introduced to regenerate NADPH from NADP+ and UDP-GIc from UDP. FDH = formate dehydrogenase. Susy = sucrose synthase. (B) Indole is converted to indoxyl by a peroxygenase; and indoxyl is converted to indican by UDP glycosyltransferase (UGT). Enzymatic cofactor recycling system is introduced to regenerate UDP-GIc from UDP. Susy = sucrose synthase.

[0052] Figure 5: Examples of enzymatic conversion of indole to indigo. (A) Indole is converted to indoxyl by an oxidoreductase; and indoxyl is spontaneously converted to indigo by exposure to air. Enzymatic cofactor recycling system is introduced to regenerate NADPH from NADP+. FDH = formate dehydrogenase. (B) Indole is converted to indoxyl by a peroxygenase; and indoxyl is spontaneously converted to indigo by exposure to air.

[0053] Figure 6: Examples of enzymatic conversion of tryptophan to indole and indican. (A) Tryptophan is converted to indole by L-tryptophan indole-lyase (TIL). Indole is then converted to indoxyl by flavin monooxygenase (FMO); and indoxyl is converted to indican by UDP glycosyltransferase (UGT). Pyr = pyruvate. Enzymatic cofactor recycling systems are introduced to regenerate NADPH from NADP+ and UDP-GIc from UDP. FDH = formate dehydrogenase. Susy = sucrose synthase. (B) Tryptophan is converted to indole by L-tryptophan indole-lyase (TIL). Indole is then converted to indoxyl by a peroxygenase; and indoxyl is converted to indican by UDP glycosyltransferase (UGT). Pyr = pyruvate. Enzymatic cofactor recycling system is introduced to regenerate UDP- GIc from UDP. Susy = sucrose synthase.

[0054] Figure 7: Enzymatic conversion of (A) 100 pM and (B) 1000 pM anthranilate to indole by activity of enzymes TkTrpD, EcTrpF, PaTrpC, and OsIGL. Concentrations of indole and anthranilate were measured at different time points of incubation. Figure 8A: Sequential conversion of anthranilic acid to IGP by enzymes TkTrpD, EcTrpF and PaTrpC with lh incubation steps in between sampling. Chromatograms were obtained through RP-HPLC and recorded at 340 nm and 260 nm.

[0055] Figure 8B: Sequential enzymatic conversion of anthranilic acid to indole. Reaction (1) conversion of anthranilic acid to NPRA by addition of TrpD; reaction (2) conversion of NPRA to CdRP by addition of TrpF; reaction (3) addition of sodium borohydride selectively reduces CdRP to reduced CdRP (rCdRP); reaction (4) conversion of CdRP to IGP by addition of TrpC; reaction (5) chemical conversion of IGP to Indole-3-aldehyde (I3A) by addition of sodium periodate; reaction (6) conversion of IGP to indole by addition of IGL.

[0056] Figure 9: Conversion of anthranilic acid to indigo in a one-pot in vitro enzymatic mixture. The concentration of indole and anthranilate were measured at different time points of incubation.

[0057] Figure 10: Conversion of ribose to IGP in a one-pot in vitro enzymatic mixture. Enzymes TkTrpD, EcTrpF, and PaTrpC were applied in all tests, while combinations of different ribokinases and phosphoribosylpyrophosphate synthase were explored. IGP was measured after 1, 2, 4, and 22 hours of incubation.

[0058] Figure 11: HPLC chromatogram recorded at 280 nm resulting from conversion of ribose and anthranilic acid after overnight incubation at 25°C by an in vitro enzymatic mixture containing HoRK, HoPRS, TkTrpD, EcTrpF, PaTrpC, OsIGL, NIFMO, and PtUGTlmut87.

[0059] Figure 12: Effect of temperature on the conversion of anthranilic acid and PRPP to IGP using enzymes TkTrpD, TmTrpF, and SsTrpC.

[0060] Figure 13: Effect of enzyme ratio on the conversion of anthranilic acid and PRPP to IGP in one-pot in vitro enzymatic mixture using enzymes TkTrpD, TmTrpF, and SsTrpC. (A) Fixed concentration of TmTrpF and SsTrpC, while varying the concentration of TkTrpD. (B) Fixed concentration of TmTrpD and SsTrpC, while varying the concentration of TkTrpF. (C) Fixed concentration of TmTrpD and TmTrpF, while varying the concentration of SsTrpC.

[0061] Figure 14: Optimization of IGP formation (A) Baseline experiment (B) Single spike with 5 mM PRPP after 60 minutes (C) Spiking with fresh 7 mM anthranilate and 14 mM PRPP every 30 minutes (D) Spiking with 4 mM anthranilate and 5 mM PRPP every 60 minutes (E) Spiking with 20 mM PRPP every 60 minutes (F) Spiking with 20 mM PRPP every 30 minutes (G) Spiking with 5 pM SsTrpC every 60 minutes (H) repeat of G, spiking with 5 pM SsTrpC and 15 mM PRPP every 60 minutes.

[0062] Figure 15: Effect of temperature on the conversion of IGP to indigo using enzymes ZmBXl, NIFMO, and MvFDH. Figure 16: Effect of enzyme ratio on the conversion of IGP to indigo using enzymes ZmBXl, NIFMO, and MvFDH. (A) Fixed concentration of NIFMO, and MvFDH, while varying the concentration of ZmBXl. (B) Fixed concentration of ZmBXl and MvFDH, while varying the concentration of NIFMO. (C) Fixed concentration of ZmBXland NIFMO, while varying the concentration of MvFDH.

[0063] Figure 17: Conversion of tryptophan to indican in a one-pot in vitro enzymatic mixture containing VcTIL, NIFMO, and PtUGTlmut87 with different tryptophan concentrations (1, 5 and 10 mM).

[0064] Abbreviations, terms, and definitions:

[0065] Amino acid sequence identity: The term "sequence identity" as used herein, indicates a quantitative measure of the degree of similarity between two amino acid sequences of essentially equal length. The two sequences to be compared must be aligned to give a best possible fit, by means of the insertion of gaps or alternatively, truncation at the ends of the protein sequences. The sequence identity can be calculated as ((Nref- Ndif)100) / (Nref), wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Sequence identity calculations are preferably automated using the BLAST program e.g. the BLASTP program (Pearson W.R and D.J. Lipman (1988)) (www.ncbi.nlm.nih.gov / cgi-bin / BLAST). Sequence alignment may be performed using program MAFFT24 (Multiple Alignment using Fast Fourier Transform; Katoh et al 2019) using default parameters (SCORING MATRIX: blosum62, gap opening penalty: 1.53, gap extension penalty 0.123).

[0066] Preferably, the numbers of substitutions, insertions, additions or deletions of one or more amino acid residues in the polypeptide as compared to its comparator polypeptide is limited, i.e. no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 insertions, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additions, and no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 deletions. Preferably the substitutions are conservative amino acid substitutions: limited to exchanges within members of group 1: Glycine, Alanine, Valine, Leucine, Isoleucine; group 2: Serine, Cysteine, Selenocysteine, Threonine, Methionine; group 3: Proline; group 4: Phenylalanine, Tyrosine, Tryptophan; Group 5: Aspartate, Glutamate, Asparagine, Glutamine; Group 6: Histidine. Lysine, Arginine.

[0067] The term 'one pot' reaction or 'one pot' synthesis as used herein means a chemical reaction or synthetic process in which all the required reactants, reagents, and steps are combined and carried out within a single reaction vessel or container.

[0068] The term 'sequential one-pot synthesis' as used herein means a chemical reaction or synthetic process in which all the required reactants, reagents, and steps are combined and carried out within a single reaction vessel or container, wherein the reactants and / or reagents are added sequentially to the reaction vessel or container - i.e. one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0069] Anthranilic acid is an aromatic acid with the formula C6H4(NH2)(CO2H); the molecule consists of a benzene ring, ortho-substituted with a carboxylic acid and an amine, as illustrated in formula I. The anion [C6H4(NH2)(CO2)]_, obtained by the deprotonation of anthranilic acid, is called anthranilate. Formula I.

[0070] The two terms: anthranilic acid and anthranilate are used interchangeably in the context of the present invention. Anthranilic acid is the actual compound added to the reaction mixtures, while anthranilate is formed in solution (pKa of 4.8).

[0071] Detailed description of the invention:

[0072] The present invention provides a novel bio-based enzymatic method for indole synthesis, and optionally followed by further enzymatic step(s) for indican and / or indigo synthesis. By the sequential action of several enzymes, basic building blocks are converted to indole, and optionally to indican and / or indigo, as illustrated in Figure 2.

[0073] I. Synthesis of indole from ribose-5-phosphate and anthranilate

[0074] In one aspect, the present invention concerns an in vitro method of producing indole, comprising the steps

[0075] (i) providing ribose-5-phosphate (R5P),

[0076] (ii) enzymatically converting R5P to phosphoribosyl pyrophosphate (PRPP),

[0077] (iii) enzymatically converting PRPP to N-phosphoribosyl anthranilate (N-PRA),

[0078] Civ) enzymatically converting N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D- ribulose 5-phosphate (CdRP),

[0079] (v) enzymatically converting CdRP to indole-3-glycerol phosphate (IGP), and

[0080] (vi) enzymatically converting IGP to indole.

[0081] The in vitro method is illustrated in Figure 3A. For the synthesis of indole, the substrates are ribose-5-phosphate and anthranilate, and cofactor ATP is further needed. Ribose-5-phosphate is needed in step (i); anthranilate is needed in step (iii); and ATP needed in step (ii).

[0082] In one embodiment, the synthesis of indole, comprises providing ribose-5-phosphate, anthranilate and ATP, for the relevant process steps, as disclosed above.

[0083] In one embodiment, ribose-5-phosphate (R5P) in step (i) is provided by

[0084] (I) enzymatically converting ribose to R5P, or

[0085] (II) enzymatically converting glucose or starch to glucose-6-phosphate (G6P), enzymatically converting G6P to 6-phosphogluconate (6PG), enzymatically converting 6PG to ribulose 5-phosphate (Ru5P), and enzymatically converting Ru5P to R5P.

[0086] In one embodiment, R5P is in step (i) provided by enzymatic conversion of ribose to R5P. In one preferred embodiment, this is facilitated by a ribokinase (RK). ATP is needed as cofactor. This is illustrated in Figure 3B. Hence, in one embodiment, in step (i), ribose- 5-phosphate is synthesized from ribose and ATP using a ribokinase. The ATP is converted to ADP. In one embodiment, ribose and ATP are in step (i) enzymatically converted to ribose-5-phosphate (R5P) by a polypeptide having ribokinase activity (EC 2.7.1.15). The amino acid sequence of said polypeptide having ribokinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 2, 4, or 6. Preferably, the amino acid sequence of said polypeptide having ribokinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 6. In one embodiment, the ribokinase is selected from SEQ ID NO. 2, 4, and 6, preferably SEQ ID NO 6.

[0087] In another embodiment, R5P is in step (i) provided by conversion of glucose to R5P. Specifically by enzymatic conversion of glucose to glucose-6-phosphate (G6P), enzymatic conversion of G6P to 6-phosphogluconate (6PG), enzymatic conversion of 6PG to ribulose 5-phosphate (Ru5P), and enzymatic conversion Ru5P to R5P. In one preferred embodiment, this is facilitated by a hexokinase (HK), a glucose 6-phosphate dehydrogenase (G6PDH), a 6-phosphogluconate dehydrogenase (6PGDH), and a ribose 5-phosphate isomerase (RPI). ATP and NAD+ are needed as cofactors. Hence, in one embodiment, in step (i), ribose-5-phosphate is synthesized from glucose, ATP and NAD+ using a hexokinase (HK), a glucose 6-phosphate dehydrogenase (G6PDH), a 6- phosphogluconate dehydrogenase (6PGDH), and a ribose 5-phosphate isomerase (RPI). The ATP is converted to ADP. The NAD+ is converted to NADH. This is illustrated in Figure 3C.

[0088] In one embodiment, in step (i) glucose and ATP are enzymatically converted to glucose-6-phosphate (G6P) by a polypeptide having hexokinase activity (EC 2.7. 1.1). The amino acid sequence of said polypeptide having hexokinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 74 or 76. In one embodiment, the hexokinase is selected from SEQ ID NO. 74 and 76, G6P and NAD+are enzymatically converted to 6-phosphoglucoronate (6PG) by a polypeptide having glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49). The amino acid sequence of said polypeptide having glucose 6- phosphate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 78 or 80. In one embodiment, the glucose 6-phosphate dehydrogenase is selected from SEQ ID NO. 78 and 80, 6PG and NAD+ are enzymatically converted to ribulose-5-phosphate (Ru5P) by a polypeptide having 6-phosphogluconate dehydrogenase activity (EC 1.1.1.44). The amino acid sequence of said polypeptide having 6-phosphogluconate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 82 or 84. In one embodiment, the 6- phosphogluconate dehydrogenase is selected from SEQ ID NO. 82 and 84, and Ru5P is enzymatically converted to R5P by a polypeptide having ribose 5- phosphate isomerase activity (EC 5.3.1.6). The amino acid sequence of said polypeptide having ribose 5-phosphate isomerase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 86 or 88. In one embodiment, the ribose 5-phosphate isomerase is selected from SEQ ID NO. 86 and 88.

[0089] In another embodiment, R5P is in step (i) provided by conversion of starch to glucose, and further conversion of the glucose to R5P as disclosed above. This is illustrated in Figure 3D. In one such embodiment, the enzymatic conversion of starch to glucose is facilitated by a polypeptide having alpha-amylase activity (E.C. 3.2.1.1) and / or a polypeptide having glucoamylase activity (E.C. 3.2.1.3). The amino acid sequence of said polypeptide having alpha-amylase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 90. In one embodiment, the alpha-amylase is SEQ ID NO. 90. The amino acid sequence of said polypeptide having glucoamylase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 92. In one embodiment, the glucoamylase is SEQ ID NO. 92.

[0090] In another embodiment, R5P is in step (i) provided by conversion of starch to R5P. Specifically by enzymatic conversion of starch to glucose-l-phosphate (G1P), enzymatic conversion of G1P to glucose-6-phosphate (G6P), enzymatic conversion of G6P to 6- phosphogluconate (6PG), enzymatic conversion 6PG to ribulose 5-phosphate (Ru5P), and enzymatic conversion Ru5P to R5P. In one preferred embodiment, this is facilitated by an alpha-glucan phosphorylase (aGP), a phosphoglucomutase (PGM), a glucose 6- phosphate dehydrogenase (G6PDH), a 6-phosphogluconate dehydrogenase (6PGDH), and a ribose 5-phosphate isomerase (RPI). ATP and NADH are needed as cofactors. Hence, in one embodiment, in step (i), ribose-5-phosphate is synthesized from starch, ATP and NADP using an alpha-glucan phosphorylase (aGP), a phosphoglucomutase (PGM), a glucose 6-phosphate dehydrogenase (G6PDH), a 6-phosphogluconate dehydrogenase (6PGDH), and a ribose 5-phosphate isomerase (RPI). The ATP is converted to ADP. The NAD+is converted to NADH. This is illustrated in Figure 3E.

[0091] In one embodiment, in step (i) starch is enzymatically converted to glucose-l-phosphate (G1P) by a polypeptide having alpha-glucan phosphorylase activity (EC 2.4. 1.1). The amino acid sequence of said polypeptide having alpha-glucan phosphorylase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 94. In one embodiment, the alpha-glucan phosphorylase is SEQ ID NO. 94, G1P is enzymatically converted to glucose-6-phosphate (G6P) by a polypeptide having phosphoglucomutase activity (EC 2.7.5. 1). The amino acid sequence of said polypeptide having phosphoglucomutase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 96. In one embodiment, the phosphoglucomutase is SEQ ID NO. 96,

[0092] R5P and NAD+ are enzymatically converted to 6-phosphoglucoronate (6PG) by a polypeptide having glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49). The amino acid sequence of said polypeptide having glucose 6- phosphate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 82 or 84. In one embodiment, the glucose 6-phosphate dehydrogenase is selected from SEQ ID NO. 82 and 84, 6PG and NAD+ are enzymatically converted to ribulose-5-phosphate (Ru5P) by a polypeptide having 6-phosphogluconate dehydrogenase activity (EC 1.1.1.44). The amino acid sequence of said polypeptide having 6-phosphogluconate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 86 or 88. In one embodiment, the 6- phosphogluconate dehydrogenase is selected from SEQ ID NO. 86 and 88, and Ru5P is enzymatically converted to R5P by a polypeptide having ribose 5- phosphate isomerase activity (EC 5.3. 1.6). The amino acid sequence of said polypeptide having ribose 5-phosphate isomerase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 90 or 92. In one embodiment, the ribose 5-phosphate isomerase is selected from SEQ ID NO. 90 and 92.

[0093] In one embodiment, step (ii) is facilitated by a phosphoribosylpyrophosphate synthase (PRS). ATP needed as cofactor. In step (ii), phosphoribosyl pyrophosphate is synthesized from R5P and ATP using a phosphoribosylpyrophosphate synthase. The ATP is converted to AMP. In one embodiment, R5P and ATP are in step (ii) enzymatically converted to phosphoribosyl pyrophosphate (PRPP) by a polypeptide having phosphoribosylpyrophosphate synthase activity (EC 2.7.6.1). The amino acid sequence of said polypeptide having phosphoribosylpyrophosphate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 8, 10, or 12. Preferably, the amino acid sequence of said polypeptide having phosphoribosylpyrophosphate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 12. In one embodiment, the phosphoribosylpyrophosphate synthase is selected from SEQ ID NO. 8, 10, and 12, preferably SEQ ID NO 12.

[0094] In one embodiment, step (iii) is facilitated by a N-phosphoribosyl anthranilate synthase (trpD). In step (iii), N-phosphoribosyl anthranilate (N-PRA) is synthesized from PRPP and anthranilate using a N-phosphoribosyl anthranilate synthase. In one embodiment, PRPP and anthranilate are in step (iii) enzymatically converted to N-phosphoribosyl anthranilate (N-PRA) by a polypeptide having N-phosphoribosyl anthranilate synthase activity (EC 2.4.2.18). The amino acid sequence of said polypeptide having N- phosphoribosyl anthranilate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 14, 16, or 18. Preferably, the amino acid sequence of said polypeptide having N-phosphoribosyl anthranilate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 14. In one embodiment, the N-phosphoribosyl anthranilate synthase is selected from SEQ ID NO. 14, 16 and 18, preferably SEQ ID NO 14.

[0095] In one embodiment, step (iv) is facilitated by an N-(5'-phosphoribosyl)anthranilate isomerase (trpF). In one embodiment, N-PRA is in step (iv) enzymatically converted to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP) by a polypeptide having N-(5'-phosphoribosyl)anthranilate isomerase activity (EC 5.3.1.24). The amino acid sequence of said polypeptide having N-(5'-phosphoribosyl)anthranilate isomerase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 20 or 22. Preferably, the amino acid sequence of said polypeptide having N-(5'- phosphoribosyl)anthranilate isomerase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 20. In one embodiment, the N-(5'- phosphoribosyl)anthranilate isomerase is selected from SEQ ID NO. 20 and 22, preferably SEQ ID NO 20.

[0096] In one embodiment, step (v) is facilitated by an indole-3-glycerol phosphate synthase (trpC). In one embodiment, CdRP is in step (v) enzymatically converted to indole-3- glycerol phosphate (IGP) by a polypeptide having indole-3-glycerol phosphate synthase activity (EC 4.1.1.48). The amino acid sequence of said polypeptide having indole-3- glycerol phosphate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 24 or 26. Preferably, the amino acid sequence of said polypeptide having indole-3-glycerol phosphate synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 26. In one embodiment, the indole-3-glycerol phosphate synthase is selected from SEQ ID NO. 24 and 26, preferably SEQ ID NO 26.

[0097] In one embodiment, step (vi) is facilitated by an indole-3-glycerol-phosphate lyase (IGL). In one embodiment, IGP is in step (vi) enzymatically converted to indole by a polypeptide having indole-3-glycerol-phosphate lyase activity (EC 4.1.2.8). The amino acid sequence of said polypeptide having indole-3-glycerol-phosphate lyase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 28 or 30. Preferably, the amino acid sequence of said polypeptide having indole-3-glycerol- phosphate lyase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 28. In one embodiment, the indole-3-glycerol-phosphate lyase is selected from SEQ ID NO. 28 and 30, preferably SEQ ID NO 28.

[0098] In one preferred embodiment, in the in vitro method of producing indole comprises all steps (i), (ii). (iii), (iv), (v), and (vi), wherein step (i) is facilitated by a ribokinase (R.K), step (ii) is facilitated by a phosphoribosylpyrophosphate synthase (PRS), step (iii) is facilitated by a N-phosphoribosyl anthranilate synthase (trpD), step (iv) is facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase (trpF), step (v) is facilitated by an indole- 3-glycerol phosphate synthase (trpC), and step (vi) is facilitated by an indole-3-glycerol- phosphate lyase (IGL).

[0099] In one preferred embodiment, in the in vitro method of producing indole comprises all steps (i), (ii). (iii), (iv), (v), and (vi); wherein step (i) is facilitated by a polypeptide having ribokinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 6; step (ii) is facilitated by a polypeptide having phosphoribosylpyrophosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 12; step (iii) is facilitated by a polypeptide having N-phosphoribosyl anthranilate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 14; step (iv) is facilitated by a polypeptide having N-(5'-phosphoribosyl)anthranilate isomerase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 20; step (v) is facilitated by a polypeptide having indole-3-glycerol phosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 26; and step (vi) is facilitated by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 28.

[0100] In one embodiment, the present invention provides an in vitro method of producing indole, comprising (I) providing substrates ribose-5-phosphate and anthranilate, cofactor ATP, and enzymes phosphoribosylpyrophosphate synthase, N-phosphoribosyl anthranilate synthase, N-(5'-phosphoribosyl)anthranilate isomerase, indole-3-glycerol phosphate synthase, and indole-3-glycerol-phosphate lyase, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indole.

[0101] In one embodiment, the present invention provides an in vitro method of producing indole, comprising (I) providing substrates ribose and anthranilate, cofactor ATP, and enzymes ribokinase, phosphoribosylpyrophosphate synthase, N-phosphoribosyl anthranilate synthase, N-(5'-phosphoribosyl)anthranilate isomerase, indole-3-glycerol phosphate synthase, and indole-3-glycerol-phosphate lyase, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indole.

[0102] In one embodiment, the present invention provides an in vitro method of producing indole, comprising (I) providing substrates glucose and anthranilate, cofactors ATP and NAD+, and enzymes hexokinase, glucose 6-phosphate dehydrogenase, 6- phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, phosphoribosylpyrophosphate synthase, N-phosphoribosyl anthranilate synthase, N-(5'- phosphoribosyl)anthranilate isomerase, indole-3-glycerol phosphate synthase, and indole-3-glycerol-phosphate lyase, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indole.

[0103] In one embodiment, the present invention provides an in vitro method of producing indole, comprising (I) providing substrates starch and anthranilate, cofactors ATP and NAD+, and enzymes alphaamylase, glucoamylase, hexokinase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, phosphoribosylpyrophosphate synthase, N-phosphoribosyl anthranilate synthase, N-(5'- phosphoribosyl)anthranilate isomerase, indole-3-glycerol phosphate synthase, and indole-3-glycerol-phosphate lyase, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indole.

[0104] In one embodiment, the present invention provides an in vitro method of producing indole, comprising (I) providing substrates starch and anthranilate, cofactors ATP and NAD+, and enzymes alpha-glucan phosphorylase, phosphoglucomutase, glucose 6- phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, ribose 5-phosphate isomerase, phosphoribosylpyrophosphate synthase, N-phosphoribosyl anthranilate synthase, N-(5'-phosphoribosyl)anthranilate isomerase, indole-3-glycerol phosphate synthase, and indole-3-glycerol-phosphate lyase, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indole. Ribose is a simple carbohydrate sugar with molecular formula C5H10O5. For the present invention, ribose may be provided directly in the form of ribose monomers, and be converted into ribose-5 phosphate as disclosed herein. Alternatively, step (i) as disclosed herein may be facilitated by enzymatically converting starch to ribose-5-phosphate as disclosed herein. Alternatively, glucose may be provided and converted into R5P, as disclosed herein. Ribose, glucose, and starch are sugar monomers and polymers widely available to the person skilled in the art.

[0105] Anthranilic acid is an aromatic acid with the formula C6H4(NH2)(CO2H). The anion [C5H4( N H2XCO2)] - , obtained by the deprotonation of anthranilic acid, is called anthranilate. Many routes to anthranilic acid have been described. The biosynthesis of anthranilate results from the shikimate pathway. Industrially it is produced from phthalic anhydride. Methyl-anthranilate is a relatively cheap and accessible compound that can be converted to anthranilic acid in a single enzymatic step. Hence the anthranilate provided and needed in step (iii) may be provided directly in the form of anthranilic acid, or as antranilate precursor which may be enzymatically converted to anthranilate.

[0106] The co-factor ATP may be regenerated from ADP and AMP by enzymatic cofactor recycling system using an ATP-regenerating enzyme (EC 2.7.4.1), such as a phosphate kinase (EC 2.7.4.1), preferably a Class 2 polyphosphate kinase (PPK2) (EC 2.7.4.1).

[0107] In one embodiment, ADP kinase (PPK2-I) in combination with a Polyphosphate (PolyP) facilitates regeneration of ATP from step (i) of the method. In one embodiment, ADP is enzymatically converted to ATP by a polypeptide having ADP kinase activity (EC 2.7.4.1). The amino acid sequence of said polypeptide having ADP kinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 58, 60 or 62. Preferably, the amino acid sequence of said polypeptide having ADP kinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 58. In one embodiment, the ADP kinase is selected from SEQ ID NO. 58, 60, and 62, preferably SEQ ID NO 58.

[0108] In one embodiment, AMP kinase (PPK2-II) in combination with a Polyphosphate (PolyP) facilitates regeneration of AMP from step (ii) of the method. In one embodiment, AMP is enzymatically converted to ATP by a polypeptide having AMP kinase activity (EC 2.7.4.1). The amino acid sequence of said polypeptide having AMP kinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 58, 60, or 62. Preferably, the amino acid sequence of said polypeptide having ADP kinase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 62. In one embodiment, the AMP kinase is selected from SEQ ID NO. 58, 60, and 62, preferably SEQ ID NO 62. 1

[0109] The co-factor NAD(P)+may be regenerated from NAD(P)H by enzymatic cofactor recycling system using a NAD(P)+-regenerating enzyme, such as a glutathione-disulfide reductase (EC 1.8.1.7).

[0110] In one embodiment, glutathione disulfide in combination with NAD(P)H facilitates regeneration of NAD(P)+. In one embodiment, NAD(P)H is enzymatically converted to NAD(P)+by a polypeptide having a glutathione-disulfide reductase activity (EC 1.8.1.7). The amino acid sequence of said polypeptide having glutathione-disulfide reductase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 98. In one embodiment, the glutathione-disulfide reductase is SEQ ID NO. 98.

[0111] In one preferred embodiment, in the in vitro method of producing indole comprises all steps (i), (ii). (iii), (iv), (v), and (vi), and further comprises co-factor recycling steps as disclosed herein; wherein step (i) is facilitated by a ribokinase (R.K), step (ii) is facilitated by a phosphoribosylpyrophosphate synthase (PRS), step (iii) is facilitated by a N- phosphoribosyl anthranilate synthase (trpD), step (iv) is facilitated by a N-(5'- phosphoribosyl)anthranilate isomerase (trpF), step (v) is facilitated by an indole-3- glycerol phosphate synthase (trpC), and step (vi) is facilitated by an indole-3-glycerol- phosphate lyase (IGL); and further wherein ATP co-factor recycling is facilitated by an ADP kinase and an AMP kinase.

[0112] In one embodiment, some or all the enzyme steps for in vitro production of indole as disclosed herein are performed in a 'one pot' reaction.

[0113] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) as disclosed herein are performed in a one-pot reaction. In a further preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi)as weHasthe ATP-co-factor regeneration from ADP and AMP, as disclosed herein, are performed in a one-pot reaction.

[0114] The in vitro biosynthesis is preferably performed at temperature and pH conditions optimal for the enzymes used.

[0115] In one embodiment, the incubation temperature applied should be in the range 20- 65°C, such as 20-50°C, such as 20-40°C, preferably in the range 25-35°C, such as preferably around 30°C. In one embodiment, the incubation pH applied should be in the range pH 5-9, such as pH 5.5-8.5, such as preferably pH 6-8, such as preferably around pH 7.5.

[0116] The enzymatic reaction may take place in buffered solution for stabilizing the enzymes, as a person skilled in the art would recognize and routinely optimize.

[0117] In one embodiment, all enzyme steps for in vitro production of indole as disclosed herein are performed in a one-pot reaction. In one preferred embodiment, steps (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a one-pot reaction. In a further embodiment, steps (ii), (iii), (iv), (v), and (vi), as well as the regeneration systems as disclosed herein are performed in a one-pot reaction.

[0118] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a one-pot reaction. In a further embodiment, steps (i), (ii), (iii), (iv), (v), and (vi),as weHasthe regeneration systems as disclosed herein are performed in a one-pot reaction.

[0119] In one embodiment, sequential one-pot synthesis may be used, where the enzymes, substrates and / or co-factors are added to a reactor one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0120] II. Synthesis of indoxyl from indole

[0121] In another aspect, the present invention provides an in vitro method for producing indican and / or indigo, said method comprising step (a) of enzymatically converting indole to indoxyl, as illustrated in Figures 4 and 5.

[0122] In one embodiment, step (a) is facilitated by an oxidizing enzyme (ox enz). In one embodiment, the oxidizing enzyme is selected from an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin-containing monooxygenase (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1), and an unspecific peroxygenase (EC 1.11.2.1).

[0123] In one embodiment, step (a) is facilitated by a flavin-containing monooxygenase (FMO). NADPH is needed as cofactor / redox partner. In step (a), indoxyl is synthesized from indole and NADPH using a flavin-containing monooxygenase. In one embodiment, indole and NADPH are in step (a) enzymatically converted to indoxyl and NADP+ by a polypeptide having flavin-containing monooxygenase activity (EC 1.14.13.8). The amino acid sequence of said polypeptide having flavin-containing monooxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 32, 34, or 36. Preferably, the amino acid sequence of said polypeptide having flavin- containing monooxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 32. In one embodiment, the flavin-containing monooxygenase is selected from SEQ ID NO. 32, 34, and 36, preferably SEQ ID NO. 32.

[0124] In another embodiment, step (a) is facilitated by a cytochrome P450 enzyme. NADPH is needed as cofactor. In step (a), indoxyl is synthesized from indole and NADPH using a cytochrome P450 enzyme (EC 1.14.14.1). In one embodiment, indole and NADPH are in step (a) enzymatically converted to indoxyl and NADP+ by a polypeptide having P450 enzyme activity (EC 1.14.14.1). The amino acid sequence of said polypeptide having P450 enzyme activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 38 or 40. Preferably, the amino acid sequence of said polypeptide having P450 enzyme activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 38. In one embodiment, the cytochrome P450 enzyme is selected from SEQ ID NO. 38 and 40, preferably SEQ ID NO. 38

[0125] The co-factor NADPH may be regenerated from NADP+ by enzymatic cofactor recycling system using an NADPH-regenerating enzyme, such as a dehydrogenase, preferably a formate dehydrogenase (FDH) (EC 1.17.1.10).

[0126] In one embodiment, formate dehydrogenase (FDH) in combination with formate facilitates regeneration of NADPH from step (a) of the method. In one embodiment, NADP+ is enzymatically converted to NADPH by a polypeptide having formate dehydrogenase activity (EC 1.17.1.10). The amino acid sequence of said polypeptide having formate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56. Preferably, the amino acid sequence of said polypeptide having formate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56. In one embodiment, the formate dehydrogenase is selected from SEQ ID NO. 56, preferably SEQ ID NO. 56.

[0127] In another embodiment, step (a) is facilitated by a peroxygenase enzyme. Hydrogenperoxide is needed as cofactor. In step (a), indoxyl is synthesized from indole and H2O2 using a peroxygenase (EC 1.11.2.1). In one embodiment, indole and H2O2 are in step (a) enzymatically converted to indoxyl and H2O by a polypeptide having peroxygenase activity (EC 1.11.2.1). The amino acid sequence of said polypeptide having peroxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 42 or 44. Preferably, the amino acid sequence of said polypeptide having peroxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 42. In one embodiment, the peroxygenase enzyme is selected from SEQ ID NO. 42 and 44, preferably SEQ ID NO. 42. in. Synthesis of indican and / or indigo

[0128] Ill.i Synthesis from indole

[0129] In one embodiment, the present invention provides an in vitro method for producing indican and / or indigo. Indican and / or indigo may be produced from indole, as illustrated in Figures 4 and 5.

[0130] In one embodiment, in vitro production of indican and / or indigo comprises

[0131] (a) enzymatically converting indole to indoxyl, as disclosed in section II, and (bl) enzymatically converting indoxyl to indican, and / or (b2) converting indoxyl to indigo by exposure to oxygen.

[0132] In one embodiment, step (bl) is facilitated by a glycosyltransferase (GT) (EC 2.4), such as a UDP-glycosyltransferase (EC 2.4.1), preferably a glycosyltransferase as disclosed in Bidart et al 2023 and WO 2023 / 161230.

[0133] In a preferred embodiment, indoxyl is in step (bl) enzymatically converted to indican by a polypeptide having glycosyltransferase activity (EC 2.4). In one embodiment, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 48, 50 or 52. In one embodiment, the glycosyltransferase is selected from SEQ ID NO. 48, 50 and 52.

[0134] Preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises one or more amino acid residue substitutions selected from single mutation E75P, Q86K, Q86R, S110V, I188L, G222D, F381V, T388A, S413K, and G430K with respect to SEQ ID NO. 46, and / or selected from double mutation T146C+M148C, P190C+A198C, D193C+N196C, G296L+V297G, and T388C+A399C with respect to SEQ ID NO. 46, and / or selected from a combination of one or more of the single and / or double mutations; and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0135] More preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46; and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0136] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0137] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0138] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0139] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, or (iv) amino acid residue substitutions T388C, and A399C with respect to SEQ ID NO. 46, or

[0140] (v) amino acid residue substitutions T146C, M148C, F381V, and T388A with respect to SEQ ID NO. 46, or

[0141] (vi) amino acid residue substitutions T146C, M148C, and T388A with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0142] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0143] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0144] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0145] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, or and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0146] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, T388A, S413K, and G430K with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0147] In one embodiment, step (bl) is facilitated by a glycosyltransferase (GT), as disclosed above. Preferably, the glycosyltransferase is a UDP-dependent glycosyltransferase. UDP-GIc is used in such reaction for attaching the glucose moiety to the indoxyl compound, hence forming indican. In step (bl), indican is thereby synthesized from indoxyl and UDP-GIc using such UDP-glycosyltransferase. The UDP-GIc is converted to UDP.

[0148] The UDP-GIc may be regenerated from UDP by an enzymatic recycling system using a sucrose synthase enzyme (EC 2.4.1.13).

[0149] In one embodiment, sucrose synthase (SuSy) in combination with sucrose facilitates regeneration of UDP-GIc from step (bl) of the method. In one embodiment, UDP is enzymatically converted to UDP-GIc by a polypeptide having sucrose synthase activity (EC 2.4.1.13). The amino acid sequence of said polypeptide having sucrose synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54. Preferably, the amino acid sequence of said polypeptide having sucrose synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54. In one embodiment, the sucrose synthase is selected from SEQ ID NO. 54, preferably SEQ ID NO 54.

[0150] In one embodiment, in vitro production of indican comprises

[0151] (a) enzymatically converting indole to indoxyl, as disclosed in section II, and (bl) enzymatically converting indoxyl to indican, as disclosed in section Ill.i.

[0152] In one embodiment, the present invention provides an in vitro method of producing indican, comprising (I) providing indole, an oxidizing enzyme and its relevant cofactor, as well as a glycosyltransferase enzyme and UDP-GIc, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indican.

[0153] In one preferred embodiment, the present invention provides an in vitro method of producing indican, comprising (I) providing indole, an oxidizing enzyme and its relevant cofactor, as well as a glycosyltransferase enzyme and UDP-GIc, wherein the indole is provided as disclosed in section I; (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indican.

[0154] In one embodiment, step (b2) is facilitated by exposure to oxygen. Upon oxygen exposure, the indoxyl will spontaneously dimerize and form indigo.

[0155] In one embodiment, in vitro production of indigo comprises

[0156] (a) enzymatically converting indole to indoxyl, as disclosed in section II, and (b2) converting indoxyl to indigo by exposure to oxygen, as disclosed in section Ill.i.

[0157] In one embodiment, the present invention provides an in vitro method of producing indigo, comprising (I) providing indole, an oxidizing enzyme and its relevant cofactor, (II) mixing said substrate, cofactor, and enzyme, and (III) letting the mixture react at a selected temperature and time period to produce indican.

[0158] In one preferred embodiment, the present invention provides an in vitro method of producing indigo, comprising (I) providing indole, an oxidizing enzyme and its relevant cofactor, wherein the indole is provided as disclosed in section I; (II) mixing said substrate, cofactor, and enzyme, and (III) letting the mixture react at a selected temperature and time period to produce indican. Ill.ii Synthesis from ribose-5-phosphate and anthranilate

[0159] In a further aspect, the present invention provides an in vitro method of producing indican and / or indigo comprising steps (i), (ii), (iii), (iv), (v), and (iv) as disclosed in section I, combined with step (a) as disclosed in section II, and further combined with step (bl) and / or (b2) as disclosed section Ill.i, wherein indole is an intermediate compound.

[0160] Specifically, in one preferred embodiment, the present invention provides an in vitro method of producing indican and / or indigo, comprising the steps

[0161] (i) providing ribose-5-phosphate (R5P),

[0162] (ii) enzymatically converting R5P to phosphoribosyl pyrophosphate (PRPP),

[0163] (iii) enzymatically converting PRPP to N-phosphoribosyl anthranilate (N-PRA),

[0164] (iv) enzymatically converting N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D- ribulose 5-phosphate (CdRP),

[0165] (v) enzymatically converting CdRP to indole-3-glycerol phosphate (IGP), and

[0166] (vi) enzymatically converting IGP to indole, and further comprising the steps

[0167] (a) enzymatically converting indole to indoxyl, and

[0168] (bl) enzymatically converting indoxyl to indican and / or (b2) converting indoxyl to indigo by exposure to oxygen.

[0169] Steps (i), (ii), (iii), (iv), (v), and (vi) are preferably facilitated by enzymes as disclosed in section I, steps (a) is preferably facilitated by an enzyme as disclosed in section II, and step (bl) is preferably facilitated by an enzyme as disclosed in section Ill.i. Any of the specifics listed in sections I, II, and Ill.i for each step equally applies here in this aspect of indican and / or indigo synthesis from ribose-5-phosphate and anthranilate

[0170] In one preferred embodiment, in the in vitro method of producing indole comprises all steps (i), (ii). (iii), (iv), (v), (vi), (a), and (bl) and further comprises co-factor recycling steps as disclosed previously herein.

[0171] In one preferred embodiment, in the in vitro method of producing indigo comprises all steps (i), (ii). (iii), (iv), (v), (vi), (a), and (b2) and further comprises co-factor recycling steps as disclosed previously herein.

[0172] In one preferred embodiment, the present invention concerns an in vitro method of producing indican, comprising the steps

[0173] - providing ribose-5-phosphate, anthranilate, ATP, and UDP-GIc, and providing NADPH if an oxidoreductase is used as the oxidizing enzyme

[0174] - performing enzymatic conversions: (ii) R5P to phosphoribosyl pyrophosphate (PRPP), facilitated by a phosphoribosylpyrophosphate synthase;

[0175] (iii) PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a N- phosphoribosyl anthranilate synthase;

[0176] (iv) N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP), facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase;

[0177] (v) CdRP to indole-3-glycerol phosphate (IGP), facilitated by an indole-3-glycerol phosphate synthase;

[0178] (vi) IGP to indole, facilitated by an indole-3-glycerol-phosphate lyase;

[0179] (a) indole to indoxyl, facilitated by an oxidizing enzyme; and

[0180] (bl) indoxyl to indican, facilitated by a glycosyltransferase; and optionally

[0181] - regeneration of ATP, by providing PolyPn and wherein the ATP-regeneration is facilitated by an ADP kinase and an AMP kinase, and / or

[0182] - regeneration of UDP-GIc, by providing sucrose, and wherein the UDP-GIc-regeneration is facilitated by a sucrose synthase and / or

[0183] - regeneration of NADPH, if an oxidoreductase is used as the oxidizing enzyme, by providing formate, and wherein the NADPH-regeneration is facilitated by a formate dehydrogenase;

[0184] - preferably, ribose-5-phosphate (R5P) is provided by conversion of ribose, glucose or starch to R5P, as disclosed herein.

[0185] In one preferred embodiment, the present invention concerns an in vitro method of producing indican, comprising the steps

[0186] - providing ribose-5-phosphate, anthranilate, ATP, NADPH, and UDP-GIc

[0187] - performing enzymatic conversions:

[0188] (ii) R5P to phosphoribosyl pyrophosphate (PRPP), facilitated by a polypeptide having phosphoribosylpyrophosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 12;

[0189] (iii) PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a polypeptide having N-phosphoribosyl anthranilate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 14;

[0190] (iv) N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP), facilitated by a polypeptide having N-(5'-phosphoribosyl)anthranilate isomerase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 20;

[0191] (v) CdRP to indole-3-glycerol phosphate (IGP), facilitated by a polypeptide having indole-3-glycerol phosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 26; (vi) IGP to indole, facilitated by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 28;

[0192] (a) indole to indoxyl, facilitated by a polypeptide having oxidizing activity and having at least 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO. 32; and

[0193] (bl) indoxyl to indican, facilitated by a polypeptide having glycosyltransferase activity and having at least 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO. 46 and wherein the amino acid sequence of said polypeptide having glycosyltransferase activity comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises either (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46, and optionally

[0194] - regeneration of ATP, by providing PolyPn and wherein the ATP-regeneration is facilitated by a polypeptide having ADP kinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 60, and a polypeptide having AMP kinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 60, and / or

[0195] - regeneration of NADPH, by providing formate, and wherein the NADPH-regeneration is facilitated by a polypeptide having formate dehydrogenase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56, and / or

[0196] - regenaration of UDP-GIc, by providing sucrose, and wherein the UDP-GIc-regeneration is facilitated by a polypeptide having sucrose synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54,

[0197] - preferably ribose-5-phosphate (R.5P) is provided by conversion of ribose to R.5P, wherein ribose to ribose-5-phosphate (R.5P), facilitated by a polypeptide having ribokinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 6.

[0198] In one embodiment, all enzyme steps for in vitro production of indican as disclosed herein are performed in a one-pot reaction.

[0199] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a one-pot reaction. In a further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (bl) as well as the regeneration systems as disclosed herein are performed in a one-pot reaction.

[0200] In such one pot reaction, intermediate products need not be purified or isolated before proceeding to the next enzymatic conversion step.

[0201] In one embodiment, sequential one-pot synthesis may be used, where the enzymes, substrates and / or co-factors are added to a reactor one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0202] In one embodiment, a sequential one-pot process is performed, wherein one or more of the enzymes, substrates and / or co-factors are added sequentially. One such sequential one-pot process may comprise multiple phases, wherein each phase has different process conditions - such as different temperature and / or pH conditions.

[0203] In one specific embodiment, a sequential one-pot process is performed for producing indican, wherein enzymes relevant for steps (iii), (iv), and (v) as disclosed herein for producing IGP are combined in a reactor in a first phase of the one-pot reaction at a first specified process condition, followed by later addition of enzymes relevant for steps (vi), (a), and (bl) as disclosed herein to the reactor for producing indican in a second phase of the one-pot reaction at a second specified process condition; wherein the first and second specified process conditions refer to two different temperatures; and wherein

[0204] • step (iii) comprises a N-phosphoribosyl anthranilate synthase (TrpD) having at least 90 % sequence identity to SEQ ID NO. 14,

[0205] • step (iv) comprises a N-(5'-phosphoribosyl)anthranilate isomerase (trpF) having at least 90 % sequence identity to SEQ ID NO. 22,

[0206] • step (v) comprises an indole-3-glycerol phosphate synthase (trpC) having at least 90 % sequence identity to SEQ ID NO. 24,

[0207] • step (vi) comprises an indole-3-glycerol-phosphate lyase (IGL) having at least 90 % sequence identity to SEQ ID NO. 30,

[0208] • step (a) comprises a flavin-containing monooxygenase (FMO) having at least 90 % sequence identity to SEQ ID NO. 32, and

[0209] • step (bl) comprises a glycosyltransferase having at least 75% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46. In one such specific embodiment, the first specified process condition is preferably a temperature between 50-60 °C, more preferably between 50-55 °C, and the second specified reaction condition is preferably a temperature between 25-35 °C, more preferably between 25-30 °C.

[0210] In one preferred embodiment, the present invention concerns an in vitro method of producing indigo, comprising the steps

[0211] - providing ribose, anthranilate, and ATP, and providing NADPH if an oxidoreductase is used as the oxidizing enzyme;

[0212] - performing enzymatic conversions:

[0213] (ii) R5P to phosphoribosyl pyrophosphate (PRPP), facilitated by a phosphoribosylpyrophosphate synthase,

[0214] (iii) PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a N- phosphoribosyl anthranilate synthase,

[0215] (iv) N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP), facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase,

[0216] (v) CdRP to indole-3-glycerol phosphate (IGP), facilitated by an indole-3-glycerol phosphate synthase,

[0217] (vi) IGP to indole, facilitated by an indole-3-glycerol-phosphate lyase, and

[0218] (a) indole to indoxyl, facilitated by an oxidizing enzyme; and

[0219] - allowing the indoxyl to be exposed to air for conversion of indoxyl to indigo; and optionally

[0220] - regeneration of ATP, by providing PolyPn and wherein the ATP-regeneration is facilitated by an ADP kinase and an AMP kinase, and / or

[0221] - regeneration of NADPH, if an oxidoreductase is used as the oxidizing enzyme, by providing formate, and wherein the NADPH-regeneration is facilitated by a formate dehydrogenase,

[0222] - preferably, ribose-5-phosphate (R5P) is provided by conversion of ribose, glucose or starch to R5P, as disclosed herein.

[0223] In one preferred embodiment, the present invention concerns an in vitro method of producing indigo, comprising the steps

[0224] - providing ribose, anthranilate, ATP, and NADPH;

[0225] - performing enzymatic conversions:

[0226] (ii) R5P to phosphoribosyl pyrophosphate (PRPP), facilitated by a polypeptide having phosphoribosylpyrophosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 12, (iii) PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a polypeptide having N-phosphoribosyl anthranilate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 14,

[0227] (iv) N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP), facilitated by a polypeptide having N-(5'-phosphoribosyl)anthranilate isomerase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 20,

[0228] (v) CdRP to indole-3-glycerol phosphate (IGP), facilitated by a polypeptide having indole-3-glycerol phosphate synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 26,

[0229] (vi) IGP to indole, facilitated by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 28, and

[0230] (a) indole to indoxyl, facilitated by a polypeptide having oxidizing activity and having at least 70, 75, 80, 85, 90 or 95% sequence identity to SEQ ID NO. 32; and

[0231] - allowing the indoxyl to be exposed to air for conversion of indoxyl to indigo; and optionally

[0232] - regeneration of ATP, by providing PolyPn and wherein the ATP-regeneration is facilitated by a polypeptide having ADP kinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 60, and a polypeptide having AMP kinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 60, and / or

[0233] - regeneration of NADPH, by providing formate, and wherein the NADPH-regeneration is facilitated by a polypeptide having formate dehydrogenase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56,

[0234] - preferably ribose-5-phosphate (R5P) is provided by conversion of ribose to R5P, wherein ribose to ribose-5-phosphate (R5P), facilitated by a polypeptide having ribokinase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 6.

[0235] In one embodiment, all enzyme steps for in vitro production of indigo as disclosed herein are performed in a one-pot reaction.

[0236] In one embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a one-pot reaction. In a further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) as well as the regeneration systems as disclosed herein are performed in a one-pot reaction. In such one pot reaction, intermediate products need not be purified or isolated before proceeding to the next enzymatic conversion step.

[0237] In one embodiment, sequential one-pot synthesis may be used, where the enzymes, substrates and / or co-factors are added to a reactor one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0238] In one embodiment, a sequential one-pot process is performed, wherein one or more of the enzymes, substrates and / or co-factors are added sequentially. One such sequential one-pot process may comprise multiple phases, wherein each phase has different process conditions - such as different temperature and / or pH conditions.

[0239] In one specific embodiment, a sequential one-pot process is performed for producing indigo, wherein enzymes relevant for steps (iii), (iv), and (v) as disclosed herein for producing IGP are combined in a reactor in a first phase of the one-pot reaction at a first specified process condition, followed by later addition of enzymes relevant for steps (vi), (a) as disclosed herein to the reactor for producing indoxyl in a second phase of the one- pot reaction at a second specified process condition; wherein the first and second specified process conditions refer to two different temperatures; and wherein the second specified process condition facilitates formation of indigo by exposure of the indoxyl to air; and wherein

[0240] • step (iii) comprises a N-phosphoribosyl anthranilate synthase (TrpD) having at least 90 % sequence identity to SEQ ID NO. 14,

[0241] • step (iv) comprises a N-(5'-phosphoribosyl)anthranilate isomerase (trpF) having at least 90 % sequence identity to SEQ ID NO. 22,

[0242] • step (v) comprises an indole-3-glycerol phosphate synthase (trpC) having at least 90 % sequence identity to SEQ ID NO. 24,

[0243] • step (vi) comprises an indole-3-glycerol-phosphate lyase (IGL) having at least 90 % sequence identity to SEQ ID NO. 30,

[0244] • step (a) comprises a flavin-containing monooxygenase (FMO) having at least 90 % sequence identity to SEQ ID NO. 32, and

[0245] In one such specific embodiment, the first specified process condition is preferably a temperature between 50-60 °C, more preferably between 50-55 °C, and the second specified reaction condition is preferably a temperature between 25-35 °C, more preferably between 25-30 °C.

[0246] III. iii Synthesis of indigo from indican

[0247] In one embodiment, indigo may be produced from indican by the steps (b3) enzymatically converting indican to indoxyl, and (b2) converting indoxyl to indigo by exposure to oxygen, wherein said indican is produced as disclosed herein in section IH.i or Ill.ii.

[0248] Beta-glucosidase (BGL) catalyzes the cleavage of glycoside bonds and is in step (b3) applied to remove glucose from indican. Such removal of the (protecting) sugar molecule will convert the compound back to its reactive form - i.e. indoxyl, which may in its reactive form spontaneously dimerize under aerobic conditions.

[0249] In one embodiment, step (b3) is facilitated by a beta-glucosidase (BLG). In one embodiment, the beta-glucosidase is selected from the group of enzymes classified as EC 3.2.1.21.

[0250] In step (b3), indoxyl is synthesized from indican using a beta-glucosidase. In one embodiment, indican is in step (b3) enzymatically converted to indoxyl by a polypeptide having beta-glucosidase activity (EC 3.2.1.21). The amino acid sequence of said polypeptide having beta-glucosidase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 70 or 72. Preferably, the amino acid sequence of said polypeptide having beta-glucosidase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 72. In one embodiment, the betaglucosidase is selected from SEQ ID NO. 70 and 72, preferably SEQ ID NO. 72.

[0251] In one embodiment, the present invention concerns an in vitro method of producing indigo, comprising the steps

[0252] - providing ribose, anthranilate, and ATP, and providing NADPH if an oxidoreductase is used as the oxidizing enzyme;

[0253] - performing enzymatic conversions:

[0254] (ii) R5P to phosphoribosyl pyrophosphate (PRPP), facilitated by a phosphoribosylpyrophosphate synthase,

[0255] (iii) PRPP to N-phosphoribosyl anthranilate (N-PRA), facilitated by a N- phosphoribosyl anthranilate synthase,

[0256] (iv) N-PRA to l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate (CdRP), facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase,

[0257] (v) CdRP to indole-3-glycerol phosphate (IGP), facilitated by an indole-3-glycerol phosphate synthase,

[0258] (vi) IGP to indole, facilitated by an indole-3-glycerol-phosphate lyase,

[0259] (a) indole to indoxyl, facilitated by an oxidizing enzyme,

[0260] (bl) indoxyl to indican, facilitated by a glycosyltransferase, and

[0261] (b3) enzymatically converting indican to indoxyl; and

[0262] - allowing the indoxyl to be exposed to air for conversion of indoxyl to indigo; and optionally

[0263] - regeneration of ATP, by providing PolyPn and wherein the ATP-regeneration is facilitated by an ADP kinase and an AMP kinase, and / or - regeneration of UDP-GIc, by providing sucrose, and wherein the UDP-GIc-regeneration is facilitated by a sucrose synthase and / or

[0264] - regeneration of NADPH, if an oxidoreductase is used as the oxidizing enzyme, by providing formate, and wherein the NADPH-regeneration is facilitated by a formate dehydrogenase,

[0265] - preferably, ribose-5-phosphate (R.5P) is provided by conversion of ribose, glucose or starch to R.5P, as disclosed herein.

[0266] IV. Synthesis of indican from tryptophan

[0267] In another aspect, the present invention provides an in vitro method for producing indican. Indican may be enzymatically produced from tryptophan, as illustrated in Figure 6.

[0268] In one embodiment, in vitro production of indican comprises

[0269] (t) enzymatically converting tryptophan to indole,

[0270] (a) enzymatically converting indole to indoxyl, and

[0271] (bl) enzymatically converting indoxyl to indican.

[0272] For the synthesis of indole, the substrate is tryptophan, and cofactors NADPH and UDP- GIc are further needed in steps (a) and (bl), respectively. The NADPH and UDP-GIc may be regenerated, as illustrated in Figure 6.

[0273] In one embodiment, step (t) is facilitated by a L-tryptophan indole lyase (TIL). In step (i), indole is synthesized from tryptophan using an L-tryptophan indole lyase. In one embodiment, tryptophan is in step (i) enzymatically converted to indole and pyruvate by a polypeptide having L-tryptophan indole lyase activity (EC 4.1.99.1). The amino acid sequence of said polypeptide having L-tryptophan indole lyase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 64, 66, or 68. Preferably, the amino acid sequence of said polypeptide having L-tryptophan indole lyase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 64. In one embodiment, the L-tryptophan indole lyase is selected from SEQ ID NO. 64, 66, and 68, preferably SEQ ID NO. 64.

[0274] In one embodiment, step (a) is facilitated by a flavin-containing monooxygenase (FMO). NADPH is needed as cofactor / redox partner. In step (a), indoxyl is synthesized from indole and NADPH using a flavin-containing monooxygenase. In one embodiment, indole and NADPH are in step (a) enzymatically converted to indoxyl and NADP+ by a polypeptide having flavin-containing monooxygenase activity (EC 1.14.13.8). The amino acid sequence of said polypeptide having flavin-containing monooxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 32, 34, or 36. Preferably, the amino acid sequence of said polypeptide having flavin- containing monooxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 32. In one embodiment, the flavin-containing monooxygenase is selected from SEQ ID NO. 32, 34, and 36, preferably SEQ ID NO. 32.

[0275] In another embodiment, step (a) is facilitated by a cytochrome P450 enzyme. NADPH is needed as cofactor. In step (a), indoxyl is synthesized from indole and NADPH using a cytochrome P450 enzyme (EC 1.14.14.1). In one embodiment, indole and NADPH are in step (a) enzymatically converted to indoxyl and NADP+ by a polypeptide having P450 enzyme activity (EC 1.14.14.1). The amino acid sequence of said polypeptide having P450 enzyme activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 38 or 40. Preferably, the amino acid sequence of said polypeptide having P450 enzyme activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 38. In one embodiment, the cytochrome P450 enzyme is selected from SEQ ID NO. 38 and 40, preferably SEQ ID NO. 38

[0276] The co-factor NADPH may be regenerated from NADP+ by enzymatic cofactor recycling system using an NADPH-regenerating enzyme, such as a dehydrogenase, preferably a formate dehydrogenase (FDH) (EC 1.17.1.10).

[0277] In one embodiment, formate dehydrogenase (FDH) in combination with formate facilitates regeneration of NADPH from step (a) of the method. In one embodiment, NADP+ is enzymatically converted to NADPH by a polypeptide having formate dehydrogenase activity (EC 1.17.1.10). The amino acid sequence of said polypeptide having formate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56. Preferably, the amino acid sequence of said polypeptide having formate dehydrogenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56. In one embodiment, the formate dehydrogenase is selected from SEQ ID NO. 56, preferably SEQ ID NO. 56.

[0278] In another embodiment, step (a) is facilitated by a peroxygenase enzyme. Hydrogenperoxide is needed as cofactor. In step (a), indoxyl is synthesized from indole and H2O2 using a peroxygenase (EC 1.11.2.1). In one embodiment, indole and H2O2 are in step (a) enzymatically converted to indoxyl and H2O by a polypeptide having peroxygenase activity (EC 1.11.2.1). The amino acid sequence of said polypeptide having peroxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 42 or 44. Preferably, the amino acid sequence of said polypeptide having peroxygenase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 42. In one embodiment, the peroxygenase enzyme is selected from SEQ ID NO. 42 and 44, preferably SEQ ID NO. 42. In one embodiment, step (bl) is facilitated by a glycosyltransferase (GT) (EC 2.4), such as a UDP-glycosyltransferase (EC 2.4.1), preferably a glycosyltransferase as disclosed in Bidart et al 2023 and WO 2023 / 161230.

[0279] In a preferred embodiment, indoxyl is in step (bl) enzymatically converted to indican by a polypeptide having glycosyltransferase activity (EC 2.4). In one embodiment, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 48, 50 or 52. In one embodiment, the glycosyltransferase is selected from SEQ ID NO. 48, 50 and 52.

[0280] Preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises one or more amino acid residue substitutions selected from single mutation E75P, Q86K, Q86R, S110V, I188L, G222D, F381V, T388A, S413K, and G430K with respect to SEQ ID NO. 46, and / or selected from double mutation T146C+M148C, P190C+A198C, D193C+N196C, G296L+V297G, and T388C+A399C with respect to SEQ ID NO. 46, and / or selected from a combination of one or more of the single and / or double mutations; and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0281] More preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46; and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0282] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0283] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0284] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0285] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, or

[0286] (iv) amino acid residue substitutions T388C, and A399C with respect to SEQ ID NO. 46, or

[0287] (v) amino acid residue substitutions T146C, M148C, F381V, and T388A with respect to SEQ ID NO. 46, or (vi) amino acid residue substitutions T146C, M148C, and T388A with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0288] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0289] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0290] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0291] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46, or and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0292] Most preferably, the amino acid sequence of said polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, T388A, S413K, and G430K with respect to SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity has increased thermal stability compared to SEQ ID NO. 46.

[0293] In one embodiment, step (bl) is facilitated by a glycosyltransferase (GT), as disclosed above. Preferably, the glycosyltransferase is a UDP-dependent glycosyltransferase. UDP-GIc is used in such reaction for attaching the glucose moiety to the indoxyl compound, hence forming indican. In step (bl), indican is thereby synthesized from indoxyl and UDP-GIc using such UDP-glycosyltransferase. The UDP-GIc is converted to UDP.

[0294] The UDP-GIc may be regenerated from UDP by an enzymatic recycling system using a sucrose synthase enzyme (EC 2.4.1.13).

[0295] In one embodiment, sucrose synthase (SuSy) in combination with sucrose facilitates regeneration of UDP-GIc from step (bl) of the method. In one embodiment, UDP is enzymatically converted to UDP-GIc by a polypeptide having sucrose synthase activity (EC 2.4.1.13). The amino acid sequence of said polypeptide having sucrose synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54. Preferably, the amino acid sequence of said polypeptide having sucrose synthase activity preferably has at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54. In one embodiment, the sucrose synthase is selected from SEQ ID NO. 54, preferably SEQ ID NO 54.

[0296] In one embodiment, the present invention provides an in vitro method of producing indican, comprising (I) providing tryptophan, NADPH, UDP-GIc, an oxidizing enzyme as well as a glycosyltransferase enzyme, (II) mixing said substrates, cofactor, and enzymes, and (III) letting the mixture react at a selected temperature and time period to produce indican.

[0297] In one preferred embodiment, the present invention concerns an in vitro method of producing indican, comprising the steps

[0298] - providing tryptophan, and UDP-GIc, and providing NADP if a oxidoreductase is used as the oxidizing enzyme

[0299] - performing enzymatic conversions:

[0300] (t) enzymatically converting tryptophan to indole, facilitated by an L-tryptophan indole lyase;

[0301] (a) indole to indoxyl, facilitated by an oxidizing enzyme; and

[0302] (bl) indoxyl to indican, facilitated by a glycosyltransferase; and optionally

[0303] - regeneration of UDP-GIc, by providing sucrose, and wherein the UDP-GIc-regeneration is facilitated by a sucrose synthase and / or

[0304] - regeneration of NADPH, if an oxidoreductase is used as the oxidizing enzyme, by providing formate, and wherein the NADPH-regeneration is facilitated by a dehydrogenase.

[0305] In one preferred embodiment, the present invention concerns an in vitro method of producing indican, comprising the steps

[0306] - providing tryptophan, NADPH, and UDP-GIc

[0307] - performing enzymatic conversions:

[0308] (t) enzymatically converting tryptophan to indole, facilitated by a polypeptide having L-tryptophan indole lyase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 64;

[0309] (a) indole to indoxyl, facilitated by a polypeptide having oxidizing activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 32; and

[0310] (bl) indoxyl to indican, facilitated by a polypeptide having glycosyltransferase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 52; and optionally - regeneration of NADPH, by providing formate, and wherein the NADPH-regeneration is facilitated by a polypeptide having formate dehydrogenase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 56, and / or

[0311] - regeneration of UDP-GIc, by providing sucrose, and wherein the UDP-GIc-regeneration is facilitated by a polypeptide having sucrose synthase activity and having at least 70, 75, 80, 85, 90, or 95 % sequence identity to SEQ ID NO. 54.

[0312] V. Compositions of the invention

[0313] In one embodiment, the present invention provides composition A, comprising

[0314] (i) a ribokinase,

[0315] (ii) a phosphoribosylpyrophosphate synthase,

[0316] (iii) a N-phosphoribosyl anthranilate synthase,

[0317] (iv) a N-(5'-phosphoribosyl)anthranilate isomerase,

[0318] (v) an indole-3-glycerol phosphate synthase, and

[0319] (vi) an indole-3-glycerol-phosphate lyase; and optionally an ADP kinase and an AMP kinase.

[0320] In one embodiment, composition A further comprises ribose, anthranilate, ATP, and optionally PolyPn. When combined with substrates ribose and anthranilate, cofactor ATP, and optionally substrate PolyPn, the composition A is suitable for preparing indole.

[0321] In one embodiment, the present invention provides composition B, comprising

[0322] (i) a ribokinase,

[0323] (ii) a phosphoribosylpyrophosphate synthase,

[0324] (iii) a N-phosphoribosyl anthranilate synthase,

[0325] (iv) a N-(5'-phosphoribosyl)anthranilate isomerase,

[0326] (v) an indole-3-glycerol phosphate synthase,

[0327] (vi) an indole-3-glycerol-phosphate lyase, and

[0328] (a) an oxidizing enzyme selected from an oxidoreductase and a peroxygenase, such as selected from a flavin-containing monooxygenase (FMO), cytochrome P450 enzyme, an unspecific peroxygenase; and optionally an ADP kinase and an AMP kinase, and / or a formate dehydrogenase if the oxidizing enzyme is an oxidoreductase.

[0329] In one embodiment, composition B further comprises ribose, anthranilate, ATP, and NADPH if the oxidizing enzyme is an oxidoreductase, and optionally PolyPn and / or formate. When combined with substrates ribose, anthranilate, and co-factors ATP and NADPH, and optionally substrates PolyPn and / or formate, the composition B is suitable for preparing indigo.

[0330] In one embodiment, the present invention provides composition C, comprising

[0331] (i) a ribokinase,

[0332] (ii) a phosphoribosylpyrophosphate synthase,

[0333] (iii) a N-phosphoribosyl anthranilate synthase,

[0334] (iv) a N-(5'-phosphoribosyl)anthranilate isomerase,

[0335] (v) an indole-3-glycerol phosphate synthase,

[0336] (vi) an indole-3-glycerol-phosphate lyase,

[0337] (a) an oxidizing enzyme selected from an oxidoreductase and a peroxygenase, such as selected from a flavin-containing monooxygenase (FMO), cytochrome P450 enzyme, an unspecific peroxygenase, and

[0338] (bl) a glycosyltransferase; and optionally an ADP kinase and an AMP kinase, and / or a sucrose synthase, and / or a formate dehydrogenase if the oxidizing enzyme is an oxidoreductase.

[0339] In one embodiment, composition C further comprises ribose, anthranilate, UDP-GIc, ATP, and NADPH if the oxidizing enzyme is an oxidoreductase, and optionally PolyPn, and / or sucrose and / or formate. When combined with substrates ribose, anthranilate, and UDP- GIc, and co-factor ATP, and cofactor NADPH if the oxidizing enzyme is an oxidoreductase, and optionally substrates PolyPn, formate, and / or sucrose, the composition C is suitable for preparing indican.

[0340] In one embodiment, the present invention provides composition D, comprising

[0341] (i) a L-tryptophan indole lyase,

[0342] (a) an oxidizing enzyme selected from a flavin-containing monooxygenase (FMO), an unspecific peroxygenase (UPO), and a cytochrome P450 enzyme (P450), and

[0343] (bl) a glycosyltransferase; and optionally a sucrose synthase and / or a formate dehydrogenase if the oxidizing enzyme is an oxidoreductase.

[0344] In one embodiment, composition D further comprises tryptophan, and UDP-GIc, and NADPH if the oxidizing enzyme is an oxidoreductase, and optionally sucrose and / or formate. When combined with substrates tryptophan and UDP-GIc, and co-factor NADPH if the oxidizing enzyme is an oxidoreductase, and optionally substrates formate and / or sucrose, the composition D is suitable for preparing indican. VI One-pot or multi-pot enzymatic cascade reaction

[0345] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a one-pot reaction. In a further preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) as well as the ATP regeneration systems, as disclosed herein, are performed in a one-pot reaction.

[0346] In another preferred embodiment, the enzymatic cascade of producing indole is performed in a two-pot system, where steps (i), (ii), (iii), (iv), (v), and (vi) as disclosed herein for producing indole are performed in a first pot reaction, while the ATP regeneration systems, as disclosed herein, are performed in a second pot reaction.

[0347] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a one-pot reaction.

[0348] In a further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (bl) as well as one or more of the ATP, NADPH, and UDP-glc regeneration systems as disclosed herein are performed in a one-pot reaction.

[0349] In another further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (bl) as well as two or more of the ATP, NADPH, and UDP-glc regeneration systems as disclosed herein are performed in a one-pot reaction.

[0350] In another further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (bl) as well as the ATP, NADPH, and UDP-glc regeneration systems as disclosed herein are performed in a one-pot reaction.

[0351] In another preferred embodiment, the enzymatic cascade of producing indican is performed in a multi-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a one-pot reaction, while the ATP, NADPH, and UDP-glc regeneration systems, as disclosed herein, are performed in separate one or more pot reaction(s).

[0352] In another preferred embodiment, the enzymatic cascade of producing indican is performed in a multi-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a first pot reaction, while one or more of the ATP, NADPH, and UDP-glc regeneration systems, as disclosed herein, are performed in a second pot reaction.

[0353] In another preferred embodiment, the enzymatic cascade of producing indican is performed in a two-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a first pot reaction, while at least the ATP regeneration system as discloed herein is performed in a second pot.

[0354] In another preferred embodiment, the enzymatic cascade of producing indican is performed in a multi-pot system, wherein steps (i), (ii), (iii), (iv), (v), (vi), (a) and (bl) as disclosed herein for producing indican are performed in a first pot, the ATP regeneration system as disclosed herein is performed in a second pot, the NADPH regeneration systems as disclosed herein is performed in the first pot, the second pot or a third pot, and the UDP-glc regeneration systems as disclosed herein is performed in the first pot, the second pot, the third pot, or a fourth pot.

[0355] In one preferred embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a one-pot reaction. In a further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) as well as the relevant cofactor regeneration systems as disclosed herein are performed in a one-pot reaction. In one such embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2), as well as the ATP regeneration, and the NADPH regeneration if an oxidoreductase is used in step (a), as disclosed herein, are all performed in a one-pot reaction.

[0356] In another preferred embodiment, the enzymatic cascade of producing indigo is performed in a multi-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a one-pot reaction, while the ATP and NADPH regeneration systems, as disclosed herein, are performed in a separate one or two pot reaction(s).

[0357] In another preferred embodiment, the enzymatic cascade of producing indigo is performed in a two-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a first pot reaction, while one or both of the ATP and NADPH regeneration systems, as disclosed herein, are performed in a second pot reaction.

[0358] In another preferred embodiment, the enzymatic cascade of producing indigo is performed in a two-pot system, where steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a first pot reaction, while at least the ATP regeneration system as disclosed herein is performed in a second pot.

[0359] In another preferred embodiment, the enzymatic cascade of producing indigo is performed in a multi-pot system, wherein steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) as disclosed herein for producing indigo are performed in a first pot, the ATP regeneration system as disclosed herein is performed in a second pot, the NADPH regeneration systems as disclosed herein is performed in the first pot, the second pot or a third pot.

[0360] In a preferred embodiment, steps (t), (a) and (bl) as disclosed herein for producing indican are performed in a one-pot reaction.

[0361] In a further embodiment, steps (t), (a), and (bl) as well as one or more of the NADPH, and UDP-glc regeneration systems as disclosed herein are performed in a one-pot reaction.

[0362] In one embodiment, two or more of the enzymatic conversion steps take place in a one- pot reaction. In one embodiment, three or more of the enzymatic conversion steps take place in a one-pot reaction. In one embodiment, four or more of the enzymatic conversion steps take place in a one-pot reaction. In one embodiment, five or more of the enzymatic conversion steps take place in a one-pot reaction. In one embodiment, six or more of the enzymatic conversion steps take place in a one-pot reaction. In one embodiment, seven or more of the enzymatic conversion steps take place in a one-pot reaction. This may be combined with one or more of the ATP, NADPH, and UDP-glc regeneration systems, as disclosed herein.

[0363] In one preferred embodiment, all enzyme steps for in vitro production of indican and / or indigo as disclosed herein are performed in a one-pot reaction. In such one pot reaction, intermediate products need not be purified or isolated before proceeding to the next enzymatic conversion step.

[0364] In one embodiment, sequential one-pot synthesis may be used, where the enzymes, substrates and / or co-factors are added to a reactor one or more at a time, in order to optimize reaction conditions for selected reaction steps.

[0365] One-pot biosynthesis improves the efficiency, as various reactions or transformations can take place sequentially or concurrently within the same reaction vessel, allowing for the formation of complex molecules or materials in a more efficient manner. This is much desired in order to avoid lengthy separation and purification processes of the intermediate compounds, which may save time and resources, while increasing yields.

[0366] Designing effective one-pot synthesis can be challenging due to the need to manage multiple reactions and intermediates within the same reaction environment while avoiding unwanted side reactions or complications. The present invention has overcome these challenges and provides a solution where multiple enzymatic reactions are successfully combined in the biosynthesis of indole, and optionally indican and / or indigo.

[0367] The enzymes disclosed herein for the in vitro method may be obtained from commercial manufacturers, or may be microbially produced (and optionally purified), and be directly applied in the in vitro method of the present invention. A person skilled in the art would know how to microbially produce such enzymes based on the nucleotide and amino acid sequences provided herein.

[0368] The products and intermediate compounds produced by the method of the present invention may be detected by HLPC-UV, LC-MS, NMR, or similar equipment as recognized by a person skilled in the art.

[0369] VII List of enzymes

[0370] Table 1 provides a list of enzymes suitable for use in the present invention.

[0371] *The DNA sequences in the sequence listing encoding these enzymes are synthetic constructs prepared from the amino acid sequences of the enzymes originating from the organisms as listed here in table 1.

[0372] VIII Numbered embodiments

[0373] Numbered embodiment 1. An in vitro method of producing indole, and optionally producing indican and / or indigo, comprising the steps

[0374] (i) providing ribose-5-phosphate (R5P),

[0375] (ii) enzymatically converting the R5P to phosphoribosyl pyrophosphate (PRPP),

[0376] (iii) enzymatically converting the PRPP to N-phosphoribosyl anthranilate (N-PRA),

[0377] (iv) enzymatically converting the N-PRA to l-(2-carboxyphenylamino)-l-deoxy- D-ribulose 5-phosphate (CdRP),

[0378] (v) enzymatically converting the CdRP to indole-3-glycerol phosphate (IGP), and

[0379] (vi) enzymatically converting the IGP to indole, and optionally comprising the steps

[0380] (a) enzymatically converting the indole to indoxyl, and

[0381] (bl) enzymatically converting the indoxyl to indican, and / or (b2) converting the indoxyl to indigo by exposure to oxygen.

[0382] Numbered embodiment 2. The in vitro method according to Numbered embodiment 1, wherein the ribose-5-phosphate (R5P) in step (i) is provided by

[0383] (I) enzymatically converting ribose to R5P, or

[0384] (II) enzymatically converting glucose or starch to glucose-6-phosphate (G6P), enzymatically converting G6P to 6-phosphogluconate (6PG), enzymatically converting 6PG to ribulose 5-phosphate (Ru5P), and enzymatically converting Ru5P to R5P. Numbered embodiment 3. The in vitro method according to Numbered embodiment

[0385] 1 or 2, wherein step (ii) is facilitated by a phosphoribosylpyrophosphate synthase (PRS), step (iii) is facilitated by a N-phosphoribosyl anthranilate synthase (trpD), step (iv) is facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase (trpF), step (v) is facilitated by an indole-3-glycerol phosphate synthase (trpC), and step (vi) is facilitated by an indole-3-glycerol-phosphate lyase (IGL).

[0386] Numbered embodiment 4. The in vitro method according to Numbered embodiment

[0387] 2 or 3, wherein for (I) the conversion of ribose to R5P is facilitated by a ribokinase (RK), and for (II) the conversion of glucose to G6P is facilitated by a hexokinase (HK); the conversion of starch to G6P is facilitated by an alphaamylase, a glucoamylase, and a hexokinase (HK), or by an alpha-glucan phosphorylase (aGP) and a phosphoglucomutase (PGM); the conversion of G6P to 6PG is facilitated by a glucose 6-phosphate dehydrogenase (G6PDH); the conversion of 6PG to Ru5P is facilitated by a 6-phosphogluconate dehydrogenase (6PGDH), and the conversion of Ru5P to R5P is facilitated by a ribose 5-phosphate isomerase (RPI).

[0388] Numbered embodiment 5. The in vitro method according to Numbered embodiment

[0389] 3 or 4, wherein

[0390] • the amino acid sequence of said phosphoribosylpyrophosphate synthase (PRS) has at least 90 % sequence identity to SEQ ID NO. 8, 10, or 12,

[0391] • the amino acid sequence of said N-phosphoribosyl anthranilate synthase (trpD) has at least 90 % sequence identity to SEQ ID NO. 14, 16, or 18,

[0392] • the amino acid sequence of said N-(5'-phosphoribosyl)anthranilate isomerase (trpF) has at least 90 % sequence identity to SEQ ID NO. 20 or 22,

[0393] • the amino acid sequence of said indole-3-glycerol phosphate synthase (trpC) has at least 90 % sequence identity to SEQ ID NO. 24 or 26, and

[0394] • the amino acid sequence of said indole-3-glycerol-phosphate lyase (IGL) has at least 90 % sequence identity to SEQ ID NO. 28 or 30.

[0395] Numbered embodiment 6. The in vitro method according to Numbered embodiment

[0396] 4 or 5, wherein

[0397] • the amino acid sequence of said ribokinase (RK) has at least 90 % sequence identity to SEQ ID NO. 2, 4 or 6,

[0398] • the amino acid sequence of said hexokinase (HK) has at least 90 % sequence identity to SEQ ID NO. 74 or 76,

[0399] • the amino acid sequence of said alpha-amylase has at least 90 % sequence identity to SEQ ID NO. 90, • the amino acid sequence of said glucoamylase has at least 90 % sequence identity to SEQ ID NO. 92,

[0400] • the amino acid sequence of said alpha-glucan phosphorylase (aGP) has at least 90 % sequence identity to SEQ ID NO. 94,

[0401] • the amino acid sequence of said phosphoglucomutase (PGM) has at least 90 % sequence identity to SEQ ID NO. 96,

[0402] • the amino acid sequence of said glucose 6-phosphate dehydrogenase (G6PDH) has at least 90 % sequence identity to SEQ ID NO. 78 or 80.

[0403] • the amino acid sequence of said 6-phosphogluconate dehydrogenase (6PGDH) has at least 90 % sequence identity to SEQ ID NO. 82 or 84, and

[0404] • the amino acid sequence of said ribose 5-phosphate isomerase (R.PI) has at least 90 % sequence identity to SEQ ID NO. 86 or 88.

[0405] Numbered embodiment 7. The in vitro method according to any one of Numbered embodiments 2-6, wherein steps (i) and (ii) are further facilitated by an ATP- regenerating enzyme, such as a phosphate kinase, preferably a Class 2 polyphosphate kinase (PPK2) (EC 2.7.4.1).

[0406] Numbered embodiment 8. The in vitro method according to any one of Numbered embodiments 1-7 comprising steps (i), (ii), (iii), (iv), (v), (iv), (a) and (b2), wherein indole is an intermediate compound, and wherein indigo is produced.

[0407] Numbered embodiment 9. The in vitro method according to any one of Numbered embodiments 1-8 comprising steps (i), (ii), (iii), (iv), (v), (iv), (a) and (bl), wherein indole is an intermediate compound, and wherein indican is produced.

[0408] Numbered embodiment 10. The in vitro method according to Numbered embodiment 8 or 9, wherein step (a) is facilitated by an oxidizing enzyme, such as an oxidizing enzyme selected from an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin- containing monooxygenase (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1), and an unspecific peroxygenase (EC 1.11.2.1).

[0409] Numbered embodiment 11. The in vitro method according to Numbered embodiment 10, wherein the oxidizing enzyme is an oxidoreductase and step (a) is further facilitated by a NADPH-regenerating enzyme, such as a dehydrogenase, preferably a formate dehydrogenase (FDH) (EC 1.17.1.10).

[0410] Numbered embodiment 12. The in vitro method according to any one of Numbered embodiments 9-11, wherein step (bl) is facilitated by a glycosyltransferase (GT) (EC 2.4.1), such as a UDP-glycosyltransferases, preferably a glycosyltransferase wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0411] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0412] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0413] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46.

[0414] Numbered embodiment 13. The in vitro method according to any one of Numbered embodiments 1-12 wherein two or more of the enzymatic conversions take place in a one-pot reaction, preferably wherein all enzymatic reactions take place in a one-pot reaction.

[0415] Numbered embodiment 14. An in vitro method of producing indican, comprising the steps:

[0416] (t) enzymatically converting tryptophan to indole,

[0417] (a) enzymatically converting indole to indoxyl, and

[0418] (bl) enzymatically converting indoxyl to indican.

[0419] Numbered embodiment 15. The in vitro method according to Numbered embodiment 14, wherein step (t) is facilitated by a L-tryptophan indole lyase (TIL), step (a) is facilitated by an oxidizing enzyme, such as an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin-containing monooxygenase (FMO) (EC 1.14.13.8), an unspecific peroxygenase (EC 1.11.2.1), or P450 (EC 1.14.14.1), and step bl is facilitated by a glycosyltransferase (GT) (EC 2.4.1), such as a UDP- glycosyltransferases, preferably a glycosyltransferase wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises

[0420] (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or

[0421] (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or

[0422] (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46.

[0423] Numbered embodiment 16. The in vitro method according to Numbered embodiment 14 or 15 wherein two or more of the enzymatic conversions take place in a one-pot reaction, preferably wherein all enzymatic reactions take place in a one-pot reaction. EXAMPLES

[0424] In the following, selected enzymes have been tested to demonstrate the combined in vitro biosynthesis of indole, and further biosynthesis of indican and indigo.

[0425] Example I: Expression and purification of enzymes

[0426] Gene synthesis and cloning were carried out by Biomatik (USA). DNA sequences of his- tagged proteins were codon optimized for expression in E. coli BL21(DE3) and cloned into pET28a with Ncol and Xhol restriction enzymes. The plasmids were transformed into E. coli BL21 Star(DE3) (Fisher Scientific) and transformants were stored as glycerol stocks at -70°C.

[0427] Overexpression of the gene of interest was carried out by the addition of 250 pM IPTG to the E. coli cultures that had reached OD600 = 0.8 - 1.0 in 2xYT medium at 37°C (200 rpm). Thereafter, the cultures were incubated for 20 hours at 20°C (200 rpm) to assist in soluble expression. The cultures were harvested and stored at -20°C until further use.

[0428] For purification, the cell pellet was resuspended in 50 mM Na-phosphate buffer (pH = 7.4) and lysis was performed by sonication. After the cell debris was removed by centrifugation, the cleared lysate was purified using immobilized metal affinity chromatography on an AKTA Pure with a Histrap FF column (Cytiva). Thereafter, the buffer was changed to a 25 mM HEPES (pH = 8) buffer and aliquots were flash-frozen in liquid nitrogen and stored at -70°C until further use.

[0429] Example II: One-pot anthranilate to indole

[0430] II. I Indole formation

[0431] In a one-pot reaction, substrates, enzymes and other reagents were mixed in concentrations as listed in table 2. The reaction started from anthranilate and PRPP, and all enzymes for conversion to indole were added. The reaction mixture was incubated at 25°C for 24 hours. The concentration of anthranilate and indole were measured by RP- HPLC-DAD at different timepoints, as indicated in Figures 7A and 7B. Here, Figure 7A shows conversion of 100 pM anthranilate and Figure 7B shows conversion of 1 mM anthranilate

[0432] II. ii Chromoatograms confirming stepwise IGP synthesis from anthranilate and PRPP

[0433] Anthranilate (1 mM) and PRPP (5 mM) were mixed and enzymes (1 mg / mL) were added sequentially with 1 hour incubation steps. Used enzymes, TkTrpD, EcTrpF, and PaTrpC, were purified according to the method described in Example I. Chromatograms obtained by RP-HPLC-DAD are shown in Figure 8 at 340 nm, where anthranilate moiety absorbs best, and at 260 nm where the indole moiety absorbs.

[0434] According to Figure 8A, after the addition of TrpD, conversion of anthranilic acid to NPRA was seen, but not 100% yet. TrpF was added after that and should convert NPRA to CdRP, but no new peak appears. This was expected since TrpF is an isomerase and the compounds are too similar to separate by the used HPLC method. Then, after the addition of TrpC, the peak of NPRA / CdRP disappeared at 340 nm which indicates that the anthranilate moiety was not present anymore. Moreover, a new peak appeared that absorbs at 260 nm (and not at 340 nm). Therefore, it is concluded that IGP has formed from the CdRP through the enzymatic activity of TrpC.

[0435] According to Figure 8B, after the addition of TrpD, conversion of anthranilic acid to NPRA was seen, but not 100% yet (1). TrpF was added after that and should convert NPRA to CdRP, but no new peak appears (2). This was expected since TrpF is an isomerase and the compounds are too similar to separate by the used HPLC method. The addition of sodium borohydride selectively reduces CdRP to reduced CdRP (rCdRP) resulting in a new peak (3). Then, after the addition of TrpC, the peak of NPRA / CdRP disappeared at 340 nm which indicates that the anthranilate moiety was not present anymore. Moreover, a new peak appeared that absorbs at 260 nm (and not at 340 nm) (4). Therefore, it is concluded that IGP has formed from the CdRP through the enzymatic activity of TrpC. IGP is chemically converted to Indole-3-aldehyde (I3A) by sodium periodate resulting in a new peak matching retention time with commercial I3A (5). The addition of IGL results in indole as is confirmed by indole as a reference compound (6).

[0436] Example III: One-pot anthranilate to indigo

[0437] In a one-pot reaction, substrates, enzymes, and other reagents were mixed in concentrations as listed in Table 3. The reaction started from anthranilate and PRPP, and all enzymes for conversion to indoxyl were added, with further spontaneous dimerization of the indoxyl to indigo by air exposure. The reaction mixture was incubated at 25°C for 24 hours and its contents were measured directly from the reaction mixture by sequential injection on RP-HPLC. The concentration of anthranilate and indole were measured at different timepoints, as indicated in Figure 9. Accumulation of indole was observed within the first 200 minutes, indicating that NIFMO is the apparent bottleneck step in this setup. Blue color formation and no residual indole further indicated that indigo was formed from anthranilate.

[0438] Example IV: One-pot ribose to IGP

[0439] In a one-pot reaction, substrates, enzymes, and other reagents were mixed in concentrations as listed in Tables 4. Different kinases were tested without the ATP- recycling. Also thermophilic enzymes were exploited at a higher incubation temperature. The reaction started from ribose and anthranilate, and all enzymes for conversion to IGP were added. The amounts of IGP produced were measured after 1, 2, 4 and 22 hours, as indicated in Figure 10.

[0440] In Figure 10 it is seen that HoRK and HoPRS work well. MtPRS and EcPRS unfortunately did not work well, as they were both not pure at all and that shows in figure 10, as reactions involving these enzymes has lower IGP measurements. hRK and LmRK did work.

[0441] Example V: Complete biosynthesis of indican

[0442] In a one-pot reaction, substrates, enzymes, and other reagents were mixed in concentrations as listed in Table 5. The reaction started from ribose and anthranilate, and all enzymes for conversion to indican were added. The reaction mixture was incubated at 25°C and a sample was taken for analysis by RP-HPLC after 20 hours. The results are shown in Figure 11, demonstrating succesful formation of indican. Example VI: Sequential one-pot

[0443] TkTrpD used in the previous examples is from a thermophilic organism, while EcTrpF and PaTrpC are from mesophilic organisms. Performing enzymatic reactions at elevated temperatures is desirable; therefore other more thermostable TrpF and TrpC enzymes were tested in combination with TkTrpD.

[0444] The melting temperatures (Tm) of the enzymes were determined by differential scanning fluorometry and given in Table 6.

[0445] Table 6. Enzymes used in this study. Gibbs free energy (AAG) values were determined with the eQuilibrator web app (eQuilibrator.weizmann.ac.il). Melting temperatures (Tm) were determined by differential scanning fluorometry (DSF).

[0446] Enzyme Enzyme AAG activity (kJ / mol)^

[0447] TkTrpD (SEQ ID NO. 14) TrpD -65.8±7.1 l.OxlO1173.8±0.5

[0448] TmTrpF (SEQ ID NO. 22) TrpF -9.8±2.6 50 Not detected^

[0449] SsTrpC (SEQ ID NO. 24) TrpC -72.0±11.8 5.0xl01287.3±0.7

[0450] ZmBXl (SEQ ID NO. 30) IGL 19.2±5.7 4.3xl0442.8±0.8

[0451] NIFMO (SEQ ID NO. 32) FMO -436±14.1 2.7xl07550.9 ±0.4

[0452] MvFDH (SEQ ID NO. 56) FDH -13.6±6.4 2.4xl0265.7±0.3

[0453] [a] Theoretical AAG values at pH 8 and ionic strength of 0.03 M [b] No increase in fluorescence observed by DSF up to 95°C.

[0454] The stability of TkTrpD and SsTrpC was high with apparent Tmof 73.8°C and 87.3°C, respectively. Moreover, no unfolding was observed for TmTrpF which indicates that TmTrpF is still active after prolonged exposure to heat. NIFMO and MvFDH are of mesophilic origin but are relatively stable with observed melting temperatures of 50.9 and 65.7°C. The relatively low Tmfor ZmBXl was expected and can be seen as a bottleneck in this cascade with regards to optimal temperature.

[0455] Therefore, a sequential one-pot process was assessed, also referred herein to as a two- phase one-pot process. First, the formation of IGP from anthranilate and PR.PP was carried out by the three enzymes from thermophilic origin (TkTrpD, TmTrpF, and SsTrpC), and thereafter the final two steps and cofactor recycling were carried out by the enzymes from mesophilic origin (ZmIGL, NIFMO, and MvFDH) in the same pot, allowing to adjustment of temperature for optimal performance of each phase.

[0456] VI. i Optimization of phase I - temperature

[0457] Substrates, enzymes, and other reagents were mixed in a one-pot reaction in concentrations as listed in Table 7, total volume 50pl, triplicates. The reaction started from anthranilate and PRPP, and enzymes for conversion to IGP were added. IGP was quantified by chemically converting it to indole-3-aldehyde (I3A) using 100 mM sodium periodate in 100 mM acetic acid, and I3A was measured by RP-HPLC-DAD. The reaction mixtures were incubated at a temperature range between 50 and 70°C. Samples were taken at selected time points and the relative IGP formation is plotted over time (Figure 12).

[0458] As seen in Figure 12, after 120 minutes of reaction time, the concentration of IGP decreases indicating thermal instability of the compound at elevated temperatures. Of course, this also occurs before the 120-minute mark but the flux in IGP formation is high enough to counteract the degradation. The optimal temperature for phase I was determined to be approximately 55°C.

[0459] VI. ii Optimization of phase I - enzyme ratio

[0460] Substrates, enzymes, and other reagents were mixed in a one-pot reaction in concentrations as listed in Tables 8, 9, and 10, total volume 500 pl. The reaction started from anthranilate and PRPP, and enzymes for conversion to IGP were added. The reaction mixtures were incubated at 55°C as determined optimal for Phase I (see above). IGP was quantified after chemically converting to indole-3-aldehyde (I3A) using 100 mM sodium periodate. The concentration of I3A was measured by RP-HPLC-DAD at different time points. The relative IGP formation was plotted after 4 hours (Figure 13).

[0461] As seen in Figure 13, TrpC appears to be the bottleneck enzyme in phase I of the cascade.

[0462] VI. Hi Further process optimization of phase I - spiking Initially, optimal conditions and enzyme ratios were determined for 10 mM anthranilate, at 53°C and a ratio of 4: 1: 12 (TkTrpD : TmTrpF : SsTrpC) resulting in the plot shown in Figure 14A. Here, nearly 6 mM of IGP is formed in 90 minutes. A decrease in IGP formation is observed and it was suspected this is due to the thermal instability of PRPP. In Figure 14B, the reaction mixture was supplemented with fresh PRPP at 60 minutes, nearly 10 mM IGP formed in 150 minutes, as opposed to the control sample that did not reach full conversion. To improve the titer of IGP, attempts were made to spike the reaction mixture with fresh anthranilate as well as PRPP. Plotted in Figures 14C and 14D are reaction mixtures that were spiked every 30 and 60 minutes, respectively. Surprisingly, this did not improve the final titer of IGP which is limited to 10 mM. It was apparent that spiking with anthranilate does not improve product titers, and therefore it was decided to start with higher substrate concentrations as that was found to not be detrimental to activity. Figures 14E and 14F show the conversion of 50 mM anthranilate to IGP spiked every 60 and 30 minutes, respectively. Again, it was observed that the titer can be improved by the frequent addition of PRPP reaching nearly 30 mM IGP (8.61 g / L). However, the rate of IGP formation still slows down within an hour suspected due to poor operational stability of one or multiple enzymes. Since the consumption of anthranilate is not drastically hampered and TmTrpF remained active after prolonged heat exposure, the stability of SsTrpC was questioned. This is in line with the accumulation of the compound eluting at 2.67 min on HPLC chromatograms, CdRP, the substrate for SsTrpC. Figure 14G compares spiking with both PRPP and TrpC every hour with spiking with solely PRPP indicating a clear increase in titer when adding fresh TrpC. In Figure 14H, the results of the same experiment are shown, demonstrating the repeatability of the setup. Even though the melting temperature of SsTrpC is exceptionally high at 87.3°C, the operational stability is lacking.

[0463] To conclude, spiking with anthranilate was unsuccessful, but spiking with PRPP and SsTrpC when starting from higher substrate concentration improved IGP titers.

[0464] VI. iv Optimization of phase II - temperature

[0465] In one-pot reactions, substrates, enzymes, and other reagents were mixed in concentrations as listed in Table 11, total volume 50 pL in triplicates. The reaction started from in situ generated IGP, and all enzymes for conversion to indigo were added. The reaction mixtures were incubated at a temperature range between 20 and 40°C. Indigo was quantified spectrophotometrically at different time points (Figure 15).

[0466] As seen in Figure 15, at 35 and 40°C, a decrease in indigo formation is observed, which likely is attributed to thermal instability of ZmBXl or the operating stability of ZmBXl, NIFMO, or MvFDH. For subsequent experiments, a reaction temperature of 30°C will be used.

[0467] VI. v Optimization of phase II - enzyme ratio

[0468] In one-pot reactions, substrates, enzymes, and other reagents were mixed in concentrations as listed in Tables 12, 13, and 14. The reaction started from in situ generated IGP, and all enzymes for conversion to indigo were added. The reaction mixtures were incubated at 30°C as determined optimal for Phase II. Indigo was quantified spectrophotometrically at 620 nm. Here, the relative indigo formation is plotted after 4 hours (Figure 16).

[0469] As seen in Figure 16, the results indicate that NIFMO is the bottleneck enzyme in Phase II. while MvFDH is the most efficient in this cascade .

[0470] VI. vi Indigo synthesis - Phase I and Phase II combined in a one-pot reaction

[0471] IGP was synthesized in situ (phase I) using the optimal process conditions as determined herein. A reaction mixture containing anthranilate and PRPP was incubated at 55°C and pH 8 with enzymes TkTrpD, TmTrpF, and SsTrpC, with spiking of PRPP and SsTrpC every 30 and 60 minutes, respectively. After Phase I, it was confirmed that IGP had formed, and Phase II was initiated by addition of the remaining enzymes: ZmBXl, NIFMO, and MvFDH, as well as the cofactors, NADP+ and PLP, and formic acid to regenerate NADPH. The reaction mixture was now incubated at 30°C as determined optimal for Phase II.

[0472] Example VII: One-pot tryptophan to indican

[0473] In a one-pot reaction, substrates, enzymes, and other reagents were mixed in concentrations as listed in Table 15. The reaction started from tryptophan, and all enzymes for conversion to indican were added. The reaction mixtures were incubated at 25°C and samples for HPLC analysis were taken at timepoints; 5, 10, 20, 30, 60, 90, and 120 minutes. Indican concentration was measured and plotted as indicated in Figure 17, demonstrating successful formation of indican from tryptophan. References

[0474] Bidart G, Teze D, Jansen C, et al. Chemoenzymatic indican for sustainable light-driven denim dyeing. Research Square; 2023. DOI: 10.21203 / rs.3.rs-2416810 / vl.

[0475] PCT / EP2023 / 054318: Thermostable glycosyltransferase variants.

Claims

CLAIMS1. An in vitro method of producing indole, and optionally producing indican and / or indigo, comprising the steps(i) providing ribose-5-phosphate (R5P),(ii) enzymatically converting the R5P to phosphoribosyl pyrophosphate (PRPP) with a polypeptide having phosphoribosylpyrophosphate synthase activity (EC 2.7.6.1),(iii) providing anthranilate and enzymatically converting the PRPP to N- phosphoribosyl anthranilate (N-PRA) with a polypeptide having N-phosphoribosyl anthranilate synthase activity (trpD; EC 2.4.2.18),(iv) enzymatically converting the N-PRA to l-(2-carboxyphenylamino)-l-deoxy- D-ribulose 5-phosphate (CdRP) with a polypeptide having N-(5'- phosphoribosyl)anthranilate isomerase activity (EC 5.3.1.24),(v) enzymatically converting the CdRP to indole-3-glycerol phosphate (IGP) with a polypeptide having indole-3-glycerol phosphate synthase activity (EC 4.1.1.48), and(vi) enzymatically converting the IGP to indole with a polypeptide having indole- 3-glycerol-phosphate lyase activity (EC 4.1.2.8), wherein the in vitro method is a one-pot reaction for steps (ii)-(vi), wherein intermediate products are not purified or isolated before proceeding to the next enzymatic conversion step.

2. The in vitro method according to claim 1, further comprising the steps(a) enzymatically converting the indole to indoxyl with an oxidizing enzyme wherein the activity of said enzyme is selected from among oxidoreductase activity (EC 1), monooxygenase activity (EC 1.14), flavin-containing monooxygenase (FMO) activity (EC 1.14.13.8), P450 activity (EC 1.14.14.1), and unspecific peroxygenase activity (EC 1.11.2.1), and(bl) providing a glycosyl doner and enzymatically converting the indoxyl to indican with a polypeptide having glycosyltransferase (GT) activity (EC 2.4.1), and / or (b2) converting the indoxyl to indigo by exposure to oxygen. wherein indole is an intermediate compound, and wherein indican and / or indigo is produced.

3. The in vitro method according to claim 1 or 2, wherein the ribose-5-phosphate (R5P) in step (i) is provided by(I) enzymatically converting ribose to R5P with a polypeptide having ribokinase activity (EC 2.7.1.15), or(II) enzymatically converting glucose to glucose-6-phosphate (G6P) with a polypeptide having hexokinase activity (EC 2.7.1.1), enzymatically converting G6P to 6-phosphogluconate (6PG) with a polypeptide having glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49), enzymatically converting 6PG to ribulose 5-phosphate (Ru5P) with a polypeptide having 6-phosphogluconate dehydrogenase activity (EC 1.1.1.44), and enzymatically converting Ru5P to R5P with a polypeptide having ribose 5-phosphate isomerase activity (EC 5.3.1.6).

4. The in vitro method according to any one of claims 1-3, wherein• the amino acid sequence of said polypeptide having phosphoribosylpyrophosphate synthase activity has at least 90 % sequence identity to SEQ ID NO. 8, 10, or 12,• the amino acid sequence of said polypeptide having N-phosphoribosyl anthranilate synthase activity has at least 90 % sequence identity to SEQ ID NO. 14, 16, or 18,• the amino acid sequence of said polypeptide having N-(5'- phosphoribosyl)anthranilate isomerase activity has at least 90 % sequence identity to SEQ ID NO. 20 or 22,• the amino acid sequence of said polypeptide having indole-3-glycerol phosphate synthase activity has at least 90 % sequence identity to SEQ ID NO. 24 or 26, and• the amino acid sequence of said polypeptide having indole-3-glycerol- phosphate lyase activity has at least 90 % sequence identity to SEQ ID NO. 28 or 30.

5. The in vitro method according to claim 3 or 4, wherein• the amino acid sequence of said polypeptide having ribokinase activity has at least 90 % sequence identity to SEQ ID NO. 2, 4 or 6,• the amino acid sequence of said polypeptide having glucose 6-phosphate dehydrogenase activity has at least 90 % sequence identity to SEQ ID NO. 78 or 80.• the amino acid sequence of said polypeptide having 6-phosphogluconate dehydrogenase activity has at least 90 % sequence identity to SEQ ID NO. 82 or 84, and• the amino acid sequence of said polypeptide having ribose 5-phosphate isomerase activity has at least 90 % sequence identity to SEQ ID NO. 86 or 88.

6. The in vitro method according to any one of claims 1-5, wherein steps (i) and (ii) are further facilitated by an ATP-regenerating enzyme, such as a polypeptide having phosphate kinase activity, preferably a Class 2 polyphosphate kinase (PPK2) (EC 2.7.4.1).

7. The in vitro method according to any one of claims 2-6, wherein step (a) is facilitated by a polypeptide having oxidoreductase activity and is further facilitated by a NADPH- regenerating enzyme, such as a polypeptide having dehydrogenase activity, preferably a formate dehydrogenase (FDH) (EC 1.17.1.10).

8. The in vitro method according to any one of claims 2-7, wherein step (bl) is facilitated by a UDP-glycosyltransferase, and the glycosyl doner is UDP-glucose.

9. The in vitro method according to claim 8, wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises(i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or(ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or(iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46.

10. The in vitro method according to any one of claims 1-9, wherein all enzymes, substrates and co-factors are combined in the one-pot reaction at the same time.

11. The in vitro method according to any one of claims 1-9, wherein the in vitro method is a sequential one-pot reaction, wherein one or more enzymes, substrates and / or cofactors are added sequentially.

12. The in vitro method according to claim 11, comprising steps (i), (ii), (iii), (iv), (v),(iv), (a), and (b2), wherein indole is an intermediate compound, wherein indigo is produced, and wherein the sequential one-pot reaction comprises a first phase and a second phase, wherein steps (iii), (iv) and (v) are performed in the first phase at a temperature between 50-55 °C, and steps (vi), (a), and (b2) are performed in the second phase at a temperature between 25-30 °C, wherein formation of indigo is facilitated by exposure of the indoxyl to air in the second phase; and wherein• step (iii) is facilitated by a polypeptide having N-phosphoribosyl anthranilate synthase activity and at least 90 % sequence identity to SEQ ID NO. 14,• step (iv) is facilitated by a polypeptide having N-(5'- phosphoribosyl)anthranilate isomerase activity and at least 90 % sequence identity to SEQ ID NO. 22,• step (v) is facilitated by a polypeptide having indole-3-glycerol phosphate synthase activity and at least 90 % sequence identity to SEQ ID NO. 24,• step (vi) is facilitated by a polypeptide having indole-3-glycerol-phosphate lyase activity and at least 90 % sequence identity to SEQ ID NO. 30, andstep (a) is facilitated by a polypeptide having flavin-containing monooxygenase activity and at least 90 % sequence identity to SEQ ID NO. 32.

13. The in vitro method according to claim 11, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a), and (bl), wherein indole is an intermediate compound, wherein indican is produced, and wherein the sequential one-pot reaction comprises a first phase and a second phase, wherein steps (iii), (iv) and (v) are performed in the first phase at a temperature between 50-55 °C, and steps (vi), (a), and (bl) are performed in the second phase at a temperature between 25-30 °C, wherein• step (iii) is facilitated by a N-phosphoribosyl anthranilate synthase (TrpD) having at least 90 % sequence identity to SEQ ID NO. 14,• step (iv) is facilitated by a N-(5'-phosphoribosyl)anthranilate isomerase (trpF) having at least 90 % sequence identity to SEQ ID NO. 22,• step (v) is facilitated by an indole-3-glycerol phosphate synthase (trpC) having at least 90 % sequence identity to SEQ ID NO. 24,• step (vi) is facilitated by an indole-3-glycerol-phosphate lyase (IGL) having at least 90 % sequence identity to SEQ ID NO. 30, and• step (a) is facilitated by a flavin-containing monooxygenase (FMO) having at least 90 % sequence identity to SEQ ID NO. 32, and• step (bl) s facilitated by a glycosyltransferase having at least 75% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or (ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or (iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46.

14. An in vitro method of producing indican, comprising the steps:(t) providing tryptophan and enzymatically converting tryptophan to indole with a polypeptide having L-tryptophan indole lyase activity (EC 4.1.99.1),(a) enzymatically converting indole to indoxyl with an oxidizing enzyme wherein the activity of said enzyme is selected from among oxidoreductase activity (EC 1), monooxygenase activity (EC 1.14), flavin-containing monooxygenase (FMO) activity (EC 1.14.13.8), P450 activity (EC 1.14.14.1), and unspecific peroxygenase activity (EC 1.11.2.1), and(bl) providing a glycosyl doner and enzymatically converting indoxyl to indican with a polypeptide having glycosyltransferase (GT) activity (EC 2.4.1), wherein the in vitromethod is a one-pot reaction, wherein intermediate products are not purified or isolated before proceeding to the next enzymatic conversion step.

15. The in vitro method according to claim 14, wherein step (bl) is facilitated by a UDP- glycosyltransferases, and the glycosyl doner is UDP-glucose.

16. The in vitro method according to claim 15, wherein the amino acid sequence of said glycosyltransferase has at least 75% sequence identity with SEQ ID NO. 46, and wherein said amino acid sequence of said glycosyltransferase comprises amino acid residue substitutions E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K with respect to SEQ ID NO. 46, and further comprises (i) amino acid residue substitutions F381V, T388C, and A399C with respect to SEQ ID NO. 46, or(ii) amino acid residue substitutions F381V, and T388A with respect to SEQ ID NO. 46, or(iii) amino acid residue substitution T388A with respect to SEQ ID NO. 46.

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