Biocatalytic pathways of indole and optional indigo glycosides and / or indigo

Abstract

This invention relates to the in vitro enzymatic biosynthesis of indole, and optionally the further production conversion of indole to indigo glycosides and / or indigo; preferably provided in the form of a one-pot solution.

Description

Technical Field

[0001] This invention relates to the in vitro enzymatic biosynthesis of indole, and optionally the further conversion of indole into indigo glycosides and / or indigo; preferably provided as a simple one-pot reaction. This invention offers a more environmentally friendly alternative to current industrial processes for colored fabrics and other products. Background Technology

[0002] As is well known, indigo is a dye primarily used in denim. Its inherent properties give denim its distinctive faded appearance. The chemical synthesis of indigo was a major breakthrough in the late 19th century, leading to an exponential increase in the production of this dye, which had previously been extracted from plants. Although chemical synthesis has been optimized, petroleum-based synthetic routes have remained similar for decades.

[0003] Blue denim is traditionally dyed with chemically synthesized indigo under harsh and environmentally challenging conditions. As the final step in the synthesis, indigo is spontaneously formed from indophenol through air oxidation, but for dyeing purposes, it also needs to be dissolved with a strong reducing agent (such as Na₂S₂O₄), which is equally environmentally challenging. Figure 1 ).

[0004] In the context of sustainable development, several attempts have been made to develop scalable, bio-based methods for indigo production. However, microbial methods relying on whole-cell indigo production are challenged by the poor solubility of indigo and economic viability.

[0005] Glycosyltransferase (GT) is considered part of green biotechnology and could replace current industrial processes in blue denim production. Figure 1 Specifically, the hydroxyl groups of chemically synthesized indophenol can be glycosylated by GT, thereby protecting the reactive functional groups and producing stable, soluble (colorless) indoside molecules. The indoside can then be hydrolyzed back to indophenol by β-glucosidase (BGL), which then spontaneously oxidizes to form blue indigo directly on the fabric. Figure 1 This is a "green" alternative to traditional industrial processes, providing an alternative solution for the final step in the indigo dyeing process.

[0006] This invention provides a completely bio-based solution that eliminates the need for chemical synthesis of substrates and avoids the use of any irritating chemicals. Summary of the Invention

[0007] This invention provides a novel bio-based enzymatic method for the synthesis of indole, optionally followed by further enzymatic steps for the synthesis of indoside and / or indigo. Through the sequential action of several enzymes, basic building blocks are converted into indole, and optionally into indoside and / or indigo. These basic building blocks are readily available and / or can be produced from renewable resources.

[0008] In one aspect, the present invention provides an in vitro method for producing indole and optionally indigo glycosides and / or indigo, comprising the following steps: (i) Provides ribose-5-phosphate (R5P); (ii) The R5P is enzymatically converted into phosphoribosyl pyrophosphate (PRPP), preferably by phosphoribosyl pyrophosphate synthase (PRS); (iii) PRPP is enzymatically converted to N-phospribosyl anthranilic acid (N-PRA) under the promotion of N-phospribosyl anthranilic acid synthase (trpD). (iv) Enzymatically converting N-PRA to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP), preferably by promoting it via N-(5'-phospribosyl)-o-aminobenzoic acid isomerase (trpF); (v) Enzymatically converting CdRP to indole-3-glycerophosphate (IGP), preferably promoted by indole-3-glycerophosphate synthase (trpC); and (vi) IGP is enzymatically converted to indole, preferably by indole-3-glycerol-phosphate lyase (IGL); And optionally include the following steps (a) Enzymatically converting indole to indolephenol, and (b1) Enzymatically converting indophenol to indole, and / or (b2) converting indophenol to indole by exposure to oxygen.

[0009] In one implementation, ribose-5-phosphate (R5P) in step (i) is provided via the following steps: (I) Converting riboproteins to R5P via enzymatic reaction, or (II) Enzymatically convert glucose or starch into glucose-6-phosphate (G6P), enzymatically convert G6P into 6-phosphogluconic acid (6PG), enzymatically convert 6PG into ribulose-5-phosphate (Ru5P), and enzymatically convert Ru5P into R5P.

[0010] In one such embodiment (I), the conversion of ribose to R5P is promoted by ribokinase (RK). In one such embodiment (II), hexokinase (HK) promotes the conversion of glucose to G6P; α-amylase, glucosylamylase, and hexokinase (HK) or α-glucan phosphorylase (aGP) and phosphoglucose mutase (PGM) promote the conversion of starch to G6P; glucose-6-phosphate dehydrogenase (G6PDH) promotes the conversion of G6P to 6PG; 6-phosphoglucate dehydrogenase (6PGDH) promotes the conversion of 6PG to Ru5P; and ribose-5-phosphate isomerase (RPI) promotes the conversion of Ru5P to R5P.

[0011] In a 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.

[0012] In a preferred embodiment, the in vitro method of the present invention is a method for producing indole, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a), and (b1), wherein indole is an intermediate compound.

[0013] Preferably, step (a) is promoted by an oxidase, such as an oxidase selected from oxidoreductase (EC 1), monooxygenase (EC 1.14), flavin-containing monooxygenase (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1) and nonspecific peroxidase (EC 1.11.2.1).

[0014] In another embodiment, the in vitro method also includes a cofactor regeneration system, such as ATP regenerating enzyme, NADPH regenerating enzyme, or NAD(P) regenerating enzyme. + - Regenerase and / or UDP-Glc regenerase.

[0015] Preferably, step (b1) is promoted by a glycosyltransferase (GT) (EC 2.4.1), such as UDP-glycosyltransferase, 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 substitutions of amino acid residues E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues of SEQ ID NO.46, and further comprises: (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, or Furthermore, the polypeptide with glycosyltransferase activity described therein exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0016] In one aspect, the present invention relates to a one-pot process for producing indole and optionally indigo glycosides and / or indigo.

[0017] In a preferred embodiment, steps (ii), (iii), (iv), (v) and (vi) disclosed herein for the production of indole are carried out in a one-pot reaction; optionally further comprising a regeneration system in the one-pot reaction disclosed herein.

[0018] In a preferred embodiment, the steps (i), (ii), (iii), (iv), (v) and (vi) disclosed herein for the production of indole are carried out in a one-pot reaction; optionally further comprising a regeneration system in the one-pot reaction disclosed herein.

[0019] In one implementation, all enzymatic reactions occur in a one-pot reaction.

[0020] In one embodiment, a continuous one-pot synthesis is used, wherein enzymes, substrates and / or cofactors are added to the reactor one or more at a time to optimize the reaction conditions of the selected reaction steps.

[0021] On the other hand, a method for producing indigo glycosides in vitro is provided, comprising the following steps: (t) The tryptophan is enzymatically converted to indole, preferably by L-tryptophan indole lyase (TIL); (a) The indole is enzymatically converted to indophenol, preferably by an oxidase, such as an oxidoreductase (EC 1), a monooxygenase (EC 1.14), a flavin-containing monooxygenase (FMO) (EC 1.14.13.8), a nonspecific peroxidase (EC 1.11.2.1), or P450 (EC 1.14.14.1); and (b1) The indophenol is enzymatically converted to indole, preferably by a glycosyltransferase (GT) (EC 2.4.1), such as UDP-glycosyltransferase, preferably a glycosyltransferase, wherein the amino acid sequence of the glycosyltransferase has at least 75% sequence identity with SEQ ID NO.46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO.46, and further comprises: (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, Furthermore, the polypeptide with glycosyltransferase activity described therein exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0022] On one hand, the present invention relates to a one-pot process for producing indigo glycosides from tryptophan, wherein all enzymatic reactions are carried out in a single pot. Attached Figure Description

[0023] Brief description of the attached diagram: Figure 1 The "traditional process" involves chemically synthesized indigo and the addition of a reducing agent (such as sodium dithionite, Na₂S₂O₄) to the indigo dyeing vat to reduce it into soluble, dyeable leucoindigo. In the "chemical enzymatic method," indophenol is glycosylated to produce indoside as a product. Indoside is stable and can be stored. During the dyeing step, the glucose groups on the fabric are removed, allowing the regenerated indophenol to oxidize into indigo on the fabric. GT = glycosyltransferase, BGL = β-glycosylhydrolase.

[0024] Figure 2 Overview of the enzymatic pathways for the biosynthesis of indole, indigo glycosides, and indigo. R5P = ribose-5-phosphate, PRPP = ribose-phosphophosphate, N-PRA = N-phosphoribosyl-o-aminobenzoic acid, CdRP = 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate, IGP = indole-3-glycerol-phosphate, G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, 6PG = 6-phosphogluconic acid, Ru5P = ribulose-5-phosphate.

[0025] Figure 3 : (A) Example of enzymatic conversion of ribose-5-phosphate (R5P) and anthranilate to indole. R5P is converted to phosphoribosyl pyrophosphate (PRPP) by phosphoribosyl pyrophosphate synthase (PRS); PRPP is converted to phosphoribosyl pyrophosphate (N-PRA) by N-phospribosyl anthranilate synthase (trpD); N-PRA is converted to 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate (CdRP) by N-phospribosyl anthranilate isomerase (trpF); CdRP is converted to indole-3-glycerol-phosphate (IGP) by indole-3-glycerol-phosphate synthase (trpC); IGP is converted to indole by indole-3-glycerol-phosphate lyase (IGL). The enzymatic cofactor cycle system can be used to regenerate ATP from AMP. PPK2-II = AMP kinase. PolyP = polyphosphate. Anth = anthranilate. PPi = inorganic pyrophosphate. G3P = glycerol-3-phosphate.

[0026] (B) An example of ribose being converted to ribose-5-phosphate (R5P) by ribokinase (RK). The enzymatic cofactor cycle can be used to regenerate ATP from ADP. PPK2-I = ADP kinase. PolyP = polyphosphate.

[0027] (C) Example of glucose conversion to ribose-5-phosphate (R5P). Glucose is converted to glucose-6-phosphate (G6P) by hexokinase (HK), G6P is converted to 6-phosphoglucuronide (6PG) by glucose-6-phosphate dehydrogenase (G6PDH), 6PG is converted to ribulose-5-phosphate (Ru5P) by 6-phosphoglucate dehydrogenase (6PGDH), and Ru5P is converted to R5P by ribose-5-phosphate isomerase (RPI). The enzymatic cofactor cycle system can 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.

[0028] (D) Example of starch conversion to ribose-5-phosphate (R5P). Starch is converted to glucose by α-amylase and glucosylamylase; glucose is converted to glucose-6-phosphate (G6P) by hexokinase (HK); G6P is converted to 6-phosphoglucuronide (6PG) by glucose-6-phosphate dehydrogenase (G6PDH); 6PG is converted to ribulose-5-phosphate (Ru5P) by 6-phosphoglucate dehydrogenase (6PGDH); and Ru5P is converted to R5P by ribose-5-phosphate isomerase (RPI). The enzymatic cofactor cycle system can 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.

[0029] (E) Example of starch conversion to ribose-5-phosphate (R5P). Starch is converted to glucose-1-phosphate (G1P) by α-glucan phosphorylase (aGP), G1P is converted to glucose-6-phosphate (G6P) by phosphogluconomutase (PGM), G6P is converted to 6-phosphoglucuronide (6PG) by glucose-6-phosphate dehydrogenase (G6PDH), 6PG is converted to ribulose-5-phosphate (Ru5P) by 6-phosphoglucate dehydrogenase (6PGDH), and Ru5P is converted to R5P by ribose-5-phosphate isomerase (RPI). The enzymatic cofactor cycle system can be used to regenerate NAD(P) from NAD(P)H. + GR = Glutathione disulfide reductase. GSSG = Glutathione disulfide. GSH = Glutathione. Pi = Inorganic phosphate.

[0030] Figure 4 Example of indole conversion to indole glycoside. (A) Indole is converted to indolephenol by an oxidoreductase; indolephenol is converted to indole glycoside by UDP-glycosyltransferase (UGT). An enzyme-cofactor recycling system is introduced to convert NADP... + Regeneration of NADPH and regeneration of UDP-Glc from UDP. FDH = formate dehydrogenase. SuSy = sucrose synthase. (B) Indole is converted to indolephenol by peroxidase; indolephenol is converted to indoleglycoside by UDP glycosyltransferase (UGT). An enzymatic cofactor cycle is introduced to regenerate UDP-Glc from UDP. SuSy = sucrose synthase.

[0031] Figure 5 Example of the enzymatic conversion of indole to indigo. (A) Indole is converted to indolephenol by an oxidoreductase; indolephenol, when exposed to air, spontaneously converts to indigo. An enzyme cofactor cycle system is introduced to convert NADP... + Regenerating NADPH. FDH = formate dehydrogenase. (B) Indole is converted to indophenol by peroxidase; indophenol is spontaneously converted to indigo upon exposure to air.

[0032] Figure 6 Examples of the enzymatic conversion of tryptophan to indole and indole glycoside. (A) Tryptophan is converted to indole by L-tryptophan indole lyase (TIL). Indole is then converted to indole by flavin monooxygenase (FMO); indole is converted to indoleglycoside by UDP glycosyltransferase (UGT). Pyr = pyruvate. An enzymatic cofactor recycling system is introduced to convert NADP... +Regeneration of NADPH and regeneration of UDP-Glc 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 indolephenol by peroxidase; indolephenol is converted to indoleglycoside by UDP glycosyltransferase (UGT). Pyr = pyruvate. An enzymatic cofactor cycle is introduced to regenerate UDP-Glc from UDP. SuSy = sucrose synthase.

[0033] Figure 7 (A) 100 μM and (B) 1000 μM anthranilate were converted to indole via the activities of enzymes TkTrpD, EcTrpF, PaTrpC, and OsIGL. The concentrations of indole and anthranilate were measured at different time points during culture.

[0034] Figure 8A Anthranilic acid was sequentially converted to IGP using enzymes TkTrpD, EcTrpF, and PaTrpC, with a 1-hour incubation step between sampling. Chromatograms were obtained by RP-HPLC and recorded at 340 nm and 260 nm.

[0035] Figure 8B : Continuous enzymatic conversion of anthranilic acid to indole. Reaction (1) converts anthranilic acid to NPRA by adding TrpD; reaction (2) converts NPRA to CdRP by adding TrpF; reaction (3) selectively reduces CdRP to reduced CdRP (rCdRP) by adding sodium borohydride; reaction (4) converts CdRP to IGP by adding TrpC; reaction (5) chemically converts IGP to indole-3-aldehyde (I3A) by adding sodium periodate; reaction (6) converts IGP to indole by adding IGL.

[0036] Figure 9 The conversion of anthranilic acid to indigo was performed in a one-pot in vitro enzyme mixture. The concentrations of indole and anthranilate were measured at different incubation time points.

[0037] Figure 10 Riboose was converted to IGP in a one-pot in vitro enzyme mixture. TkTrpD, EcTrpF, and PaTrpC enzymes were used in all assays, while different combinations of ribokinases and phosphoribosyl pyrophosphate synthases were explored. IGP was measured after 1, 2, 4, and 22 hours of incubation.

[0038] Figure 11The HPLC chromatogram recorded at 280 nm is generated by the transformation of ribose and anthranilic acid at 25 °C after overnight incubation with an in vitro enzyme mixture containing HoRK, HoPRS, TkTrpD, EcTrpF, PaTrpC, OsIGL, NIFMO and PtUGT1mut87.

[0039] Figure 12 Effect of temperature on the conversion of anthranilic acid and PRPP to IGP using TkTrpD, TmTrpF, and SsTrpC enzymes.

[0040] Figure 13 The effect of enzyme ratios on the conversion of anthranilic acid and PRPP to IGP in an in vitro one-pot enzyme mixture using enzymes TkTrpD, TmTrpF, and SsTrpC. (A) TmTrpF and SsTrpC at fixed concentrations, while the concentration of TkTrpD was varied. (B) TmTrpD and SsTrpC at fixed concentrations, while the concentration of TkTrpF was varied. (C) TMT RPD and TmTrpF at fixed concentrations, while the concentration of SsTrpC was varied.

[0041] Figure 14 Optimization of IGP formation: (A) Baseline experiment; (B) Single addition of 5 mM PRPP after 60 minutes; (C) Addition of fresh 7 mM o-aminobenzoate and 14 mM PRPP every 30 minutes; (D) Addition of 4 mM o-aminobenzoate and 5 mM PRPP every 60 minutes; (E) Addition of 20 mM PRPP every 60 minutes; (F) Addition of 20 mM PRPP every 30 minutes; (G) Addition of 5 μM SsTrpC every 60 minutes; (H) Repeat G, adding 5 μM SsTrpC and 15 mM PRPP every 60 minutes.

[0042] Figure 15 Effect of temperature on the conversion of IGP to indigo using enzymes ZmBX1, NIFMO, and MvFDH.

[0043] Figure 16 The effect of enzyme ratios on the conversion of IGP to indigo using enzymes ZmBX1, NIFMO, and MvFDH. (A) With NIFMO and MvFDH concentrations fixed, the concentration of ZmBX1 was varied. (B) With ZmBX1 and MvFDH concentrations fixed, the concentration of NIFMO was varied. (C) With ZmBX1 and NIFMO concentrations fixed, the concentration of MvFDH was varied.

[0044] Figure 17In a one-pot in vitro enzyme mixture containing VcTIL, NlFMO, and PtUGT1mut87, tryptophan with different concentrations (1, 5, and 10 mM) was converted to indigo glycoside.

[0045] Abbreviations, terms and definitions: Amino acid sequence identity: The term “sequence identity” as used herein refers to a quantitative measure of the similarity between two amino acid sequences of substantially equal length. The two sequences to be compared must be aligned to obtain the best match, which can be achieved by inserting gaps or truncating the protein sequence ends. Sequence identity can be calculated as ((Nref - Ndif)100) / (Nref), where Ndif is the total number of dissimilar residues in the two sequences at the time of alignment, and Nref is the number of residues in one of the sequences. Sequence identity calculation is preferably performed automatically using a BLAST program, such as BLASTP (Pearson W.R and DJ Lipman (1988)) (www.ncbi.nlm.nih.gov / cgi-bin / BLAST). Sequence alignment can be performed using the MAFFT24 program (multiple alignment using Fast Fourier Transform; Katoh et al. 2019) with default parameters (scoring matrix: blosum62, gap opening penalty: 1.53, gap extension penalty: 0.123).

[0046] Preferably, compared to its comparative polypeptide, the number of substitutions, insertions, additions, or deletions of one or more amino acid residues in the 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 conserved amino acid substitutions: limited to exchanges between members within each group, Group 1: glycine, alanine, valine, leucine, isoleucine; Group 2: serine, cysteine, selenocysteine, threonine, methionine; Group 3: proline; Group 4: phenylalanine, tyrosine, tryptophan; Group 5: aspartic acid, glutamic acid, asparagine, glutamine; Group 6: histidine, lysine, arginine.

[0047] As used in this article, the term "one-pot" reaction or "one-pot" synthesis refers to a chemical reaction or synthetic process in which all the required reactants, reagents and steps are mixed and carried out in a single reaction vessel or container.

[0048] As used in this article, the term "continuous one-pot synthesis" refers to a chemical reaction or synthetic process in which all the necessary reactants, reagents and steps are combined and carried out in a single reaction vessel or container, wherein 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 the reaction conditions of the selected reaction steps.

[0049] anthranilic acid is an aromatic acid with the molecular formula C6H4(NH2)(CO2H); the molecule consists of a benzene ring, with the ortho-position substituted by carboxylic acids and amines, as shown in chemical formula I. Anion [C6H4(NH2)(CO2)] - It is obtained by deprotonation of an-aminobenzoic acid and is called an-aminobenzoate.

[0050] Chemical formula I.

[0051] The two terms, anthranilic acid and anthranilate, are used interchangeably in the context of this invention. Anthranilic acid is the actual compound added to the reaction mixture, while anthranilate is formed in solution (pKa 4.8). Detailed Implementation

[0052] This invention provides a novel bio-based enzymatic method for indole synthesis, optionally followed by further enzymatic steps for the synthesis of indigo glycosides and / or indigo. Figure 2 As shown, through the sequential action of several enzymes, the basic building blocks are converted into indole, and optionally into indigo glycosides and / or indigo.

[0053] I. Synthesis of indole from ribose-5-phosphate and o-aminobenzoate On one hand, the present invention relates to a method for producing indole in vitro, comprising the following steps: (i) Provides ribose-5-phosphate (R5P), (ii) R5P is enzymatically converted into phosphoribosyl pyrophosphate (PRPP). (iii) PRPP is enzymatically converted into N-phosphoribosyl anthranilic acid (N-PRA). (iv) N-PRA was enzymatically converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP). (v) CdRP is enzymatically converted to indole-3-glycerophosphate (IGP), and (vi) IGP is enzymatically converted into indole.

[0054] In vitro methods such as Figure 3 As shown in Figure A.

[0055] For the synthesis of indole, the substrates are ribose-5-phosphate and anthranilate, and the cofactor ATP is also required. Ribose-5-phosphate is required in step (i); anthranilate is required in step (iii); and ATP is required in step (ii).

[0056] In one embodiment, the synthesis of indole includes providing ribose-5-phosphate, o-aminobenzoate, and ATP for the relevant process steps, as described above.

[0057] In one implementation, ribose-5-phosphate (R5P) in step (i) is provided via the following steps: (I) Converting riboproteins to R5P via enzymatic reaction, or (II) Enzymatically convert glucose or starch into glucose-6-phosphate (G6P), enzymatically convert G6P into 6-phosphogluconic acid (6PG), enzymatically convert 6PG into ribulose-5-phosphate (Ru5P), and enzymatically convert Ru5P into R5P.

[0058] In one implementation, R5P is provided in step (i) via the enzymatic conversion of ribose to R5P. In a preferred implementation, this is promoted by ribokinase (RK). ATP is required as a cofactor. This is in Figure 3 As described in section B. Therefore, in one embodiment, in step (i), ribokinase is used to synthesize ribose-5-phosphate from ribose and ATP. ATP is converted to ADP. In one embodiment, ribose and ATP are enzymatically converted to ribose-5-phosphate (R5P) in step (i) by a polypeptide with ribokinase activity (EC 2.7.1.15). The amino acid sequence of the polypeptide with ribokinase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO.2, SEQ ID NO.4, or SEQ ID NO.6. Preferably, the amino acid sequence of the polypeptide with ribokinase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO.6. In one embodiment, the ribokinase is selected from SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.6, preferably SEQ ID NO.6.

[0059] In another embodiment, R5P is provided in step (i) by converting glucose into R5P. Specifically, this is achieved through the enzymatic conversion of glucose to glucose-6-phosphate (G6P), G6P to gluconic acid 6-phosphate (6PG), 6PG to ribulose 5-phosphate (Ru5P), and Ru5P to R5P. In a preferred embodiment, this is promoted by hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), gluconic acid 6-phosphate dehydrogenase (6PGDH), and ribose-5-phosphate isomerase (RPI). ATP and NAD are required. + As a cofactor. Therefore, in one embodiment, in step (i), hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphate gluconate dehydrogenase (6PGDH), and ribose-5-phosphate isomerase (RPI) are used to extract glucose, ATP, and NAD. + Ribose-5-phosphate is synthesized. ATP is converted to ADP. NAD + Converted to NADH. This is in Figure 3 This is explained in section C.

[0060] In one implementation, 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 the polypeptide having hexokinase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 74 or SEQ ID NO. 76. In one embodiment, the hexokinase is selected from SEQ ID NO. 74 and SEQ ID NO. 76. -G6P and NAD + A polypeptide having glucose-6-phosphate dehydrogenase activity (EC 1.1.1.49) is enzymatically converted to gluconic acid 6-phosphate (6PG). 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 with SEQ ID NO. 78 or SEQ ID NO. 80. In one embodiment, the glucose-6-phosphate dehydrogenase is selected from SEQ ID NO. 78 and SEQ ID NO. 80. -6PG and NAD +The polypeptide is enzymatically converted to ribulose-5-phosphate (Ru5P) by a polypeptide having 6-phosphoglucate dehydrogenase activity (EC 1.1.1.44). The amino acid sequence of the polypeptide having 6-phosphoglucate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 82 or SEQ ID NO. 84. In one embodiment, the 6-phosphoglucate dehydrogenase is selected from SEQ ID NO. 82 and SEQ ID NO. 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 with SEQ ID NO. 86 or SEQ ID NO. 88. In one embodiment, the ribose-5-phosphate isomerase is selected from SEQ ID NO. 86 and SEQ ID NO. 88.

[0061] In another embodiment, R5P in step (i) is provided by converting starch into glucose, and further converting glucose into R5P, as described above. This is in Figure 3 As shown in D. In one such embodiment, a polypeptide having α-amylase activity (e.g., EC3.2.1.1) and / or a polypeptide having glucosylase activity (e.g., EC3.2.1.3) promotes the enzymatic conversion of starch to glucose. The amino acid sequence of said polypeptide having α-amylase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 90. In one embodiment, the α-amylase is SEQ ID NO. 90. The amino acid sequence of said polypeptide having glucosylase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 92. In one embodiment, the glucosylase is SEQ ID NO. 92.

[0062] In another embodiment, R5P is provided in step (i) by the conversion of starch to R5P. Specifically, this is achieved by enzymatically converting starch to glucose-1-phosphate (G1P), G1P to glucose-6-phosphate (G6P), G6P to 6-phosphoglucuronide (6PG), 6PG to ribulose-5-phosphate (Ru5P), and Ru5P to R5P. In a preferred embodiment, this is promoted by α-glucan phosphorylase (aGP), phosphogluconomutase (PGM), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and ribose-5-phosphate isomerase (RPI). ATP and NADH are required as cofactors. Therefore, in one embodiment, in step (i), ribose-5-phosphate is synthesized from starch, ATP, and NADP using α-glucan phosphorylase (aGP), phosphoglucose mutase (PGM), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphoglucate dehydrogenase (6PGDH), and ribose-5-phosphate isomerase (RPI). ATP is converted to ADP. NAD + Converted to NADH. This is in Figure 3 This is explained in section E.

[0063] In one implementation, in step (i) Starch is enzymatically converted to glucose-1-phosphate (G1P) by a polypeptide having α-glucan phosphorylase activity (EC 2.4.1.1). The amino acid sequence of the polypeptide having α-glucan phosphorylase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 94. In one embodiment, the α-glucan phosphorylase is SEQ ID NO. 94. -G1P is enzymatically converted to glucose-6-phosphate (G6P) by a polypeptide having phosphoglucose mutase activity (EC 2.7.5.1). The amino acid sequence of said polypeptide having phosphoglucose mutase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 96. In one embodiment, the phosphoglucose mutase is SEQ ID NO. 96. -R5P and NAD +A polypeptide having glucose-6-phosphate dehydrogenase activity (EC 1.1.1.49) is enzymatically converted to gluconic acid 6-phosphate (6PG). 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 with SEQ ID NO. 82 or 84. In one embodiment, the glucose-6-phosphate dehydrogenase is selected from SEQ ID NO. 82 and SEQ ID NO. 84. -6PG and NAD + The polypeptide is enzymatically converted to ribulose-5-phosphate (Ru5P) by a polypeptide having 6-phosphoglucate dehydrogenase activity (EC 1.1.1.44). The amino acid sequence of the polypeptide having 6-phosphoglucate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 86 or SEQ ID NO. 88. In one embodiment, the 6-phosphoglucate dehydrogenase is selected from SEQ ID NO. 86 and SEQ ID NO. 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 with SEQ ID NO. 90 or SEQ ID NO. 92. In one embodiment, the ribose-5-phosphate isomerase is selected from SEQ ID NO. 90 and SEQ ID NO. 92.

[0064] In one embodiment, step (ii) is promoted by phosphoribosyl pyrophosphate synthase (PRS). ATP is required as a cofactor. In step (ii), phosphoribosyl pyrophosphate is synthesized from R5P and ATP using phosphoribosyl pyrophosphate synthase. ATP is converted to AMP. In one embodiment, R5P and ATP are enzymatically converted to phosphoribosyl pyrophosphate (PRPP) in step (ii) by a polypeptide having phosphoribosyl pyrophosphate synthase activity (EC 2.7.6.1). The amino acid sequence of said polypeptide having phosphoribosyl pyrophosphate synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 12. Preferably, the amino acid sequence of said polypeptide having phosphoribosyl pyrophosphate synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 12. In one embodiment, the phosphoribosyl pyrophosphate synthase is selected from SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.12, preferably SEQ ID NO.12.

[0065] In one embodiment, step (iii) is promoted by N-phospribosyl-anaminobenzoic acid synthase (trpD). In step (iii), N-phospribosyl-anaminobenzoic acid (N-PRA) is synthesized from PRPP and anaminobenzoate using N-phospribosyl-anaminobenzoic acid synthase. In one embodiment, PRPP and anaminobenzoate are enzymatically converted to N-phospribosyl-anaminobenzoic acid (N-PRA) in step (iii) by a polypeptide having N-phospribosyl-anaminobenzoic acid synthase activity (EC 2.4.2.18). The amino acid sequence of said polypeptide having N-phospribosyl-anaminobenzoic acid synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 14, SEQ ID NO. 16, or SEQ ID NO. 18. Preferably, the amino acid sequence of the polypeptide having N-phospribosyl-o-aminobenzoic acid synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 14. In one embodiment, the N-phospribosyl-o-aminobenzoic acid synthase is selected from SEQ ID NO. 14, SEQ ID NO. 16, and SEQ ID NO. 18, preferably SEQ ID NO. 14.

[0066] In one embodiment, step (iv) is promoted by N-(5'-phospribosyl)-o-aminobenzoic acid isomerase (trpF). In one embodiment, N-PRA is enzymatically converted in step (iv) to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP) by a polypeptide having N-(5'-phospribosyl)-o-aminobenzoic acid isomerase activity (EC 5.3.1.24). The amino acid sequence of the polypeptide having N-(5'-phospribosyl)-o-aminobenzoic acid isomerase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 20 or SEQ ID NO. 22. Preferably, the amino acid sequence of the polypeptide having N-(5'-phospribosyl)-o-aminobenzoic acid isomerase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 20. In one embodiment, the N-(5'-phospribosyl)-o-aminobenzoic acid isomerase is selected from SEQ ID NO. 20 and SEQ ID NO. 22, preferably SEQ ID NO. 20.

[0067] In one embodiment, step (v) is promoted by indole-3-glycerol phosphate synthase (trpC). In one embodiment, CdRP is enzymatically converted to indole-3-glycerol phosphate (IGP) in step (v) by a polypeptide having indole-3-glycerol phosphate synthase activity (EC 4.1.1.48). The amino acid sequence of the polypeptide having indole-3-glycerol phosphate synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 24 or SEQ ID NO. 26. Preferably, the amino acid sequence of the polypeptide having indole-3-glycerol phosphate synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 26. In one embodiment, the indole-3-glycerol phosphate synthase is selected from SEQ ID NO. 24 and SEQ ID NO. 26, preferably SEQ ID NO. 26.

[0068] In one embodiment, step (vi) is promoted by indole-3-glycerol-phosphate lyase (IGL). In one embodiment, IGL is enzymatically converted to indole by a polypeptide having indole-3-glycerol-phosphate lyase activity (EC 4.1.2.8) in step (vi). 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 with SEQ ID NO. 28 or SEQ ID NO. 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 with SEQ ID NO. 28. In one embodiment, the indole-3-glycerol-phosphate lyase is selected from SEQ ID NO. 28 and SEQ ID NO. 30, preferably SEQ ID NO. 28.

[0069] In a preferred embodiment, the method for producing indole in vitro comprises all steps (i), (ii), (iii), (iv), (v), and (vi), wherein step (i) is promoted by ribokinase (RK), step (ii) is promoted by phosphoribosyl pyrophosphate synthase (PRS), step (iii) is promoted by N-phospribosyl-o-aminobenzoic acid synthase (trpD), step (iv) is promoted by N-(5'-phospribosyl)o-aminobenzoic acid isomerase (trpF), step (v) is promoted by indole-3-glycerol phosphate synthase (trpC), and step (vi) is promoted by indole-3-glycerol-phosphate lyase (IGL).

[0070] In a preferred embodiment, the method for producing indole in vitro comprises all steps (i), (ii), (iii), (iv), (v), and (vi); wherein step (i) is promoted by a polypeptide having ribokinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 6; step (ii) is promoted by a polypeptide having phosphoribosyl pyrophosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 12; step (iii) is promoted by a polypeptide having N-phospribosyl anthranilate synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 14; and step (iv) is promoted by a polypeptide having N-(5'-phospribosyl) anthranilate isomerase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 14. NO. 20 is promoted by a polypeptide having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity; step (v) is promoted by a polypeptide having indole-3-glycerol phosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 26; and step (vi) is promoted by a polypeptide having indole-3-glycerol phosphate lyase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 28.

[0071] In one embodiment, the present invention provides a method for producing indole in vitro, comprising (I) providing a substrate ribose-5-phosphate and anthranilate, a cofactor ATP and enzymes phosphoribosyl pyrophosphate synthase, N-phospribosyl anthranilate synthase, N-(5'-phospribosyl) anthranilate isomerase, indole-3-glycerol phosphate synthase and indole-3-glycerol phosphate lyase, (II) mixing the substrate, cofactor and enzyme, and (III) reacting the mixture at a selected temperature and time to produce indole.

[0072] In one embodiment, the present invention provides a method for producing indole in vitro, comprising (I) providing substrates ribose and anthranilate, cofactor ATP and enzymes ribokinase, phosphoribosyl pyrophosphate synthase, N-phospribosyl anthranilate synthase, N-(5'-phospribosyl) anthranilate isomerase, indole-3-glycerol phosphate synthase and indole-3-glycerol phosphate lyase, (II) mixing said substrates, cofactors and enzymes, and (III) reacting the mixture at a selected temperature and time to produce indole.

[0073] In one embodiment, the present invention provides a method for in vitro production of indole, comprising (I) providing substrates glucose and o-aminobenzoate, cofactor ATP and NAD. + (II) mixing the substrate, cofactor and enzyme, and (III) reacting the mixture at a selected temperature and time to generate indole.

[0074] In one embodiment, the present invention provides a method for in vitro production of indole, comprising (I) providing substrates starch and anthranilate, cofactor ATP and NAD. + And the enzymes α-amylase, glucosylamylase, hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphate glucuronide dehydrogenase, ribose-5-phosphate isomerase, phosphoribosyl pyrophosphate synthase, N-phospribosyl-o-aminobenzoic acid synthase, N-(5'-phospribosyl)o-aminobenzoic acid isomerase, indole-3-glycerol phosphate synthase and indole-3-glycerol-phosphate lyase, (II) mixing the substrate, cofactor and enzyme, and (III) reacting the mixture at a selected temperature and time to generate indole.

[0075] In one embodiment, the present invention provides a method for in vitro production of indole, comprising (I) providing substrate starch and anthranilate, cofactor ATP and NAD. + The enzymes α-glucan phosphorylase, phosphoglucose mutase, glucose-6-phosphate dehydrogenase, 6-phosphoglucuronide dehydrogenase, ribose-5-phosphate isomerase, phosphoribosyl pyrophosphate synthase, N-phospribosyl-o-aminobenzoic acid synthase, N-(5'-phospribosyl)o-aminobenzoic acid isomerase, indole-3-glycerol phosphate synthase and indole-3-glycerol phosphate lyase, (II) mix the substrate, cofactor and enzyme, and (III) react the mixture at a selected temperature and time to generate indole.

[0076] Ribose is a simple carbohydrate sugar with the molecular formula C5H. 10 O5. For the purposes of this invention, ribose can be provided directly in the form of ribose monomers and converted into the ribose-5-phosphate disclosed herein. Alternatively, step (i) disclosed herein can be promoted by enzymatically converting amylase into the ribose-5-phosphate disclosed herein. Alternatively, as disclosed herein, glucose can be provided and converted into R5P. Ribose, glucose, and starch are sugar monomers and polymers widely available to those skilled in the art.

[0077] Anthranilic acid is an aromatic acid with the molecular formula C6H4(NH2)(CO2H). The anion [C6H4(NH2)(CO2)]- is obtained by deprotonation of anthranilic acid and is called anthranilate. Many synthetic pathways for anthranilic acid have been described. The biosynthesis of anthranilate originates from the shikimic acid pathway. Industrially, it is produced from phthalic anhydride. Methyl anthranilate is a relatively inexpensive and readily available compound that can be converted to anthranilic acid in a single enzymatic step. Therefore, the anthranilate provided and required in step (iii) can be provided directly in the form of anthranilic acid or as a precursor of anthranilate, which can be enzymatically converted to anthranilate.

[0078] The cofactor ATP can be regenerated from ADP and AMP via an enzymatic cofactor recycling system using ATP regenerating enzymes (EC 2.7.4.1), such as phosphokinases (EC 2.7.4.1), preferably polyphosphokinase 2 (PPK2) (EC 2.7.4.1).

[0079] In one embodiment, the combination of ADP kinase (PPK2-I) and polyphosphate (PolyP) promotes the 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 with SEQ ID NO. 58, SEQ ID NO. 60, or SEQ ID NO. 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 with SEQ ID NO. 58. In one embodiment, the ADP kinase is selected from SEQ ID NO. 58, SEQ ID NO. 60, and SEQ ID NO. 62, preferably SEQ ID NO. 58.

[0080] In one embodiment, the combination of AMP kinase (PPK2-II) and polyphosphate (PolyP) promotes the 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 with SEQ ID NO. 58, SEQ ID NO. 60, or SEQ ID NO. 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 with SEQ ID NO. 62. In one embodiment, the AMP kinase is selected from SEQ ID NO. 58, SEQ ID NO. 60, and SEQ ID NO. 62, preferably SEQ ID NO. 62.

[0081] Cofactor NAD(P) + It can be regenerated from NAD(P)H via an enzymatic cofactor recycling system, which uses NAD(P)H. + - Regenerating enzymes, such as glutathione disulfide reductase (EC 1.8.1.7).

[0082] In one embodiment, the combination of glutathione disulfide and NAD(P)H promotes the regeneration of NAD(P)+. In another embodiment, NAD(P)H is enzymatically converted to NAD(P) by a polypeptide having glutathione disulfide reductase activity (EC 1.8.1.7). + The amino acid sequence of the polypeptide having glutathione disulfide reductase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 98. In one embodiment, the glutathione disulfide reductase is SEQ ID NO. 98.

[0083] In a preferred embodiment, the method for producing indole in vitro comprises all steps (i), (ii), (iii), (iv), (v), and (vi), and further comprises the cofactor recycling step disclosed herein; wherein step (i) is promoted by ribokinase (RK), step (ii) is promoted by phosphoribosyl pyrophosphate synthase (PRS), step (iii) is promoted by N-phospribosyl-o-aminobenzoic acid synthase (trpD), step (iv) is promoted by N-(5'-phospribosyl)o-aminobenzoic acid isomerase (trpF), step (v) is promoted by indole-3-glycerol phosphate synthase (trpC), and step (vi) is promoted by indole-3-glycerol phosphate lyase (IGL); and wherein ATP cofactor recycling is promoted by ADP kinase and AMP kinase.

[0084] In one embodiment, some or all of the enzymatic steps disclosed herein for the in vitro production of indole are carried out in a one-pot reaction.

[0085] In a preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) disclosed herein are carried out in a one-pot reaction. In a further preferred embodiment, as disclosed herein, steps (i), (ii), (iii), (iv), (v), and (vi), as well as the regeneration of ATP cofactors from ADP and AMP, are carried out in a one-pot reaction.

[0086] In vitro biosynthesis is preferably carried out under the optimal temperature and pH conditions for the enzymes used.

[0087] In one embodiment, the incubation temperature should be in the range of 20-65°C, for example 20-50°C, for example 20-40°C, preferably in the range of 25-35°C, for example preferably about 30°C. In one embodiment, the incubation pH should be in the range of pH 5-9, for example pH 5.5-8.5, for example preferably pH 6-8, for example preferably about pH 7.5.

[0088] Enzymatic reactions can be carried out in buffer solutions used to stabilize enzymes, as those skilled in the art will recognize, and are routinely optimized.

[0089] In one embodiment, all the enzymatic steps disclosed herein for the in vitro production of indole are carried out in a one-pot reaction.

[0090] In a preferred embodiment, steps (ii), (iii), (iv), (v), and (vi) disclosed herein for the production of indole are carried out in a one-pot reaction. In another embodiment, steps (ii), (iii), (iv), (v), and (vi), as well as the regeneration system disclosed herein, are carried out in a one-pot reaction.

[0091] In a preferred embodiment, steps (i), (ii), (iii), (iv), (v), and (vi) disclosed herein for the production of indole are carried out in a one-pot reaction. In another embodiment, steps (i), (ii), (iii), (iv), (v), and (vi), as well as the regeneration system disclosed herein, are carried out in a one-pot reaction.

[0092] In one embodiment, a continuous one-pot synthesis can be used, wherein one or more enzymes, substrates and / or cofactors are added to the reactor one at a time to optimize the reaction conditions of the selected reaction steps.

[0093] II. Synthesis of indolephenol from indole In another aspect, the present invention provides an in vitro method for producing indigo glycosides and / or indigo, the method comprising the step (a) enzymatically converting indole to indolephenol, such as... Figure 4 and 5 As shown.

[0094] In one embodiment, step (a) is promoted by an oxidase (ox enz). In one embodiment, the oxidase is selected from oxidoreductases (EC 1), monooxygenases (EC 1.14), flavin-containing monooxygenases (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1) and nonspecific peroxidases (EC 1.11.2.1).

[0095] In one embodiment, a flavin-containing monooxygenase (FMO) promotes step (a). NADPH is required as a cofactor / redox chaperone. In step (a), indole is synthesized from indole and NADPH using a flavin-containing monooxygenase. In one embodiment, indole and NADPH are enzymatically converted into indole and NADP by a polypeptide having flavin-containing monooxygenase activity (EC 1.14.13.8) in step (a). + The amino acid sequence of the polypeptide having flavin monooxygenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32, SEQ ID NO. 34, or SEQ ID NO. 36. Preferably, the amino acid sequence of the polypeptide having flavin monooxygenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32. In one embodiment, the flavin monooxygenase is selected from SEQ ID NO. 32, SEQ ID NO. 34, and SEQ ID NO. 36, preferably SEQ ID NO. 32.

[0096] In another embodiment, step (a) is promoted by a cytochrome P450 enzyme. NADPH is required as a cofactor. In step (a), indole is synthesized from indole and NADPH using a cytochrome P450 enzyme (EC 1.14.14.1). In one embodiment, indole and NADPH are enzymatically converted into indole and NADP by a peptidase having P450 enzyme activity (EC 1.14.14.1) in step (a). + The amino acid sequence of the polypeptide having P450 enzyme activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 38 or SEQ ID NO. 40. Preferably, the amino acid sequence of the polypeptide having P450 enzyme activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 38. In one embodiment, the cytochrome P450 enzyme is selected from SEQ ID NO. 38 and SEQ ID NO. 40, preferably SEQ ID NO. 38.

[0097] The cofactor NADPH can be obtained from NADP through an enzymatic cofactor recycling system. + For regeneration, the system uses NADPH-regenerating enzymes, such as dehydrogenases, preferably formate dehydrogenase (FDH) (EC 1.17.1.10).

[0098] In one embodiment, the combination of formate dehydrogenase (FDH) and formate promotes the regeneration of NADPH from step (a) of the method. In one embodiment, NADP... + The polypeptide is enzymatically converted to NADPH by a polypeptide having formate dehydrogenase activity (EC 1.17.1.10). The amino acid sequence of the polypeptide having formate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56. Preferably, the amino acid sequence of the polypeptide having formate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56. In one embodiment, the formate dehydrogenase is selected from SEQ ID NO. 56, preferably SEQ ID NO. 56.

[0099] In another embodiment, step (a) is promoted by a peroxidase. Hydrogen peroxide is required as a cofactor. In step (a), indophenol is synthesized from indole and H2O2 using a peroxidase (EC 1.11.2.1). In one embodiment, indole and H2O2 are enzymatically converted to indophenol and H2O by a polypeptide having peroxidase (EC 1.11.2.1) activity in step (a). The amino acid sequence of the polypeptide having peroxidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 42 or SEQ ID NO. 44. Preferably, the amino acid sequence of the polypeptide having peroxidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 42. In one embodiment, the peroxidase is selected from SEQ ID NO. 42 and SEQ ID NO. 44, preferably SEQ ID NO. 42.

[0100] III. Synthesis of indigo glycosides and / or indigo III.i Synthesis from indole In one embodiment, the present invention provides an in vitro method for producing indigo glycosides and / or indigo. For example... Figure 4 and Figure 5 As shown, indole and / or indigo can be produced from indole.

[0101] In one embodiment, the in vitro production of indigo glycosides and / or indigo includes (a) As disclosed in Part II, the indole enzyme is converted to indolephenol, and (b1) Enzymatically converting indophenol to indole, and / or (b2) converting indophenol to indole by exposure to oxygen.

[0102] In one embodiment, step (b1) is promoted by a glycosyltransferase (GT) (EC 2.4), such as UDP-glycosyltransferase (EC 2.4.1), preferably the glycosyltransferase disclosed in Bidart et al. 2023 and WO 2023 / 161230.

[0103] In a preferred embodiment, indophenol is enzymatically converted to indoleglycoside by a polypeptide (EC 2.4) having glycosyltransferase activity in step (b1). 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 with SEQ ID NO. 48, SEQ ID NO. 50, or SEQ ID NO. 52. In one embodiment, the glycosyltransferase is selected from SEQ ID NO. 48, SEQ ID NO. 50, and SEQ ID NO. 52.

[0104] Preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises a single mutation with substitution of one or more amino acid residues selected from the group consisting of E75P, Q86K, Q86R, S110V, I188L, G222D, F381V, T388A, S413K, and G430K relative to SEQ ID NO. 46, and / or with substitution of one or more amino acid residues relative to SEQ ID NO. 46. NO.46 is a double mutation selected from one or more of the following amino acid residue substitutions: T146C+M148C, P190C+A198C, D193C+N196C, G296L+V297G and T388C+A399C, and / or a combination of one or more single mutations and / or double mutations; and wherein the polypeptide having glycosyltransferase activity is thermally more stable than SEQ ID NO.46.

[0105] More preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues of SEQ ID NO. 46, and wherein the polypeptide having glycosyltransferase activity has enhanced thermostability compared to SEQ ID NO. 46.

[0106] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO. 46, and further comprises... (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, or (iv) Substitution of T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (v) Substitution of T146C, M148C, F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (vi) Substitution of amino acid residues T146C, M148C, and T388A relative to SEQ ID NO.46, Furthermore, the polypeptide containing glycosyltransferase activity exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0107] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO. 46, and further comprises... (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, or Furthermore, the polypeptide containing glycosyltransferase activity exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0108] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, T388A, S413K, and G430K relative to the amino acid residues of SEQ ID NO. 46, and wherein the polypeptide having glycosyltransferase activity has enhanced thermostability compared to SEQ ID NO. 46.

[0109] In one embodiment, as described above, a glycosyltransferase (GT) facilitates step (b1). Preferably, the glycosyltransferase is a UDP-dependent glycosyltransferase. UDP-Glc is used in this reaction to link the glucose moiety to an indophenol compound, thereby forming indinoside. In step (b1), indinoside is synthesized from indophenol and UDP-Glc using this UDP-glycosyltransferase. UDP-Glc is converted to UDP.

[0110] UDP-Glc can be regenerated from UDP using an enzyme cycle system that utilizes sucrose synthase (EC 2.4.1.13).

[0111] In one embodiment, the combination of sucrase synthase (SuSy) and sucrose promotes the regeneration of UDP-Glc from step (b1) of the method. In one embodiment, UDP is enzymatically converted to UDP-Glc by a polypeptide having sucrase synthase activity (EC 2.4.1.13). The amino acid sequence of said polypeptide having sucrase synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54. Preferably, the amino acid sequence of said polypeptide having sucrase synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54. In one embodiment, the sucrase synthase is selected from SEQ ID NO. 54, preferably SEQ ID NO. 54.

[0112] In one implementation, the in vitro production of indigo glycosides includes... (a) As disclosed in Part II, the indole enzyme is converted to indolephenol, and (b1) As described in Part III.i, indophenol is enzymatically converted to indinoside.

[0113] In one embodiment, the present invention provides an in vitro method for producing indigo glycosides, comprising (I) providing indole, an oxidase and its associated cofactors, as well as a glycosyltransferase and UDP-Glc, (II) mixing the substrate, cofactors and enzymes, and (III) reacting the mixture at a selected temperature and time to produce indigo glycosides.

[0114] In a preferred embodiment, the present invention provides an in vitro method for producing indigo glycosides, comprising (I) providing indole, an oxidase and its associated cofactors, as well as a glycosyltransferase and UDP-Glc, wherein the indole is prepared as disclosed in Part I; (II) mixing the substrate, cofactors and enzyme; and (III) reacting the mixture at a selected temperature and time to produce indigo glycosides.

[0115] In one implementation, step (b2) is facilitated by exposure to oxygen. Once exposed to oxygen, indophenol spontaneously dimers and forms indigo.

[0116] In one implementation, the in vitro production of indigo includes... (a) As disclosed in Part II, the indole enzyme is converted to indolephenol, and (b2) As disclosed in Part III.i, indophenol is converted to indigo by exposure to oxygen.

[0117] In one embodiment, the present invention provides a method for in vitro production of indigo, comprising (I) providing indole, an oxidase and its associated cofactor, (II) mixing the substrate, cofactor and enzyme, and (III) reacting the mixture at a selected temperature and time to produce indigo glycoside.

[0118] In a preferred embodiment, the present invention provides a method for in vitro production of indigo, comprising (I) providing indole, an oxidase and an associated cofactor, wherein the indole is provided as disclosed in Part I; (II) mixing the substrate, cofactor and enzyme; and (III) reacting the mixture at a selected temperature and time to produce indigo glycosides.

[0119] III.ii Synthesis from ribo-5-phosphate and anthranilate In another aspect, the present invention provides an in vitro method for producing indole and / or indigo, comprising steps (i), (ii), (iii), (iv), (v) and (iv) as disclosed in Part I, combined with step (a) disclosed in Part II, and further combined with steps (b1) and / or (b2) disclosed in Part III.i, wherein indole is an intermediate compound.

[0120] Specifically, in a preferred embodiment, the present invention provides an in vitro method for producing indigo glycosides and / or indigo, comprising the following steps: (i) Provides ribose-5-phosphate (R5P), (ii) R5P is enzymatically converted into phosphoribosyl pyrophosphate (PRPP). (iii) PRPP is enzymatically converted into N-phosphoribosyl anthranilic acid (N-PRA). (iv) N-PRA was enzymatically converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP). (v) CdRP is enzymatically converted to indole-3-glycerophosphate (IGP), and (vi) IGP is enzymatically converted into indole. And also includes the following steps (a) Enzymatically converting indole to indolephenol, and (b1) Enzymatically converting indophenol to indoleglycoside by exposure to oxygen and / or (b2) converting indophenol to indigo.

[0121] Steps (i), (ii), (iii), (iv), (v), and (vi) are preferably promoted by the enzymes disclosed in Part I, step (a) is preferably promoted by the enzymes disclosed in Part II, and step (b1) is preferably promoted by the enzymes disclosed in Part III.i. Any details of each step listed in Parts I, II, and III.i also apply to this aspect of the synthesis of indigo glycosides and / or indigo from ribose-5-phosphate and o-aminobenzoate.

[0122] In a preferred embodiment, the method for producing indole in vitro includes all steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) and further includes a cofactor recycling step as disclosed above herein.

[0123] In a preferred embodiment, the method for producing indigo in vitro includes all steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) and further includes a cofactor recycling step as disclosed above herein.

[0124] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo glycosides, comprising the following steps: It provides ribose-5-phosphate, anthranilate, ATP, and UDP-Glc, and if an oxidoreductase is used as an oxidase, it provides NADPH. - Perform enzymatic conversion: (ii) Conversion of R5P to phosphoribosyl pyrophosphate (PRPP) promoted by phosphoribosyl pyrophosphate synthase; (iii) Conversion of PRPP to N-phospribosyl anthranilic acid (N-PRA) promoted by N-phospribosyl anthranilic acid synthase; (iv) The conversion of N-PRA to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP) is promoted by N-(5'-phospribosyl)-o-aminobenzoic acid isomerase; (v) Conversion of CdRP to indole-3-glycerophosphate (IGP) promoted by indole-3-glycerophosphate synthase (vi) IGP to indole mediated by indole-3-glycerol-phosphate lyase; (a) The oxidase-promoted conversion of indole to indophenol; and (b1) The conversion of indophenol to indinoside promoted by glycosyltransferase; And optional - ATP regeneration is achieved by providing PolyPn, and ATP regeneration is promoted by ADP kinase and AMP kinase, and / or -Regeneration of UDP-Glc by providing sucrose, wherein UDP-Glc regeneration is promoted by sucrose synthase, and / or - NADPH regeneration, if an oxidoreductase is used as an oxidase, is achieved by providing formic acid, and where NADPH regeneration is promoted by formic acid dehydrogenase; - Preferably, ribose-5-phosphate (R5P) is provided by converting ribose, glucose or starch into R5P, as disclosed herein.

[0125] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo glycosides, comprising the following steps: - Provides ribose-5-phosphate, anthranilate, ATP, NADPH and UDP-Glc - Perform enzymatic conversion: (ii) R5P is converted to phosphoribosyl pyrophosphate (PRPP) by a polypeptide having phosphoribosyl pyrophosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO. 12; (iii) PRPP is converted to N-phospribosyl anthranilic acid (N-PRA) by a polypeptide having N-phospribosyl anthranilic acid synthase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.14. (iv) N-PRA is converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP) by a polypeptide having N-(5'-phospribosyl)-o-aminobenzoic acid isomerase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO. 20; (v) CdRP is converted to indole-3-glycerophosphate (IGP) by a polypeptide having indole-3-glycerophosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.26. (vi) IGP is converted to indole by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.28. (a) The conversion of indole to indophenol, facilitated by a polypeptide having oxidative activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32; and (b1) The conversion of indophenol to indole, promoted by a polypeptide having glycosyltransferase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, wherein the amino acid sequence of said polypeptide having glycosyltransferase activity comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to amino acid residues in SEQ ID NO. 46, and further comprises (i) substitutions of F381V, T388C, and A399C relative to amino acid residues in SEQ ID NO. 46, or (ii) substitutions of F381V and T388A relative to amino acid residues in SEQ ID NO. 46, or (iii) substitution of T388A relative to amino acid residues in SEQ ID NO. 46, and wherein said polypeptide having glycosyltransferase activity exhibits enhanced thermostability compared to SEQ ID NO. 46. And optionally - ATP regeneration, through the provision of PolyP n And wherein ATP regeneration is promoted by a polypeptide having ADP kinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 60 and a polypeptide having AMP kinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 60, and / or - NADPH regeneration is achieved by providing formic acid, and a polypeptide having formic acid dehydrogenase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56 promotes NADPH regeneration, and / or - UDP-Glc regeneration is achieved by providing sucrose, wherein a polypeptide having sucrose synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54 promotes UDP-Glc regeneration. Preferably, ribose-5-phosphate (R5P) is provided by the conversion of ribose to R5P, wherein the conversion of ribose to ribose-5-phosphate (R5P) is promoted by a polypeptide having ribokinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 6.

[0126] In one embodiment, all the enzymatic steps disclosed herein for the in vitro production of indigo glycosides are carried out in a one-pot reaction.

[0127] In a preferred embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein for the production of indigo glycosides are carried out in a one-pot reaction. In another embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein, along with the regeneration system, are carried out in a one-pot reaction.

[0128] In this one-pot reaction, the intermediate products do not need to be purified or separated before proceeding to the next enzymatic conversion step.

[0129] In one embodiment, a continuous one-pot synthesis can be used, wherein one or more enzymes, substrates and / or cofactors are added to the reactor one at a time to optimize the reaction conditions of the selected reaction steps.

[0130] In one embodiment, a continuous one-pot process is performed, wherein one or more enzymes, substrates, and / or cofactors are added sequentially. Such a continuous one-pot process may include multiple stages, each with different process conditions, such as different temperature and / or pH conditions.

[0131] In one specific embodiment, indigo glycosides are produced in a continuous one-pot process, wherein, under first specific process conditions, in the first stage of the one-pot reaction, enzymes associated with steps (iii), (iv), and (v) disclosed herein for the production of IGP are combined in a reactor, and subsequently, under second specific process conditions, in the second stage of the one-pot reaction, enzymes associated with steps (vi), (a), and (b1) disclosed herein are added to the reactor for the production of indigo glycosides; wherein the first specific process conditions and the second specified process conditions refer to two different temperatures; and wherein ●Step (iii) includes N-phosphopribosyl-o-aminobenzoic acid synthase (TrpD) having at least 90% sequence identity with SEQ ID NO.14. ●Step (iv) includes an N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (trpF) having at least 90% sequence identity with SEQ ID NO.22. ●Step (v) includes indole-3-glycerol phosphate synthase (trpC) having at least 90% sequence identity with SEQ ID NO.24. ●Step (vi) includes an indole-3-glycerol-phosphate lyase (IGL) having at least 90% sequence identity with SEQ ID NO.30. ●Step (a) comprises a flavin-containing monooxygenase (FMO) having at least 90% sequence identity with SEQ ID NO.32, and ● Step (b1) comprises a glycosyltransferase having at least 75% sequence identity with SEQ ID NO.46, wherein the amino acid sequence of said glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K and G430K relative to amino acid residues of SEQ ID NO.46, and further comprises (i) substitutions of F381V, T388C and A399C relative to amino acid residues of SEQ ID NO.46, or (ii) substitutions of F381V and T388A relative to amino acid residues of SEQ ID NO.46, or (iii) substitution of T388A relative to amino acid residues of SEQ ID NO.46.

[0132] In one such embodiment, the first specific process condition is preferably a temperature between 50-60°C, more preferably between 50-55°C, and the second specific reaction condition is preferably a temperature between 25-35°C, more preferably between 25-30°C.

[0133] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo, comprising the following steps: - Provides ribose, anthranilate, and ATP, and if an oxidoreductase is used as an oxidase, provides NADPH. - Perform enzymatic conversion: (ii) R5P is converted to phosphoribosyl pyrophosphate (PRPP), which is promoted by phosphoribosyl pyrophosphate synthase. (iii) PRPP is converted to N-phospribosyl-anaminobenzoic acid (N-PRA), which is promoted by N-phospribosyl-anaminobenzoic acid synthase. (iv) N-PRA is converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP), promoted by N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase. (v) CdRP is converted to indole-3-glycerophosphate (IGP), which is promoted by indole-3-glycerophosphate synthase. (vi) IGP is converted to indole, promoted by indole-3-glycerol-phosphate lyase, and (a) Indole is converted to indophenol, promoted by oxidases; and - Expose indophenol to air to convert it into indigo; And optional - ATP regeneration, through the provision of PolyP n And in which ATP regeneration is promoted by ADP kinase and AMP kinase, and / or - NADPH regeneration, if an oxidoreductase is used as the oxidase, is achieved by providing formic acid, and where NADPH regeneration is promoted by formic acid dehydrogenase. - Preferably, ribose-5-phosphate (R5P) is provided by converting ribose, glucose or starch into R5P, as disclosed herein.

[0134] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo, comprising the following steps: - Provides ribose, anthranilate, ATP, and NADPH - Perform enzymatic conversion: (ii) R5P is converted to phosphoribosyl pyrophosphate (PRPP) by a polypeptide having phosphoribosyl pyrophosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 12. (iii) PRPP is converted to N-phospribosyl anthranilic acid (N-PRA) by a polypeptide having N-phospribosyl anthranilic acid synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 14. (iv) N-PRA is converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP), promoted by a polypeptide having N-(5'-phospribosyl)-o-aminobenzoic acid isomerase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 20. (v) CdRP is converted to indole-3-glycerophosphate (IGP) by a polypeptide having indole-3-glycerophosphate synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 26. (vi) IGP is converted to indole, promoted by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 28, and (a) The conversion of indole to indophenol, facilitated by a polypeptide having oxidative activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32; and - Expose indophenol to air to convert it into indigo; And optional - ATP regeneration, through the provision of PolyP n And wherein ATP regeneration is promoted by a polypeptide having ADP kinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 60 and a polypeptide having AMP kinase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 60, and / or - NADPH regeneration is achieved by providing formic acid, wherein NADPH regeneration is promoted by a polypeptide having formic acid dehydrogenase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56. - Preferably, ribose-5-phosphate (R5P) is provided by the conversion of ribose to R5P, wherein the conversion of ribose to ribose-5-phosphate (R5P) is promoted by a polypeptide having ribokinase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO. 6.

[0135] In one embodiment, all the enzymatic steps of the in vitro production of indigo disclosed herein are carried out in a one-pot reaction.

[0136] In one embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) disclosed herein for the production of indigo are carried out in a one-pot reaction. In another embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) disclosed herein, along with the regeneration system, are carried out in a one-pot reaction.

[0137] In this one-pot reaction, the intermediate products do not need to be purified or separated before proceeding to the next enzymatic conversion step.

[0138] In one embodiment, a continuous one-pot synthesis can be used, wherein one or more enzymes, substrates and / or cofactors are added to the reactor one at a time to optimize the reaction conditions of the selected reaction steps.

[0139] In one embodiment, a continuous one-pot process is performed, wherein one or more enzymes, substrates, and / or cofactors are added sequentially. Such a continuous one-pot process may include multiple stages, each with different process conditions, such as different temperature and / or pH conditions.

[0140] In one specific embodiment, indigo is produced in a continuous one-pot process, wherein, under first specific process conditions, in the first stage of the one-pot reaction, enzymes associated with steps (iii), (iv), and (v) disclosed herein for the production of IGP are combined in a reactor, and subsequently, under second specific process conditions, in the second stage of the one-pot reaction, enzymes associated with steps (vi) and (a) disclosed herein are added to a reactor for the production of indophenol; wherein the first specific process conditions and the second specific process conditions refer to two different temperatures; and wherein the second specific process conditions facilitate the formation of indigo by exposing indophenol to air; and wherein ●Step (iii) includes N-phosphopribosyl-o-aminobenzoic acid synthase (TrpD) having at least 90% sequence identity with SEQ ID NO.14. ●Step (iv) includes an N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (trpF) having at least 90% sequence identity with SEQ ID NO.22. ●Step (v) includes indole-3-glycerol phosphate synthase (trpC) having at least 90% sequence identity with SEQ ID NO.24. ●Step (vi) includes an indole-3-glycerol-phosphate lyase (IGL) having at least 90% sequence identity with SEQ ID NO.30. ●Step (a) comprises a flavin-containing monooxygenase (FMO) having at least 90% sequence identity with SEQ ID NO.32, and In one such embodiment, the first specific process condition is preferably a temperature between 50-60°C, more preferably between 50-55°C, and the second specific reaction condition is preferably a temperature between 25-35°C, more preferably between 25-30°C.

[0141] III.iii Synthesis of indigo from indigo glycosides In one implementation, indigo can be produced from indigo glycosides via the following steps. (b3) Converting indigo glycosides to indophenol, and (b2) Indophenol is converted into indigo by exposure to oxygen. The indigo glycosides mentioned herein are produced as disclosed in Parts III.i or III.ii of this document.

[0142] β-glucosidase (BGL) catalyzes the cleavage of glycosidic bonds and is used in step (b3) to remove glucose from indoside. This removal of the (protective) sugar molecule converts the compound back to its reactive form—indophenol—which can spontaneously dimerize under aerobic conditions.

[0143] In one embodiment, step (b3) is promoted by β-glucosidase (BLG). In one embodiment, the β-glucosidase is selected from the enzyme group classified as EC 3.2.1.21.

[0144] In step (b3), indophenol is synthesized from indinoside using a β-glucosidase. In one embodiment, in step (b3), indinoside is enzymatically converted to indophenol by a polypeptide having β-glucosidase activity (EC 3.2.1.21). The amino acid sequence of said polypeptide having β-glucosidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 70 or SEQ ID NO. 72. Preferably, the amino acid sequence of said polypeptide having β-glucosidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 72. In one embodiment, the β-glucosidase is selected from SEQ ID NO. 70 and SEQ ID NO. 72, preferably SEQ ID NO. 72.

[0145] In one embodiment, the present invention relates to an in vitro method for producing indigo, comprising the following steps: - Provides ribose, anthranilate, and ATP, and if an oxidoreductase is used as an oxidase, provides NADPH. - Perform enzymatic conversion: (ii) The conversion of R5P to phosphoribosyl pyrophosphate (PRPP) is promoted by phosphoribosyl pyrophosphate synthase. (iii) The conversion of PRPP to N-phosphoribosyl-anaminobenzoic acid (N-PRA) is promoted by N-phosphoribosyl-anaminobenzoic acid synthase. (iv) N-PRA is converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP) by N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase. (v) CdRP is converted to indole-3-glycerophosphate (IGP) by indole-3-glycerophosphate synthase. (vi) IGP is converted to indole by indole-3-glycerol-phosphate lyase. (a) The conversion of indole to indolephenol is promoted by oxidase. (b1) The conversion of indophenol to indoleglycoside is promoted by glycosyltransferase, and (b3) Enzymatically converting indigoglycosides to indophenol; and - Expose indophenol to air to convert it into indigo; And optionally - ATP regeneration, through the provision of PolyP n And in which ATP regeneration is promoted by ADP kinase and AMP kinase, and / or -Regeneration of UDP-Glc by providing sucrose, wherein UDP-Glc regeneration is promoted by sucrose synthase, and / or - NADPH regeneration, if an oxidoreductase is used as the oxidase, is achieved by providing formic acid, and where NADPH regeneration is promoted by formic acid dehydrogenase. - Preferably, ribose-5-phosphate (R5P) is provided by converting ribose, glucose or starch into R5P, as disclosed herein.

[0146] IV. Synthesis of indigo glycosides from tryptophan In another aspect, the present invention provides a method for the in vitro production of indigo glycosides. For example... Figure 6 As shown, indigo glycosides can be produced by tryptophan enzymes.

[0147] In one implementation, the in vitro production of indigo glycosides includes: (t) converts tryptophan to indole via enzymatic activation. (a) Enzymatically converting indole to indolephenol, and (b1) Enzymatic conversion of indophenol to indoleglycoside.

[0148] For the synthesis of indole, the substrate is tryptophan, and the cofactors NADPH and UDP-Glc are required in steps (a) and (b1), respectively. NADPH and UDP-Glc can be regenerated, such as... Figure 6 As shown.

[0149] In one embodiment, step (t) is promoted by L-tryptophan indole lyase (TIL). In step (i), indole is synthesized from tryptophan using L-tryptophan indole lyase. In one embodiment, tryptophan is enzymatically converted to indole and pyruvate in step (i) 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 with SEQ ID NO. 64, SEQ ID NO. 66, or SEQ ID NO. 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 with SEQ ID NO. 64. In one embodiment, the L-tryptophan indole lyase is selected from SEQ ID NO.64, SEQ ID NO.66 and SEQ ID NO.68, preferably SEQ ID NO.64.

[0150] In one embodiment, a flavin-containing monooxygenase (FMO) facilitates step (a). NADPH is required as a cofactor / redox chaperone. In step (a), indole is synthesized from indole and NADPH using a flavin-containing monooxygenase. In one embodiment, indole and NADPH are enzymatically converted into indole and NADP by a peptidase having flavin-containing monooxygenase activity (EC 1.14.13.8) in step (a). + The amino acid sequence of the polypeptide having flavin monooxygenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32, SEQ ID NO. 34, or SEQ ID NO. 36. Preferably, the amino acid sequence of the polypeptide having flavin monooxygenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32. In one embodiment, the flavin monooxygenase is selected from SEQ ID NO. 32, SEQ ID NO. 34, and SEQ ID NO. 36, preferably SEQ ID NO. 32.

[0151] In another embodiment, step (a) is promoted by a cytochrome P450 enzyme. NADPH is required as a cofactor. In step (a), indole is synthesized from indole and NADPH using a cytochrome P450 enzyme (EC 1.14.14.1). In one embodiment, indole and NADPH are enzymatically converted into indole and NADP by a polypeptide (EC 1.14.14.1) having P450 enzyme activity in step (a). + The amino acid sequence of the polypeptide having P450 enzyme activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 38 or SEQ ID NO. 40. Preferably, the amino acid sequence of the polypeptide having P450 enzyme activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 38. In one embodiment, the cytochrome P450 enzyme is selected from SEQ ID NO. 38 and SEQ ID NO. 40, preferably SEQ ID NO. 38.

[0152] The cofactor NADPH can be obtained from NADP through an enzymatic cofactor recycling system. + For regeneration, the system uses NADPH regenerating enzymes, such as dehydrogenases, preferably formate dehydrogenase (FDH) (EC 1.17.1.10).

[0153] In one embodiment, the combination of formate dehydrogenase (FDH) and formate promotes the regeneration of NADPH from step (a) of the method. In one embodiment, NADP... + The polypeptide is enzymatically converted to NADPH by a polypeptide having formate dehydrogenase activity (EC 1.17.1.10). The amino acid sequence of the polypeptide having formate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56. Preferably, the amino acid sequence of the polypeptide having formate dehydrogenase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56. In one embodiment, the formate dehydrogenase is selected from SEQ ID NO. 56, preferably SEQ ID NO. 56.

[0154] In another embodiment, step (a) is promoted by a peroxidase. Hydrogen peroxide is required as a cofactor. In step (a), indophenol is synthesized from indole and H2O2 using a peroxidase (EC 1.11.2.1). In one embodiment, indole and H2O2 are enzymatically converted to indophenol and H2O by a polypeptide having peroxidase activity (EC 1.11.2.1) in step (a). The amino acid sequence of the polypeptide having peroxidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 42 or SEQ ID NO. 44. Preferably, the amino acid sequence of the polypeptide having peroxidase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 42. In one embodiment, the peroxidase is selected from SEQ ID NO. 42 and SEQ ID NO. 44, preferably SEQ ID NO. 42.

[0155] In one embodiment, step (b1) is promoted by a glycosyltransferase (GT) (EC 2.4), such as UDP-glycosyltransferase (EC 2.4.1), preferably the glycosyltransferase disclosed in Bidart et al. 2023 and WO 2023 / 161230.

[0156] In a preferred embodiment, indophenol is enzymatically converted to indoleglycoside by a polypeptide having glycosyltransferase activity (EC 2.4) in step (b1). In one embodiment, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 48, SEQ ID NO. 50, or SEQ ID NO. 52. In one embodiment, the glycosyltransferase is selected from SEQ ID NO. 48, SEQ ID NO. 50, and SEQ ID NO. 52.

[0157] Preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70, 75, 80, 85, 90, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises one or more amino acid residue substitutions selected from single mutations E75P, Q86K, Q86R, S110V, I188L, G222D, F381V, T388A, S413K, and G430K relative to SEQ ID NO. 46, and / or selected from double mutations T146C+M148C, P190C+A198C, D193C+N196C, G296L+V297G relative to SEQ ID NO. 46, and / or selected from combinations of one or more single mutations and / or double mutations; and wherein the polypeptide having glycosyltransferase activity has enhanced thermostability compared to SEQ ID NO. 46.

[0158] More preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues of SEQ ID NO. 46, and wherein the polypeptide having glycosyltransferase activity has enhanced thermostability compared to SEQ ID NO. 46.

[0159] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO. 46, and further comprises... (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, or (iv) Substitution of T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (v) Substitution of T146C, M148C, F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (vi) Substitution of amino acid residues T146C, M148C, and T388A relative to SEQ ID NO.46, Furthermore, the polypeptide containing glycosyltransferase activity exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0160] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO. 46, and further comprises... (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) Substitution of T388A with respect to the amino acid residue in SEQ ID NO.46, or Furthermore, the polypeptide containing glycosyltransferase activity exhibits enhanced thermal stability compared to SEQ ID NO.46.

[0161] Most preferably, the amino acid sequence of the polypeptide having glycosyltransferase activity has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, T388A, S413K, and G430K relative to the amino acid residues of SEQ ID NO. 46, and wherein the polypeptide having glycosyltransferase activity has enhanced thermostability compared to SEQ ID NO. 46.

[0162] In one embodiment, as described above, a glycosyltransferase (GT) facilitates step (b1). Preferably, the glycosyltransferase is a UDP-dependent glycosyltransferase. UDP-Glc is used in this reaction to link the glucose moiety to an indophenol compound, thereby forming indinoside. In step (b1), indinoside is synthesized from indophenol and UDP-Glc using this UDP-glycosyltransferase. UDP-Glc is converted to UDP.

[0163] UDP-Glc can be regenerated from UDP using an enzyme recycling system that utilizes sucrose synthase (EC 2.4.1.13).

[0164] In one embodiment, the combination of sucrase synthase (SuSy) and sucrose promotes the regeneration of UDP-Glc from step (b1) of the method. In one embodiment, UDP is enzymatically converted to UDP-Glc by a polypeptide (EC 2.4.1.13) having sucrase synthase activity. The amino acid sequence of said polypeptide having sucrase synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54. Preferably, the amino acid sequence of said polypeptide having sucrase synthase activity preferably has at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54. In one embodiment, the sucrase synthase is selected from SEQ ID NO. 54, preferably SEQ ID NO. 54.

[0165] In one embodiment, the present invention provides a method for in vitro production of indigo glycosides, comprising (I) providing tryptophan, NADPH, UDP-GLC, an oxidase, and a glycosyltransferase, (II) mixing the substrate, cofactor, and enzyme, and (III) allowing the mixture to react at a selected temperature and time to produce indigo glycosides.

[0166] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo glycosides, comprising the following steps: - Provides tryptophan and UDP-Glc, and NADP if the oxidoreductase is used as an oxidase. - Perform enzymatic conversion: (t) Tryptophan is enzymatically converted to indole, which is promoted by L-tryptophan indole lyase; (a) Indole is converted to indophenol, promoted by oxidases; and (b1) The conversion of indophenol to indoleglycoside is promoted by glycosyltransferase; And optional -Regeneration of UDP-Glc by providing sucrose, wherein UDP-Glc regeneration is promoted by sucrose synthase, and / or - NADPH regeneration, if an oxidoreductase is used as an oxidase, is achieved by providing formic acid, and where NADPH regeneration is promoted by a dehydrogenase.

[0167] In a preferred embodiment, the present invention relates to a method for in vitro production of indigo glycosides, comprising the following steps: - Provides tryptophan, NADPH, and UDP-Glc. - Perform enzymatic conversion: (t) Tryptophan is enzymatically converted to indole by a polypeptide having L-tryptophan indole lyase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 64. (a) The conversion of indole to indophenol, facilitated by a polypeptide having oxidative activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 32; and (b1) Indophenol is converted to indoleglycoside by a polypeptide having glycosyltransferase activity and having at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.52. And optional - NADPH regeneration is achieved by providing formic acid, and a polypeptide having formic acid dehydrogenase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 56 promotes NADPH regeneration, and / or - UDP-Glc is regenerated by providing sucrose, wherein a polypeptide having sucrose synthase activity and having at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO. 54 promotes UDP-Glc regeneration.

[0168] V. The compositions of the present invention In one embodiment, the present invention provides composition A, comprising... (i) ribokinase, (ii) Phosphoribose pyrophosphate synthase (iii) N-phosphoribosyl-o-aminobenzoic acid synthase (iv) N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (v) indole-3-glycerol phosphate synthase, and (vi) Indole-3-glycerol-phospholyase; And optional ADP kinase and AMP kinase.

[0169] In one embodiment, composition A further comprises ribose, anthranilate, ATP, and optionally PolyP. n When combined with substrates ribose and anthranilate, cofactor ATP, and optional substrate PolyP... n When combined, composition A is suitable for preparing indole.

[0170] In one embodiment, the present invention provides composition B, comprising... (i) ribokinase, (ii) Phosphoribose pyrophosphate synthase (iii) N-phosphoribosyl-o-aminobenzoic acid synthase (iv) N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (v) Indole-3-glycerol phosphate synthase, (vi) Indole-3-glycerol-phosphate lyase, and (a) An oxidase selected from oxidoreductases and peroxidases, such as flavin-containing monooxygenase (FMO), cytochrome P450 enzyme, and nonspecific peroxidase. And optional ADP kinase and AMP kinase, and / or If the oxidase is an oxidoreductase, then it is formate dehydrogenase.

[0171] In one embodiment, composition B further comprises ribose, anthranilate, ATP and NADPH (if the oxidase is an oxidoreductase), and optionally PolyP. n And / or formic acid. When used with substrates ribose, anthranilate, and cofactors ATP and NADPH, and optionally the substrate PolyP n Composition B, when combined with / or formic acid, is suitable for the preparation of indigo.

[0172] In one embodiment, the present invention provides composition C, comprising... (i) ribokinase, (ii) Phosphoribose pyrophosphate synthase (iii) N-phosphoribosyl-o-aminobenzoic acid synthase (iv) N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (v) Indole-3-glycerol phosphate synthase, (vi) Indole-3-glycerol-phosphate lyase, (a) Oxidases selected from oxidoreductases and peroxidases, such as flavin-containing monooxygenases (FMOs), cytochrome P450 enzymes, nonspecific peroxidases, and (b1) Glycosyltransferases; And optional ADP kinase and AMP kinase, and / or Sucrose synthase, and / or If the oxidase is an oxidoreductase, then it is formate dehydrogenase.

[0173] In one embodiment, composition C further comprises ribose, anthranilate, UDP-Glc, ATP and NADPH (if the oxidase is a redox enzyme), and optionally PolyP. n And / or sucrose and / or formic acid. When used with substrate ribose, anthranilate and UDP-Glc, cofactors ATP and NADPH (if the oxidase is an oxidoreductase), and optionally substrate PolyP. n Composition C is suitable for the preparation of indigo glycosides when combined with formic acid and / or sucrose.

[0174] In one embodiment, the present invention provides composition D, comprising... (i) L-tryptophan indole lyase, (a) Oxidases selected from flavin monooxygenase (FMO), nonspecific peroxidase (UPO), and cytochrome P450 enzyme (P450), and (b1) Glycosyltransferases; And optional Sucrose synthase and / or If the oxidase is an oxidoreductase, then it is formate dehydrogenase.

[0175] In one embodiment, composition D further comprises tryptophan, UDP-Glc, and NADPH (if the oxidase is an oxidoreductase), and optionally sucrose and / or formic acid. When combined with the substrates tryptophan and UDP-Glc, the cofactor NADPH (if the oxidase is an oxidoreductase), and optionally the substrates formic acid and / or sucrose, composition D is suitable for the preparation of indigo glycosides.

[0176] VI. One-pot or multi-pot enzymatic cascade reactions In a preferred embodiment, the steps (i), (ii), (iii), (iv), (v), and (vi) disclosed herein for the production of indole are carried out in a one-pot reaction. In another preferred embodiment, the steps (i), (ii), (iii), (iv), (v), and (vi) disclosed herein, along with the ATP regeneration system, are carried out in a one-pot reaction.

[0177] In another preferred embodiment, the enzymatic cascade for the production of indole is carried out in a two-pot system, wherein steps (i), (ii), (iii), (iv), (v) and (vi) disclosed herein for the production of indole are carried out in the first-pot reaction, while the ATP regeneration system disclosed herein is carried out in the second-pot reaction.

[0178] In a preferred embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b1) disclosed herein for the production of indigo glycosides are carried out in a one-pot reaction.

[0179] In another embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein, along with one or more ATP, NADPH, and UDP-Glc regeneration systems, are carried out in a single-pot reaction.

[0180] In another further embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein, as well as two or more of the ATP, NADPH, and UDP-Glc regeneration systems, are carried out in a one-pot reaction.

[0181] In another further embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b1) disclosed herein, as well as the ATP, NADPH and UDP-Glc regeneration system, are carried out in a one-pot reaction.

[0182] In another preferred embodiment, the enzymatic cascade for the production of indigo glycosides is carried out in a multipot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein for the production of indigo glycosides are carried out in a one-pot reaction, while the ATP, NADPH, and UDP-Glc regeneration system disclosed herein is carried out in a separate one-pot or multipot reaction.

[0183] In another preferred embodiment, the enzymatic cascade for the production of indigo glycosides is carried out in a multipot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b1) disclosed herein for the production of indigo glycosides are carried out in the first pot reaction, while one or more of the ATP, NADPH, and UDP-Glc regeneration systems disclosed herein are carried out in the second pot reaction.

[0184] In another preferred embodiment, the enzymatic cascade for the production of indigo glycosides is carried out in a two-pot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b1) disclosed herein for the production of indigo glycosides are carried out in the first pot reaction, while at least the ATP regeneration system disclosed herein is carried out in the second pot.

[0185] In another preferred embodiment, the enzymatic cascade for the production of indigo glycosides is carried out in a multi-pot system, wherein - In the first pot, the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b1) disclosed herein for the production of indigo glycosides are carried out. -The ATP regeneration system disclosed in this paper is carried out in the second pot. - The NADPH regeneration system disclosed in this paper is carried out in the first, second, or third pot, and - The UDP-Glc regeneration system disclosed in this paper is carried out in the first, second, third, or fourth pot.

[0186] In a preferred embodiment, the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) disclosed herein for the production of indigo are carried out in a one-pot reaction. In another embodiment, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2), as well as the associated cofactor regeneration system disclosed herein, are carried out in a one-pot reaction. In such an embodiment, as disclosed herein, steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2), as well as ATP regeneration and NADPH regeneration (if an oxidoreductase is used in step (a)), are all carried out in a one-pot reaction.

[0187] In another preferred embodiment, the enzymatic cascade for producing indigo is carried out in a multipot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a), and (b2) for producing indigo disclosed herein are carried out in a one-pot reaction, while the ATP and NADPH regeneration system disclosed herein is carried out in a separate one-pot or two-pot reaction.

[0188] In another preferred embodiment, the enzymatic cascade for producing indigo is carried out in a two-pot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) disclosed herein for producing indigo are carried out in the first pot reaction, while one or both of the ATP and NADPH regeneration systems disclosed herein are carried out in the second pot reaction.

[0189] In another preferred embodiment, the enzymatic cascade for producing indigo is carried out in a two-pot system, wherein the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) disclosed herein for producing indigo are carried out in the first pot reaction, while at least the ATP regeneration system disclosed herein is carried out in the second pot.

[0190] In another preferred embodiment, the enzymatic cascade for producing indigo is carried out in a multi-pot system, wherein - In the first pot, the steps (i), (ii), (iii), (iv), (v), (vi), (a) and (b2) disclosed herein for the production of indigo are carried out. -The ATP regeneration system disclosed in this paper is carried out in the second pot. - The NADPH regeneration system disclosed in this paper is carried out in the first, second, or third pot.

[0191] In a preferred embodiment, the steps (t), (a) and (b1) disclosed herein for the production of indigo glycosides are carried out in a one-pot reaction.

[0192] In another embodiment, the steps (t), (a), and (b1) disclosed herein, along with one or more NADPH and UDP-Glc regeneration systems, are carried out in a single-pot reaction.

[0193] In one embodiment, two or more enzymatic conversion steps occur in a one-pot reaction. In one embodiment, three or more enzymatic conversion steps occur in a one-pot reaction. In one embodiment, four or more enzymatic conversion steps occur in a one-pot reaction. In one embodiment, five or more enzymatic conversion steps occur in a one-pot reaction. In one embodiment, six or more enzymatic conversion steps occur in a one-pot reaction. In one embodiment, seven or more enzymatic conversion steps occur in a one-pot reaction. This can be combined with one or more of the ATP, NADPH, and UDP-Glc regeneration systems disclosed herein.

[0194] In a preferred embodiment, all the enzymatic steps disclosed herein for the in vitro production of indigo glycosides and / or indigo are carried out in a one-pot reaction. In such a one-pot reaction, the intermediate products do not need to be purified or separated before proceeding to the next enzymatic conversion step.

[0195] In one embodiment, a continuous one-pot synthesis can be used, wherein one or more enzymes, substrates and / or cofactors are added to the reactor one at a time to optimize the reaction conditions of the selected reaction steps.

[0196] One-pot biosynthesis increases efficiency because various reactions or transformations can occur sequentially or simultaneously in the same reaction vessel, allowing for the formation of complex molecules or materials in a more efficient manner. This is essential to avoid lengthy separation and purification processes of intermediate compounds, saving time and resources while increasing yield.

[0197] Designing efficient one-pot synthesis methods is challenging because it requires managing multiple reactions and intermediates within the same reaction environment while avoiding side reactions or complexities. This invention overcomes these challenges and provides a solution in which multiple enzymatic reactions are successfully integrated in the biosynthesis of indole and optionally indigo glycosides and / or indigo.

[0198] The enzymes disclosed herein for use in in vitro methods are available from commercial manufacturers or may be microbially produced (and optionally purified), and can be directly applied to the in vitro methods of this invention. Those skilled in the art will know how to produce such enzymes microorganisms based on the nucleotide and amino acid sequences provided herein.

[0199] The products and intermediate compounds produced by the method of this invention can be detected by HLPC-UV, LC-MS, NMR or similar equipment recognized by those skilled in the art.

[0200] VII. Enzyme List Table 1 provides a list of enzymes suitable for use in this invention.

[0201]

[0202] The DNA sequences encoding these enzymes in the sequence listing are synthetic constructs prepared from the amino acid sequences of the enzymes produced by the organisms listed in Table 1.

[0203] VIII. Numbering Implementation Scheme Implementation Scheme 1. An in vitro method for producing indole and optionally indigo glycosides and / or indigo, comprising the following steps: (i) Provides ribose-5-phosphate (R5P), (ii) R5P is enzymatically converted into phosphoribosyl pyrophosphate (PRPP). (iii) PRPP is enzymatically converted into N-phosphoribosyl anthranilic acid (N-PRA). (iv) N-PRA was enzymatically converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP). (v) CdRP is enzymatically converted to indole-3-glycerophosphate (IGP), and (vi) IGP is enzymatically converted into indole. And optionally include the following steps (a) Enzymatically converting indole to indolephenol, and (b1) Enzymatically converting indophenol to indole, and / or (b2) converting indophenol to indole by exposure to oxygen.

[0204] Implementation Scheme 2. The in vitro method according to Implementation Scheme 1, wherein the ribose-5-phosphate (R5P) in step (i) is provided by means of (I) Converting riboproteins to R5P via enzymatic reaction, or (II) Enzymatically convert glucose or starch into glucose-6-phosphate (G6P), enzymatically convert G6P into 6-phosphogluconic acid (6PG), enzymatically convert 6PG into ribulose-5-phosphate (Ru5P), and enzymatically convert Ru5P into R5P.

[0205] Implementation Scheme 3. The in vitro method according to Implementation Scheme 1 or 2, wherein... Step (ii) is promoted by phosphoribosyl pyrophosphate synthase (PRS). Step (iii) is promoted by N-phosphoribosyl-o-aminobenzoic acid synthase (trpD). Step (iv) is promoted by N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (trpF). Step (v) is promoted by indole-3-glycerol phosphate synthase (trpC), and Step (vi) is promoted by indole-3-glycerol-phosphate lyase (IGL).

[0206] Implementation Scheme 4. The in vitro method according to Implementation Scheme 2 or 3, wherein For (I) the conversion of ribose to R5P promoted by ribokinase (RK), and For (II), glucose is converted to G6P by hexokinase (HK); starch is converted to G6P by α-amylase, glucosylamylase and hexokinase (HK) or α-glucan phosphorylase (aGP) and phosphoglucose mutase (PGM); G6P is converted to 6PG by glucose-6-phosphate dehydrogenase (G6PDH); 6PG is converted to RU5P by 6-phosphoglucate dehydrogenase (6PGDH); and RU5P is converted to R5P by ribose-5-phosphate isomerase (RPI).

[0207] Implementation Scheme 5. The in vitro method according to Implementation Scheme 3 or 4, wherein ● The amino acid sequence of the phosphoribosyl pyrophosphate synthase (PRS) described herein has at least 90% sequence identity with SEQ ID NO. 8, SEQ ID NO. 10, or SEQ ID NO. 12. ● The amino acid sequence of the N-phosphoribosyl anthranilate synthase (trpD) has at least 90% sequence identity with SEQ ID NO.14, SEQ ID NO.16, or SEQ ID NO.18. ● The amino acid sequence of the N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (trpF) has at least 90% sequence identity with SEQ ID NO. 20 or SEQ ID NO. 22. ● The amino acid sequence of the indole-3-glycerol phosphate synthase (trpC) has at least 90% sequence identity with SEQ ID NO.24 or SEQ ID NO.26, and ●The amino acid sequence of the indole-3-glycerol-phosphate lyase (IGL) has at least 90% sequence identity with SEQ ID NO.28 or SEQ ID NO.30.

[0208] Implementation Scheme 6. The in vitro method according to Implementation Scheme 4 or 5, wherein... ● The amino acid sequence of the ribokinase (RK) has at least 90% sequence identity with SEQ ID NO.2, SEQ ID NO.4, or SEQ ID NO.6. ● The amino acid sequence of the hexokinase (HK) has at least 90% sequence identity with SEQ ID NO. 74 or SEQ ID NO. 76. ●The amino acid sequence of the α-amylase has at least 90% sequence identity with SEQ ID NO. 90. ●The amino acid sequence of the glucosylamylase described has at least 90% sequence identity with SEQ ID NO.92. ● The amino acid sequence of the α-glucan phosphorylase (aGP) has at least 90% sequence identity with SEQ ID NO. 94. ● The amino acid sequence of the glucose-1,4-phosphate mutase (PGM) has at least 90% sequence identity with SEQ ID NO. 96. ●The amino acid sequence of the glucose-6-phosphate dehydrogenase (G6PDH) has at least 90% sequence identity with SEQ ID NO.78 or SEQ ID NO.80.

[0209] ● The amino acid sequence of the 6-phosphoglucate dehydrogenase (6PGDH) has at least 90% sequence identity with SEQ ID NO. 82 or SEQ ID NO. 84, and ●The amino acid sequence of the ribose 5-phosphoisomerase (RPI) has at least 90% sequence identity with SEQ ID NO.86 or SEQ ID NO.88.

[0210] Scheme 7. The in vitro method according to any one of Schemes 2-6, wherein steps (i) and (ii) are further promoted by an ATP regenerating enzyme, such as a phosphokinase, preferably a class 2 polyphosphokinase (PPK2) (EC 2.7.4.1).

[0211] Scheme 8. An in vitro method according to any one of Schemes 1-7, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a), and (b2), wherein indole is an intermediate compound, and wherein indigo is produced.

[0212] Scheme 9. An in vitro method according to any one of Schemes 1-8, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a), and (b1), wherein indole is an intermediate compound, and wherein indole is produced.

[0213] Implementation Scheme 10. An in vitro method according to Implementation Scheme 8 or 9, wherein step (a) is promoted by an oxidase, such as an oxidase selected from oxidoreductase (EC 1), monooxygenase (EC 1.14), flavin-containing monooxygenase (FMO) (EC 1.14.13.8), P450 (EC 1.14.14.1) and nonspecific peroxidase (EC 1.11.2.1).

[0214] Scheme 11. The in vitro method according to Scheme 10, wherein the oxidase is an oxidoreductase, and step (a) is further promoted by an NADPH regenerating enzyme, such as a dehydrogenase, preferably formate dehydrogenase (FDH) (EC1.17.1.10).

[0215] Scheme 12. An in vitro method according to any one of Schemes 9-11, wherein step (b1) is promoted by a glycosyltransferase (GT) (EC 2.4.1), such as UDP-glycosyltransferase, 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 substitutions of amino acid residues E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to SEQ ID NO. 46, and further comprises: (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) T388A is substituted relative to the amino acid residue of SEQ ID NO.46.

[0216] Scheme 13. The in vitro method according to any one of Schemes 1-12, wherein two or more enzymatic transformations occur in a one-pot reaction, preferably wherein all enzymatic reactions occur in a one-pot reaction.

[0217] Implementation Scheme 14. An in vitro method for producing indigo glycosides, comprising the following steps: (t) converts tryptophan to indole via enzymatic activation. (a) Enzymatically converting indole to indolephenol, and (b1) Indophenol is enzymatically converted to indoleglycoside.

[0218] Implementation Scheme 15. The in vitro method according to Implementation Scheme 14, wherein Step (t) is promoted by L-tryptophan indole lyase (TIL). Step (a) is promoted by oxidases, such as oxidoreductases (EC 1), monooxygenases (EC 1.14), flavin-containing monooxygenases (FMOs) (EC 1.14.13.8), nonspecific peroxidases (EC 1.11.2.1), or P450 (EC 1.14.14.1), and Step b1 is promoted by a glycosyltransferase (GT) (EC 2.4.1), such as a UDP-glycosyltransferase, preferably a glycosyltransferase, wherein the amino acid sequence of the glycosyltransferase has at least 75% sequence identity with SEQ ID NO.46, and wherein the amino acid sequence of the glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K, and G430K relative to the amino acid residues in SEQ ID NO.46, and further comprises: (i) Substitution of F381V, T388C and A399C with respect to the amino acid residues in SEQ ID NO.46, or (ii) Substitution of F381V and T388A with respect to the amino acid residues in SEQ ID NO.46, or (iii) T388A is substituted relative to the amino acid residue of SEQ ID NO.46.

[0219] Scheme 16. The in vitro method according to Scheme 14 or 15, wherein two or more enzymatic transformations occur in a one-pot reaction, preferably wherein all enzymatic reactions occur in a one-pot reaction.

[0220] Example The selected enzymes were tested below to demonstrate the in vitro biosynthesis of indole, as well as the further biosynthesis of indigo glycosides and indigo.

[0221] Example I: Enzyme Expression and Purification Gene synthesis and cloning were performed by Biomatik (USA). The DNA sequence of the his-tagged protein was codon-optimized for expression in *E. coli* BL21(DE3) and cloned into pET28a using NcoI and XhoI restriction endonucleases. The plasmid was transformed into *E. coli* BL21 Star(DE3) (Fisher Scientific) and the transformants were stored as glycerol at -70°C.

[0222] Overexpression of the target gene was achieved by adding 250 µM IPTG to an E. coli culture, which reached OD200 in 2xYT medium at 37°C (200 rpm). 600 =0.8-1.0. Subsequently, the culture was incubated at 20°C (200 rpm) for 20 hours to aid soluble expression. The culture was harvested and stored at -20°C until further use.

[0223] For purification, the cell pellet was resuspended in 50 mM sodium phosphate buffer (pH = 7.4) and lysed by sonication. After removing cell debris by centrifugation, the clarified lysate was purified by immobilized metal affinity chromatography on an AKTA Pure column equipped with a Histrap FF (Cytiva) column. Subsequently, the buffer was replaced with 25 mM HEPES (pH = 8) buffer, aliquots were rapidly frozen in liquid nitrogen, and stored at -70°C until further use.

[0224] Example II: One-pot production of indole from o-aminobenzoate II.i Indole Formation In a one-pot reaction, the substrate, enzymes, and other reagents were mixed at the concentrations listed in Table 2. The reaction began with anthranilate and PRPP, followed by all enzymes used for the conversion to indole. The reaction mixture was incubated at 25°C for 24 hours. Figure 7 As shown in Figures A and 7B, the concentrations of o-aminobenzoate and indole were measured at different time points using RP-HPLC-DAD. Figure 7 A shows the conversion of 100 µM o-aminobenzoate. Figure 7 B shows the conversion of 1 mM anthranilate.

[0225]

[0226] II.ii Chromatograms confirming the stepwise synthesis of IGP from anthranilate and PRPP Anthranilate (1 mM) and PRPP (5 mM) were mixed, and then enzymes (1 mg / mL) were added sequentially, followed by incubation for 1 hour. The enzymes TkTrpD, EcTrpF, and PaTrpC used were purified according to the method described in Example I. Figure 8 shows the chromatograms obtained by RP-HPLC-DAD, where the anthranilate fraction showed the best absorption at 340 nm, and the indole fraction showed the best absorption at 260 nm.

[0227] according to Figure 8A Upon addition of TrpD, the conversion of anthranilic acid to NPRA was observed, but not 100%. Subsequently, TrpF was added; TrpF should have converted NPRA to CdRP, but no new peak appeared. This was expected, as TrpF is an isomerase, and the compounds are too similar to be separated by the HPLC method used. Then, upon addition of TrpC, the NPRA / CdRP peak disappeared at 340 nm, indicating that the anthranilic acid fraction was no longer present. Furthermore, a new peak with absorption at 260 nm (instead of 340 nm) appeared. Therefore, it can be inferred that CdRP formed IGP under the enzymatic activity of TrpC.

[0228] according to Figure 8B Upon addition of TrpD, anthranilic acid was observed to be converted to NPRA, but not 100% (1). TrpF was subsequently added, which should have converted NPRA to CdRP, but no new peak appeared (2). This was expected, as TrpF is an isomerase and the compounds were too similar to be separated by the HPLC method used. The addition of sodium borohydride selectively reduced CdRP to reduced CdRP (rCdRP), producing a new peak (3). Then, upon addition of TrpC, the NPRA / CdRP peak disappeared at 340 nm, indicating that the anthranilic acid fraction was no longer present. Furthermore, a new peak with absorption at 260 nm (instead of 340 nm) appeared (4). Therefore, it can be inferred that CdRP formed IGP under the enzymatic activity of TrpC. Sodium periodate chemically converted IGP to indole-3-aldehyde (I3A), producing a new peak with a retention time matching that of commercially available I3A (5). The addition of IGL produces indole, which is confirmed by indole as a reference compound (6).

[0229] Example III: One-pot conversion of o-aminobenzoate to indigo In a one-pot reaction, the substrate, enzyme, and other reagents were mixed at concentrations listed in Table 3. The reaction began with anthranilate and PRPP, followed by the addition of all enzymes used for the conversion to indophenol. Indophenol further spontaneously dimerized to indigo by exposure to air. The reaction mixture was incubated at 25°C for 24 hours, and its content was measured directly from the reaction mixture by sequential injection using RP-HPLC. Figure 9 As shown, the concentrations of an-aminobenzoate and indole were measured at different time points. Indole accumulation was observed within the first 200 minutes, indicating that NIFMO was a significant bottleneck step in this setup. The formation of blue and the absence of residual indole further suggest that indigo is formed from an-aminobenzoate.

[0230]

[0231] Example IV: One-pot conversion of ribose to IGP In a one-pot reaction, substrates, enzymes, and other reagents were mixed at concentrations listed in Table 4. Different kinases were tested without ATP cycling. Thermostats were also used at higher incubation temperatures. The reaction began with ribose and anthranilate, followed by all enzymes used for conversion to IGP. Figure 10 As shown, the amount of IGP generated was measured after 1, 2, 4 and 22 hours.

[0232]

[0233] from Figure 10 As can be seen, HoRK and HoPRS work very well. Unfortunately, MtPRS and EcPRS do not work well because they are not pure, such as... Figure 10 As shown, reactions involving these enzymes have lower IGP measurements. hRK and LmRK do indeed play a role.

[0234] Example V: Complete biosynthesis of indigo glycosides In a one-pot reaction, the substrate, enzyme, and other reagents were mixed at the concentrations listed in Table 5. The reaction began with ribose and anthranilate, followed by all enzymes used for the conversion to indigoglycoside. The reaction mixture was incubated at 25°C for 20 hours, and samples were taken for RP-HPLC analysis. The results are as follows: Figure 11 As shown, this indicates that indigo glycoside was successfully formed.

[0235]

[0236] Example VI: Continuous One-Pot Method The TkTrpD used in the previous examples was derived from thermophilic organisms, while EcTrpF and PaTrpC were derived from mesophilic organisms. Enzymatic reactions are expected to occur at high temperatures; therefore, other more thermostable TrpF and TrpC enzymes were tested in combination with TkTrpD.

[0237] The melting temperature (Tm) of the enzyme was determined by differential scanning fluorometry and is listed in Table 6.

[0238]

[0239] [a] Theoretical pH value of 8 and ionic strength of 0.03 M No increase in fluorescence was observed in G [b] by DSF detection at temperatures up to 95°C.

[0240] TkTrpD and SsTrpC have high stability, and the apparent T m The melting temperatures were 73.8 °C and 87.3 °C, respectively. Furthermore, no unfolding of TmTrpF was observed, indicating that TmTrpF remains active after prolonged exposure to heat. NIFMO and MvFDH are thermophilic but relatively stable, with observed melting temperatures of 50.9 °C and 65.7 °C, respectively. ZmBX1 exhibits a relatively low T... m This is expected and can be considered the bottleneck for the optimal temperature in this cascade.

[0241] Therefore, a sequential one-pot method, also referred to in this paper as a two-stage one-pot method, was evaluated. First, the formation of IGP from anthranilate and PRPP was carried out using three enzymes (TkTrpD, TmTrpF, and SsTrpC) derived from thermophilic organisms. Subsequently, the final two steps and cofactor cycling were carried out in the same pot using enzymes (ZmIGL, NIFMO, and MvFDH) derived from mesophilic organisms, allowing for temperature regulation to achieve optimal performance at each stage.

[0242] VI.i. Stage I Optimization - Temperature The substrate, enzyme, and other reagents were mixed in a one-pot reaction at the concentrations listed in Table 7, in triplicate of a total volume of 50 µl. The reaction began with anthranilate and PRPP, followed by the addition of the enzyme for conversion to IGP. IGP was quantified by chemically converting it to indole-3-aldehyde (I3A) using 100 mM sodium periodate dissolved in 100 mM acetic acid, and I3A was measured by RP-HPLC-DAD. The reaction mixture was incubated at temperatures ranging from 50 to 70 °C. Samples were taken at selected time points, and a graph showing the relative formation of IGP over time was plotted. Figure 12 ).

[0243]

[0244] like Figure 12 As shown, the concentration of IGP decreased after 120 minutes of reaction, indicating the thermal instability of the compound at high temperatures. Of course, this also occurred before the 120-minute labeling, but the IGP generation flux was high enough to offset the degradation. The optimal temperature for the first stage was determined to be approximately 55°C.

[0245] VI.ii Stage I Optimization - Enzyme Ratio The substrate, enzyme, and other reagents were mixed in a one-pot reaction at the concentrations listed in Tables 8, 9, and 10, with a total volume of 500 μl. The reaction began with anthranilate and PRPP, followed by the addition of the enzyme for conversion to IGP. The reaction mixture was incubated at 55 °C, the optimal temperature determined for Phase I (see above). IGP was quantified after being chemically converted to indole-3-aldehyde (I3A) by 100 mM sodium periodate. The concentration of I3A at different time points was determined by RP-HPLC-DAD. The relative amount of IGP generated after 4 hours was plotted. Figure 13 ).

[0246] like Figure 13 As shown, TrpC appears to be the bottleneck enzyme in stage I of the cascade reaction.

[0247]

[0248]

[0249]

[0250] VI.iii Further process optimization of stage I - spiking Initially, the optimal conditions and enzyme ratio for 10 mM o-aminobenzoate were determined at 53 °C and a ratio of 4:1:12 (TkTrpD:TmTrpF:SsTrpC). Figure 14 Figure A shows an IGP of nearly 6 mM formed within 90 minutes. The reduced IGP formation was observed, suspected to be due to the thermal instability of PRPP. Figure 14 In sample B, the reaction mixture was replenished with fresh PRPP at 60 minutes, resulting in the formation of nearly 10 mM IGP within 150 minutes, in contrast to the control sample which did not achieve complete conversion. To increase the IGP titer, fresh anthranilate and PRPP were added to the reaction mixture. Figure 14 Figures C and 14D show the results of the reaction mixture with feed every 30 minutes and 60 minutes, respectively. Surprisingly, this did not increase the final titer of IGP, which was limited to 10 mM. Clearly, adding o-aminobenzoate did not increase the product titer, so it was decided to start with a higher substrate concentration, as this was found not to be detrimental to activity. Figure 14 E and 14F show the conversion of 50 mM an-aminobenzoate to IGP, fed every 60 and 30 minutes, respectively. Again, frequent addition of PRPP was observed to increase the titer to nearly 30 mM IGP (8.61 g / L). However, the rate of IGP formation still slowed down over one hour, suspected to be due to poor operational stability of one or more enzymes. The stability of SsTrpC is questionable, as the consumption of an-aminobenzoate was not significantly hindered and TmTrpF remained active after prolonged heat exposure. This is consistent with the accumulation of the compound with a retention time of 2.67 minutes (i.e., the substrate CdRP of SsTrpC) in the HPLC chromatogram. Figure 14 G compared the simultaneous hourly supplementation of PRPP and TrpC with the hourly supplementation of PRPP alone, showing that the titer increased significantly when fresh TrpC was added. Figure 14 H shows the results of the same experiment, demonstrating the reproducibility of the setup. Although the melting temperature of SsTrpC is very high, reaching 87.3℃, its operational stability is still insufficient.

[0251] In summary, the addition of o-aminobenzoate was unsuccessful, but when starting from higher substrate concentrations, the addition of PRPP and SsTrpC increased the IGP titer.

[0252] Phase II Optimization - Temperature In a one-pot reaction, the substrate, enzyme, and other reagents were mixed at the concentrations listed in Table 11, in triplicate, with a total volume of 50 µL. The reaction began with in-situ generated IGP, along with all enzymes used for the conversion to indigo. The reaction mixture was incubated at temperatures ranging from 20 to 40 °C. Indigo was quantified spectrophotometrically at different time points. Figure 15 ).

[0253] like Figure 15 As shown, a reduction in indigo formation was observed at 35 °C and 40 °C, which may be attributed to the thermal instability of ZmBX1 or the operational stability of ZmBX1, NIFMO, or MvFDH. For subsequent experiments, a reaction temperature of 30 °C will be used.

[0254]

[0255] Phase II Optimization - Enzyme Ratio In a one-pot reaction, the substrate, enzyme, and other reagents were mixed at concentrations listed in Tables 12, 13, and 14. The reaction began with in-situ generated IGP, followed by the addition of all enzymes used for the conversion to indigo. The reaction mixture was incubated at 30°C, which was determined to be the optimal temperature for stage II. Indigo was quantified spectrophotometrically at 620 nm. Here, the relative amount of indigo produced after 4 hours is plotted (…). Figure 16 ).

[0256] like Figure 16 As shown, the results indicate that NIFMO is the bottleneck enzyme in the second stage. MvFDH is the most efficient enzyme in this cascade.

[0257]

[0258]

[0259]

[0260] VI.vi Indigo Synthesis - Combination of Stage I and Stage II in a One-Pot Reaction IGP was synthesized in situ using the optimal process conditions determined in this paper (Stage I). The reaction mixture containing anthranilate and PRPP was incubated with enzymes TkTrpD, TmTrpF, and SsTrpC at 55°C and pH 8, with PRPP and SsTrpC added every 30 and 60 minutes, respectively. After Stage I, IGP formation was confirmed by the addition of the remaining enzymes: ZmBX1, NIFMO, and MvFDH, along with the cofactor NADP. + PLP and formic acid are used to regenerate NADPH, thereby initiating the second stage. The reaction mixture is now incubated at 30°C, which has been determined to be the optimal temperature for stage II.

[0261] Example VII: One-pot conversion of tryptophan to indigo glycosides In a one-pot reaction, the substrate, enzyme, and other reagents were mixed at concentrations listed in Table 15. The reaction began with tryptophan, followed by all enzymes used for the conversion to indigoglycoside. The reaction mixture was incubated at 25°C, and samples were taken at different time points for HPLC analysis: 5 min, 10 min, 20 min, 30 min, 60 min, 90 min, and 120 min. Figure 17 As shown, the concentration of indigo glycosides was measured and plotted, demonstrating the successful formation of indigo glycosides from tryptophan.

[0262]

[0263] References Bidart G, Teze D, Jansen C, et al. Chemoenzymatic indican forsustainable light-driven denim dyeing. Research Square; 2023. DOI: 10.21203 / rs.3.rs-2416810 / v1. PCT / EP2023 / 054318: Thermostable glycosyltransferase variants.

Claims

1. An in vitro method for producing indole and optionally indigo glycosides and / or indigo, comprising the following steps: (i) Provides ribose-5-phosphate (R5P), (ii) Using a polypeptide with phosphoribosyl pyrophosphate synthase activity (EC 2.7.6.1), R5P is enzymatically converted to phosphoribosyl pyrophosphate (PRPP). (iii) Provide anthranilate and use anthranilate synthase activity with N-phosphoribosyl anthranilate synthase (trpD); The polypeptide (EC 2.4.2.18) enzymatically converts PRPP to N-phosphoribosyl anthranilic acid (N-PRA). (iv) Using a polypeptide with N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase activity (EC 5.3.1.24), N-PRA was enzymatically converted to 1-(2-carboxyphenylamino)-1-deoxy-D-ribulose 5-phosphate (CdRP). (v) CdRP was enzymatically converted to indole-3-glycerophosphate (IGP) using a polypeptide with indole-3-glycerophosphate synthase activity (EC 4.1.1.48), and (vi) IGP was enzymatically converted to indole using a polypeptide with indole-3-glycerol-phosphate lyase activity (EC 4.1.2.8). The in vitro method described therein is a one-pot reaction for steps (ii)-(vi), wherein the intermediate product is not purified or separated before proceeding to the next enzymatic conversion step.

2. The in vitro method according to claim 1, further comprising the following steps: (a) The indole enzyme is used to convert indole to indophenol using an oxidase, wherein the activity of the enzyme is selected from oxidoreductase activity (EC1), monooxygenase activity (EC 1.14), flavin-containing monooxygenase (FMO) activity (EC 1.14.13.8), P450 activity (EC 1.14.14.1), and nonspecific peroxidase activity (EC 1.11.2.1), and (b1) Providing a glycosyl donor and using a polypeptide with glycosyltransferase (GT) activity (EC 2.4.1) to enzymatically convert indophenol to indigo glycoside, and / or (b2) converting indophenol to indigo by exposure to oxygen. Indole is an intermediate compound, and indigo glycosides and / or indigo are produced therein.

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

4. The in vitro method according to any one of claims 1-3, wherein, ● The amino acid sequence of the polypeptide having phosphoribosyl pyrophosphate synthase activity has at least 90% sequence identity with SEQ ID NO. 8, SEQ ID NO. 10 or SEQ ID NO.

12. ● The amino acid sequence of the polypeptide having N-phospribosyl anthranilate synthase activity has at least 90% sequence identity with SEQ ID NO. 14, SEQ ID NO. 16 or SEQ ID NO.

18. ● The amino acid sequence of the polypeptide having N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase activity has at least 90% sequence identity with SEQ ID NO. 20 or SEQ ID NO.

22. ● The amino acid sequence of the polypeptide having indole-3-glycerol phosphate synthase activity has at least 90% sequence identity with SEQ ID NO. 24 or SEQ ID NO. 26, and ●The amino acid sequence of the polypeptide having indole-3-glycerol-phosphate lyase activity has at least 90% sequence identity with SEQ ID NO.28 or 30.

5. The in vitro method according to claim 3 or 4, wherein, ● The amino acid sequence of the polypeptide having ribokinase activity has at least 90% sequence identity with SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.

6. ● The amino acid sequence of the polypeptide having glucose-6-phosphate dehydrogenase activity has at least 90% sequence identity with SEQ ID NO. 78 or 80. ● The amino acid sequence of the polypeptide having 6-phosphoglucate dehydrogenase activity has at least 90% sequence identity with SEQ ID NO. 82 or 84, and ● The amino acid sequence of the polypeptide having ribose 5-phosphate isomerase activity has at least 90% sequence identity with SEQ ID NO. 86 or SEQ ID NO.

88.

6. The in vitro method according to any one of claims 1-5, wherein steps (i) and (ii) are further promoted by an ATP regenerating enzyme, said ATP regenerating enzyme being, for example, a polypeptide having phosphokinase activity, preferably a class 2 polyphosphokinase (PPK2) (EC2.7.4.1).

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

8. The in vitro method according to any one of claims 2-7, wherein step (b1) is promoted by UDP-glycosyltransferase and the glycosyl donor is UDP-glucose.

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

46.

10. The in vitro method according to any one of claims 1-9, wherein all enzymes, substrates and cofactors are simultaneously combined in a one-pot reaction.

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

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 indole is produced, and wherein the continuous one-pot reaction comprises a first stage and a second stage, wherein steps (iii), (iv), and (v) are carried out in the first stage at a temperature between 50 and 55°C, and steps (vi), (a), and (b2) are carried out in the second stage at a temperature between 25 and 30°C, wherein indole formation is promoted in the second stage by exposing indolephenol to air; and wherein... ●Step (iii) is promoted by a polypeptide having N-phospribosyl o-aminobenzoic acid synthase activity and having at least 90% sequence identity with SEQ ID NO.

14. ●Step (iv) is promoted by a polypeptide having N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase activity and having at least 90% sequence identity with SEQ ID NO.

22. ●Step (v) is promoted by a polypeptide having indole-3-glycerol phosphate synthase activity and having at least 90% sequence identity with SEQ ID NO.

24. ●Step (vi) is promoted by a polypeptide having indole-3-glycerol-phosphate lyase activity and having at least 90% sequence identity with SEQ ID NO. 30, and ●Step (a) is promoted by a polypeptide having flavin monooxygenase activity and having at least 90% sequence identity with SEQ ID NO.

32.

13. The in vitro method according to claim 11, comprising steps (i), (ii), (iii), (iv), (v), (iv), (a), and (b1), wherein indole is an intermediate compound, wherein indigo glycoside is produced, and wherein the continuous one-pot reaction comprises a first stage and a second stage, wherein steps (iii), (iv), and (v) are carried out in the first stage at a temperature of 50-55°C, and steps (vi), (a), and (b1) are carried out in the second stage at a temperature of 25-30°C, wherein... ●Step (iii) is promoted by N-phospribosyl-o-aminobenzoic acid synthase (TrpD), which has at least 90% sequence identity with SEQ ID NO.

14. ●Step (iv) is promoted by N-(5'-phosphoribosyl)-o-aminobenzoic acid isomerase (trpF) having at least 90% sequence identity with SEQ ID NO.

22. ●Step (v) is promoted by indole-3-glycerol phosphate synthase (trpC), which has at least 90% sequence identity with SEQ ID NO.

24. ●Step (vi) is promoted by indole-3-glycerol-phosphate lyase (IGL) having at least 90% sequence identity with SEQ ID NO.30, and ●Step (a) is promoted by a flavin-containing monooxygenase (FMO) having at least 90% sequence identity with SEQ ID NO.32, and ● Step (b1) is promoted by a glycosyltransferase having at least 75% sequence identity with SEQ ID NO.46, wherein the amino acid sequence of said glycosyltransferase comprises substitutions of E75P, Q86K, S110V, I188L, G222D, G296L, V297G, S413K and G430K relative to amino acid residues of SEQ ID NO.46, and further comprises (i) substitutions of F381V, T388C and A399C relative to amino acid residues of SEQ ID NO.46, or (ii) substitutions of F381V and T388A relative to amino acid residues of SEQ ID NO.46, or (iii) substitution of T388A relative to amino acid residues of SEQ ID NO.

46.

14. An in vitro method for producing indigo glycosides, comprising the following steps: (t) Provide tryptophan and use a polypeptide with L-tryptophan indole lyase activity (EC 4.1.99.1) to enzymatically convert tryptophan to indole. (a) The indole enzyme is used to convert indole to indophenol using an oxidase, wherein the activity of the enzyme is selected from oxidoreductase activity (EC1), monooxygenase activity (EC 1.14), flavin-containing monooxygenase (FMO) activity (EC 1.14.13.8), P450 activity (EC 1.14.14.1), and nonspecific peroxidase activity (EC 1.11.2.1), and (b1) Provide a glycosyl donor and use a polypeptide with glycosyltransferase (GT) activity (EC 2.4.1) to enzymatically convert indophenol to indole, wherein the in vitro method is a one-pot reaction and the intermediate is not purified or separated before proceeding to the next enzymatic conversion step.

15. The in vitro method according to claim 14, wherein step (b1) is promoted by UDP-glycosyltransferase and the glycosyl donor is UDP-glucose.

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

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