DE102015016339A1 - Process for the preparation of halogenated indoxyl derivatives in transgenic plants - Google Patents

Abstract

The present invention relates to a transgenic plant cell comprising a heterologous tryptophanase (EC 4.1.99.1) and a heterologous cytochrome P450 (CYP450). The present invention also relates to the use of such a transgenic plant cell which has the activity of a tryptophanase (EC 4.1.99.1) and the activity of a cytochrome P450 (CYP450) for the preparation of halogenated indoxyl derivatives from tryptophan. The transgenic plant cell according to the invention makes it possible for the first time to produce halogenated indoxyl derivatives in a plant. The present invention further relates to a process for the preparation of halogenated indoxyl derivatives from tryptophan as substrate and enzymatic conversion of halogenated tryptophan to halogenated indole by a tryptophanase (EC 4.1.99.1) and enzymatic conversion of the halogenated indole to halogenated indoxyl by a cytochrome P450.

Description

The present invention relates to a transgenic plant cell comprising a heterologous tryptophanase (EC 4.1.99.1) and a heterologous cytochrome P450 (CYP450). The present invention also relates to the use of such a transgenic plant cell which has the activity of a tryptophanase (EC 4.1.99.1) and the activity of a cytochrome P450 (CYP450) for the preparation of halogenated indoxyl derivatives from tryptophan.

Moreover, the present invention relates to a process for the preparation of halogenated indoxyl derivatives from tryptophan as substrate and enzymatic conversion of halogenated tryptophan to halogenated indole by a tryptophanase (EC 4.1.99.1) and enzymatic conversion of the halogenated indole to halogenated indoxyl by a cytochrome P450.

The derived from plant natural dyes dye indigo (IUPUC name: (2E) -2- (3-oxo-1H-indol-2-ylidene) -1H-indole-3-one) has a great historical significance, he did not For a long time the only possibility for permanent blue coloring of textiles and other objects ( Balfour-Paul, J. (1998). Indigo. (London: British Museum Press) ). Indigo in this form does not occur in nature, but is formed by fermentative processes of plant material from uncolored precursors with indoxyl basic structure. In the case of plants, the most important precursor is indikan (IUPAC name: (2S, 3R, 4S, 5R, 6R) -2- (6-chloroindol-1-yl) oxy-6- (hydroxymethyl) oxane-3,4, 5-triol), the β-D-glucoside of 3-hydroxyindole (Indoxyl), a secondary metabolite expressed in Polygonum sp. ( Maier et al., Phytochemistry 29 (1990), 817-819 ), Indigofera tinctoria, Baphicacan thus cusia, and probably in most other indigo-staining plants (Hadders, Systematic Distribution and Occurrence of Indoxylglucosides Plant Analysis, G. Klein, ed (Vienna: Springer) (1933), 1062-1063 ).

Indoxyl derivatives are used today mainly as substrates for biochemical detection reactions. They are currently the most widely used chromogenic substrates for microbiological culture media. Enzymatic reactions in which the sugar residue is split off from the indoxyl lead to oxidative dimerization and formation of the highly colored indigo ( Holt and Withers, Proc. Roy. Soc. B, 148 (1958), 520 ). By using substrates with different sugar units, it is thus possible to achieve specificities towards certain enzymes and thus detection reactions for bacterial classes. By using variably halogenated Indoxylmolekülen a variation in the type and intensity of staining can be achieved. The most well-known substrate of this type is X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside), which is used for the detection of the lacZ reporter gene in molecular biological studies ( Horwitz et al., J. Med. Chem. 7 (1964), 574 ).

Since it is the halogenated derivatives of the indicator, such as magenta-Glc (5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside) or Salmon-Glu (6-chloro-3-indolyl-β- D-glucopyranoside; IUPAC name: (2S, 3R, 4S, 5R, 6R) -2- (6-chloroindol-1-yl) oxy-6- (hydroxymethyl) oxanes-3,4,5-triol) Natural products, their extraction must be based on complex chemical syntheses.

Robertson Robertson, Journal of the Chemical Society (Resumed) (1927), 1937-1943 ) developed the chemical synthesis of indikan in 1927. The starting substance for this was methyl indoxylate, which was glycosylated with acetobromoglucose in acetone and potassium hydroxide. The resulting glycoside was then deacetylated in methanol with potassium hydroxide and the ester hydrolyzed. Subsequently, the deprotected product was decarboxylated under acetic anhydride and sodium acetate at 160 ° C. Finally, a deacetylation with ammonia dissolved in methanol followed, resulting in the free Indikan.

Robertson synthesized 6-bromo-indicin in 1929 using the 6-bromo-indoxylic acid methyl ester according to the same reaction scheme ( Robertson and Waters, J. Synthesis of Glucosides. Part VII. The Synthesis of 6-Bromoindicans. Chem. Soc. 2239-2243 (1929) ). The problem with this approach is firstly the step of glycosylation, since indoxyl is very reactive and can form various by-products even under exclusion of oxygen, also under strong basic conditions elimination is possible, which can drastically reduce the yield. In particular, the decarboxylation, which is carried out at 160 ° C, is not suitable for sensitive sugar compounds, which side reactions and fragmentations are favored. For X-Glu (5-bromo-4-chloro-1H-indol-3-yl β-D-glucopyranoside) a yield of 15% could be achieved with this approach. A newer variant of the indicator synthesis was published in 2013 by Böttcher and Thiem (Europ. J. Org. Chem. 2014 (2013), 564-574) presented. This is based on the approach of Robertson ( Robertson and Waters, J. Synthesis of Glucosides. Part VII. The Synthesis of 6-Bromoindicans. Chem. Soc. 2239-2243 (1929) ), where the deacetylation at milder temperatures (90-110 ° C) and a shorter reaction time using silver salts. In this approach, the highest yield ever achieved was achieved (43% for X-Glu, three reaction steps). Both variants require either indoxylic acid methyl ester or indoxylic acid allyl ester. For X-Glu, the corresponding indoxylic acid allyl ester is prepared via seven reaction steps from 4-bromo-3-chloro-2-methylaniline. Common to all current chemical processes is the high use of basic chemicals and the low yield. Consequently, the halogenated indoxyl derivatives can currently only be prepared by complex chemical syntheses using partly toxic reagents. It would be desirable to provide a process for preparing halogenated indoxyl derivatives which would be independent of chemical production steps and which could be carried out in living organisms, thereby making it environmentally friendly and cost effective.

However, little is known about the biosynthesis of indoxyl derivatives in plants. In 2007, it was demonstrated for the first time that an Indian biosynthesis can be achieved by combining different genes in transgenic plants ( Warzecha et al., Plant Biotechnol. J. 5 (2007), 185-191 ). By using the maize bx1, which makes indole 3-glycerophosphate indole, the precursor for the Indian biosynthesis could be provided. The conversion of indole to indoxyl was carried out by the human cytochrome P450 monooxygenase 2A6 whose activity has already been described in the bacterial system ( Gillam et al., Biochem. Biophys. Res. Commun. 265: 469-472 ). Final glycosylation to indica was performed by endogenous glycosyltransferases of the host plant. Thus, an alternative biosynthetic pathway could be established, which allows the formation of Indikan in non-staining plants. However, halogenation does not take place in the plant. For this purpose, the precursor indole would have to be added as a halogenated derivative.

There is a need for environmentally friendly, inexpensive and simple processes for preparing the aforementioned halogenated compounds. This need is met by the subject matter as recited in the claims.

Accordingly, in a first aspect, the present invention relates to a transgenic plant cell comprising a heterologous tryptophanase (EC 4.1.99.1) and a heterologous cytochrome P450 (CYP450). In particular, the present invention relates to a process for the preparation of halogenated indoxyl derivatives from tryptophan as a substrate and enzymatic conversion of halogenated tryptophan to halogenated indole by a tryptophanase (EC 4.1.99.1) and enzymatic conversion of the halogenated indole to halogenated indoxyl by a cytochrome P450 (CYP450). , As shown in Figure 1C, the present invention relates to an artificial biosynthetic pathway for the production of halogenated indoxyl derivatives in transgenic plants. In a first step, the indole ring of the tryptophan may be substituted with chlorine, bromine, iodine or fluorine by means of a halogenase described herein, preferably a tryptophan haloase (EC 1.14.99.9) at position 4, 5, 6, or 7 of the indole ring. In FIG. 1C, the 6- and / or 7-position of the indole ring are exemplarily substituted with chlorine. Instead of chlorine, bromine, iodine and fluorine substitutions are also possible here, likewise in the 4- and / or 5-position and all combinations of the various substituents and positions. In a second step, halogenated tryptophan is converted into halogenated indole by means of a tryptophanase (EC 4.1.99.1). In a third step, the halogenated indole is converted into (a) halogenated (s) indoxyl derivative (s) by means of a cytochrome P450 (CYPP450). Finally, in a fourth reaction step, glycosyltransferases, preferably endogenous glycosyltransferase (s), can be converted into a halogenated indicator derivative (s).

The term "indoxyl derivatives" includes compounds that may be grouped together as "haloindicans". The term "haloindicene" refers to indicans (ie, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (1H-indol-3-yloxy) -tetrahydropyran-3,4,5-triol) which is substituted with one or more (e.g., 1 to 4) halogen atoms independently selected from fluoro, chloro, bromo and iodo. A preferred "haloindicine" is indian, which is substituted at its indole ring with 1 to 4 halogen atoms (more preferably with 1, 2 or 3 halogen atoms, even more preferably with 1 halogen atom), wherein the halogen atoms independently of one another from fluorine, chlorine, bromine and Iodine are selected. Preferred positions for bonding said halogen atom (s) are positions 2, 4, 5, 6 and 7 of the indole ring of the indicator. Examples of such haloindicans are (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (4-fluoro-1H- indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (4-chloro-1H-indol-3-yloxy) tetrahydropyran -3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (4-bromo-1H-indol-3-yloxy) -tetrahydropyran-3,4,5- triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (4-iodo-1H-indol-3-yloxy) -tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (5-fluoro-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) - 2- (hydroxymethyl) -6- (5-chloro-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6 - (5-bromo-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (5-iodo-1H -indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (6-fluoro-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (6-chloro-1H-indol-3-yloxy) -tetrahydropyran-3,4,5 -triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (6-bromo-1H-indol-3-yloxy) -tetrahydropyran-3,4,5-triol, (2R, 3S , 4S, 5R, 6S) -2- (hydroxymethyl) -6- (6-iodo-1 H -indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (7-fluoro-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (7-chloro-1H-indol-3-yloxy) -tetrahydropyran-3,4,5 triol, (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (7-bromo-1H-indol-3-yloxy) -tetrahydropyran-3,4,5-triol, and (2R, 3S, 4S, 5R, 6S) -2- (hydroxymethyl) -6- (7-iodo-1H-indol-3-yloxy) tetrahydropyran-3,4,5-triol. Exemplary indoxyl derivatives in the context of the present invention are 4-chloroindicene, 5-chloroindicene, 6-chloroindicene, 7-chloroindicene, 4-bromoindane, 5-bromoindane, 6-bromoindane, 7-bromoindane, 4,5-dibromoindicene, 4,6-bromoindane. Dibromo-indican, 4,7-dibromoindicine, 5,6-dibromoindicine, 5,7-dibromoindicine, 6,7-dibromoindicene, 4,5-dichloroindicene, 4,6-dichloroindicene, 4,7-dichloroindicene, 5,6-dichloroindicene, 5,7-dichloroindane, 6,7-dichloroindane, 4,5,6-trihaloindicene, 5,6,7-trihaloindicene, 4,6,7-trihaloindicene, 4,5,7-trihaloindicene, 4,5,6, 7-tetrahaloindane, isoindigo, 6,6'-haloindigo, or 7,7'-haloindigo.

The term "tryptophanase" in the context of the present invention refers to an enzyme which is capable of cleaving tryptophan and releasing indole, in particular according to the example of the "tryptophanase" TmA (SEQ ID NOs: 1 (cDNA), 2 (Protein)) in 6 given reaction scheme. The enzyme activity can be as in the publication of Newton and Snell Proc Natl Acad Sci USA 48 (1962), 1431-1439 be measured described. Tryptophanase, that is, an enzyme having tryptophanase activity, is an enzyme classified as a tryptophanase or an enzyme derived from such an enzyme and capable of cleaving tryptophan and / or halogenated tryptophan and indole and / or halogenated indole. Tryptophanase is classified with EC number EC 4.1.99.1. A tryptophanase is able to cleave tryptophan and release indole. In this reaction, L-tryptophan and a water molecule are converted to indole, pyruvate and ammonia ( Newton and Snell, Proc Natl Acad Sci USA 48 (1962), 1431-1439 ). The co-factor required by tryptophanase is pyridoxal phosphate ( Wood et al., J. Biol. Chem. 170 (1947), 313-321 ). In addition, the tryptophanase activity can be increased by potassium, ammonium and rubidium ions ( Happold and Struyvenberg, Biochem J 58 (3) (1954), 379-382 ). The reaction catalyzed by the tryptophanase is in 1 respectively. 6 shown schematically for the tryptophanase TnaA from E. coli. In the context of the present invention, in principle any tryptophanase can be used, in particular from prokaryotic or eukaryotic organisms. Examples of prokaryotic tryptophanases are described in bacteria of the genus Escherichia, Vibrio, Symblobacterium, Edwardsiella, Porphyromonas, Proteus, Alkaliphilus, Bacillus, Clostridium, Desulfotomaculum, Desulfitobacterium, Nocardioides, Oribacterium, Propionibacterium, Aeromonas, Bacteroides, Brachyspira, Burkholderia, Chromobacterium, Chryseobacterium, Citrobacter, Desulfovibrio, Dichelobacter, Enterobacter, Flavobacteria, Flavobacterium, Fusobacterium, Haemophilus, Haloarcula, Halogeometricum, Hyphomonas, Oxalobacter, Pantoea, Pasturella, Photorhabdus, Porphyromonas, Pseudovibrio, Rhizobium, Saccharomonospora, Salinibacter, Shewanella, Shigella, Spirosoma, Treponema or Yersinia ( summarized in Lee and Lee, Fems Microbiology Reviews 34 (4) (2010), 426-444 ). In the context of the present invention preferably tryptophanases from bacteria of the genus Escherichia, Vibrio, Symbiobacterium, Edwardsiella, Porphyromonas, Proteus, Alkaliphilus, Bacillus, Clostridium, Desulfotomaculum, Desulfitobacterium, Nocardioides, Oribacterium, Propionibacterium, Aeromonas, Bacteroides, Brachyspira, Burkholderia, Chromobacterium, Chryseobacterium, Citrobacter, Desulfovibrio, Enterobacter, Flavobacteria, Flavobacterium, Fusobacterium, Haemophilus, Haloarcula, Halogeometricum, Hyphomonas, Pasturella, Porphyromonas, Pseudovibrio, Rhizobium, Saccharomonospora, Shigella, Treponema or Yersinia. In a particularly preferred embodiment, the tryptophanase is of the genus Escherichia, and more preferably of Escherichia coli.

In the context of the present invention, tryptophanase is an enzyme encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1. In a particularly preferred embodiment, the tryptophanase is an enzyme comprising an amino acid sequence comprising SEQ ID NO: 2 or a sequence which is at least n% identical to SEQ ID NO: 2 and has a tryptophanase activity, where n is an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 77, 78, 79, 80 , 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any of the above SEQ ID NOs. certainly. If the sequences being compared are not the same length, the degree of identity preferably refers to either the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence, or the percentage of amino acid residues in the longer sequence longer sequence, which to the Amino acid residues in the shorter sequence are identical. The degree of sequence identity may be determined according to methods well known in the art, preferably using appropriate computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particular sequence is, for example, 80% identical to a reference sequence, default settings may be used, or preferably the settings are as follows: Matrix: BLOSUM 30; Open gap penalty: 10.0; Extend Gap Penalty: 0.05; Delay Divergent: 40; Gap Separation Distance: 8 for comparison of amino acid sequences. For nucleotide sequence comparisons, the extend gap penalty is preferably set to 5.0.

Preferably, the degree of identity is calculated over the entire length of the sequence.

• (a) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, die aus der Nucleotidsequenz der SEQ ID NO: 1 besteht;
• (b) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 2 besteht;
• (c) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 2 besteht, in welcher 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 oder 40 Aminosäurerest(e) deletiert, substituiert, inseriert und/oder hinzugefügt wurden, und eine Tryptophanase-Aktivität hat; und
• (d) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein codiert, welches eine Aminosäuresequenz mit mindestens 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 oder 99%iger Sequenzidentität zu der Aminosäuresequenz nach SEQ ID NO: 2 hat und eine Tryptophanase-Aktivität hat.

In the context of the present invention, the transgenic plant cell comprises a tryptophanase, wherein the tryptophanase is encoded by a nucleic acid molecule selected from the group consisting of:

• (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 1;
• (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
• (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues (e) have been deleted, substituted, inserted and / or added, and have tryptophanase activity; and
• (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having tryptophanase activity.

The tryptophanase used according to the invention may be a naturally occurring tryptophanase or a tryptophanase derived from a naturally occurring tryptophanase, e.g. B. by introducing mutations or other changes, which z. As the enzymatic activity, the stability, etc. change or improve, be derived.

The term "tryptophanase" or "a protein / enzyme having the activity of a tryptophanase" in the context of the present application also encompasses proteins / enzymes derived from a tryptophanase which are capable of producing indole and / or halogenated indole derivatives by an enzymatic Reaction of tryptophan and / or halogenated tryptophan derivatives to produce, but have only a low affinity for (halogenated) tryptophan as a substrate or tryptophan no longer accept as a substrate. Methods for modifying and / or improving the desired enzymatic activities of proteins / enzymes are well known to those skilled in the art and include e.g. B. a random mutagenesis or a targeted mutagenesis and subsequent selection for enzymes with the desired properties or methods of so-called "directed evolution" a. For example, for gene manipulation of prokaryotic cells, a nucleic acid molecule encoding, for example, a tryptophanase can be introduced into plasmids that allow for mutagenesis or sequence modification through recombination of DNA sequences. Standard procedure (cf. Sambrook and Russell (2001), Molecular Cloning: A Laboratory Manual, CSH Press, Cold Spring Harbor, NY, USA ) allow the exchange of bases or the addition of natural or synthetic sequences. DNA fragments can be linked by introducing adapter or linker molecules into the fragments. In addition, genetic engineering can be used to provide suitable restriction sites or to remove excess DNA or restriction sites. In cases where insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation may be used. In general, sequence analysis, restriction analysis and other methods of biochemistry and molecular biology are used as analytical methods. The resulting tryptophanase variants are then examined for their enzymatic activity, and in particular for their ability to prefer (halogenated) tryptophan as a substrate. An assay for measuring the ability of a tryptophanase to use tryptophan as a substrate is described in US Pat Newton and Snell, Proc Natl Acad Sci USA 48 (1962), 1431-1439 described. The tryptophanase used in the context of the present invention may be a natural variant of the protein or a synthesized protein, as well as a protein that is chemically synthesized or in a biological system or method was prepared with recombination. The tryptophanase may also be chemically modified, for example to increase its stability or resistance, e.g. B. compared to a temperature.

The transgenic plant cell according to the invention is further characterized in that it comprises a heterologous cytochrome P450. In the context of the present invention, the transgenic plant cell comprises a heterologous tryptophanase as described herein and a heterologous cytochrome P450 (CYP450).

The term "cytochrome P450 (CYP450)" or "a protein having the activity of a cytochrome P450 (CYP450)" in the context of the present invention refers to a protein capable of carrying an oxygen function (hydroxyl), for example, in to introduce an indole ring of tryptophan in 3-position; see. this in the 8th shown reaction scheme.

The cytochrome P450 (CYP450) activity can be as in the Publication Gillam et al., Biochemistry, 39 (45) (2000), 13817-13824 be measured described. The cytochrome P450 (CYP450), ie, a protein having cytochrome P450 (CYP450) activity, is a protein classified as cytochrome P450, or a protein derived from such a protein, and the ability has indole, preferably halogenated indole, to react in indoxyl. In the context of the present invention, in principle any cytochrome P450 can be used, in particular from eukaryotic organisms. Eukaryotic organisms may, in principle, include all mammals such as, for example, Homo sapiens, Rattus spp., Mus spp. be. In the context of the present invention, the cytochrome P450 (CYP450) may also be derived from insects. The following Table A lists some cytochrome P450 of eukaryotes which can be used in the context of the present invention: Surname Deposit number (GeneBank) Gene identifier CYP2A6 AF182275.1 GI: 6470138 CYP2E1 AF182276.1 GI: 6470140 CYP2C9 NM_000771 GI: 189242609 CYP2C19 NP_000760.1 GI: 4503219 CYP102A1 (BM3) D1041691.1 D1165400.1 BD357426.1 GI: 168358941 GI: 168322820 GI: 92248772 CYP3A4 AF182273.1 GI: 6470134

In the context of the present invention, cytochrome P450 (CYP450) is a protein encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 3 or one of the deposited nucleotide sequences set forth in Table A. In a particularly preferred embodiment, the cytochrome P450 (CYP450) is a protein which comprises an amino acid sequence which consists of SEQ ID NO: 4 or one of the deposited protein sequence mentioned in Table A, or of a sequence which is at least n% identical to SEQ ID NO: 4 or at least n% is identical to one of the deposited protein sequences listed in Table A and has a CYP450 activity, where n is an integer between 10 and 100, preferably 10, 15, 20, 25, 30 , 35, 35, 40, 45, 50, 55, 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or 99.

Preferably, the degree of identity is determined by comparing the respective sequence with the amino acid sequence of any of the above SEQ ID NOs. certainly. If the sequences being compared are not the same length, the degree of identity preferably refers to either the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence, or the percentage of amino acid residues in the longer sequence longer sequence, which are identical to the amino acid residues in the shorter sequence. The degree of sequence identity may be determined according to methods well known in the art, preferably using appropriate computer algorithms such as CLUSTAL.

When using the Clustal analysis method to determine whether a particular sequence is, for example, 80% identical to a reference sequence, default settings may be used, or preferably the settings are as follows: Matrix: BLOSUM 30; Open gap penalty: 10.0; Extend gap Penalty: 0.05; Delay Divergent: 40; Gap Separation Distance: 8 for comparison of amino acid sequences. For nucleotide sequence comparisons, the extend gap penalty is preferably set to 5.0.

Preferably, the degree of identity is calculated over the entire length of the sequence.

The cytochrome P450 used according to the invention may be a naturally occurring cytochrome P450 or a cytochrome P450 derived from a naturally occurring cytochrome P450, e.g. B. by introducing mutations or other changes, which z. As the enzymatic activity, the stability, etc. change or improve, be derived. Nakamura et al. (Arch Biochem Biophys 395 (1) (2001), 25-31) describes, for example, a mutant of human cytochrome P450 2A6 in which leucine 240 has been replaced by cysteine 240 and asparagine 297 by glutamic acid 297 and which exhibits an increase in the 3-hydroxylation of indole and, above all, improved acceptance of substituted indole derivatives. The cytochrome P450 to be used in the context of the present invention may be a natural variant of the protein or a synthetic protein, as well as a protein that has been chemically synthesized or produced in a recombinant biological process.

• (a) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, die aus der Nucleotidsequenz der SEQ ID NO: 3 besteht;
• (b) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 3 besteht;
• (c) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 4 besteht, in welcher 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 oder 40 Aminosäurerest(e) deletiert, substituiert, inseriert und/oder hinzugefügt wurden, und eine Cytochrom P450-Aktivität hat; und
• (d) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein codiert, welches eine Aminosäuresequenz mit mindestens 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 oder 99%iger Sequenzidentität zu der Aminosäuresequenz nach SEQ ID NO: 4 hat und eine Cytochrom P450-Aktivität hat.

In the context of the present invention, in addition to the tryptophanase described herein, the transgenic plant cell comprises a heterologous cytochrome P450 (CYP450), wherein the cytochrome P450 (CYP450) is encoded by a nucleic acid molecule selected from the group consisting of:

• (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 3;
• (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 3;
• (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acid residues (e) have been deleted, substituted, inserted and / or added, and have cytochrome P450 activity; and
• (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4 and having cytochrome P450 activity.

The transgenic plant cell of the present invention is further characterized as comprising a heterologous halogenase. In a preferred embodiment of the present invention, the transgenic plant cell comprises a heterologous tryptophanase as described herein, a heterologous cytochrome P450 (CYP450) as hereinbefore described, and a heterologous halogenase. The term "halogenase" in the context of the present invention refers to an enzyme capable of converting the substrate tryptophan into a halogenated tryptophan derivative, in particular according to the art 7 (Exemplary for the tryptophan halogenase SttH from Streptomyces toxytricini (SEQ ID NOs: 7 (cDNA), 8 (protein)) The enzyme activity can be determined as described in the publication of Zeng and Zhan, Biotechnol Lett 33 (8) (2011), 1607-1613 be measured described. The halogenase, that is, an enzyme having a halogenase activity, is an enzyme classified as a halogenase or an enzyme derived from such an enzyme and capable of halogenating tryptophan. In the context of the present invention, in principle any halogenase capable of halogenating the indole ring of tryptophan at positions 4, 5, 6 and / or 7 with fluorine, chlorine, bromine or iodine may be used.

In the context of the present invention, in principle any halogenase can be used, in particular from prokaryotic or eukaryotic organisms. Eukaryotic halogenases are described, e.g. Examples of prokaryotic tryptophanases are described in bacteria of the genus Lechevalieria, Streptomyces, Escherichia, eg, fungi such as Pochonia (eg the halogenase (accession number: HQ149729; gene recognition: GI: 306412957), plants such as Pisum sativum and animals. Kutzneria, Pseudomonas, Acytostelium, Microbispora In a particularly preferred embodiment, the halogenase is derived from the genus Lechevalieria and / or Streptomyces, and more preferably from the species Lechevalieria aerocolonigenes and / or Streptomyces toxytricini.

Listed below in Table B are some prokaryote halogenases which may be used in the context of the present invention: Surname description Deposit number (GeneBank) Gene identifier REBH 7 halogenase AB071405.1 GI: 22830833 Pyr 5 halogenase AY623051 GI: 53854582 stth 6 halogenase HQ844046.1 GI: 321531617 KtzQ 7 halogenase EU074211 GI: 157429075 KTZR 6 halogenase EU074211 GI: 157429076 PLT4 AF081920 GI: 4582981 PrnA 7 halogenase AF161184 GI: 5669517 RDC2 HQ149729.1 GI: 306412957 CHLA AB902922.1 GI: 666669466 MIBH 5 halogenase HM536998.1 GI: 300719244 Tha 6 halogenase EF095207.1 GI: 118213905 ThdH 6 halogenase KC182553.1 GI: 451774857

In the context of the present invention, the halogenase is preferably a tryptophan haloase. Tryptophan haloase is classified with EC number EC 1.14.19.9. A tryptophan halide is to halogenate in the tryptophan. In a particularly preferred embodiment, the tryptophan haloase is derived from the genus Lechevalieria or Streptomyces, and more preferably from the species Lechevalieria aerocolonigenes and / or Streptomyces toxytricini. The reaction catalyzed by the tryptophan halogenase is in 1C schematically for the Tryptophanhalogenase RebH from Lechevalieria aerocolonigenes and / or for the Tryptophanhalogenase SttH from Streptomyces toxytricini. In the context of the present invention, the transgenic plant cell may also comprise two, three or four halogenases, preferably tryptophan halogenases. In the context of the present invention, it is also possible to introduce two, three or four different halogens, preferably tryptophan halo genes, into the plant cell by means of one or more nucleic acid molecules so as to obtain a polyhalogenated educt, preferably a polyhalogenated tryptophan (ie halogenation at positions 4, 5, 6 and 10) / or 7 of the indole ring). For example, as shown in Table B above, the halogenase Stth (SEQ ID NOs: 7 (cDNA), 8 (protein)) described in the context of the present invention catalyses the halogenation of the indole ring of tryptophan at position 6. The halogenase RebH (SEQ ID NOs: 5 (cDNA), 6 (protein)) catalyzes the halogenation of the indole ring of tryptophan at position 7; see. this too 1C , A transgenic plant cell described herein into which two, three or four halogenases, preferably tryptophan halo genes, are introduced with one or more nucleic acid molecules can, for example, halogenate the indole ring of the tryptophan at positions 6 and 7 with fluorine, chlorine, bromine or iodine. In the context of the present invention, the halogenating positions of the indole ring can be halogenated with different halogens (ie, fluorine, chlorine, bromine, or iodine). For example. can be like in 1C Figure 3 illustrates, using the halogenases described herein (e.g., RebH and SttH), the indole ring of tryptophan at positions 6 and 7 being halogenated with chlorine. Examples of the Tryptophanhalogenasen are listed in the SEQ ID NO: 6 (Tryptophanhalogeanse RebH from Lechevalieria aerocolonigenes) or SEQ ID NO: 8 (tryptophan halogenase SttHaus Streptomyces toxytricini). In a preferred embodiment of the present invention, the tryptophan haloase is an enzyme comprising an amino acid sequence consisting of SEQ ID NO: 6 or SEQ ID NO: 8 or of a sequence which is at least n% identical to SEQ ID NO: 6 or which is at least n% identical to SEQ ID NO: 8 and has a tryptophan haloase activity, where n is an integer between 10 and 100, preferably 10, 15, 20, 25, 30, 35, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99.

Preferably, the degree of identity is determined by comparing the particular sequence with the amino acid sequence of any of the aforementioned SEQ ID NOs:. If the sequences being compared do not have the same length, the degree of identity refers either to the percentage of amino acid residues in the shorter sequence that are identical to the amino acid residues in the longer sequence or to the percentage of amino acid residues in the longer one Sequence identical to amino acid residues in the shorter sequence. The degree of sequence identity can be determined according to methods well known in the art, with computer algorithms such as e.g. B. CLUSTAL be used.

Using the CLUSTAL analysis method to determine if a particular sequence is, for example, 80% identical to a reference sequence, the default settings can be used, or the settings are preferably as follows: Matrix: BLOSUM 30; Gap penalty: 10.0; Gap extension penalty: 0.05; Delay Divergent: 40; Gap Separation Distance: 8 for comparison of amino acid sequences. For comparisons of nucleotide sequences, the gap extension penalty is preferably set to 5.0.

• (a) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, die aus der Nucleotidsequenz der SEQ ID NO: 5 oder der SEQ ID NO: 7 besteht;
• (b) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 6 oder der SEQ ID NO: 8 besteht;
• (c) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein kodiert, welches aus der Aminosäuresequenz der SEQ ID NO: 6, oder der Aminosäuresequenz der SEQ ID NO: 8 besteht, in welcher 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 oder 40 Aminosäurereste deletiert, substituiert, inseriert und/oder hinzugefügt wurden, und eine Tryptophanhalogenase-Aktivität hat; und
• (d) einem Nucleinsäuremolekül umfassend eine Nucleinsäuresequenz, das ein Protein codiert, welches eine Aminosäuresequenz mit mindestens 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 oder 99%iger Sequenzidentität zu der Aminosäuresequenz nach SEQ ID NO: 6 oder zu der Aminosäuresequenz nach SEQ ID NO: 8 hat und eine Tryptophanhalogenase-Aktivität hat.

In the context of the present invention, the transgenic plant cell comprises a heterologous tryptophanase as defined herein, a heterologous cytochrome P450 as defined herein, and a heterologous tryptophan haloase, wherein the tryptophan haloase is encoded by a nucleic acid molecule selected from the group consisting of :

• (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7;
• (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8;
• (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 8, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues have been deleted, substituted, inserted and / or added, and have tryptophan haloase activity; and
• (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the amino acid sequence of SEQ ID NO: 6 or to the amino acid sequence of SEQ ID NO: 8 and having tryptophan haloacid activity.

In a particular embodiment of the present invention, the transgenic plant cell comprises a heterologous tryptophanase as defined herein, a heterologous cytochrome P450 as defined herein, and the Lechevalieria aerocolonigenes tryptophan halogenases RebH described herein (SEQ ID NOs: 5 (cDNA), 6 (Protein)) and SttH from Streptomyces toxytricini (SEQ ID NOs: 7 (cDNA), 8 (protein)). Transgenic plants comprising the tryptophan halogenase RebH described herein can be further characterized as comprising an enzyme which catalyzes the NADH-dependent reduction of FAD to FADH2. An example of such an enzyme would be the RebF from Lechevalieria aerocolonigenes (as shown in SEQ ID NOs: 29 (cDNA) and 30 (protein)) or a structurally closely related molecule. For example, as shown in the Examples herein, there is no need in the present transgenic plant cells of the invention to co-express the RebH-corresponding reductase RebF, since the reduction equivalents required for the enzymatic reactions described herein occur in sufficient amount endogenously in the plant cell described herein , In the context of the present invention, the transgenic plant cell described herein is further characterized as comprising a glycosyltransferase. In the context of the present invention, the transgenic plant cell comprises a heterologous tryptophanase as defined herein, a heterologous cytochrome P450 (CYP450) as defined herein, and a glycosyltransferase. The present invention also relates to a transgenic plant cell, characterized in that it comprises a heterologous tryptophanase as defined herein, a heterologous cytochrome P450 (CYP450) as defined herein, a heterologous halogenase as defined herein, and a glycosyltransferase. In the context of the present invention, glucosylation is preferably performed by an endogenous glycosyltransferase. In the context of the present invention, a glycosyltransferase can also be introduced into the transgenic plant cell according to the invention by means of a heterologous expression of, for example, a glycosyltransferase gene to optimize glucosylation or to generate entirely new molecules by expression of alternative glycosyltransferases (eg galactosides or glucuronides by expression of corresponding galactosyl or glucuronyltransferase genes). In the context of the present invention, in principle any glycosyltransferase may be employed, in particular from prokaryotic or eukaryotic organisms. Eukaryotic glycosyltransferase are described, e.g. Of fungi such as Botrytis cinerea (accession number: EMR87737.1, gene identifier: GI: 472243069), plants such as e.g. B. Arabidopsis thaliana glucosyltransferase (BAD43267.1 GI: 51969150) and animals such. B. Homo sapiens glucuronyltransferase (accession number: BAA96077.1, gene identifier: GI: 8051678). In a particularly preferred embodiment, the glycosyltransferase is the arbutin synthase (accession number: AJ310148, gene identifier: GI: 13508843)

In a particularly preferred embodiment, the transgenic plant cell is genetically engineered to be transformed with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-described reaction step from tryptophan to halogenated tryptophan, and to a A nucleic acid molecule which encodes an enzyme capable of catalyzing the above-described reaction step from halogenated tryptophan to halogenated indole or with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-described reaction step from halogenated tryptophan to halogenated indole , and a nucleic acid molecule encoding an enzyme capable of catalyzing the above-described halogenated indole to halogenated indoxyl reaction step, or a nucleic acid molecule encoding an enzyme capable of converting the above-described reaction step from tryptophan to halogenated one Tryptophan, with a nucleic acid molecule encoding an enzyme capable of catalyzing the above-described reaction step of halogenated tryptophan to halogenated indole, and with a nucleic acid molecule encoding an enzyme capable of the above-described Rea Catalyst step from halogenated indole to halogenated indoxyl.

Methods of producing the transgenic (genetically modified) plant cell described above are known in the art. Accordingly, we generally transform the plant cell with a DNA construct which allows the expression of the respective enzyme in the plant cell. Such a construct will normally comprise the particular coding sequence associated with regulatory sequences enabling transcription and translation in the particular host cell, e.g. As a promoter and / or enhancer and / or a transcription terminator and / or ribosome binding sites, etc.

In the context of the present invention, the plant cell used in the method of the present invention is a transgenic plant cell derived from a plant cell that does not naturally produce halogenated indoxyl derivatives, but has been genetically engineered as described herein to produce halogenated indoxyl derivatives. and expressing the enzyme (s) capable of catalyzing the formation of halogenated indoxyl derivatives.

In the context of the present invention, the term "heterologous" means that the plant cell is genetically engineered to contain a foreign nucleic acid molecule or molecules that encode an enzyme as defined above, i. H. For example, a tryptophanase (EC 4.1.99.1), encoded. The term "foreign" in this context means that the nucleic acid molecule naturally does not occur in the plant cell. This means that it does not occur in the same structure or at the same place in the plant cell. In a preferred embodiment, the foreign nucleic acid molecule is a recombinant molecule comprising a promoter and a coding sequence encoding the corresponding enzyme, i. H. a tryptophanase (EC 4.1.99.1), wherein the promoter which directs the expression of the coding sequence is heterologous with respect to the coding sequence. Heterologous in this context means that the promoter is not the promoter that naturally controls the expression of the coding sequence, but is a promoter that naturally controls the expression of another coding sequence, i. H. it is from another gene or is a synthetic promoter or a chimeric promoter. Preferably, the promoter is a promoter which is heterologous to the plant cell, i. H. a promoter that does not naturally occur in the particular plant cell. More preferably, the promoter is an inducible promoter. Promoters for the control of expression in plant cells are well known to those skilled in the art.

In another preferred embodiment, the nucleic acid molecule is foreign to the plant cell in that the encoded enzyme, i. H. For example, the tryptophanase (EC 4.1.99.1), not found in the plant cell, d. H. is not naturally expressed by the plant cell if it is not genetically modified. In other words, for example, the encoded tryptophanase (EC 4.1.99.1) is heterologous with respect to the plant cell.

In the context of the present invention, the term "heterologous" means that the plant cell within the regulatory region, which controls the expression of an enzyme as defined above naturally occurring in the plant cell, is genetically engineered to increase the expression of the particular enzyme in the plant Comparison with a corresponding non-genetically modified organism. The meaning of the term "higher expression" is described in more detail below.

Such a modification of a regulatory region can be achieved by methods known to those skilled in the art. An example is the replacement of the naturally occurring promoter by a promoter that allows for higher expression, or the modification of the naturally occurring promoter to have higher expression. Accordingly, in this embodiment, the plant cell within the regulatory region of the gene encoding an enzyme as defined above contains a foreign nucleic acid molecule that does not naturally occur in the plant cell higher expression of the enzyme compared to a corresponding non-genetically modified plant cell.

The foreign nucleic acid molecule may be present in the plant cell in extrachromosomal form, e.g. As a plasmid, or stable in (the) chromosome (s) are integrated. In principle, the foreign nucleic acid molecule can be introduced into the nuclear genome of a plant cell or into the organelle genome, such as plastids, chloroplasts, mitochondria. Methods for introducing the foreign nucleic acid molecule (s) are well known to those skilled in the art. Stable integration is preferred. In the context of the present invention, the nucleic acid molecules encoding, for example, a tryptophanase (EC 4.1.99.1) comprise a protein targeting signal sequence which ensures localization in a particular cell compartment, such as the vacuole or plastid / chloroplast. In the context of the present invention, the foreign Nucleic acid molecule comprises one or more nucleic acid molecules of protein targeting signal sequence (s) which, for example, mediate cytosolic localization of tryptophanase.

Exemplary protein targeting signal sequence (s) in the present invention are SEQ ID Nos: 26 (as encoded by the nucleic acid sequence of SEQ ID NO: 25) or 32 (as encoded by the nucleic acid sequence of SEQ ID NO: 31) in the context of the present invention For example, the proteins described herein (ie, tryptophanase, halogenase, cytochrome P450 (CYP450) and / or glycosyltransferase) can be expressed in chloroplasts and / or the cytosol. In a particular embodiment, the proteins described herein are expressed in the plant compartment / plant compartment described / described in Example 3; For example, co-localization of tryptophanase and cytochrome P450 (CYP450) in the chloroplast and / or localization of tryptophanase in the cytosol and localization of cytochrome P450 (CYP450) in the cytosol.

The term "transgenic" in this context preferably means that the plant cell is transgenic in the sense that it has additionally been genetically engineered to express an enzyme as defined above. The term "transgenic" in one embodiment means that the plant cell is genetically engineered to contain one or more foreign nucleic acid molecules encoding one or more enzymes as defined herein, i. H. For example, a tryptophanse (EC 4.1.99.1), a cytochrome P450 and / or a halogenase, preferably a tryptophan analogue (EC 1.14.99.9) encoded.

As far as the definition of the term "foreign nucleic acid molecule" is concerned, the same applies as stated above.

In another embodiment, the term "transgenic" means that the plant cell within the regulatory region, which controls the expression of an enzyme as defined above naturally occurring in the plant cell, is genetically altered to increase the expression of the respective enzyme in comparison to cause a corresponding non-genetically modified organism. The meaning of the term "higher expression" is described in more detail below.

Such a modification of a regulatory region can be achieved by methods known to those skilled in the art. An example is the replacement of the naturally occurring promoter by a promoter that allows for higher expression, or the modification of the naturally occurring promoter to have higher expression. Accordingly, in this embodiment, the plant cell within the regulatory region of the gene encoding one or more of the enzymes defined herein contains a foreign nucleic acid molecule that does not naturally occur in the plant cell and that is non-genetically higher in expression of the enzyme compared to a corresponding one altered plant cell leads.

Preferably, such an organism / microorganism is characterized in that the expression / activity of one of the enzymes defined herein, for example the tryptophan haloase in the transgenic plant cell, is higher in comparison with a corresponding non-genetically modified plant cell. A "higher" expression / activity means that the expression / activity of the enzyme, i. H. For example, in the tryptophan halo, the genetically modified plant cell is at least 10%, preferably at least 20%, more preferably at least 30% or 50%, more preferably at least 70% or 80%, and most preferably at least 90% or 100% higher than in the corresponding non-genetically modified plant cell. In more preferred embodiments, the increase in expression / activity may be at least 150%, at least 200%, or at least 500%. In particularly preferred embodiments, the expression is at least 10-fold, more preferably at least 100-fold, and more preferably at least 1000-fold higher than in the corresponding non-genetically modified plant cell.

The term "higher" expression / activity also encompasses the fact that the corresponding non-genetically modified plant cell does not express a corresponding enzyme, ie, for example, no tryptophan haloase, so that the corresponding expression / activity in the non-genetically modified plant cell is zero.

As for the methods for measuring the degree of expression or activity, the same applies as stated above.

The term "plant cell" as used in the context of the present invention generally refers to any type of eukaryotic plant. The term also includes transgenic plants or cell aggregates of such plant cells as plant cell cultures, tissues or calli.

In a preferred embodiment, the plant cell is derived from a plant. In principle, any plant can be used. Preferred plants are plants of the families Solanaceae, Loganiaceae, Apocnaceae, Rosaceae, Asteraceae, Caryophyllaceae, Asclepiadaceae, Rubiaceae, Acanthaceae, Polygonaceae, Brassicacea or Fabaceae. In a particularly preferred embodiment, the plant belongs to the family Solanaceae, in particular to the genus Nicotiana, Solanum, and more preferably to the species Nicotiana tabacum, Nicotiana glauca, Nicotiana rustica, Nicotiana benthamiana, Solanum tuberosum or Solanum lycopersicum.

In the context of the present invention, the transgenic plant cell is selected from the group consisting of Rauvolfia serpentina, Rauvolfia tetraphylla, Rauvolfia verticillata, Catharanthus roseus, Dianthus caryophyllus, Rosa spp., Petunia hybrida, Gossypium hirsutum, Gossypium herbaceum, Daucus carota, Olea europea, Zea mays, Oryza sativa, Arabidopsis thaliana, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium, Wolffia arrhiza or Wolffia globosa.

• (a) das Züchten der hierin beschriebenen, transgenen Pflanzenzelle, der hierin beschriebenen transgenen Pflanze oder der hierin beschriebenen pflanzlichen Zellkultur in einem geeigneten Medium; und
• (b) das Gewinnen der halogenierten Indoxylderivaten aus den transformierten Pflanzenzellen und/oder dem Medium der Zellkultur.

The present invention also relates to a process for the preparation of halogenated indoxyl derivatives from tryptophan, comprising the steps:

• (a) culturing the transgenic plant cell described herein, the transgenic plant described herein or the plant cell culture described herein in a suitable medium; and
• (b) recovering the halogenated indoxyl derivatives from the transformed plant cells and / or the medium of the cell culture.

Also included in the invention is a halogenated indoxyl derivative obtainable / manufactured by a process of the present invention. For example, halogenated indoxyl derivatives can be selected from the group consisting of halogenated indole, halogenated indoxyl, halogenated Indikan and halogenated indigo obtained by the inventive method / manufactured. Exemplary indoxyl derivatives in the context of the present invention are 4-chloroindicene, 5-chloroindicene, 6-chloroindicene, 7-chloroindicene, 4-bromoindane, 5-bromoindane, 6-bromoindane, 7-bromoindane, 4,5-dibromoindicene, 4,6-bromoindane. Dibromo-indican, 4,7-dibromoindicine, 5,6-dibromoindicine, 5,7-dibromoindicine, 6,7-dibromoindicene, 4,5-dichloroindicene, 4,6-dichloroindicene, 4,7-dichloroindicene, 5,6-dichloroindicene, 5,7-dichloroindane, 6,7-dichloroindane, 4,5,6-trihaloindicene, 5,6,7-trihaloindicene, 4,6,7-trihaloindicene, 4,5,7-trihaloindicene, 4,5,6, 7-tetrahaloindane, isoindigo, 6,6'-haloindigo, or 7,7'-haloindigo.

In the context of the method of the present invention, it is also conceivable that the method according to the invention is carried out in a manner in which two types of plant cells are used, i. H. a type which produces, for example, halogenated tryptophan and a type which uses the halogenated tryptophan produced by the first type of plant cell to convert it to halogenated indole and / or to halogenated indoxyl by means of one of the enzymes defined above.

When the method according to the invention is carried out using plant cells which provide the respective enzyme activity (s), the plant cells are cultured under suitable culture conditions, for example in a suitable medium, which allow the enzyme reaction (s) to be established. The specific culture conditions depend on the specific plant cell, but are well known to those skilled in the art. The culture conditions are generally chosen to allow the expression of the genes that encode the enzymes for the respective reactions. The skilled person various methods are known to improve the expression of certain genes in certain phases of the culture and vote such. B. by induction of gene expression by means of chemical inducers or by a temperature shift. Further, various media are known to those skilled in the art to cultivate the transgenic plant cells in the culture method described herein. Examples of suitable Medium are, for example, the LS medium ( Linsmaier and Skoog Physiol. Plant. 18 (1965), 100-127 ) or the MS medium ( Murashige and Skoog Physiol. Plant. 15 (1962), 473 ).

In another preferred embodiment, the plant cell used in the method according to the invention is a transgenic plant or plant cell culture. In principle, every possible plant, d. H. a monocot plant or a dicotyledonous plant. Preferred plants are plants of the families Solanaceae, Loganiaceae, Apocnaceae, Rosaceae, Asteraceae, Caryophyllaceae, Asclepiadaceae, Rubiaceae, Acanthaceae, Polygonaceae, Brassicacea or Fabaceae. In a particularly preferred embodiment, the plant belongs to the family Solanaceae, in particular to the genus Nicotiana, Solanum, and more preferably to the species Nicotiana tabacum, Nicotiana glauca, Nicotiana rustica, Nicotiana benthamiana, Solanum tuberosum or Solanum lycopersicum.

In the context of the present invention, the plant is selected from the group consisting of Rauvolfia serpentina, Rauvolfia tetraphylla, Rauvolfia verticillata, Catharanthus roseus, Dianthus caryophyllus, Rosa spp., Petunia hybrida, Gossypium hirsutum, Gossypium herbaceum, Daucus carota, Olea europea, Zea mays , Oryza sativa, Arabidopsis thaliana, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia, Polygonum tinctorium, Wolffia arrhiza or Wolffia globosa.

• (a) sie ist fähig, Tryptophan in halogeniertes Tryptophan umzusetzen; und
• (b) sie exprimiert ein Enzym, das die Aktivität einer Halogenase, vorzugsweise einer Tryptophanhalogenase (EC 1.14.19.9) aufweist,

für die Herstellung von halogeniertem Tryptophan.The present invention also relates to the use of a transgenic plant cell characterized by the following features:

• (a) it is capable of converting tryptophan to halogenated tryptophan; and
• (b) it expresses an enzyme which has the activity of a halogenase, preferably a tryptophan haloase (EC 1.14.19.9),

for the production of halogenated tryptophan.

That is, the present invention also relates to the use of a transgenic plant cell according to the invention for the production of halogenated tryptophan.

• (a) sie ist fähig halogeniertes Tryptophan in halogeniertes Indol umzusetzen; und
• (b) sie exprimiert ein Enzym, das die Aktivität einer Tryptophanase (EC 4.1.99.1) aufweist,

für die Herstellung von halogeniertem Indol.The present invention also relates to the use of a transgenic plant cell characterized by the following features:

• (a) it is capable of converting halogenated tryptophan to halogenated indole; and
• (b) it expresses an enzyme which has the activity of a tryptophanase (EC 4.1.99.1),

for the production of halogenated indole.

In the context of the present invention, for the production of halogenated indole it is also possible to use a transgenic plant cell which is characterized in that it expresses an enzyme which has the activity of a halogenase, preferably a tryptophan haloase (EC 1.14.19.9) and expresses an enzyme in that it has the activity of a tryptophanase (EC 4.1.99.1).

That is, the present invention also relates to the use of the plant cell according to the invention for the production of halogenated indole.

• (a) sie ist fähig (ein) halogenierte(s) Indol in (ein) halogenierte(s) Indoxylderivat(e) umzusetzen; und
• (b) sie exprimiert ein Enzym, das die Aktivität eines Cytochrom P450 aufweist,

für die Herstellung von (einem) halogenierten Indoxylderivat(en).In the context of the present invention, the use of a transgenic plant cell characterized by the following features is also described:

• (a) it is capable of reacting a halogenated (s) indole with halogenated (s) indoxyl derivative (s); and
• (b) it expresses an enzyme that has the activity of a cytochrome P450,

for the preparation of (a) halogenated indoxyl derivative (s).

In the context of the present invention, for the preparation of halogenated indoxyl derivatives it is also possible to use a transgenic plant cell which is characterized in that it expresses an enzyme which has and / or has the activity of a halogenase, preferably a tryptophan haloase (EC 1.14.19.9) Enzyme expressing the activity of a tryptophanase (EC 4.1.99.1).

• (a) sie ist fähig (ein) halogenierte(s) Indoxylderivat(e) in (ein) halogenierte(s) Indikanderivat(e) umzusetzten
• (b) sie exprimiert ein Enzym, das die Aktivität einer Glycosyltransferase aufweist,

für die Herstellung von (einem) halogenierten Indikanderivat(en).That is, the present invention also relates to the use of the plant cell according to the invention for the production of (a) halogenated indoxyl derivative (s). The present invention also relates to the use of a transgenic plant cell which is characterized by the following features:

• (a) it is capable of converting a halogenated (s) indoxyl derivative (s) to a halogenated indicator derivative (s)
• (b) it expresses an enzyme which has the activity of a glycosyltransferase,

for the preparation of (a) halogenated indicator derivative (s).

That is, the present invention also relates to the use of a transgenic plant cell for the production of halogenated indicator derivative (s). In the context of the present invention, the transgenic plant cell according to the invention is characterized in that it is capable of converting halogenated indole into halogenated indoxyl and / or of converting halogenated indoxyl into halogenated indan.

Description of the figures:

1 shows the schematic representation of the reaction reactions A: conversion of indole-3-glycerol phosphate to tryptophan by tryptophan synthase. B: Reaction of indole-3-glycerol phosphate by recombinant BX1 to indole and its conversion by recombinant CYP2A6 into indoxyl and subsequently by endogenous glycosyltransferase (GT) in Indikan. The latter can be converted to indigo by splitting off sugar. C: New, artificial biosynthetic pathway to halogenated indoxyl derivatives bswp. Indikanderivaten. Exemplified here are the chlorination at the 6- and / or 7-position of the indole ring. Instead of chlorine, bromine, iodine and fluorine substitutions are also possible here, likewise in the 4- and / or 5-position and all combinations of the various substituents and positions.

2 shows HPLC analysis of the leaf extracts metabolites after transient expression of bx1, 2A6 and each rebH or stth. The localization of the 7-halogenase RebH or the 6-halogenase Stth in the cytosol (A) or the chloroplast (B) correlated with the biosynthesis of 7-chlorotryptophan (Rt 5.2 min) or 6-chlorotryptophan (Rt 5.8 min ). Furthermore, small amounts of the indicator synthesized by CYP2A6 and BX1 (Rt 3.5 min) could be detected in some samples. However, no halogenated indole or Indikanderivate were detectable. WT: untreated wild type.

3 Figure 2 shows the IC-MS analysis of the leaf extracts metabolites after transient expression of rebH or stth and infiltration of a potassium bromide solution. A: The localization of the 7-halogenase RebH or the 6-halogenase Stth led to the biosynthesis of 7-bromotryptophan (Rt 2.40 min, m / z 283, ESI +) or 6-bromotryptophan (Rt 2.40 min, m / z 283, ESI +). B: The mass spectra of the metabolites synthesized by RebH and Stth exhibited two mass peaks of the same intensity of 283 and 285, which are characteristic of a mono-brominated tryptophan molecule, with positive ionization.

4 shows the HPLC analysis of the metabolites from leaf extracts after transient expression of tnaA, cyp2A6 and each rebH or stth. The localization of the tryptophanase TnaA in the cytosol or the chloroplast (CP) led to the biosynthesis of idol (Rt 8.0 min) and small amounts of indican (Rt 3.5 min). The co-localization of CYP2A6 in the cytosol or the chloroplasts led to a significantly increased Indikanbiosynthese. The further modification of the synthetic route by introducing the 7-halogenase RebH resulted in the accumulation of 7-chloroindole (Rt 9.3 min) and 7-chloroindicene (Rt 7.0 min). Accordingly, 6-chloroindole (Rt 9.7 min) and 6-chloroindicene (Rt 7.0 min) were synthesized by the activity of the 6-halogenase Stth. By way of example, the cytosolic localization of TnaA and CYP2A6 is shown with simultaneous localization of the halogenases in chloroplasts. Rt 5.2 min: 7-chlorotryptophan; Rt 5.8 min: 6-chlorotryptophan. EV: Negative Control (Empty Vector)

5 LC-MS analysis of leaf extracts metabolites after transient expression of tnaA, cyp2A6 and each rebH or stth. A: The co-localization of TnaA and CYP2A6 in the chloroplasts led to a significantly increased indigenous biosynthesis (Rt 1.69 min, m / z 294 ESI-) compared to tryptophanase alone or the negative control (EV). The simultaneous activity of TnaA, CYP2A6 and the 7-halogenase RebH in chloroplasts led to the biosynthesis of 7-chloroindicene (Rt 2.42 min, m / z 328 ESI-). The translocation of the 6-halogenase Stth into the chloroplasts with cytosolic localization of TnaA and CYP2A6 correlated with the accumulation of 6-chloroindicene (Rt 2.39 min, m / z 328 ESI-). Another non-halogenated endogenous metabolite (m / z 328) eluted after 3.39 min and 3.42 min, respectively. Furthermore, in TnaA / CYP2A6 samples, low levels of chloroindicene were detected by diffusion of chlorindole synthesized in adjacent leaves. B: The mass spectra of 7-chloroindicene (7-halogenase RebH) and 6-chloroindicene (6-halogenase Stth) showed the m / z values expected for a mono-chlorinated Indian molecule of 328 and 330 in an intensity ratio of 3: 1.

6 shows the reaction scheme of the enzyme tryptophanase, which is able to convert (halogenated) tryptophan into (halogenated) indole.

7 Figure 9 shows the reaction scheme of the halogenase, preferably a tryptophan haloase capable of converting tryptophan into a halogenated tryptophan derivative.

8th shows the reaction scheme of a cytochrome P450 (CYP450).

The following examples serve to illustrate the invention.

The following standard techniques were used in the examples:

1. Cloning method

For the cloning of the DNA constructs described herein, the following target vectors as well as domesticated DNA sequences, such as promoters, etc., from the GoldenBraid database (http://www.gbcloning.org) were used: pUPD, pDGB1_alpha1, pDGB1_alpha2, pDGB2_alpha1, pDGB2_alpha2, pDGB2_omega1, pDGB2_omega2R, pP35S (GB0030), pTnos (GB0037), pPUbq3 (GB0272), pTAct2 (GB0210), pbfp_CT (GB0034), pYFP_CT (GB0024).

2. Plant material

The Nicotiana benthamiana (N. benthamiana) plants used for transient gene expression were cultivated in the greenhouse at 23 ° C, 60% humidity and 16 h exposure time.

3. Bacterial strains and cultivation conditions

For the cloning of the DNA constructs E. coli DH5α (New England Biolabs, Ipswich, MA, USA) or E. coli TOP10 (Thermo Fisher Scientific, Waltham, USA) was used which was incubated at 37 ° C in Luria-Bertani (LB ) Medium were cultivated. The medium was designed according to the plasmid-mediated resistance as described previously ( Sarrion-Perdigones et al., PLoS One 6 (2011), e21622 ), mixed with antibiotics. The transient transformation of N. benthamiana was carried out using Agrobacterium tumefaciens EHA105 cells which were incubated at 28 ° C. in LB medium containing 50 mg / L rifampicin and 50 mg / L spectinomycin and 100 μM 3,5-dimethoxy-4-hydroxyacetophenone ( Acetosyringone) were cultured.

4. Chemicals

Reference substances for the analytical measurements were indole (Merck KGaA, Darmstadt, Germany), Indikan (AppliChem Darmstadt, Germany), 6-chloroindole (Alfa Aesar, Ward Hill, MA, USA) and 7-chloroindole (Alfa Aesar Ward Hill, MA , USA).

Example 1: Cloning of the DNA constructs

The tnaA gene encoding the tryptophanase (TnaA) (Gene ID: 948221; SEQ ID NOs: 1 (cDNA), 2 (protein)) was prepared from genomic DNA of E. coli DH5α cells by polymerase chain reaction (PCR) with the oligonucleotides 5'-CGCCGTCTCACTCGAGCCGAAAACTTTAAACATCTCCCTGAACCGTT-3 '(SEQ ID NO: 9) and 5'-CGCCGTCTCGCTCGAAGCTTAAACTTCTTTAAGTTTTGCGGTGAAGTG-3' (SEQ ID NO: 10) amplified under standard conditions ( Sambrook and Russell, Molecular Cloning, 3rd edition. Cold Spring Harbor, Cold Spring Harbor Laboratory Press: 8.18-18.24 (2001 ).

At the same time, the GoldenBraid overhangs required for further cloning and the flanking BsmBI restriction cleavage sites were added to the gene sequence.

For cytochrome P450 (CYP) 2A6 L240C / N297Q ( Nakamura et al., Arch Biochem Biophys 395 (1) (2001), 25-31 ) DNA sequence was synthesized. The required GoldenBraid overhangs as well as BsmBI restriction sites were added by exclusion of the endogenous signal sequence by PCR with the oligonucleotides 5'-GACTCGTCTCGCTCGAGCCTCCGTGTGGCAGCAAAGGAAATCCAA-3 '(SEQ ID NO: 11) and 5'-GCCGCGTCTCTCTCGAAGCCTACCTAGGCAAGAATGACATG-3' (SEQ ID NO: 12). The endogenous signal sequence of CYP 2A6 was cloned in several reaction steps. For this purpose, in each case 40 pmol of the two oligonucleotides 5'- CTCGAATGCTCGCTTCTGGTATGTTATTAGTGGCTTTACTGGTTTGCTTGACTGTCATGGTACTAGCC-3 '(SEQ ID NO: 13) and 5'-CTCGGGCTAGTACCATGACAGTCAAGCAAACCAGTAAAGCCACTAATAACATACCAGAAGCGAGCATT-3' (SEQ ID NO: 14) in 1 × ligase buffer (Promega, Madison, WI, USA) in a reaction volume of 20 ul for 5 min at 95 C. and then incubated for 2 h at room temperature (RT). 2 μL of the thus hybridized oligonucleotides were phosphorylated using T4 polynucleotide kinase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

The gene of flavin-dependent tryptophan 7-halogenase RebH (GeneBank AB071405.1, GI: 22830833; (SEQ ID NOs: 5 (cDNA), 6 (protein)) was amplified by PCR to attach all relevant overhangs and BsmBI sites, as well The amplification was carried out in four separate PCR batches using the following oligonucleotides: 5'-AGCGTCTCACTCGAGCCTCCGGCAAGATTGACAAGATCCTC-3 '(SEQ ID NO: 15), 5'-TGCGTCTCTGAGTCTCCGGGTCGAGGTGCCACAT-3' (SEQ ID NO: 16) , 5'-GACGTCTCGACTCAGCCCCTCAACAGGAT-3 '(SEQ ID NO: 17), 5'-TCCGTCTCCATTGTCTCGATCTCGCGGTTG-3' (SEQ ID NO: 18), 5'-AGCGTCTCACAATGTTCGACGACACGCGCGACTT-3 '(SEQ ID NO: 19), 5'-AGCGTCTCCAGTGTCTCGAGCAGGTTCCGCTGC -3 '(SEQ ID NO: 20), 5'-TGCGTCTCACACTGCCGAGCCTCCACGAGTT-3' (SEQ ID NO: 21), 5'-CGCGTCTCACTCGCTGCGCGGCCGTGCTGTTGCCT-3 '(SEQ ID NO: 22).

Furthermore, a modified Cauliflower Mosaic Virus 35S promoter (CaMV P35S_ATG) was modified with GoldenBraid overhangs using the oligonucleotides 5'-CGTACGTCTCTCTCGGGAGACTAGAGCCAAGCTGATCTCCTTTGCACTAGAGCCAAGC-3 '(SEQ ID NO: 23) and 5'-ACTACGTCTCGCTCGGGCTGAGGAAGCCATTTCGACTAGAATAGTAAATTGTAATGTTGTTTGTTG-3' (SEQ ID NO: 24 ) by PCR. The template used was the GoldenBraid pP35S plasmid (GB0030).

In addition, an artificial DNA sequence encoding the chloroplast transit peptide (MASSMLSSAAVVATRASAAQASMVAPFTGLKSAASFPVTRKQNNLDITSIASNGGRVQCA; SEQ ID NO: 26 (as encoded by SEQ ID NO: 25)) of the small RuBisCo subunit was PCR-reacted with the oligonucleotides 5'-TAAGCGTCTCGCTCGAATGGCTTCTTCTATGCTTTCTTCTGC-3 (SEQ ID NO: 27) and 5'-ATTACGTCTCTCTCGGGCTCCTGCGCATTGGACTCTTCCTCCG-3 '(SEQ ID NO: 28) for attaching all relevant GoldenBraid overhangs and BsmBI restriction sites.

All previously described and amplified by PCR DNA fragments were separated by electrophoresis Sambrook and Russell 2001b ( Sambrook, JG and Russell, DW (2001), The Basic Polymerase Chain Reaction. Molecular Cloning, 3rd edition. Cold Spring Harbor, Cold Spring Harbor Laboratory Press: 8: 8.18-18.24) and then purified using the PureLink Quick Gel Extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. This was followed by a GoldenBraid reaction for the ligation of the individual DNA fragments into the Universal Domesticator under the reaction conditions described (US Pat. Sarrion-Perdigones et al., Plant Physiol 162 (2013), 1618-1631 ).

Deviating from this protocol, 3 μL of the phosphorylation mixture was used to domesticate the endogenous 2A6 signal sequence. The entire GoldenBraid reaction mixture was used for the transformation of E. coli DH5α cells by means of heat shock ( Sambrook and Russell Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press: 1.105-101.111 ) and purified from the same using the EZNA Plasmid DNA Mini Kit I (Omega Bio-Tek, Norcross, GA, USA). The gene sequences were checked for error-free amplification by subsequent sequencing.

For the production of a functional tnaA transcription unit, the gene was co-administered with the CaMV P35S_ATG and the nopaline terminator (TNos) in a GoldenBraid reaction ( Sarrion-Perdigones et al., PLoS One 6 (2011), e21622 ) ligated into the pDGB2α1 vector. Another transcription construct consisted of CaMV P35S, cp, tnaA and TNos.

Furthermore, two 2A6 L240C / N297Q transcriptional units composed of the CaMV P35S, 2A6 L240C / N297Q, TNos and either the endogenous 2A6 signal sequence or cp were each ligated into a pDGB2α2 vector. Subsequently, the 2A6 L240C / N297Q transcription unit with the endogenous 2A6 signal sequence was ligated into the pDGB2Ω1 vector with either the tnaA construct without any signal sequence or with cp signal sequence. Furthermore, the tnaA and 2A6 L240C / N297Q transcription units, both with integrated cp sequence, were fused in a pDGB2Ω1 vector.

For the production of functional tryptophan halogenase transcription units, the coding sequences of the flavin-dependent tryptophan 6-halogenase Stth (GeneBank HQ844046.1, GI: 321531617) and the flavin reductase RebF (GeneBank AB071405.1, GI: 22830831), with flanking BsaI restriction sites and all relevant overhangs synthetically produced. Internal BsaI and BsmBI interfaces have been removed. The gene sequences rebH and stth were each cloned with CaMV P35S_ATG, yfp (pYFP_CT) and TNos in a GoldenBraid reaction in the pDGB1α1 vector. Furthermore, rebF was ligated with CaMV P35S_ATG, bfp (pbfp_CT) and TNos into the pDGB2α2 vector. Each of the halogenase constructs prepared in this way was subsequently fused with the reductase transcription unit in a pDGB2Ω2R vector.

For the localization of the halogenases in chloroplasts, additional transcription constructs consisting of rebH or stth and CaMV P35S, cp, yfp and TNos were prepared. Furthermore, rebF was ligated into the pDGB1α2 vector together with the ubiquitin3 promoter (pPUbq3), cp, bfp and the actin terminator (pPAct2). Subsequently, in each case one of the two halogenase transcription units with the rebF construct was cloned into the pDGB2Ω2R vector.

All newly generated plasmids were digested by use of appropriate restriction endonucleases (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions and checked after electrophoretic separation.

Example 2: Transient production of indigo derivatives in Nicotiana benthamiana

Plasmid preparations of the previously described tryptophanase 2A6 L240C / N297Q and the halogenase reductase GoldenBraid constructs were used for the transformation of A. tumefaciens by heat shock. The cells were then centrifuged and placed in infiltration buffer consisting of 10 mM MES (AppliChem, Darmstadt, Germany), 10 mM MgSO4 (AppliChem, Darmstadt, Germany) and 100 μM 3,5-dimethoxy-4-hydroxyacetophenone (Sigma-Aldrich, St. Louis, MO, USA) with a pH of 5.5 to an OD600 of 1. For parallel transformation with the tryptophanase 2A6 L240C / N297Q and the halogenase reductase constructs, the respective bacterial suspensions were mixed in the ratio 1: 1. Cells were infiltrated into the leaf underside of three to four week old N. benthamiana plants using a needleless syringe. To study the bromination of tryptophan by the halogenases, a 40 mM potassium bromide solution was infiltrated into the leaves. All infiltrations were carried out in three biological replicates and the plants were then incubated overnight at RT without exposure. The further cultivation was carried out at 26 ° C, 60% humidity and 12 h exposure time. The accumulation of the recombinant enzymes was checked by fluorescence of the YFP fused to the halogenases four days after infiltration using the Axioskop 40 microscope (Zeiss). The leaf samples were then frozen in liquid nitrogen. For the extraction of the metabolites to be investigated, 100 mg of triturated leaf material were mixed with 200 ml of 80% (v / v) methanol and the cells were digested for 30 min in a Sonorex ultrasonic bath (Bandelin, Berlin, Germany). The supernatant was cleaned by centrifuging twice at 4 ° C from remaining leaf pieces. 10 μL of the leaf extract was purified on a ZORBAX Bonus-RP (C14, 4.6 x 150 mm, particle size 5 μm) column (Agilent Technologies, Santa Clara, CA) using the 1260 Infinity HPLC instrument (Agilent Technologies, Santa Clara, CA , USA) at a column temperature of 30 ° C and a flow rate of 1 mL / min. The detection was carried out by means of a diode array detector (DAD) at 280 nm, wherein also UV spectra were recorded between 200-400 nm. The plant extracts were separated using 0.1% (v / v) formic acid and methanol using a non-linear gradient composed as follows: Time [min] 0.1% (v / v) formic acid methanol 0.0-3.0 70% 30% isocratic 3.0-5.0 30% 70% gradient 5.0-10.0 30% 70% isocratic 10.0 to 11.0 0% 100% gradient 11.0 to 15.0 0% 100% isocratic 15.0 to 16.0 70% 30% gradient 16.0 to 20.0 70% 30% isocratic

Further analysis of the newly synthesized metabolites was performed by ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS). For this purpose, 5 .mu.l leaf extract were assayed using an Acquity BEH (C18, 2.1.times.50 mm, particle size 1.7 .mu.m) UPLC column (Waters, Milford, MA, USA) using the Acquity UPLC device (Waters, Milford, MA, USA) separated. The analysis was performed at a column temperature of 40 ° C and a flow rate of 0.25 mL / min using 0.1% (v / v) formic acid and methanol through a non-linear gradient composed as follows: Time [min] 0.1% (v / v) formic acid methanol 0.0 90% 10% initial 0.0-2.5 70% 30% gradient 2.5-5.0 70% 30% isocratic 5.01 to 7.5 0% 100% isocratic 7.51 to 10.0 90% 10% isocratic

After chromatographic separation, the metabolites were analyzed directly by the connected Micromass Quattro Premier triple quadrupole mass spectrometer (Waters, Milford, MA, USA). The mass spectrometric analysis was performed by alternating positive (ESI +) and negative (ESI) electron spray ionization with a capillary voltage of 2.75 kV (ESI +) and 3 kV (ESI-), respectively. The samples were analyzed at a desolvation temperature of 400 ° C using nitrogen gas at a gas flow rate of 800 L / h, a source temperature of 120 ° C, a cone voltage of 25 V and a cone gas flow of 10 L / h ,

Example 3: Characterization of transiently transformed Nicotiana benthamiana plants

As stated above, it was demonstrated for the first time in 2007 that Indiosbiosynthesis can be achieved by combining different genes in transgenic plants ( Warzecha et al., Plant Biotechnol. J. 5 (2007), 185-191 ). However, the formation of halogenated indoxyl derivatives could not be detected in the plants. Based on the work on Indikanbiosynthese first attempts were made to modify the biosynthesis of Indikan by BX1 and CYP2A6 by means of halogenases. For this purpose, a large number of plant expression vectors by means of modular assembly ( Sarrion-Perdigones et al., PLoS One 6 (2012), e21622 ) joined together. The functional characterization was carried out after mobilization of the plasmids in agrobacteria and transient expression in Nicotiana benthamiana plants. In the transient method, the halogenases RebH and Stth were each co-localized with BX1 and CYP2A6. In no case could halogenated indole or halogenated indicator derivatives be detected ( 2 ). On the contrary, the indole produced by BX1 from indole-3-glycerol phosphate was probably halogenated by the tryptophan halogens in the preferred 2-position and thus removed from the indoxyl biosynthesis.

Only by the modification of the combinatorial biosynthetic pathway by integration of an additional enzyme, d. H. by introducing a tryptophanase, significant amounts of 6- or 7-chloroindicene or 6- or 7-bromoindane could be obtained.

In order to first generate halogenated tryptophan in an alternative biosynthetic pathway, which then serves as a substrate for recombinant tryptophanase TnaA, plant expression vectors were generated which allow variable localization of TnaA and the halogenases RebH and Stth in different plant cell compartments. To further metabolize the resulting halogenated indole derivatives, CYP2A6 was also co-expressed.

The investigations of the plant extracts by means of thin-layer chromatography and HPLC clearly show that the combination of TnaA and CYP2A6 significantly higher levels of indican can be formed in plants, as with BX1 and CYP2A6. In addition, by incorporating the halogenases, it was possible for the first time to generate halogenated indicator derivatives in planta. Both the cytosol (with ER membrane-bound P450) and the chloroplast were identified as compartments with the highest yield. Moreover, in chloroplasts, there was no need to coexpress the corresponding reductases. This means that reduction equivalents (NADPH and FADH) can be obtained from photosynthesis. The cellular localization of the recombinant enzymes was very flexible. For example, efficient co-localization of TnaA and CYP2A6 in the chloroplasts or the cytosol as well as in the separation of the two enzymes into chloroplasts or the cytosol resulted in an efficient Indican biosynthesis. Furthermore, it was possible to produce halogenated indicator derivatives by co-localization of all enzymes in chloroplasts or the cytosol. Similarly, the translocation of the halogenases in the Chloroplasts with simultaneous cytosolic activity of TnaA and CYP2A6 for the accumulation of Chlorindikan in the plants.

In addition to the specific 7-chlorination by RebH and 6-chlorination by Stth, bromination of the indole ring could also be generated by additional infiltration of a potassium bromide solution ( 3 to 5 ).

This is followed by a sequence protocol according to WIPO St. 25. This can be downloaded from the official publication platform of the DPMA.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

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Claims (11)

Transgenic plant cell comprising a heterologous tryptophanase (EC 4.1.99.1) and a heterologous cytochrome P450 (CYP450). The transgenic plant cell of claim 1, wherein the tryptophanase is E. coli. Transgenic plant cell according to claim 1 or claim 2, characterized in that it comprises a heterologous halogenase. A transgenic plant cell according to claim 3, wherein the halogenase is from a bacterium of the genus Lechevalieria or the genus Streptomyces. A transgenic plant cell according to claim 3 or claim 4, wherein the halogenase is a tryptophan haloase (EC 1.14.19.9). A transgenic plant cell according to any one of claims 1 to 5, wherein the tryptophanase, the cytochrome P450 2A6 and the halogenase have been introduced into the transgenic plant cell by one or more nucleic acid molecules. A transgenic plant cell according to any one of claims 3 to 6, wherein the tryptophan haloase is encoded by a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8; (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 in which one to 40 amino acid residues have been deleted, substituted, inserted and / or added, and has tryptophan haloase activity; and (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8 and having tryptophan haloacid activity. A transgenic plant cell according to any one of claims 1 to 7, wherein the tryptophanase is encoded by a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 1; (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2; (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2 in which one to 40 amino acid residues have been deleted, substituted, inserted and / or added, and have tryptophanase activity; and (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having tryptophanase activity. A transgenic plant cell according to any one of claims 1 to 8, wherein the cytochrome P450 (CYP450) is encoded by a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule comprising a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 3; (b) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4; (c) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 4 in which one to 40 amino acid residues have been deleted, substituted, inserted and / or added, and cytochrome P450 2A6 activity Has; and (d) a nucleic acid molecule comprising a nucleic acid sequence encoding a protein having an amino acid sequence of at least 75% sequence identity to the amino acid sequence of SEQ ID NO: 4 and having cytochrome P450 (CYP450) activity. Transgenic plant or plant cell culture containing a transgenic plant cell according to any one of claims 1 to 9. Process for the preparation of halogenated indoxyl derivatives from tryptophan, comprising the steps: (a) culturing the transformed plant cell of any one of claims 1 to 9, the transgenic plant of claim 10 or the plant cell culture of claim 10 in a suitable medium; and (b) recovering the halogenated indoxyl derivatives from the transformed plant cell and / or cell culture medium.

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