Yeast cells and methods for production of tryptophan derivatives
AssigneeDanmarks Tekniske Universitet
InventorsIrina Borodina, Nicholas Milne, Steven van der Hoek, Morten Otto Alexander Sommer, Jérémy Christophe Daniel Armetta, Philip Tinggaard Thomsen
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Abstract
The present disclosure relates to methods for production of 4-hydroxytryptamine and derivatives thereof in a yeast cell. Herein are also disclosed methods for production of halogenated tryptophans and derivatives thereof in a cell. Herein are also disclosed methods for production of methylated tryptamine. The disclosure also provides nucleic acid constructs and cells useful for performing the present methods.
Claims118
A cell capable of producing a halogenated tryptophan, wherein the halogenated tryptophan is a tryptophan substituted with one, two or three halogen atoms, and optionally derivatives thereof, in the presence of a halogen or derivatives thereof, said cell expressing at least one of:
- a tryptophan-2-halogenase (EC 1.14.14), preferably a heterologous tryptophan-2-halogenase such as CcCmdE (SEQ ID NO:
- 48) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a tryptophan-5-halogenase (EC 1.14.19.58), preferably a heterologous tryptophan-5-halogenase such as SrPyrH (SEQ ID NO:
- 32) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a tryptophan-6-halogenase (EC 1.14.19.59), preferably a heterologous tryptophan-6-halogenase such as SttH (SEQ ID NO:
- 33), SaThaI (SEQ ID NO:
- 51), or KtzR (SEQ ID NO:
- 54), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-6-halogenase is SttH (SEQ ID NO:
- 33) or a functional variant thereof having at least 80% homology thereto,
- a tryptophan-7-halogenase (EC 1.14.19.9), preferably a heterologous tryptophan-7-halogenase such as LaRebH (SEQ ID NO:
- 34), PfPrnA (SEQ ID NO:
- 50), or KtzQ (SEQ ID NO:
- 53), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-7-halogenase is LaRebH (SEQ ID NO:
- 34) or a functional variant thereof having at least 80% homology thereto, or
- a tryptophan halogenase, preferably a heterologous tryptophan halogenase such as DdChlA (SEQ ID NO:
- 52), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- and optionally expressing a flavin reductase (EC 1.5.1.30), preferably a heterologous flavin reductase, such as LaRebF (SEQ ID NO:
- 35) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- whereby the cell is capable of converting tryptophan into a halogenated tryptophan and optionally derivatives thereof, a dihalogenated tryptophan and optionally derivatives thereof, or a trihalogenated tryptophan and optionally derivatives thereof,
- preferably wherein the cell is a microorganism or a plant cell.
The cell according to any one of the preceding claims, wherein the derivative of tryptophan is 2,5-dihalogenated, 2,6-dihalogenated, 2,7-dihalogenated, 5,6-dihalogenated, 5,7-dihalogenated or 6,7-dihalogenated by two halogens independently selected from the group consisting of fluorine, bromine, iodine and chlorine.
The cell according to any one of the preceding claims, wherein the derivative of tryptophan is 2,5,6-trihalogenated, 2,5,7-trihalogenated, 2,6,7-trihalogenated or 5,6,7-trihalogenated by three halogens independently selected from the group consisting of fluorine, bromine, iodine and chlorine.
The cell according to any one of the preceding claims, wherein the cell is a eukaryotic cell or a bacteria or the plant cell is a microalgae cell.
The cell according to any one of the preceding claims, wherein the cell is a yeast cell belonging to the genus of Saccharomyces, such as S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, such as K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, such as C. utilis, C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, such as P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus, such as C. aerius, Debaromyces, such as D. hansenii, Hansenula, Pichia, such as P. pastoris, Yarrowia, such as Y. lipolytica, Zygosaccharomyces, such as Z. bailii, Torulaspora, such as T. delbrueckii, Schizosaccharomyces, such as S. pombe, Brettanomyces, such as B. bruxellensis, Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, and Trichoderma.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2-halogenated, a 5-halogenated, a 6-halogenated, or a 7-halogenated tryptophan with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5-dihalogenated, a 2,6-dihalogenated, a 2,7-dihalogenated, a 5,6-dihalogenated, a 5,7-dihalogenated or a 6,7-dihalogenated tryptophan with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5,6-trihalogenated, a 2,5,7-trihalogenated, a 2,6,7-trihalogenated or a 5,6,7-trihalogenated tryptophan with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, further expressing a tryptophan decarboxylase (EC 4.1.1.105), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the tryptophan decarboxylase is capable of converting the halogenated tryptophan to a corresponding halogenated tryptamine, whereby the cell is capable of producing the halogenated tryptamine and optionally derivatives thereof, wherein the halogenated tryptamine or derivative thereof is a tryptamine or a tryptamine derivative substituted with one, two or three halogen atoms.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2-halogenated tryptamine, a 5-halogenated tryptamine, a 6-halogenated tryptamine, and/or a 7-halogenated tryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5-dihalogenated tryptamine, a 2,6-dihalogenated tryptamine, a 2,7-dihalogenated tryptamine, a 5,6-dihalogenated tryptamine, a 5,7-dihalogenated tryptamine, and/or a 6,7-dihalogenated tryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5,6-trihalogenated tryptamine, a 2,5,7-trihalogenated tryptamine, a 2,6,7-trihalogenated tryptamine and/or a 5,6,7-trihalogenated tryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, additionally expressing an indole N-methyltransferase (EC 2.1.1.49), preferably a heterologous indole N-methyltransferase, such as OcINMT (SEQ ID NO:
- 36) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the indole N-methyltransferase is capable of converting the halogenated tryptamine to a corresponding halogenated N-methyltryptamine, a corresponding halogenated N,N-dimethyltryptamine, and/or a corresponding halogenated N,N,N-trimethyltryptamine, whereby the cell is capable of producing the halogenated N-methyltryptamine, the halogenated N,N-dimethyltryptamine, the halogenated N,N,N-trimethyltryptamine, and optionally derivatives thereof, wherein the halogenated N-methyltryptamine, the halogenated N,N-dimethyltryptamine, the halogenated N,N,N-trimethyltryptamine, or derivatives thereof, are N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, or derivatives thereof, substituted with one, two or three halogen atoms.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2-halogenated N-methyltryptamine, a 2-halogenated N,N-dimethyltryptamine and/or a 2-halogenated N,N,N-trimethyltryptamine, a 5-halogenated N-methyltryptamine, a 5-halogenated N,N-dimethyltryptamine and/or a 5-halogenated N,N,N-trimethyltryptamine, a 6-halogenated N-methyltryptamine, a 6-halogenated N,N-dimethyltryptamine and/or a 6-halogenated N,N,N-trimethyltryptamine, and/or a 7-halogenated N-methyltryptamine, a 7-halogenated N,N-dimethyltryptamine and/or a 7-halogenated N,N,N-trimethyltryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5-dihalogenated N-methyltryptamine, a 2,5-dihalogenated N,N-dimethyltryptamine and/or a 2,5-dihalogenated N,N,N-trimethyltryptamine, a 2,6-dihalogenated N-methyltryptamine, a 2,6-dihalogenated N,N-dimethyltryptamine and/or a 2,6-dihalogenated N,N,N-trimethyltryptamine, a 2,7-dihalogenated N-methyltryptamine, a 2,7-dihalogenated N,N-dimethyltryptamine and/or a 2,7-dihalogenated N,N,N-trimethyltryptamine, a 5,6-dihalogenated N-methyltryptamine, a 5,6-dihalogenated N,N-dimethyltryptamine and/or a 5,6-dihalogenated N,N,N-trimethyltryptamine, a 5,7-dihalogenated N-methyltryptamine, a 5,7-dihalogenated N,N-a dimethyltryptamine and/or a 5,7-dihalogenated N,N,N-trimethyltryptamine, and/or a 6,7-dihalogenated N-methyltryptamine, a 6,7-dihalogenated N,N-dimethyltryptamine and/or a 6,7-dihalogenated N,N,N-trimethyltryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the cell is capable of producing a 2,5,6-trihalogenated N-methyltryptamine, a 2,5,6-trihalogenated N,N-dimethyltryptamine and/or a 2,5,6-trihalogenated N,N,N-trimethyltryptamine, a 2,5,7-trihalogenated N-methyltryptamine, a 2,5,7-trihalogenated N,N-dimethyltryptamine and/or a 2,5,7-trihalogenated N,N,N-trimethyltryptamine, a 2,6,7-trihalogenated N-methyltryptamine, a 2,6,7-trihalogenated N,N-dimethyltryptamine and/or a 2,6,7-trihalogenated N,N,N-trimethyltryptamine, a 5,6,7-trihalogenated N-methyltryptamine, a 5,6,7-trihalogenated N,N-dimethyltryptamine and/or a 5,6,7-trihalogenated N,N,N-trimethyltryptamine, preferably with a titer of at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more.
The cell according to any one of the preceding claims, wherein the total titer of all produced halogenated compounds is at least 0.25 mg/L, such as at least 0.3 mg/L, such as at least 0.4 mg/L, such as at least 0.5 mg/L, such as at least 0.75 mg/L, such as at least 1 mg/L, such as at least 1.5 mg/L, such as at least 2.5 mg/L, such as at least 5.0 mg/L, such as at least 10 mg/L, such as at least 15 mg/L, such as at least 20 mg/L, such as 25 mg/L, such as at least 50 mg/L, such as at least 100 mg/L, such as at least 250 mg/L, such as at least 500 mg/L, such as at least 750 mg/L, such as at least 1 g/L, such as at least 2 g/L, such as at least 3 g/L, such as at least 4 g/L, such as at least 5 g/L, such as at least 6 g/L, such as at least 7 g/L, such as at least 8 g/L, such as at least 9 g/L, such as at least 10 g/L, such as at least 20 g/L, such as at least 30 g/L or more, wherein the total titer is the sum of the titers of the 2-halogenated, 5-halogenated, 6-halogenated, 7-halogenated, 2,5-dihalogenated, 2,6-dihalogenated, 2,7-dihalogenated, 5,6-dihalogenated, 5,7-dihalogenated, 6,7-dihalogenated, 2,5,6-trihalogenated, 2,5,7-trihalogenated, 2,6,7-trihalogenated and 5,6,7-trihalogenated tryptophans, tryptamines, N-methyltryptamines, N,N-dimethyltryptamines and N, N, N-trimethyltryptamines.
The cell according to any one of the preceding claims, wherein one or more of the genes encoding the tryptophan-2-halogenase, the tryptophan-5-halogenase, the tryptophan-6-halogenase, the tryptophan-7-halogenase, the flavin reductase, the tryptophan decarboxylase and/or the indole N-methyltransferase is under the control of an inducible promoter.
The cell according to any one of the preceding claims, wherein one or more of the genes encoding the tryptophan-2-halogenase, the tryptophan-5-halogenase, the tryptophan-6-halogenase, the tryptophan-7-halogenase, the flavin reductase, the tryptophan decarboxylase and/or the indole N-methyltransferase is codon-optimised for the cell.
The cell according to any one of the preceding claims, wherein one or more of the genes encoding the tryptophan-2-halogenase, the tryptophan-5-halogenase, the tryptophan-6-halogenase, the tryptophan-7-halogenase, the flavin reductase, the tryptophan decarboxylase and/or the indole N-methyltransferase is present in high copy number.
The cell according to any one of the preceding claims, wherein one or more of the genes encoding the tryptophan-2-halogenase, the tryptophan-5-halogenase, the tryptophan-6-halogenase, the tryptophan-7-halogenase, the flavin reductase, the tryptophan decarboxylase and/or the indole N-methyltransferase is integrated in the genome of the cell.
The cell according to any one of the preceding claims, wherein one or more of the genes encoding the tryptophan-2-halogenase, the tryptophan-5-halogenase, the tryptophan-6-halogenase, the tryptophan-7-halogenase, the flavin reductase, the tryptophan decarboxylase and/or the indole N-methyltransferase is expressed from a vector such as a plasmid.
The cell according to any one of the preceding claims, wherein the halogen is chlorine, fluorine, bromine or iodine, and wherein the cell is capable of producing, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, said cell expressing:
- the tryptophan-5-halogenase SrPyrH (SEQ ID NO:
- 32), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, and
- the flavin reductase LaRebF (SEQ ID NO:
- 35), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
The cell according to any one of the preceding claims, wherein the halogen is chlorine, fluorine, bromine or iodine, and wherein the cell is capable of producing, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, said cell expressing:
- the tryptophan-6-halogenase SttH (SEQ ID NO:
- 33), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, and
- the flavin reductase LaRebF (SEQ ID NO:
- 35), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
The cell according to any one of the preceding claims, wherein the halogen is chlorine, fluorine, bromine or iodine, and wherein the cell is capable of producing, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptophan and/or, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptamine, said cell expressing:
- the tryptophan-5-halogenase SrPyrH (SEQ ID NO:
- 32), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- the flavin reductase LaRebF (SEQ ID NO:
- 35), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, and
- the tryptophan decarboxylase CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
The cell according to any one of the preceding claims, wherein the halogen is chlorine, fluorine, bromine or iodine, and wherein the cell is capable of producing, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptophan and/or, respectively, a chlorinated, a fluorinated, a brominated or a iodinated tryptamine, said cell expressing:
- the tryptophan-6-halogenase SttH (SEQ ID NO:
- 33), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- the flavin reductase LaRebF (SEQ ID NO:
- 35), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, and
- the tryptophan decarboxylase CrTDC (SEQ ID NO:
- 1), or a functional variant thereof having at least at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
A method of producing a halogenated tryptophan wherein the halogenated tryptophan is a tryptophan substituted with one, two or three halogen atoms, and optionally derivatives thereof, in a cell, preferably wherein the cell is a microorganism or a plant cell, said method comprising the steps of providing a cell and incubating said cell in the presence of a halogen, wherein the cell expresses at least one of:
- a tryptophan-2-halogenase (EC 1.14.14), preferably a heterologous tryptophan-2-halogenase such as CcCmdE (SEQ ID NO:
- 48) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a tryptophan-5-halogenase (EC 1.14.19.58), preferably a heterologous tryptophan-5-halogenase such as SrPyrH (SEQ ID NO:
- 32) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a tryptophan-6-halogenase (EC 1.14.19.59), preferably a heterologous tryptophan-6-halogenase such as SttH (SEQ ID NO:
- 33), SaThaI (SEQ ID NO:
- 51), or KtzR (SEQ ID NO:
- 54), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-6-halogenase is SttH (SEQ ID NO:
- 33) or a functional variant thereof having at least 80% homology thereto,
- a tryptophan-7-halogenase (EC 1.14.19.9), preferably a heterologous tryptophan-7-halogenase such as LaRebH (SEQ ID NO:
- 34), PfPrnA (SEQ ID NO:
- 50), or KtzQ (SEQ ID NO:
- 53), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-7-halogenase is LaRebH (SEQ ID NO:
- 34) or a functional variant thereof having at least 80% homology thereto, or
- a tryptophan halogenase, preferably a heterologous tryptophan halogenase such as DdChlA (SEQ ID NO:
- 52), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- and optionally a flavin reductase, preferably a heterologous flavin reductase (EC:
- EC 1.5.1.30), such as LaRebF (SEQ ID NO:
- 35), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
A nucleic acid construct comprising at least one of:
- a polynucleotide encoding a tryptophan-2-halogenase (EC 1.14.14),
- preferably a heterologous tryptophan-2-halogenase such as CcCmdE (SEQ ID NO:
- 48) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a polynucleotide encoding a tryptophan-5-halogenase (EC 1.14.19.58), preferably a heterologous tryptophan-5-halogenase such as SrPyrH (SEQ ID NO:
- 32) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a polynucleotide encoding a tryptophan-6-halogenase (EC 1.14.19.59), preferably a heterologous tryptophan-6-halogenase such as SttH (SEQ ID NO:
- 33), SaThaI (SEQ ID NO:
- 51), or KtzR (SEQ ID NO:
- 54), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-6-halogenase is SttH (SEQ ID NO:
- 33) or a functional variant thereof having at least 80% homology thereto,
- a polynucleotide encoding a tryptophan-7-halogenase (EC 1.14.19.9), preferably a heterologous tryptophan-7-halogenase such as LaRebH (SEQ ID NO:
- 34), PfPrnA (SEQ ID NO:
- 50), or KtzQ (SEQ ID NO:
- 53), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, more preferably the tryptophan-7-halogenase is LaRebH (SEQ ID NO:
- 34) or a functional variant thereof having at least 80% homology thereto, or
- a polynucleotide encoding a tryptophan halogenase, preferably a heterologous tryptophan halogenase such as DdChlA (SEQ ID NO:
- 52), or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- and optionally a polynucleotide encoding a flavin reductase (EC 1.5.1.30), preferably a heterologous flavin reductase, such as LaRebF (SEQ ID NO:
- 35) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
The nucleic acid construct according to any one of 49 to 58, wherein the polynucleotide encoding the heterologous indole N-methyltransferase comprises or consists of SEQ ID NO:
- 41 or a variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
A cell capable of producing N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine, preferably wherein the cell is a microorganism or a plant cell, said cell expressing:
- a tryptophan decarboxylase (EC 4.1.1.105), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine;
- and
- an indole N-methyltransferase, preferably a heterologous indole N-methyltransferase (EC 2.1.1.49), such as OcINMT (SEQ ID NO:
- 36) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the indole N-methyltransferase is capable of converting tryptamine to N-methyltryptamine, to N,N-dimethyltryptamine, and/or to N,N,N-trimethyltryptamine,
- whereby the cell is capable of producing N-methyltryptamine, N,N-dimethyltryptamine, and/or N,N,N-trimethyltryptamine.
A method for producing N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine in a cell, preferably wherein the cell is a microorganism or a plant cell, said method comprising the steps of providing a cell and incubating said cell in a medium, wherein the cell expresses:
- a tryptophan decarboxylase (EC 4.1.1.105), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine;
- and
- an indole N-methyltransferase, preferably a heterologous indole N-methyltransferase (EC 2.1.1.49), such as OcINMT (SEQ ID NO:
- 36) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the indole N-methyltransferase is capable of converting tryptamine to N-methyltryptamine, to N,N-dimethyltryptamine, and/or to N,N,N-trimethyltryptamine.
A nucleic acid construct for modifying a cell, wherein the cell is as defined in any one of the preceding claims, said construct comprising:
- a first polynucleotide encoding a tryptophan decarboxylase (EC 4.1.1.105) (SEQ ID NO:
- 6), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a first polynucleotide comprising or consisting of SEQ ID NO:
- 6 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto
- a seventh polynucleotide encoding an indole N-methyltransferase, preferably a heterologous indole N-methyltransferase (EC 2.1.1.49), such as OcINMT (SEQ ID NO:
- 36) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a seventh polynucleotide comprising or consisting of SEQ ID NO:
- 36 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
A cell, preferably a yeast cell, capable of producing 4-hydroxytryptamine and optionally derivatives thereof, said cell expressing:
- a tryptophan decarboxylase (EC 4.1.1.105), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine;
- a tryptamine 4-monooxygenase (EC 1.14.99.59), preferably a heterologous tryptamine 4-monooxygenase such as PcPsiH (SEQ ID NO:
- 2) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- a cytochrome P450 reductase (EC 1.6.2.4), preferably a heterologous cytochrome P450 reductase such as PcCpr (SEQ ID NO:
- 3) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto,
- wherein the tryptamine 4-monooxygenase and the cytochrome P450 reductase together catalyse the conversion of tryptamine to 4-hydroxytryptamine,
- whereby the cell is capable of converting tryptophan to 4-hydroxytryptamine.
A method of producing 4-hydroxytryptamine and optionally derivatives thereof in a cell such as a yeast cell, said method comprising the steps of providing a cell and incubating said cell in a medium, wherein the cell expresses:
- a tryptophan decarboxylase (EC 4.1.1.105), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine;
- a tryptamine 4-monooxygenase (EC 1.14.99.59), preferably a heterologous tryptamine 4-monooxygenase such as PcPsiH (SEQ ID NO:
- 2) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, and
- a cytochrome P450 reductase (EC 1.6.2.4), preferably a heterologous cytochrome P450 reductase such as PcCpr (SEQ ID NO:
- 3) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
A nucleic acid construct for modifying a cell, wherein the cell preferably is a yeast cell, said construct comprising:
- a first polynucleotide encoding a tryptophan decarboxylase (EC 4.1.1.105) (SEQ ID NO:
- 6), preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO:
- 1) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a first polynucleotide comprising or consisting of SEQ ID NO:
- 6 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto
- a second polynucleotide encoding a tryptamine 4-monooxygenase (EC 1.14.99.59) (SEQ ID NO:
- 7), preferably a heterologous tryptamine 4-monooxygenase such as PcPsiH (SEQ ID NO:
- 2) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a second polynucleotide comprising or consisting of SEQ ID NO:
- 7 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto;
- and
- a third polynucleotide encoding a cytochrome P450 reductase (EC 1.6.2.4) (SEQ ID NO:8), preferably a heterologous cytochrome P450 reductase such as PcCpr (SEQ ID NO:
- 3) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a third polynucleotide comprising or consisting of SEQ ID NO:
- 8 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto;
- and optionally,
- a fourth polynucleotide encoding a 4-hydroxytryptamine kinase (EC 2.7.1.222), preferably a heterologous 4-hydroxytryptamine kinase such as PcPsiK (SEQ ID NO:
- 4) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a fourth polynucleotide comprising or consisting of SEQ ID NO:
- 9 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto;
- and
- a fifth polynucleotide encoding a psilocybin synthase (EC 2.1.1.345), preferably a heterologous psilocybin synthase such as PcPsiM (SEQ ID NO:
- 5) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a fifth polynucleotide comprising or consisting of SEQ ID NO:
- 10 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto;
- and/or
- a sixth polynucleotide encoding a serotonin N-acetyltransferase (EC 2.3.1.87), preferably a heterologous serotonin N-acetyltransferase such as BtAANAT (SEQ ID NO:
- 11) or a functional variant thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto, such as a sixth polynucleotide comprising or consisting of SEQ ID NO:
- 30 or a homologue thereof having at least 80% homology, such as at least 85%, such as at least 90%, such as at least 95% homology thereto.
Dependencies are inferred from the claim text.