Patent Application
20220403428
Herein are disclosed 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.
Psilocybin is a tryptamine-derived psychoactive alkaloid found in the fungal genus Psilocybe, among others, and is the active ingredient in so-called ‘magic mushrooms’. Psilocybin itself is not psychoactive—rather it is the dephosphorylated derivative psilocin that causes the hallucinogenic effect. Psilocybin is rapidly dephosphorylated to psilocin following ingestion in the mucosa by alkaline phosphatases and nonspecific esterases. Psilocin is structurally similar to human signaling molecules such as serotonin, and has been shown to bind to over 15 human serotonin-related receptors.
Clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. Preliminary results suggest that psilocybin assisted treatment may be a good candidate for managing substance addiction (Bogenschutz et al., 2015; Riaz et al., 2016), anxiety in terminally ill patients (Grob et al., 2011), cluster headaches (Tyls̆ et al., 2014), and treatment-resistant depression (Carhart-Harris et al., 2018). Psilocybin seems to be a particularly interesting candidate for “treatment resistant depression”—a term applied to the 13% of patients with Major Depressive Disorder (MDD) who relapse, in spite of four rounds of traditional treatment (Rush et al., 2006). Approximately 16 million Americans carried the MDD diagnosis in 2016, indicating a large number of people with untreated mental illness (Tice, 2017).
Unfortunately, the content of psilocybin and psilocin in hallucinogenic mushrooms is too low (0.2%-1% dry weight) to make extraction a commercially viable option (Tyls̆ et al., 2014), and chemical synthesis is complicated and expensive (Nichols and Frescas, 1999). Although the chemical synthesis of psilocybin has been improved since its discovery by Hoffman et al. in 1959, who achieved final yields of 20% of semi pure psilocybin, it continues to challenge chemists primarily due to the difficulty of the last synthetic step; the phosphorylation of psilocin (Nichols and Frescas, 1999).
Therefore, psilocybin is currently obtained mainly through complex and expensive chemical synthesis. Accordingly, there is a demand for effective and low cost production of psilocybin for pharmaceutical applications.
Halogenated compounds constitute a large fraction of pharmaceuticals on the market and in development due to the ability of halogenation to modulate the properties of a lead drug candidate. Since many natural products with therapeutic properties are synthesized from tryptamine, for example the Catharanthus roseus anticancer agent vinblastine, halogenated tryptamine derivatives might be of great therapeutic interest.
Biotechnological production of halogenated tryptamines in a cell factory could serve both as a production method and as a means for drug discovery. Halogenated tryptophan, halogenated tryptamine, halogenated N-methylated tryptamine, halogenated N,N-dimethyltryptamine and halogenated N,N,N-trimethyltryptamine are expected to have enhanced therapeutic properties compared to their non-halogenated counterparts. Accordingly, a method for effective and low cost production of these compounds for pharmaceutical applications is desirable.
DMT is a psychoactive tryptamine derivative present in a wide range of plants as well as in mammals, where it is synthesized from tryptamine by the SAM-dependent indole N-methyltransferase INMT.
The invention is as defined in the claims.
Herein is provided a method for the production of 4-hydroxytryptamine and derivatives thereof, such as psilocybin, in a yeast cell by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. The inventors have achieved improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from Psilocybe cubensis. Microbial based production of 4-hydroxytriptamine and derivatives thereof, including psilocybin, can be performed at reduced financial and environmental costs compared to methods known in the art.
In one aspect, the present invention provides a yeast cell capable of producing 4-hydroxytryptamine and optionally derivatives thereof, said cell expressing:
In another aspect, the present invention provides a method of producing 4-hydroxytryptamine and optionally derivatives thereof in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
In one aspect, the present invention provides 4-hydroxytryptamine and derivatives thereof obtainable by the methods disclosed herein.
In one aspect, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In another aspect, the present invention provides a kit of parts comprising:
Herein are also disclosed methods for the production of halogenated tryptophans and derivatives thereof in a cell by expression of a heterologous biosynthesis pathway sourced from Chondromyces crocatus, Streptomyces rugosporus, Streptomyces toxytricini, Lechevalieria aerocolonigenes, Catharanthus roseus and Oryctolagus cuniculus. De novo cell-based production of halogenated tryptophan and derivatives thereof in cells, such as yeast cells, can be performed at low financial and environmental costs.
In one aspect, is provided 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:
In another aspect, is provided 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:
In one aspect, are provided halogenated tryptophans and derivatives thereof, in particular a halogenated tryptophan, a dihalogenated tryptophan, a trihalogenated tryptophan, a halogenated tryptamine, a dihalogenated tryptamine, a trihalogenated tryptamine, a halogenated N-methyltryptamine, a halogenated N,N-dimethyltryptamine, a halogenated N,N,N-trimethyltryptamine, a dihalogenated N-methyltryptamine, a dihalogenated N,N-dimethyltryptamine, a dihalogenated N,N,N-trimethyltryptamine, a trihalogenated N-methyltryptamine, a trihalogenated N,N-dimethyltryptamine and a trihalogenated N,N,N-trimethyltryptamine obtainable by the methods disclosed herein.
In one aspect, is provided a nucleic acid construct for modifying a cell, said construct comprising at least one of:
In another aspect, the present invention provides a kit of parts comprising:
Herein is also provided 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:
whereby the cell is capable of producing N-methyltryptamine, N,N-dimethyltryptamine, and/or N,N,N-trimethyltryptamine.
Also provided herein is 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:
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 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% 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.
Also provided herein are nucleic acid constructs comprising:
Herein are disclosed yeast cells useful for production of 4-hydroxytryptamine and derivatives thereof, as well as methods, nucleic acid constructs and kits for production of 4-hydroxytryptamine and derivatives thereof.
Herein is provided a yeast cell capable of producing 4-hydroxytryptamine and optionally derivatives thereof, said cell expressing:
Herein is also provided a method of producing 4-hydroxytryptamine and optionally derivatives thereof in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
Herein is also provided 4-hydroxytryptamine, norbaeocystin, baeocystin, norpsilocin, psilocybin, psilocin, aeruginascin, dephosphorylated aeruginascin or N-acetyl-4-hydroxytryptamine obtainable by the methods disclosed herein.
Herein is also provided a nucleic acid construct for modifying a yeast cell, said construct comprising:
Herein is also provided a kit of parts comprising:
Tdc: tryptophan decarboxylase (EC 4.1.1.105)
L-tryptophan+H+→CO2+tryptamine
The tryptophan decarboxylase (Tdc) converts L-tryptophan into tryptamine.
The enzyme Tdc preferably originates from the organism Catharanthus roseus.
PsiH: Tryptamine 4-monooxygenase (EC 1.14.99.59)
tryptamine+reduced acceptor+O2=4-hydroxytryptamine+acceptor+H2O
Tryptamine 4-monooxygenase (PsiH) converts tryptamine into 4-hydroxytryptamine.
The enzyme PsiH preferably originates from the organism Psilocybe cubensis.
Cpr: cytochrome P450 reductase (EC 1.6.2.4) catalyses the reaction NADPH+H++n oxidized hemoprotein=NADP++n reduced hemoprotein Cytochrome P450 reductase (Cpr) converts oxidized hemoprotein into reduced hemoprotein.
The enzyme Cpr preferably originates from the organism Psilocybe cubensis.
PsiK: 4-hydroxy tryptamine kinase (EC 2.7.1.222)
ATP+4-hydroxytryptamine→ADP+norbaeocystin+H+
4-hydroxy tryptamine kinase (PsiK) converts 4-hydroxytryptamine into norbaeocystin.
The enzyme PsiK preferably originates from the organism Psilocybe cubensis.
AANAT: serotonin N-acetyltransferase (EC 2.3.1.87)
Acetyl-CoA+a 2-arylethylamine<=>CoA+an N-acetyl-2-arylethylamine
The enzyme catalyses conversion of a 2-arylethylamine to the corresponding N-acetyl-2-arylethylamine in the presence of acetyl-CoA. For example it catalyses the conversion of 4-hydroxytryptamine into N-acetyl-4-hydroxytryptamine.
The enzyme AANAT preferably originates from the organism Bos taurus.
PsiM: Psilocybin synthase (EC 2.1.1.345) 2 S-adenosyl-L-methionine+norbaeocystin→2 S-adenosyl-L-homocysteine+psilocybin+2 H+
PsiM enzyme catalyses 2 (or 3) consecutive N-methylation steps; Norbaeocystin->Baeocystin->Psilocybin->Aeruginascin
The enzyme PsiM preferably originates from the organism Psilocybe cubensis.
4-hydroxytryptamine derivatives: the term herein refers to compounds obtainable from 4-hydroxytryptamine, such as psilocybin, psilocin, norbaeocystin, N-acetyl-4-hydroxytryptamine, baeocystin, norpsilocin, aeruginascin and dephosphorylated aeruginascin.
Functional variant: the term is herein applied to functional variants of enzymes, i.e. modified versions of the enzyme which retain some or all the catalytic activity of the enzyme. Functional variants may have been modified by introducing mutations which confer e.g. increased activity, a change in intracellular localisation, prolonged half-life, among others, but retain the ability to perform the same enzymatic reaction as the enzymes they are derived from. The mutations resulting in modified activity may be in the genes coding for the enzymes, including in their promoter region.
CmdE: tryptophan-2-halogenase (EC 1.14.14)
tryptophan+FADH2+X−+O2+H+2-X-L-tryptophan+FAD+2 H2O,
where X is any halogen selected from the group consisting of fluorine, bromine, iodine and chlorine.
The tryptophan-2-halogenase replaces the hydrogen at position 2 of the indole ring of tryptophan with a halogen atom.
The enzyme CmdE preferably originates from the organism Chondromyces crocatus.
PyrH: tryptophan-5-halogenase (EC 1.14.19.58)
tryptophan+FADH2+X−+O2+H+5-X-L-tryptophan+FAD+2 H2O,
where X is any halogen selected from the group consisting of fluorine, bromine, iodine and chlorine.
The tryptophan-5-halogenase replaces the hydrogen at position 5 of the indole ring of tryptophan with a halogen atom.
The enzyme PyrH preferably originates from the organism Streptomyces rugosporus.
tH: tryptophan-6-halogenase (EC 1.14.19.59)
tryptophan+FADH2+X−+O2+H+6-X-L-tryptophan+FAD+2 H2O,
where X is any halogen selected from the group consisting of fluorine, bromine, iodine and chlorine.
The tryptophan-6-halogenase replaces the hydrogen at position 6 of the indole ring of tryptophan with a halogen atom.
The enzyme tH preferably originates from the organism Streptomyces toxytricini.
RebH: tryptophan-7-halogenase (EC 1.14.19.9)
tryptophan+FADH2+X−+O2+H+7-X-L-tryptophan+FAD+2 H2O,
where X is any halogen selected from the group consisting of fluorine, bromine, iodine and chlorine.
The tryptophan-7-halogenase replaces the hydrogen at position 7 of the indole ring of tryptophan with a halogen atom.
The enzyme RebH preferably originates from the organism Lechevalieria aerocolonigenes.
RebF: flavin reductase (EC 1.5.1.30)
FAD+NADH+H+→FADH2+NAD+.
The flavin reductase reduces FAD to FADH2.
The enzyme RebF preferably originates from the organism Lechevalieria aerocolonigenes.
INMT: indole N-methyltransferase (EC 2.1.1.49)
S-adenosyl-L-methionine+an amine→S-adenosyl-L-homocysteine+a methylated amine.
The indole N-methyltransferase catalyzes consecutive N-methylation steps of tryptamine.
The enzyme INMT preferably originates from the organism Oryctolagus cuniculus.
Halogenated: the term herein refers to a compound or a molecule with one or more halogen atoms introduced in the place of hydrogen, i.e. a compound substituted with one or more halogen atoms. If one halogen atom is present, the compound is halogenated or monohalogenated; if two halogen atoms are present, the compound is dihalogenated; if three halogen atoms are present, the compound is trihalogenated. In the context of the present disclosure, the halogen atom(s) may be present in position 2, 5, 6 and/or 7.
Halogenated tryptophan derivatives: the term herein refers to compounds obtainable from halogenated tryptophan, such as halogenated tryptamine, halogenated N-methyltryptamine, halogenated N,N-dimethyltryptamine and halogenated N,N,N-trimethyltryptamine.
Corresponding halogenated compound: the term herein refers to a halogenated compound derived from another halogenated compound by the action of an enzyme, e.g. a tryptophan decarboxylase, such as CrTDC, or an indole N-methyltransferase, such as OcINMT. Such a halogenated compound contains the same halogen atoms in the same positions of the indole ring as the halogenated compound from which it is derived. For example, the corresponding halogenated tryptamine of 5,6-dichlorotryptophan is 5,6-dichlorotryptamine and the corresponding halogenated N,N-dimethyltryptamine of 5-chlorotryptamine is 5-chloro-N,N-dimethyltryptamine.
Titer: the term herein refers to the concentration of a compound that accumulates inside the production host and/or in the extracellular media during cultivation of the host.
Mutation: the term herein refers to a change in nucleic acid sequence compared to the parent nucleic acid sequence. The term mutation covers single nucleotide mutations, but also insertions and deletions, i.e. any change that leads to a different nucleic acid sequence than the parent nucleic acid sequence. The term mutation thus encompasses deletions, such as deletions of a whole gene or coding sequence.
“Identity”, “similarity” and “homology” with respect to a polynucleotide (or polypeptide) is defined herein as the percentage of nucleic acids (or amino acids) in the candidate sequence that are identical with the residues of a corresponding native nucleic acids (or amino acids), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity/similarity/homology, and considering any conservative substitutions according to the NCIUB rules (hftp://www.chem.qmul.ac.uk/iubmb/misc/naseq.html; NC-IUB, Eur J Biochem (1985) 150: 1-5) as part of the sequence identity. Neither 5′ or 3′ extensions nor insertions result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art.
Yeast Cell
The present disclosure relates to a yeast cell capable of producing 4-hydroxytryptamine and optionally derivatives thereof, as outlined in
In one aspect, the present invention provides a yeast cell capable of producing 4-hydroxytryptamine and optionally derivatives thereof, said cell expressing:
wherein the tryptamine 4-monooxygenase and the cytochrome P450 reductase together catalyse the conversion of tryptamine to 4-hydroxytryptamine, whereby the yeast cell is capable of converting tryptophan to 4-hydroxytryptamine.
In some embodiments, the genus of said yeast is selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In some embodiments, the genus of said yeast is Saccharomyces or Yarrowia.
The yeast cell may be selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica. In preferred embodiments, the yeast cell is a Saccharomyces cerevisiae cell, a Saccharomyces boulardii cell or a Yarrowia lipolytica cell.
Throughout the present disclosure, it will be understood that the cells can produce the compounds of interest listed herein when incubated in a cultivation medium under conditions that enable the cell to grow and produce the desired compound. From the description of the production host cells provided herein, the skilled person will not have difficulties in identifying suitable cultivation media and conditions to achieve production.
Production of 4-Hydroxytryptamine
The yeast cell of the present disclosure can produce 4-hydroxytryptamine. This requires that the yeast cell expresses a tryptophan decarboxylase capable of converting tryptophan to tryptamine, a tryptamine 4-monooxygenase and a cytochrome P450 reductase which together are capable of converting tryptamine to 4-hydroxytryptamine.
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is a heterologous tryptophan decarboxylase. In some embodiments, the tryptophan decarboxylase is CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine.
In some embodiments, the tryptamine 4-monooxygenase (EC 1.14.99.59) is a heterologous tryptamine 4-monooxygenase. In some embodiments, the tryptamine 4-monooxygenase is PcPsiH (SEQ ID NO: 2) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the cytochrome P450 reductase (EC 1.6.2.4) is a heterologous cytochrome P450 reductase. In some embodiments, the cytochrome P450 reductase is PcCpr (SEQ ID NO: 3) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the yeast cell expresses CrTDC, PcPsiH and PcCpr as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or functional variants thereof having at least 85% homology thereto.
In some embodiments, the yeast cell is capable of producing 4-hydroxytryptamine 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 yeast cell may be further engineered to allow production of 4-hydroxytryptamine derivatives, as described herein below in detail. Such derivatives include norbaeocystin, N-acetyl-4-hydroxytryptamine, baeocystin, psilocybin, psilocin and aeruginascin. Norpsilocin is another 4-hydroxytryptamin derivative which may be obtained by spontaneous degradation of baeocystin. Hence, a yeast cell capable of producing baeocystin may also produce norpsilocin, in particular by spontaneous degradation. Psilocin may be obtained from psilocyin by spontaneous degradation.
Hence, a yeast cell capable of producing psilocybin may also produce psilocin. Aeruginascin may be spontaneously converted to dephosphorylated aeruginascin. Hence, a yeast cell capable of producing aeruginascin may also produce dephosphorylated aeruginascin.
In some embodiments, the yeast cell further expresses a cytochrome b5, such as a heterologous cytochrome b5. In some embodiments, the cytochrome b5 is the putative P. cubensis cytochrome b5 as set forth in SEQ ID NO: 42, or a functional variant thereof having at least 80% homology or identity thereto and retaining the cytochrome b5 function, such as a functional variant having at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology or identity to SEQ ID NO: 42. In some embodiments the cytochrome b5 is encoded by SEQ ID NO: 43 or a homologue thereof having at least 80% homology or identity thereto, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology or identity to SEQ ID NO: 43. Such homologues encode a protein which preferably retains the function of the original protein.
Production of Norbaeocystin
The yeast cell may be further engineered to produce norbaeocystin from 4-hydroxytryptamine. This can be achieved by introducing a 4-hydroxytryptamine kinase capable of converting 4-hydroxytryptamine to norbaeocystin.
In some embodiments, the yeast cell further expresses 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, wherein the 4-hydroxytryptamine kinase is capable of converting 4-hydroxytryptamine to norbaeocystin, whereby the yeast cell is capable of converting 4-hydroxytryptamine to norbaeocystin. The yeast cell can thus produce norbaeocystin.
In some embodiments, the 4-hydroxytryptamine kinase (EC 2.7.1.222) is a heterologous 4-hydroxytryptamine kinase. In some embodiments, the 4-hydroxytryptamine kinase is PcPsiK (SEQ ID NO: 4) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Accordingly, in some embodiments, the yeast cell is capable of producing norbaeocystin and expresses:
In some embodiments, the yeast cell expresses CrTDC, PcPsiH and PcCpr as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or functional variants thereof having at least 85% homology thereto, and further expresses PcPsiK as set forth in SEQ ID NO: 4 or a functional variant thereof having at least 85% homology thereto.
In some embodiments, the yeast cell is capable of producing norbaeocystin 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.
Production of Baeocystin and Norpsilocin
The yeast cell may be further engineered to produce baeocystin from norbaeocystin. This can be achieved by introducing a norbaeocystin N-methyl transferase (also termed psilocybin synthase; the two terms are herein used interchangeably) capable of converting norbaeocystin to baeocystin.
In some embodiments, the yeast cell further expresses a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345), preferably a heterologous norbaeocystin N-methyl transferase/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, wherein the norbaeocystin N-methyl transferase/psilocybin synthase is capable of converting norbaeocystin to baeocystin, whereby the yeast cell is capable of converting norbaeocystin to baeocystin. The yeast cell can thus produce baeocystin.
In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) is a heterologous norbaeocystin N-methyl transferase/psilocybin synthase. In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase is PcPsiM (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Accordingly, in some embodiments, the yeast cell is capable of producing baeocystin and expresses:
In some embodiments, the yeast cell expresses CrTDC, PcPsiH, PcCpr and PcPsiK as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or functional variants thereof having at least 85% homology thereto, and further expresses PcPsiM as set forth in SEQ ID NO: 5 or a functional variant thereof having at least 85% homology thereto.
In some embodiments, the yeast cell is capable of producing baeocystin 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 baeocystin produced by the cell may be further converted to norpsilocin. This may happen spontaneously, by spontaneous degradation of baeocystin to norpsilocin.
In some embodiments, the yeast cell is capable of producing norpsilocin 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.
Production of Psilocybin and Psilocin
The yeast cell may be further engineered to produce psilocybin. This can be done by expressing a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) in the yeast cell, which enzyme is capable of converting norbaeocystin to baeocystin and further to psilocybin. The resulting yeast cell can thus produce psilocybin. Psilocin may be produced as a result of the action of phosphatases in the cell, or by spontaneous degradation. Thus in some embodiments the yeast cell is capable of producing psilocin.
Accordingly, in some embodiments, the yeast cell expresses a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) as described above, 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, wherein the psilocybin synthase is capable of converting norbaeocystin to psilocybin, whereby the yeast cell is capable of converting norbaeocystin to psilocybin.
Accordingly, in some embodiments, the yeast cell is capable of producing psilocybin and expresses:
In some embodiments, the yeast cell expresses CrTDC, PcPsiH, PcCpr and PcPsiK as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or functional variants thereof having at least 85% homology thereto, and further expresses PcPsiM as set forth in SEQ ID NO: 5 or a functional variant thereof having at least 85% homology thereto.
In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) is a heterologous psilocybin synthase. In some embodiments, the psilocybin synthase is PcPsiM (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the yeast cell is capable of producing psilocybin 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.
In some embodiments, the psilocybin is converted to psilocin and the yeast cell is capable of producing psilocin 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.
If it is desirable to direct the production towards psilocin, it may be advantageous to modulate the activity of the 4-hydroxytryptamine kinase. As is shown on
Production of Aeruqinascin and Dephosphorylated Aeruqinascin
Psilocybin can be further converted to aeruginascin by the action of the norbaeocystin N-methyl transferase/psilocybin synthase. This can be done by expressing a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) in the yeast cell, which enzyme is capable of converting psilocybin to aeruginascin. The resulting yeast cell can thus produce aeruginascin. Aeruginascin may then be spontaneously dephosphorylated, yielding dephosphorylated aeruginascin. Thus in some embodiments the yeast cell is capable of producing aeruginascin and optionally dephosphorylated aeruginascin.
Accordingly, in some embodiments, the yeast cell expresses a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) as described above, 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, wherein the psilocybin synthase is capable of converting psilocybin to aeruginascin, whereby the yeast cell is capable of converting psilocybin to aeruginascin.
Accordingly, in some embodiments, the yeast cell is capable of producing aeruginascin and expresses:
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 yeast cell is capable of converting tryptophan to 4-hydroxytryptamine;
In some embodiments, the yeast cell expresses CrTDC, PcPsiH, PcCpr and PcPsiK as set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or functional variants thereof having at least 85% homology thereto, and further expresses PcPsiM as set forth in SEQ ID NO: 5 or a functional variant thereof having at least 85% homology thereto.
In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) is a heterologous psilocybin synthase. In some embodiments, the psilocybin synthase is PcPsiM (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the yeast cell is capable of producing aeruginascin 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.
In some embodiments, the aeruginascin is dephosphorylated and the yeast cell is capable of producing dephosphorylated aeruginascin 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.
Production of N-Acetyl-4-Hydroxytryptamine
The yeast cell may be further engineered to produce N-acetyl-4-hydroxytryptamine from 4-hydroxytryptamine. This can be achieved by introducing a serotonin N-acetyltransferase (EC 2.3.1.87) capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine.
In some embodiments, the yeast cell further expresses 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, wherein the serotonin N-acetyltransferase is capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine, whereby the yeast cell is capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine. The yeast cell can thus produce N-acetyl-4-hydroxytryptamine.
In some embodiments, the serotonin N-acetyltransferase (EC 2.3.1.87) is a heterologous serotonin N-acetyltransferase. In some embodiments, the serotonin N-acetyltransferase is BtAANAT (SEQ ID NO: 11) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Accordingly, in some embodiments, the yeast cell is capable of producing N-acetyl-4-hydroxytryptamine and expresses:
In addition to the enzymes necessary for producing N-acetyl-4-hydroxytryptamine, the yeast cell may also express the enzymes necessary for producing norbaeocystin, and optionally baeocystin and psilocybin, as described herein above.
In some embodiments, the yeast cell expresses CrTDC, PcPsiH and PcCpr as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, or functional variants thereof having at least 85% homology thereto, and further expresses BtAANAT as set forth in SEQ ID NO: 11 or a functional variant thereof having at least 85% homology thereto.
In some embodiments, the yeast cell is capable of producing N-acetyl-4-hydroxytryptamine 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.
Expression of Heterologous Enzymes
In some embodiments, one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase is under the control of an inducible promoter.
In some embodiments, one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase is codon-optimised for the yeast cell, as is known in the art.
In some embodiments, one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase is present in 2 to 30 copies.
In some embodiments, one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase is integrated in the genome of the yeast cell.
In some embodiments, one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase is expressed from a vector such as a plasmid.
In some embodiments, expression of one or more of the genes encoding the tryptophan decarboxylase, the tryptamine 4-monooxygenase, the cytochrome P450 reductase, the 4-hydroxytryptamine kinase, the serotonin N-acetyltransferase and/or the psilocybin synthase can be induced or repressed, for instance to obtain transient expression, as is known in the art.
Nucleic acid constructs useful for obtaining yeast cells capable of producing 4-hydroxytryptamine or derivatives thereof are described in the section “Nucleic acid construct”.
Because the present pathways require tryptophan as a first substrate, and without being bound by theory, it may be advantageous to modify the yeast cell in such a manner that tryptophan metabolism is directed towards increased tryptophan synthesis, thereby further increasing the titers of 4-hydroxytryptamine or derivatives thereof.
In some embodiments, the yeast cell further comprises one or more mutations resulting in increased availability of L-tryptophan.
In some embodiments, the one or more mutations is in one or more genes encoding a transcriptional repressor(s) of genes of the aromatic amino acid precursor pathway; the mutation may be in the coding region or in the promoter of the gene. In Saccharomyces cerevisiae, examples of such genes are ARO1, ARO2, ARO3 or ARO4. Mutations, including deletions, inactivating or partially inactivating the products of such genes may increase tryptophan availability and help further improve the titers. In some embodiments, the one or more mutations is a mutation resulting in partial or total loss of activity of the one or more transcriptional repressor(s). In some embodiments, the transcriptional repressor is Ric1 and the RIC1 gene is mutated in such a way that Ric1 function is abolished, e.g. by a deletion.
Alternatively or additionally, genes involved in tryptophan biosynthesis may be mutated or overexpressed. In S. cerevisiae, examples of such genes are TRP1, TRP2, TRP3, TRP4 or TRP5. Mutations conferring increased activity may be particularly advantageous. The genes may also be overexpressed as is known in the art, for example by taking advantage of a constitutive promoter.
In some embodiments, the yeast cell is a S. cerevisiae cell and the cell expresses a mutated ARO4 gene, where the mutation removes feedback regulation. The mutation may be in the coding region or in the promoter of the gene. In a specific embodiment, the cell expresses an Aro4 mutant having a mutation at position 229, such as a K229L substitution. The wild type sequence of ARO4 is set forth in SEQ ID NO: 12.
In some embodiments, the yeast cell overexpresses genes involved in the shikimate pathway, such as ARO1 and/or ARO2. The sequences of ARO1 and ARO2 are as set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.
In some embodiments, the yeast cell comprises mutations which increase the flux towards the shikimate pathway. For example, the yeast cell is S. cerevisiae and CDC19 (SEQ ID NO: 26) is mutated, where the mutation leads to a partial or total loss of activity. The mutation may be in the coding region or in the promoter of the gene.
In some embodiments, the yeast cell has a mutation, for example a deletion, of genes involved in tryptophan catabolism; the mutation may be in the coding region or in the promoter of the gene. For example, the yeast cell is a S. cerevisiae cell and ARO8 and/or ARO9 are deleted or mutated, where the mutation leads to a loss of function, to prevent tryptophan degradation. The sequences of ARO8 and ARO9 are set forth in SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
In some embodiments, the yeast cell comprises mutations which direct the glutamine flux towards tryptophan. For example, the yeast cell comprises a mutation or a deletion of GLT1 (SEQ ID NO: 25). In some embodiments, the yeast cell is cultivated in a medium which is supplemented with glutamine. The mutation may be in the coding region or in the promoter of the gene.
In some embodiments, the yeast cell is a S. cerevisiae cell and the cell expresses a mutated TRP2 gene, where the mutation removes feedback regulation. In a specific embodiment, the cell expresses a Trp2 mutant having a mutation at position 65 and/or 76, such as an S65R and/or an S76L substitution. The wild type sequence of TRP2 is set forth in SEQ ID NO: 16. The mutation may be in the coding region or in the promoter of the gene.
In some embodiments, the yeast cell overexpresses one or more genes involved in the tryptophan synthesis pathway, such as TRP1, TRP2, TRP3, TRP4 and/or TRP5, the sequences of which are set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively.
In some embodiments, the yeast cell overexpresses one or more genes involved in the serine pathway, such as SER1, SER2, SER3 and/or SER33, the sequences of which are set forth in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 31, respectively.
In some embodiments, the yeast cell comprises modifications which result in increased NADPH availability. For example, the yeast cell is S. cerevisiae and one or more of STB5 (SEQ ID NO: 26), POS5 (SEQ ID NO: 27) and ZWF1 (SEQ ID NO: 29) are overexpressed.
In the psilocybin biosynthetic pathway, the conversion of tryptamine to 4-hydroxytryptamine is catalyzed by the CYP PcPsiH. The catalytic cycle of monooxygenation by a CYP proceeds as follows: the CYP binds its substrate in the active site adjacent to the CYP heme co-factor, which is in ferric state. The partner
CPR supplies an electron from NADPH, which reduces the heme-complex to the ferrous state. This reduction enables the heme-complex to bind molecular oxygen, forming a ferric superoxo state. Input of a second electron is then required, which leads to cleavage of the O—O bond, and subsequently product formation. CPRs are capable of supplying this second electron, but if it is not supplied fast enough, the superoxide anion in the ferric superoxo complex is released, a process termed uncoupling.
Uncoupling of a CYP catalyzed reaction results in lack of product formation and release of superoxide anion that dismutates to hydrogen peroxide, which can be detrimental to growth.
Cytochrome b5 are a class of membrane-bound heme-proteins that often take part in CYP catalyzed reactions. Without being bound by theory, it is thought that cytochrome b5 is capable of providing a rapid input of the second electron (from NADH) in the catalytic cycle, thereby decreasing uncoupling of the CYP catalyzed reaction and increasing product formation.
In some embodiments, any of the yeast strains described herein further expresses a cytochrome b5, such as a heterologous cytochrome b5. In some embodiments, the cytochrome b5 is the putative P. cubensis cytochrome b5 as set forth in SEQ ID NO: 43, or a functional variant thereof having at least 80% homology or identity thereto and retaining the cytochrome b5 function, such as a functional variant having at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology or identity to SEQ ID NO: 43. In some embodiments the cytochrome b5 is encoded by SEQ ID NO: 42 or a homologue thereof having at least 80% homology or identity thereto, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% homology or identity to SEQ ID NO: 42. Such homologues encode a protein which preferably retains the function of the original protein.
Without being bound by theory, modifications which increase availability of S-adenosylmethionine (SAM) may further help improve the titers of the tryptamine derivatives obtainable by the methods disclosed herein. In some embodiments, the cell is modified, for example the ergosterol biosynthetic pathway is modified. In embodiments where the cell is a yeast cell, the modification can be a mutation in or a deletion of the gene encoding Erg4 (SEQ ID NO: 44), resulting in a partial or total loss of Erg4. The gene encoding Erg4 is set forth in SEQ ID NO: 45. The mutation may be in the coding region or in the promoter of the gene.
In other embodiments, the gene encoding an S-adenosylmethionine decarboxylase proenzyme is mutated or deleted so as to result in a partial or total loss of activity. S-adenosylmethionine decarboxylase catalyzes the decarboxylation of SAM into S-adenosylmethioninamine as the first step of the spermidine biosynthesis. In embodiments where the cell is a yeast cell, the modification can be a mutation in or a deletion of the gene encoding Spe2 (SEQ ID NO: 46), resulting in a partial or total loss of Spe2. The gene encoding Spe2 is set forth in SEQ ID NO: 47. The mutation may be in the coding region or in the promoter of the gene.
In some embodiments, the above modifications are combined. For example, in embodiments where the cell is a yeast cell, the yeast cell has reduced activity of Erg4 or Spe 2 (or both), and further has reduced activity of Ric1 as described herein above. ARO1 and ARO2 may also be overexpressed in such strains, which may in addition have any of the modifications described herein above.
Examples of Useful Yeast Cells
In this section a number of specific yeast cells representing specific embodiments are listed. In some embodiments, the cell is a Saccharomyces cerevisiae cell or a Saccharomyces boulardii cell. In other embodiments, the cell is a Yarrowia lipolytica cell.
In a specific embodiment, the yeast cell is capable of producing 4-hydroxytryptamine, and expresses:
Or functional variants thereof having at least 85% homology thereto. Preferably the yeast cell is a S. cerevisiae cell or a Y. lipolytica cell.
In another embodiment, the yeast cell is capable of producing norbaeocystin, and expresses:
Or functional variants thereof having at least 85% homology thereto. Preferably the yeast cell is a S. cerevisiae cell or a Y. lipolytica cell.
In another embodiment, the yeast cell is capable of baeocystin and/or psilocybin and/or psilocin and/or aeruginascin and/or dephosphorylated aeruginascin, and expresses:
Or functional variants thereof having at least 85% homology thereto. Preferably the yeast cell is a S. cerevisiae cell or a Y. lipolytica cell.
In some embodiments, the yeast cell further expresses BtAANAT (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology thereo, and is capable of producing N-acetyl-4-hydroxytryptamine. Preferably the yeast cell is a S. cerevisiae cell or a Y. lipolytica cell.
In some embodiments, one or more of the genes encoding enzymes of the aromatic amino acid precursor pathway ARO1, ARO2, TRP1, TRP3, TRP4, TRP5 have been overexpressed in the yeast cell. In some embodiments, the transcriptional repressor RIC1 is mutated and has reduced activity.
In some embodiments, the yeast cell further expresses a cytochrome b5 such as PcCyb5 as set forth in SEQ ID NO: 43.
Other Organisms
Other organisms besides yeast may also be useful as production organisms according to the present disclosure. Thus, in some embodiments the production cell is a microorganism or a plant cell. The microorganism may e.g. be a fungus or a bacteria.
Useful fungi include a fungus belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, A. nidulans, a yeast belonging to the genus of Saccharomyces, e.g. S. cerevisiae, S. kluyveri, S. bayanus, S. exiguus, S. sevazzi, S. uvarum, S. boulardii, a yeast belonging to the genus Kluyveromyces, e.g. K. lactis, K. marxianus var. marxianus, K. thermotolerans, a yeast belonging to the genus Candida, e.g. C. utilis C. tropicalis, C. albicans, C. lipolytica, C. versatilis, a yeast belonging to the genus Pichia, e.g. P. stipidis, P. pastoris, P. sorbitophila, other yeast genera such as Cryptococcus (e.g. C. aerius), Debaromyces (e.g. D. hansenii), Hansenula, Yarrowia (e.g. Y. lipolytica), Zygosaccharomyces (e.g. Z. bailii), Torulaspora (e.g. T. delbrueckii), Schizosaccharomyces (e.g. S. pombe), Brettanomyces (e.g. B. bruxellensis), Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, or Trichoderma.
Useful bacteria include bacteria belonging to the genus Bacillus (e.g. B. subtilis), a species belonging to the genus Escherichia (e.g. E. coli), a species belonging to the genus Lactobacillus (e.g. L. casei), a species belonging to the genus Lactococcus (e.g. L. lactis), a species belonging to the genus Corynebacterium (e.g. C. glutamicum), a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas (e.g. P. putida), and a species belonging to the genus Streptomyces (e.g. S. coelicolor).
Useful plants include plants belonging to the genus Arabidopsis (e.g. A. thaliana), a species belonging to the genus Zea (e.g. Z. mays), a species belonging to the genus Medicago (e.g. M. truncatula), a species belonging to the genus Nicotiana (e.g. N. tabacum) and a species belonging to the genus Glycine (e.g. G. Max).
Methods of Production of 4-Hydroxytryptamine and Derivatives Thereof
The present disclosure relates to methods for producing 4-hydroxytryptamine and derivatives thereof. The yeast cells and nucleic acid constructs described herein are useful for microbial-based production of 4-hydroxytryptamine and derivatives thereof, including psilocybin. Throughout the present disclosure, it will be understood that the cells can produce the compounds of interest listed herein when incubated in a cultivation medium under conditions that enable the cell to grow and produce the desired compound. From the description of the production host cells provided herein, and knowing the type of host cell used, the skilled person will not have difficulties in identifying suitable cultivation media and conditions to achieve production. In particular, the cultivation may be performed aerobically or anaerobically, at temperatures and at pH suitable for supporting growth of the cell. The cultivation medium should include the required nutrients, and may be supplemented with precursors as applicable. The time of cultivation will vary depending on which cell is used, but can easily be adapted by the skilled person.
In one aspect, the present invention provides a method of producing 4-hydroxytryptamine and optionally derivatives thereof in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
Yeast cells useful for producing 4-hydroxytryptamine are described herein, in particular in the section “Production of 4-hydroxytryptamine” herein above.
Herein are also provided methods for producing derivatives of 4-hydroxytryptamine.
Norbaeocystin
In some embodiments, the 4-hydroxytryptamine derivative is norbaeocystin. In such embodiments, the yeast cell further expresses 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. Yeast cells useful for production of norbaeocystin are described herein, in particular in the section “Production of norbaeocystin” herein above.
In some embodiments, the method is for producing norbaeocystin and the yeast cell further expresses 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.
In some embodiments, the present invention provides a method of producing norbaeocystin in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
Baeocystin and Norpsilocin
In some embodiments, the 4-hydroxytryptamine derivative is baeocystin. In such embodiments, the yeast cell further expresses a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345), preferably a heterologous norbaeocystin N-methyl transferase/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. The baeocystin may be further converted to norpsilocin. Hence in some embodiments the method results in production of norpsilocin. Yeast cells useful for production of baeocystin are described herein, in particular in the section “Production of baeocystin and norpsilocin” herein above.
In some embodiments, the present invention provides a method of producing baeocystin and optionally norpsilocin in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
Psilocybin and Psilocin
In some embodiments, the 4-hydroxytryptamine derivative is psilocybin. In such embodiments, the yeast cell expresses a norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345), preferably a heterologous norbaeocystin N-methyl transferase/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. Yeast cells useful for production of psilocybin are described herein, in particular in the section “Production of psilocybin and psilocin” herein above.
In some embodiments, the present invention provides a method of producing psilocybin and optionally psilocin in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
The psilocybin may be further converted spontaneously to psilocin. Hence in some embodiments the method results in production of psilocin.
Aeruginascin and Dephosphorylated Aeruginascin
The psilocybin may also be further converted to aeruginascin by the action of the norbaeocystin N-methyl transferase/psilocybin synthase. Hence in some embodiments the methods are for production of aeruginascin, which may optionally be spontaneously converted to dephosphorylated aeruginascin. Yeast cells useful for production of psilocybin are described herein, in particular in the section “Production of aeruginascin and dephosphorylated aeruginascin” herein above.
In some embodiments, the present invention provides a method of producing aeruginascin and optionally dephosphorylated aeruginascin in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
N-Acetyl-4-Hydroxytryptamine
In some embodiments, the 4-hydroxytryptamine derivative is N-acetyl-4-hydroxytryptamine. In such embodiments, the yeast cell expresses a serotonin N-acetyltransferase (EC 2.3.1.87) capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine, preferably a heterologous norbaeocystin N-methyl transferase/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. Yeast cells useful for production of psilocybin are described herein, in particular in the section “Production of N-acetyl-4-hydroxytryptamine” herein above.
In some embodiments, the present invention provides a method of producing N-acetyl-4-hydroxytryptamine in a yeast cell, said method comprising the steps of providing a yeast cell and incubating said yeast cell in a medium, wherein the yeast cell expresses:
Such yeast cells may also express, in addition to the above, a 4-hydroxytryptamine kinase and optionally a norbaeocystin N-methyl transferase/psilocybin synthase as described herein above.
Useful Enzymes for Production of 4-Hydroxytryptamine and Derivatives Thereof
Enzymes useful for the present methods, which can advantageously be introduced in the yeast cell, are described in detail herein above in the section entitled “Yeast cell”.
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine.
In some embodiments, the tryptamine 4-monooxygenase (EC 1.14.99.59) is a heterologous tryptamine 4-monooxygenase such as PcPsiH (SEQ ID NO: 2) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the cytochrome P450 reductase (EC 1.6.2.4) is a heterologous cytochrome P450 reductase such as PcCpr (SEQ ID NO: 3) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the 4-hydroxytryptamine kinase (EC 2.7.1.222) is a heterologous 4-hydroxytryptamine kinase such as PcPsiK (SEQ ID NO: 4) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) is a heterologous psilocybin synthase such as PcPsiM (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the serotonin N-acetyltransferase (EC 2.3.1.87) is a heterologous serotonin N-acetyltransferase such as BtAANAT (SEQ ID NO: 11) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the serotonin N-acetyltransferase is capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine.
In some embodiments, the medium comprises tryptophan and/or the yeast cell is capable of synthesising tryptophan.
The yeast cell may further comprise any of the modifications detailed in the section “Other modifications”. For example, in some embodiments, the yeast cell further comprises one or more mutations resulting in increased availability of L-tryptophan.
Recovering the 4-Hydroxytryptamine and/or Derivatives Thereof
The present methods may comprise a further step of recovering the compounds obtained by the methods disclosed herein. Methods for recovering the products obtained by the present invention are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. For example, the step of recovering the compound(s) may comprise separating the cell culture in a solid phase and in a liquid phase to obtain a supernatant. The supernatant can then be contacted with one or more adsorbent resins to which the compound(s) can bind, and the compound(s) can then be eluted as is known in the art. Alternatively, one or more ion exchange or reversed-phase chromatography columns can be used. Another option is to employ liquid-liquid extraction in an immiscible solvent, which may optionally be evaporated before precipitating the compound(s), or further liquid-liquid extraction may be employed.
The yeast cell is preferably as defined herein.
In some embodiments, the method is for production of 4-hydroxytryptamine and further comprises a step of recovering the 4-hydroxytryptamine.
In some embodiments, the method is for production of norbaeocystin and further comprises a step of recovering the norbaeocystin.
In some embodiments, the method is for production of baeocystin and further comprises a step of recovering the baeocystin. Optionally, the method is for production of norpsilocin and further comprises a step of recovering the norpsilocin.
In some embodiments, the method is for production of psilocybin and optionally psilocin and further comprises a step of recovering the psilocybin and optionally the psilocin.
In some embodiments, the method is for production of aeruginascin and optionally dephosphorylated aeruginascin and further comprises a step of recovering the aeruginascin and optionally the dephosphorylated aeruginascin.
In some embodiments, the method is for production of N-acetyl-4-hydroxytryptamine and further comprises a step of recovering the N-acetyl-4-hydroxytryptamine.
Titers
The present methods are useful for producing 4-hydroxytryptamine and derivatives thereof with high titers.
In some embodiments, 4-hydroxytryptamine is produced 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.
In some embodiments, norbaeocystin is produced 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.
In some embodiments, baeocystin is produced 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.
In some embodiments, norpsilocin is produced 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.
In some embodiments, psilocybin is produced 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.
In some embodiments, psilocin is produced 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.
In some embodiments, aeruginascin is produced 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.
In some embodiments, dephosphorylated aeruginascin is produced 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.
In some embodiments, N-acetyl-4-hydroxytryptamine is produced 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.
Methods for determining the titer are known in the art, for example by measuring the peak area from LC-MS analysis and comparing to the peak area of an authentic analytical standard of known concentration.
The titer of any of the above can be increased by introducing further modifications in the cell, such as any of the modifications described in “Other modifications”. In some embodiments, the titers may also or alternatively be increased by supplementing the culture medium with glutamine. In some embodiments, the medium is supplemented with at least 1 g/L glutamine, such as at least 2 g/L, such as at least 3 g/L glutamine, such as at least 4 g/L glutamine, such as at least 5 g/L glutamine, such as at least 6 g/L glutamine, such as at least 7 g/L glutamine, such as at least 8 g/L glutamine, such as at least 9 g/L glutamine, such as at least 10 g/L glutamine, or more.
4-Hydroxytryptamine and Derivatives Thereof Obtainable by the Present Methods
In one aspect, the present invention provides 4-hydroxytryptamine and derivatives thereof obtainable by a method as disclosed herein.
In some embodiments, the present invention provides norbaeocystin obtainable by the method described herein. In some embodiments, the present invention provides baeocystin obtainable by the method described herein. In some embodiments, the present invention provides norpsilocin obtainable by the method described herein. In some embodiments, the present invention provides psilocybin obtainable by the method described herein. In some embodiments, the present invention provides psilocin obtainable by the method described herein. In some embodiments, the present invention provides aeruginascin obtainable by the method described herein. In some embodiments, the present invention provides dephosphorylated aeruginascin obtainable by the method described herein. In some embodiments, the present invention provides N-acetyl-4-hydroxytryptamine obtainable by the method described herein.
Nucleic Acid Constructs
Also provided herein are nucleic acid constructs useful for engineering a yeast cell capable of producing 4-hydroxytryptamine or derivatives thereof as described above. The present nucleic acid constructs may be provided as one or more nucleic acid molecules or polynucleotides, for example they may be comprised in one or more vectors. Such nucleic acids may be introduced in the cell by methods known in the art.
It will be understood that throughout the present disclosure, the term ‘nucleic acid encoding an activity’ shall refer to a nucleic acid molecule capable of encoding a peptide, a protein or a fragment thereof having said activity. Such nucleic acid molecules may be open reading frames or genes, or fragments thereof.
In one aspect, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In some embodiments, the nucleic acid further comprises 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.
In some embodiments, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In some embodiments, the nucleic acid further comprises 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.
In some embodiments, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
Any of the above nucleic acid constructs may further comprise a sixth polynucleotide encoding a serotonin N-acetyltransferase (EC 2.3.1.87) capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine. In some embodiments, the serotonin N-acetyltransferase is 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, wherein the serotonin N-acetyltransferase is capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine, whereby the yeast cell is capable of converting 4-hydroxytryptamine to N-acetyl-4-hydroxytryptamine. The yeast cell can thus produce N-acetyl-4-hydroxytryptamine.
Thus in some embodiments, the nucleic acid construct comprises:
In other embodiments, the nucleic acid construct comprises:
In some embodiments, the nucleic acid construct comprises:
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is preferably a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase is capable of converting tryptophan to tryptamine.
In some embodiments, the tryptamine 4-monooxygenase (EC 1.14.99.59) is preferably a heterologous tryptamine 4-monooxygenase such as PcPsiH (SEQ ID NO: 2) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the cytochrome P450 reductase (EC 1.6.2.4) is preferably a heterologous cytochrome P450 reductase such as PcCpr (SEQ ID NO: 3) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the 4-hydroxytryptamine kinase (EC 2.7.1.222) is preferably a heterologous 4-hydroxytryptamine kinase such as PcPsiK (SEQ ID NO: 4) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the norbaeocystin N-methyl transferase/psilocybin synthase (EC 2.1.1.345) is a heterologous psilocybin synthase such as PcPsiM (SEQ ID NO: 5) or a functional variant thereof having at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the serotonin N-acetyltransferase (EC 2.3.1.87) is 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% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the first polynucleotide comprises or consists of SEQ ID NO: 6 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the second polynucleotide comprises or consists of SEQ ID NO: 7 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. In some embodiments, the third polynucleotide comprises or consists of SEQ ID NO: 8 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the fourth polynucleotide comprises or consists of SEQ ID NO: 9 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the fifth polynucleotide comprises or consists of SEQ ID NO: 10 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the sixth polynucleotide comprises or consists of SEQ ID NO: 30 or a homologue thereof having at least 80% homology, such as at least 85% homology, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In a preferred embodiment, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In a preferred embodiment, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In a preferred embodiment, the present invention provides a nucleic acid construct for modifying a yeast cell, said construct comprising:
In some embodiments, one or more of the first, second, third, fourth, fifth or sixth polynucleotide(s) is/are codon-optimised for said yeast cell.
In some embodiments, each of the nucleic acids encoding each of the present activities, i.e. a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase, a norbaeocystin N-methyl transferase/psilocybin synthase and a serotonin N-acetyltransferase may be designed to be integrated within the genome of the yeast cell or they may be within one or more vectors comprised within the yeast cell.
In some embodiments, one or more of the nucleic acids encoding each of the present activities may be integrated in the genome of said yeast cell. Methods for integrating a nucleic acid are well known in the art. Thus in some embodiments the activity of interest is encoded by introduction of a heterologous nucleic acid in the yeast cell. The heterologous nucleic acid encoding said activity may be codon-optimised, or may comprise features that can help improve the activity. Such modifications include, but are not limited to, the introduction of localisation signals, gain-of-function or loss-of-function mutations, fusion of the protein to a marker or a tag such as fluorescent tag, insertion of an inducible promoter, introduction of modifications conferring increased stability and/or half-life.
The introduction of the heterologous nucleic acid encoding the activity of interest can be performed by methods known in the art. The skilled person will recognise that such methods include, but are not limited to: cloning and homologous recombination-based methods. Cloning methods may involve the design and construction of a plasmid e.g. in an organism such as Escherichia coli. The plasmid may be an integrative or a non-integrative vector. Cloning-free methods comprise homologous recombination-based methods such as adaptamer-mediated PCR or gap repair. Such methods often result in integration of the heterologous nucleic acid in the genome of the yeast cell.
The nucleic acids encoding the activities of interest may be present in high copy number.
In some embodiments, the nucleic acid construct further comprises or consists of one or more vectors, such as an integrative vector or a replicative vector. In some embodiments, the vector is a high copy replicative vector.
Each of the nucleic acid sequences comprised within the present nucleic acid constructs may be present in multiple copies. In some embodiments, at least one of the nucleic acid sequences is present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies. In some embodiments, all of the nucleic acid sequences are present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies.
The nucleic acid constructs may, in addition to the first, second, third, fourth, fifth and sixth polynucleotides described above, also comprise additional polynucleotides useful for introducing additional modifications in the yeast cell, to obtain cells as described in “Other modifications”. Designing such additional polynucleotides can be performed as is known in the art.
The nucleic acid constructs may be a PCR product or a synthetic DNA molecule.
Kit of Parts
Also provided herein is a kit of parts comprising a yeast cell, and/or a nucleic acid construct as described herein, and instructions for use.
In some embodiments, the kit comprises a yeast cell that can be used in the methods described herein. In other embodiments, the kit comprises a nucleic acid construct that can be used to engineer a yeast cell useful for the methods described herein. In some embodiments, the kit comprises a yeast cell and a nucleic acid construct as described herein.
In some embodiments, the kit comprises a yeast cell capable of producing 4-hydroxytryptamine, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase and a cytochrome P450 reductase. In some embodiments, the kit comprises a yeast cell capable of producing norbaeocystin, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase and a 4-hydroxytryptamine kinase. In some embodiments, the kit comprises a yeast cell capable of producing baeocystin and optionally norpsilocin, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase, a 4-hydroxytryptamine kinase and a psilocybin synthase. In some embodiments, the kit comprises a yeast cell capable of producing psilocybin and optionally psilocin, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase, a 4-hydroxytryptamine kinase and a psilocybin synthase. In some embodiments, the kit comprises a yeast cell capable of producing aeruginascin and optionally dephosphorylated aeruginascin, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase, a 4-hydroxytryptamine kinase and a psilocybin synthase. In some embodiments, the kit comprises a yeast cell capable of producing N-acetyl-4-hydroxytryptamine, wherein the yeast cell expresses a tryptophan decarboxylase, a tryptamine monooxygenase, a cytochrome P450 reductase, a serotonin N-acetyltransferase and optionally a 4-hydroxytryptamine kinase and a psilocybin synthase. The yeast cell may be further modified as detailed in “Other modifications”.
In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a tryptophan decarboxylase, a second polynucleotide encoding a tryptamine monooxygenase and a third polynucleotide encoding a cytochrome P450 reductase. In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a tryptophan decarboxylase, a second polynucleotide encoding a tryptamine monooxygenase, a third polynucleotide encoding a cytochrome P450 reductase and a fourth polynucleotide encoding a 4-hydroxytryptamine kinase. In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a tryptophan decarboxylase, a second polynucleotide encoding a tryptamine monooxygenase, a third polynucleotide encoding a cytochrome P450 reductase, a fourth polynucleotide encoding a 4-hydroxytryptamine kinase and a fifth polynucleotide encoding a psilocybin synthase. Additionally, any of the previously cited may also comprise a sixth polynucleotide encoding a serotonin N-acetyltransferase.
In some embodiments, the kit comprises the nucleic acid construct as described herein and the yeast cell to be modified. In some embodiments, the yeast cell to be modified is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
In some embodiments, the kit comprises the yeast cell and a nucleic acid construct as described herein.
Cell Capable of Producing Halogenated Tryptophans and Derivatives Thereof
The present disclosure relates to a cell capable of producing a halogenated tryptophan and optionally derivatives thereof, as outlined in
In some aspects, the present invention provides 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:
preferably wherein the cell is a microorganism or a plant cell.
In some embodiments, the cell is a eukaryotic cell or a bacteria or the plant cell is a microalgae cell.
The eukaryotic cell may be selected from the group consisting of the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, and A. nidulans.
The bacteria may be selected from the group consisting of a species belonging to the genus Bacillus, such as B. subtilis, a species belonging to the genus Escherichia, such as E. coli, a species belonging to the genus Lactobacillus, such as L. casei, a species belonging to the genus Lactococcus, such as L. lactis, a species belonging to the genus Corynebacterium, such as C. glutamicum, a species belonging to the genus
Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas, such as P. putida, and a species belonging to the genus Streptomyces, such as S. coelicolor.
The plant may be selected from the group consisting of a species belonging to the genus Arabidopsis, such as A. thaliana, a species belonging to the genus Zea, such as Z. mays, a species belonging to the genus Medicago, such as M. truncatula, a species belonging to the genus Nicotiana, such as N. tabacum, and a species belonging to the genus Glycine, such as G. Max.
In some embodiments, the cell is a yeast cell. The yeast cell may belong 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, or belong to the genus Candida, such as C. utilis, C. tropicalis, C. albicans, C. lipolytica, C. versatilis, or belong to the genus Pichia, such as P. stipidis, P. pastoris, P. sorbitophila, or 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. In preferred embodiments, the yeast cell is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
Production of Halogenated Tryptophans
The cell of the present disclosure can produce halogenated tryptophans. This requires that the cell expresses a tryptophan halogenase and optionally a flavin reductase, whereby the cell is capable of converting tryptophan to a halogenated tryptophan. Preferably the cell is a yeast cell as described herein above.
Tryptophan Halogenase
Depending on which type of halogenation is desired, different tryptophan halogenases can be used. If a 2-halogenated compound is desired, the tryptophan halogenase is a tryptophan-2-halogenase. If a 5-halogenated compound is desired, the tryptophan halogenase is a tryptophan-5-halogenase. If a 6-halogenated compound is desired, the tryptophan halogenase is a tryptophan-6-halogenase. If a 7-halogenated compound is desired, the tryptophan halogenase is a tryptophan-7-halogenase. It is also possible to express several halogenases to obtain dihalogenated or trihalogenated compounds. By way of example, expression of a tryptophan-5-halogenase and a tryptophan-6-halogenase can be employed to obtain 5,6-dihalogenated compounds.
In some embodiments, the tryptophan halogenase is a tryptophan-2-halogenase such as a heterologous tryptophan-2-halogenase (EC 1.14.14). In some embodiments, the tryptophan-2-halogenase is CcCmdE (SEQ ID NO: 48) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-2-halogenase is capable of converting tryptophan to a 2-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-5-halogenase such as a heterologous tryptophan-5-halogenase (EC 1.14.19.58). In some embodiments, the tryptophan-5-halogenase is SrPyrH (SEQ ID NO: 32) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-5-halogenase is capable of converting tryptophan to a 5-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-6-halogenase such as a heterologous tryptophan-6-halogenase (EC 1.14.19.59). In some embodiments, the tryptophan-6-halogenase is SttH (SEQ ID NO: 33) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-6-halogenase is capable of converting tryptophan to a 6-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-6-halogenase such as a heterologous tryptophan-6-halogenase (EC 1.14.19.59). In some embodiments, the tryptophan-6-halogenase is SaThaI (SEQ ID NO: 51) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-6-halogenase is capable of converting tryptophan to a 6-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-6-halogenase such as a heterologous tryptophan-6-halogenase (EC 1.14.19.59). In some embodiments, the tryptophan-6-halogenase is KtzR (SEQ ID NO: 54) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-6-halogenase is capable of converting tryptophan to a 6-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-7-halogenase such as a heterologous tryptophan-7-halogenase (EC 1.14.19.9). In some embodiments, the tryptophan-7-halogenase is LaRebH (SEQ ID NO: 34) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-7-halogenase is capable of converting tryptophan to a 7-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-7-halogenase such as a heterologous tryptophan-7-halogenase (EC 1.14.19.9). In some embodiments, the tryptophan-7-halogenase is PfPrnA (SEQ ID NO: 50) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-7-halogenase is capable of converting tryptophan to a 7-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is a tryptophan-7-halogenase such as a heterologous tryptophan-7-halogenase (EC 1.14.19.9). In some embodiments, the tryptophan-7-halogenase is KtzQ (SEQ ID NO: 53) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan-7-halogenase is capable of converting tryptophan to a 7-halogenated tryptophan.
In some embodiments, the tryptophan halogenase is DdChlA (SEQ ID NO: 52) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan halogenase is capable of converting tryptophan to a halogenated tryptophan.
In some embodiments, the halogen is selected from the group consisting of fluorine, bromine, iodine and chlorine.
In some embodiments, the cell expresses two tryptophan halogenases independently selected from the group consisting of a heterologous tryptophan-2-halogenase, a heterologous tryptophan-5-halogenase, a heterologous tryptophan-6-halogenase and a heterologous tryptophan-7-halogenase, whereby the cell is capable of converting tryptophan to 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.
For example, the cell expresses a tryptophan-2-halogenase as described above, for example CcCmdE or a functional variant thereof; and
In some embodiments the cell expresses a tryptophan-5-halogenase as described above, for example SrPyrH or a functional variant thereof, and:
In some embodiments the cell expresses a tryptophan-6-halogenase as described above, for example SttH, SaThaI or KtzR, or a functional variant thereof, and:
In some embodiments the cell expresses a tryptophan-7-halogenase as described above, for example LaRebH, PfPrnA or KtzQ, or a functional variant thereof, and:
In some embodiments the cell expresses DdChlA or a functional variant thereof, and:
In some embodiments, the cell expresses three tryptophan halogenases independently selected from the group consisting of a heterologous tryptophan-2-halogenase, a heterologous tryptophan-5-halogenase, a heterologous tryptophan-6-halogenase and a heterologous tryptophan-7-halogenase, whereby the cell is capable of converting tryptophan to a 2,5,6-trihalogenated, a 2,5,7-trihalogenated, a 2,6,7-trihalogenated or a 5,6,7-trihalogenated tryptophan.
For example, the cell expresses a tryptophan-2-halogenase as described above, for example CcCmdE or a functional variant thereof; and a tryptophan-5-halogenase as described above, for example SrPyrH or a functional variant thereof; and
In some embodiments, the cell expresses a tryptophan-2-halogenase as described above, for example CcCmdE or a functional variant thereof; and a tryptophan-6-halogenase as described above, for example SttH, SaThaI or KtzR; and
In some embodiments, the cell expresses a tryptophan-2-halogenase as described above, for example CcCmdE or a functional variant thereof; and a tryptophan-7-halogenase as described above, for example LaRebH, PfPrnA or KtzQ; and DdChlA or a functional variant thereof.
In some embodiments, the cell expresses a tryptophan-5-halogenase as described above, for example SrPyrH or a functional variant thereof; and a tryptophan-6-halogenase as described above, for example SttH, SaThaI or KtzR or a functional variant thereof; and
In some embodiments, the cell expresses a tryptophan-5-halogenase as described above, for example SrPyrH or a functional variant thereof; and a tryptophan-7-halogenase as described above, for example LaRebH, PfPrnA or KtzQ or a functional variant thereof; and DdChIA.
In some embodiments, the cell expresses a tryptophan-6-halogenase as described above, for example SttH, SaThaI or KtzR or a functional variant thereof; DdChlA or a functional variant thereof; and a tryptophan-7-halogenase as described above, for example LaRebH, PfPrnA or KtzQ or a functional variant thereof.
Flavin Reductase
In order to increase the titer of halogenated tryptophan produced by the present cells, it may be helpful to express in the cell a flavin reductase in addition to one or more tryptophan halogenases as described herein above.
In some embodiments, the flavin reductase (EC 1.5.1.30) is a heterologous flavin reductase. In some embodiments, the flavin reductase is LaRebF (SEQ ID NO: 35) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the flavin reductase is capable of reducing FAD to FADH2.
In some embodiments, the cell expresses CcCmdE, SrPyrH, SttH and/or LaRebH, and optionally LaRebF as set forth in SEQ ID NO: 48, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively, or functional variants thereof having at least 80% homology thereto, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Titers
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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 may be further engineered to allow production of halogenated tryptophan derivatives, as described herein below in detail. Such derivatives include halogenated tryptamine, halogenated N-methyltryptamine, halogenated N,N-dimethyltryptamine and halogenated N,N,N-trimethyltryptamine.
Methods for determining the titer are known in the art, for example by measuring the peak area from LC-MS analysis and comparing to the peak area of an authentic analytical standard of known concentration.
Production of Halogenated Tryptamines
The cell may be further engineered to produce halogenated tryptamines from halogenated tryptophans. This can be achieved by introducing a tryptophan decarboxylase capable of converting the halogenated tryptophan to a corresponding halogenated tryptamine. Preferably the cell is a yeast cell as described herein above.
Tryptophan Decarboxylase
Expression of a tryptophan decarboxylase in the above cells capable of producing halogenated tryptamines results in cells capable of producing the corresponding halogenated tryptamines.
In some embodiments, the cell further 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase is capable of converting the halogenated tryptophan to a corresponding halogenated tryptamine, whereby the cell is capable converting the halogenated tryptophan to a corresponding halogenated tryptamine. The cell can thus produce the corresponding halogenated tryptamine.
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is a heterologous tryptophan decarboxylase. In some embodiments, the tryptophan decarboxylase is CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Accordingly, in some embodiments, the cell is capable of producing a halogenated tryptamine, wherein the halogenated tryptamine is a tryptamine substituted with one, two or three halogen atoms, and said cell expresses at least one of:
and said cell also 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase catalyzes the conversion of the halogenated tryptophan to a corresponding halogenated tryptamine, whereby the cell is capable of converting the halogenated tryptophan into a corresponding halogenated tryptamine,
2-halo-tryptophan can be used to produce 2-halo-tryptamine. 5-halo-tryptophan can be used to produce 5-halo-tryptamine. 6-halo-tryptophan can be used to produce 6-halo-tryptamine. 7-halo-tryptophan can be used to produce 7-halo-tryptamine.
2,5-dihalo-tryptophan can be used to produce 2,5-dihalo-tryptamine. 2,6-dihalo-tryptophan can be used to produce 2,6-dihalo-tryptamine. 2,7-dihalo-tryptophan can be used to produce 2,7-dihalo-tryptamine. 5,6-dihalo-tryptophan can be used to produce 5,6-dihalo-tryptamine. 5,7-dihalo-tryptophan can be used to produce 5,7-dihalo-tryptamine. 6,7-dihalo-tryptophan can be used to produce 6,7-dihalo-tryptamine.
2,5,6-trihalo-tryptophan can be used to produce 2,5,6-trihalo-tryptamine. 2,5,7-trihalo-tryptophan can be used to produce 2,5,7-trihalo-tryptamine. 2,6,7-trihalo-tryptophan can be used to produce 2,6,7-trihalo-tryptamine. 5,6,7-trihalo-tryptophan can be used to produce 5,6,7-trihalo-tryptamine.
In some embodiments, the cell expresses CcCmdE, SrPyrH, SttH and/or LaRebH, and optionally LaRebF as set forth in SEQ ID NO: 48, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively, or functional variants thereof having at least 80% homology thereto, and further expresses CrTDC as set forth in SEQ ID NO: 1 or a functional variant thereof having at least 80% homology thereto.
Titers
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
Methods for determining the titer are known in the art, for example by measuring the peak area from LC-MS analysis and comparing to the peak area of an authentic analytical standard of known concentration.
Production of Halogenated N-Methylated, N,N-Dimethylated and N,N,N-Trimethylated Tryptamines
The cell may be further engineered to produce halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine from halogenated tryptamine. This can be achieved by introducing an indole N-methyltransferase capable of converting the halogenated tryptamine produced by the cell to the corresponding halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine. The cell may be any of the cells described herein. Preferably the cell is a yeast cell as described herein above.
Indole N-Methyltransferase
In some embodiments, the cell is any of the cells described herein above which is capable of producing a halogenated tryptamine. In order to produce halogenated, methylated tryptamine, the cell in some embodiments further expresses 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the indole N-methyltransferase is capable of converting the halogenated tryptamine to a corresponding halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine, whereby the cell is capable of converting halogenated tryptamine to halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine. The cell can thus produce halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine.
In some embodiments, the indole N-methyltransferase (EC 2.1.1.49) is a heterologous indole N-methyltransferase. In some embodiments, the indole N-methyltransferase is OcINMT (SEQ ID NO: 36) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
Accordingly, in some embodiments, the cell is capable of producing halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine, wherein the halogenated N-methyltryptamine, the halogenated N,N-dimethyltryptamine or the halogenated N,N,N-trimethyltryptamine is N-methyltryptamine, N,N-dimethyltryptamine or N,N,N-trimethyltryptamine, respectively, substituted with one, two or three halogen atoms, and said cell expresses at least one of:
also 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase catalyzes the conversion of the halogenated tryptophan to the corresponding halogenated tryptamine, whereby the cell is capable of converting the halogenated tryptophan into the corresponding halogenated tryptamine,
2-halo-tryptamine can be used to produce 2-halogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 5-halo-tryptamine can be used to produce 5-halogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 6-halo-tryptamine can be used to produce 6-halogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 7-halo-tryptamine can be used to produce 7-halogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine.
2,5-dihalo-tryptamine can be used to produce 2,5-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 2,6-dihalo-tryptamine can be used to produce 2,6-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 2,7-dihalo-tryptamine can be used to produce 2,7-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 5,6-dihalo-tryptamine can be used to produce 5,6-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 5,7-dihalo-tryptamine can be used to produce 5,7-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 6,7-dihalo-tryptamine can be used to produce 6,7-dihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine.
2,5,6-trihalo-tryptamine can be used to produce 2,5,6-trihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 2,5,7-trihalo-tryptamine can be used to produce 2,5,7-trihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 2,6,7-trihalo-tryptamine can be used to produce 2,6,7-trihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine. 5,6,7-trihalo-tryptamine can be used to produce 5,6,7-trihalogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamine.
halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine
In some embodiments, the cell expresses CcCmdE, SrPyrH, SttH and/or LaRebH, as well as CrTDC and optionally LaRebF as set forth in SEQ ID NO: 48, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 1 and SEQ ID NO: 35, respectively, or functional variants thereof having at least 80% homology thereto, and further expresses OcINMT as set forth in SEQ ID NO: 36 or a functional variant thereof having at least 80% homology thereto.
Titers
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, the total titer of all halogenated compounds produced by the cell 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.
Methods for determining the titer are known in the art, for example by measuring the peak area from LC-MS analysis and comparing to the peak area of an authentic analytical standard of known concentration.
Expression of Heterologous Enzymes
In some embodiments, 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.
In some embodiments, 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, as is known in the art.
In some embodiments, 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 2 to 30 copies.
In some embodiments, 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.
In some embodiments, 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.
In some embodiments, expression of 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 can be induced or repressed, for instance to obtain transient expression, as is known in the art.
Nucleic acid constructs useful for obtaining yeast cells capable of halogenated tryptophan or derivatives thereof are described in the section “Nucleic acid constructs”.
Other Modifications for Increasing Titers
Because the present pathways require tryptophan as a first substrate, and without being bound by theory, it may be advantageous to modify the cell in such a manner that tryptophan metabolism is directed towards increased tryptophan synthesis, thereby further increasing the titers of the halogenated tryptophans or derivatives thereof. Examples of these modifications can be found in the section “Other modifications”.
Examples of Useful Cells
In this section a number of specific cells representing specific embodiments of cells useful for production of halogenated compounds are listed. In some embodiments, the cell is a yeast cell. Preferably the yeast cell is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
In a specific embodiment, the cell is capable of producing a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, and expresses:
Or functional variants thereof having at least 80% homology thereto. Preferably the cell is a yeast cell as described herein above.
In another embodiment, the cell is capable of producing a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, and expresses:
Or functional variants thereof having at least 80% homology thereto. Preferably the cell is a yeast cell as described herein above.
In another embodiment, the cell is capable of producing a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, and/or a chlorinated, a fluorinated, a brominated or a iodinated tryptamine, and expresses:
Or functional variants thereof having at least 80% homology thereto. Preferably the cell is a yeast cell as described herein above.
In another embodiment, the cell is capable of producing a chlorinated, a fluorinated, a brominated or a iodinated tryptophan, and/or a chlorinated, a fluorinated, a brominated or a iodinated tryptamine, and expresses:
Or functional variants thereof having at least 80% homology thereto. Preferably the cell is a yeast cell as described herein above.
Other organisms besides yeast may also be useful as production organisms according to the present disclosure. Thus, in some embodiments the production cell is a microorganism or a plant cell. The microorganism may e.g. be a eukaryotic cell, a yeast cell or a bacteria, and the plant cell may e.g. be a microalgae.
Useful eukaryotic cells include eukaryotic cells belonging to the genus of Aspergillus, e.g. A. niger, A. awamori, A. oryzae, and A. nidulans.
Useful bacteria include bacteria belonging to the genus Bacillus, such as B. subtilis, a species belonging to the genus Escherichia, such as E. coli, a species belonging to the genus Lactobacillus, such as L. casei, a species belonging to the genus Lactococcus, such as L. lactis, a species belonging to the genus Corynebacterium, such as C. glutamicum, a species belonging to the genus Acetobacter, a species belonging to the genus Acinetobacter, a species belonging to the genus Pseudomonas, such as P. putida, and a species belonging to the genus Streptomyces, such as S. coelicolor.
Useful yeast cells include 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, or belong to the genus Candida, such as C. utilis, C. tropicalis, C. albicans, C. lipolytica, C. versatilis, or belong to the genus Pichia, such as P. stipidis, P. pastoris, P. sorbitophila, or 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.
Useful plants include plants belonging to the genus Arabidopsis, such as A. thaliana, a species belonging to the genus Zea, such as Z. mays, a species belonging to the genus Medicago, such as M. truncatula, a species belonging to the genus Nicotiana, such as N. tabacum, and a species belonging to the genus Glycine, such as G. Max.
Methods of Production of Halogenated Tryptophans and Derivatives Thereof
The present disclosure relates to methods for producing halogenated tryptophans and derivatives thereof. The cells and nucleic acid constructs described herein are useful for cell-based production of halogenated tryptophans and derivatives thereof, including halogenated tryptamines, halogenated N-methylated tryptamines, halogenated N,N-dimethylated tryptamines and halogenated N,N,N-trimethylated tryptamines. Throughout the present disclosure, it will be understood that the cells can produce the compounds of interest listed herein when incubated in a cultivation medium under conditions that enable the cell to grow and produce the desired compound. From the description of the production host cells provided herein, and knowing the type of host cell used, the skilled person will not have difficulties in identifying suitable cultivation media and conditions to achieve production. In particular, the cultivation may be performed aerobically or anaerobically, at temperatures and at pH suitable for supporting growth of the cell. The cultivation medium should include the required nutrients, and may be supplemented with precursors as applicable. The time of cultivation will vary depending on which cell is used, but can easily be adapted by the skilled person.
Halogenated Tryptophans
In some aspects, the present invention provides 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:
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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the method is for producing a 2-halogenated, a 5-halogenated, a 6-halogenated and/or a 7-halogenated tryptophan.
In some embodiments, the method is for producing a 2,5-dihalogenated, a 2,6-dihalogenated, a 2,7-dihalogenated, a 5,6-dihalogenated, a 5,7-dihalogenated and/or a 6,7-dihalogenated tryptophan.
In some embodiments, the method is for producing a 2,5,6-trihalogenated, a 2,5,7-trihalogenated, a 2,6,7-trihalogenated and/or a 5,6,7-trihalogenated tryptophan.
In some embodiments, the halogen is selected from the group consisting of fluorine, bromine, iodine and chlorine.
Cells useful for producing halogenated tryptophans are described herein, in particular in the section “Production of halogenated tryptophans” herein above.
Herein are also provided methods for producing derivatives of halogenated tryptophans.
Halogenated Tryptamines
In some embodiments, the method is for producing a halogenated tryptamine, wherein the halogenated tryptophan derivative is a halogenated tryptamine. In such embodiments, the cell further 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 homology, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Cells useful for production of halogenated tryptamines are described herein, in particular in the section “Production of halogenated tryptamines” herein above.
In some embodiments, the method is for producing a halogenated tryptamine, wherein the halogenated tryptamine is a tryptamine substituted with one, two or three halogen atoms, and the cell further 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 homology, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the method is for producing a halogenated tryptamine in a cell, wherein the halogenated tryptamine is a tryptamine substituted with one, two or three halogen atoms, said method comprising the steps of providing a cell and incubating said cell in a medium, wherein the cell expresses at least one of:
In some embodiments, the method is for producing a 2-halogenated, a 5-halogenated, a 6-halogenated and/or a 7-halogenated tryptamine.
In some embodiments, the method is for producing a 2,5-dihalogenated, a 2,6-dihalogenated, a 2,7-dihalogenated, a 5,6-dihalogenated, a 5,7-dihalogenated and/or a 6,7-dihalogenated tryptamine.
In some embodiments, the method is for producing a 2,5,6-trihalogenated, a 2,5,7-trihalogenated, a 2,6,7-trihalogenated and/or a 5,6,7-trihalogenated tryptamine.
Halogenated N-Methylated, N,N-Dimethylated, and N,N,N-Trimethylated Tryptamine
In some embodiments, the halogenated tryptophan derivative is a halogenated N-methylated tryptamine, a halogenated N,N-dimethylated tryptamine, or a halogenated N,N,N-trimethylated tryptamine. In such embodiments, the cell further expresses 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Cells useful for production of halogenated N-methylated tryptamine, halogenated N,N-dimethylated tryptamine, and halogenated N,N,N-trimethylated tryptamine are described herein, in particular in the section “Production of halogenated N-methylated, N,N-dimethylated and N,N,N-trimethylated tryptamines” herein above.
In some embodiments, the method is for producing halogenated N-methylated, halogenated N,N-dimethylated and/or halogenated N,N,N-trimethylated tryptamine, wherein the halogenated N-methyltryptamine, the halogenated N,N-dimethyltryptamine or the halogenated N,N,N-trimethyltryptamine is N-methyltryptamine, N,N-dimethyltryptamine or N,N,N-trimethyltryptamine, respectively, substituted with one, two or three halogen atoms, in a cell, said method comprising the steps of providing a cell and incubating said cell in a medium, wherein the cell expresses at least one of:
also 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto, wherein the tryptophan decarboxylase catalyzes the conversion of the halogenated tryptophan to the corresponding halogenated tryptamine, whereby the cell is capable of converting the halogenated tryptophan into the corresponding halogenated tryptamine,
In some embodiments, the method is for producing a 2-halogenated N-methyltryptamine, a 2-halogenated N,N-dimethyltryptamine and/or a 2-halogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 5-halogenated N-methyltryptamine, a 5-halogenated N,N-dimethyltryptamine and/or a 5-halogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 6-halogenated N-methyltryptamine, a 6-halogenated N,N-dimethyltryptamine and/or a 6-halogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 7-halogenated N-methyltryptamine, a 7-halogenated N,N-dimethyltryptamine and/or a 7-halogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing 2,5-dihalogenated N-methyltryptamine, a 2,5-dihalogenated N,N-dimethyltryptamine and/or a 2,5-dihalogenated N,N,N-trimethyltryptamine.
In some embodiments, the method is for producing a 2,6-dihalogenated N-methyltryptamine, a 2,6-dihalogenated N,N-dimethyltryptamine and/or a 2,6-dihalogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 2,7-dihalogenated N-methyltryptamine, a 2,7-dihalogenated N,N-dimethyltryptamine and/or a 2,7-dihalogenated N,N,N-trimethyltryptamine.
In some embodiments, the method is for producing a 5,6-dihalogenated N-methyltryptamine, a 5,6-dihalogenated N,N-dimethyltryptamine and/or a 5,6-dihalogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 5,7-dihalogenated N-methyltryptamine, a 5,7-dihalogenated N,N-a dimethyltryptamine and/or a 5,7-dihalogenated N, N, N-trimethyltryptamine.
In some embodiments, the method is for producing a 6,7-dihalogenated N-methyltryptamine, a 6,7-dihalogenated N,N-dimethyltryptamine and/or a 6,7-dihalogenated N,N,N-trimethyltryptamine.
In some embodiments, the method is for 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.
In some embodiments, the method is for producing 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.
In some embodiments, the method is for producing 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.
In some embodiments, the method is for producing 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.
Useful Enzymes for Production of Halogenated Tryptophans and Derivatives Thereof
Enzymes useful for the present methods, which can advantageously be introduced in the cell, are described in detail herein above in the section entitled “Cell capable of producing halogenated tryptophans and derivatives thereof”.
In some embodiments, the tryptophan-2-halogenase (EC 1.14.14) is a heterologous tryptophan-2-halogenase such as CcCmdE (SEQ ID NO: 48) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan-5-halogenase (EC 1.14.19.58) is a heterologous tryptophan-5-halogenase such as SrPyrH (SEQ ID NO: 32) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan-6-halogenase (EC 1.14.19.59) is 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Preferably the tryptophan-6-halogenase is SttH (SEQ ID NO: 33) or a functional variant thereof having at least 80% homology thereto.
In some embodiments, the tryptophan-7-halogenase (EC 1.14.19.9) is 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Preferably the tryptophan-7-halogenase is LaRebH (SEQ ID NO: 34) or a functional variant thereof having at least 80% homology thereto.
In some embodiments, the tryptophan halogenase is a heterologous tryptophan halogenase such as DdChlA (SEQ ID NO: 52) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the flavin reductase (EC 1.5.1.30) is a heterologous flavin reductase such as LaRebF (SEQ ID NO: 35) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the indole N-methyltransferase (EC 2.1.1.49) is a heterologous indole N-methyltransferase such a OcINMT (SEQ ID NO: 36) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the medium comprises tryptophan and/or the cell is capable of synthesizing tryptophan.
The cell may further comprise any of the modifications detailed in the section “Other modifications”. For example, in some embodiments, the cell further comprises one or more mutations resulting in increased availability of L-tryptophan.
Recovering the Halogenated Tryptophans and/or Derivatives Thereof
The present methods may comprise a further step of recovering the compounds obtained by the methods disclosed herein. Methods for recovering the products obtained by the present invention are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. Methods for recovering the products obtained by the present invention are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. For example, the step of recovering the compound(s) may comprise separating the cell culture in a solid phase and in a liquid phase to obtain a supernatant. The supernatant can then be contacted with one or more adsorbent resins to which the compound(s) can bind, and the compound(s) can then be eluted as is known in the art. Alternatively, one or more ion exchange or reversed-phase chromatography columns can be used. Another option is to employ liquid-liquid extraction in an immiscible solvent, which may optionally be evaporated before precipitating the compound(s), or further liquid-liquid extraction may be employed.
The cell is preferably as defined herein.
In some embodiments, the method is for production of halogenated tryptophan(s) and further comprises a step of recovering the halogenated tryptophan(s).
In some embodiments, the method is for production of dihalogenated tryptophan(s) and further comprises a step of recovering the dihalogenated tryptophan(s).
In some embodiments, the method is for production of trihalogenated tryptophan(s) and further comprises a step of recovering the trihalogenated tryptophan(s).
In some embodiments, the method is for production of halogenated tryptamine(s) and further comprises a step of recovering the halogenated tryptamine(s).
In some embodiments, the method is for production of dihalogenated tryptamine(s) and further comprises a step of recovering the dihalogenated tryptamine(s).
In some embodiments, the method is for production of trihalogenated tryptamine(s) and further comprises a step of recovering the trihalogenated tryptamine(s).
In some embodiments, the method is for production of halogenated N-methyltryptamine, halogenated N,N-dimethyltryptamine and/or halogenated N,N,N-trimethyltryptamine, and further comprises a step of recovering the halogenated N-methyltryptamine, halogenated N,N-dimethyltryptamine and/or halogenated N,N,N-trimethyltryptamine.
In some embodiments, the method is for production of dihalogenated N-methyltryptamine, dihalogenated N,N-dimethyltryptamine and/or dihalogenated N,N,N-trimethyltryptamine, and further comprises a step of recovering the dihalogenated N-methyltryptamine, dihalogenated N,N-dimethyltryptamine and/or dihalogenated N,N,N-trimethyltryptamine.
In some embodiments, the method is for production of trihalogenated N-methyltryptamine, trihalogenated N,N-dimethyltryptamine and/or trihalogenated N,N,N-trimethyltryptamine, and further comprises a step of recovering the trihalogenated N-methyltryptamine, trihalogenated N,N-dimethyltryptamine and/or trihalogenated N,N,N-trimethyltryptamine.
Titers
The present methods are useful for producing halogenated tryptophans and derivatives thereof with high titers.
In some embodiments, 2-halogenated, 5-halogenated, 6-halogenated, or 7-halogenated tryptophan is produced 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.
In some embodiments, 2,5-dihalogenated, 2,6-dihalogenated, 2,7-dihalogenated, 5,6-dihalogenated, 5,7-dihalogenated or 6,7-dihalogenated tryptophan is produced 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.
In some embodiments, 2,5,6-trihalogenated, 2,5,7-trihalogenated, 2,6,7-trihalogenated or 5,6,7-trihalogenated tryptophan is produced 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.
In some embodiments, 2-halogenated, 5-halogenated, 6-halogenated, or 7-halogenated tryptamine is produced 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.
In some embodiments, 2,5-dihalogenated, 2,6-dihalogenated, 2,7-dihalogenated, 5,6-dihalogenated, 5,7-dihalogenated or 6,7-dihalogenated tryptamine is produced 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.
In some embodiments, 2,5,6-trihalogenated, 2,5,7-trihalogenated, 2,6,7-trihalogenated or 5,6,7-trihalogenated tryptamine is produced 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.
In some embodiments, 2-halogenated N-methyltryptamine, 2-halogenated N,N-dimethyltryptamine, 2-halogenated N,N,N-trimethyltryptamine, 5-halogenated N-methyltryptamine, 5-halogenated N,N-dimethyltryptamine, 5-halogenated N,N,N-trimethyltryptamine, 6-halogenated N-methyltryptamine, 6-halogenated N,N-dimethyltryptamine, 6-halogenated N,N,N-trimethyltryptamine, 7-halogenated N-methyltryptamine, 7-halogenated N,N-dimethyltryptamine, or 7-halogenated N,N,N-trimethyltryptamine is produced 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.
In some embodiments, 2,5-dihalogenated N-methyltryptamine, 2,5-dihalogenated N,N-dimethyltryptamine, 2,5-dihalogenated N,N,N-trimethyltryptamine, 2,6-dihalogenated N-methyltryptamine, 2,6-dihalogenated N,N-dimethyltryptamine, 2,6-dihalogenated N,N,N-trimethyltryptamine, 2,7-dihalogenated N-methyltryptamine, 2,7-dihalogenated N,N-dimethyltryptamine, 2,7-dihalogenated N,N,N-trimethyltryptamine, 5,6-dihalogenated N-methyltryptamine, 5,6-dihalogenated N,N-dimethyltryptamine, 5,6-dihalogenated N,N,N-trimethyltryptamine, 5,7-dihalogenated N-methyltryptamine, 5,7-dihalogenated N,N-a dimethyltryptamine, 5,7-dihalogenated N,N,N-trimethyltryptamine, 6,7-dihalogenated N-methyltryptamine, 6,7-dihalogenated N,N-dimethyltryptamine, or 6,7-dihalogenated N,N,N-trimethyltryptamine is produced 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.
In some embodiments, 2,5,6-trihalogenated N-methyltryptamine, 2,5,6-trihalogenated N,N-dimethyltryptamine, 2,5,6-trihalogenated N,N,N-trimethyltryptamine, 2,5,7-trihalogenated N-methyltryptamine, 2,5,7-trihalogenated N,N-dimethyltryptamine, 2,5,7-trihalogenated N,N,N-trimethyltryptamine, 2,6,7-trihalogenated N-methyltryptamine, 2,6,7-trihalogenated N,N-dimethyltryptamine, 2,6,7-trihalogenated N,N,N-trimethyltryptamine, 5,6,7-trihalogenated N-methyltryptamine, 5,6,7-trihalogenated N,N-dimethyltryptamine, or 5,6,7-trihalogenated N,N,N-trimethyltryptamine is produced 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.
In some embodiments, the total titer of all halogenated compounds produced 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 produced.
Methods for determining the titer are known in the art, for example by measuring the peak area from LC-MS analysis and comparing to the peak area of an authentic analytical standard of known concentration.
Halogenated Tryptophans and Derivatives Thereof Obtainable by the Present Methods
In one aspect, the present invention provides halogenated tryptophans and derivatives thereof obtainable by a method as disclosed herein.
In some embodiments, the present invention provides halogenated tryptophans obtainable by the method described herein. In some embodiments, the present invention provides dihalogenated tryptophans obtainable by the method described herein. In some embodiments, the present invention provides trihalogenated tryptophans obtainable by the method described herein. In some embodiments, the present invention provides halogenated tryptamines obtainable by the method described herein. In some embodiments, the present invention provides dihalogenated tryptamines obtainable by the method described herein. In some embodiments, the present invention provides trihalogenated tryptamines obtainable by the method described herein. In some embodiments, the present invention provides halogenated N-methyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides dihalogenated N-methyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides trihalogenated N-methyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides halogenated N,N-dimethyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides dihalogenated N,N-dimethyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides trihalogenated N,N-dimethyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides halogenated N,N,N-trimethyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides dihalogenated N,N,N-trimethyltryptamines obtainable by the method described herein. In some embodiments, the present invention provides trihalogenated N,N,N-trimethyltryptamines obtainable by the method described herein.
Nucleic Acid Constructs
Also provided herein are nucleic acid constructs useful for engineering a cell capable of producing halogenated tryptophan or derivatives thereof as described above. The present nucleic acid constructs may be provided as one or more nucleic acid molecules or polynucleotides, for example they may be comprised in one or more vectors. Such nucleic acids may be introduced in the cell by methods known in the art.
It will be understood that throughout the present disclosure, the term ‘nucleic acid encoding an activity’ shall refer to a nucleic acid molecule capable of encoding a peptide, a protein or a fragment thereof having said activity. Such nucleic acid molecules may be open reading frames or genes or fragments thereof.
In some aspects, the present invention provides a nucleic acid construct for modifying a cell, said construct comprising at least one of:
and optionally a polynucleotide encoding a flavin reductase, preferably a heterologous flavin reductase (EC 1.5.1.30), such as LaRebF (SEQ ID NO: 35) or a functional variant thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nucleic acid further comprises a polynucleotide encoding 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In one aspect, the present invention provides a nucleic acid construct for modifying a cell, said construct comprising at least one of:
and also comprising a polynucleotide encoding 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the nucleic acid further comprises a polynucleotide encoding 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 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In one aspect, the present invention provides a nucleic acid construct for modifying a cell, said construct comprising at least one of:
also comprising a polynucleotide encoding 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%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto,
In some embodiments, the tryptophan-2-halogenase (EC 1.14.14) is a heterologous tryptophan-2-halogenase such as CcCmdE (SEQ ID NO: 48) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 49 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan-5-halogenase (EC 1.14.19.58) is a heterologous tryptophan-5-halogenase such as SrPyrH (SEQ ID NO: 32) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 37 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan-6-halogenase (EC 1.14.19.59) is 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 thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 38, SEQ ID NO: 56, or SEQ ID NO: 59, respectively, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Preferably the tryptophan-6-halogenase is SttH (SEQ ID NO: 33) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 38 or a homologue thereof having at least 80% thereto.
In some embodiments, the tryptophan-7-halogenase (EC 1.14.19.9) is 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 thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 39. SEQ ID NO: 55, or SEQ ID NO: 58, respectively, or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto. Preferably the tryptophan-7-halogenase is LaRebH (SEQ ID NO: 34) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 39.
In some embodiments, the tryptophan halogenase is a heterologous tryptophan halogenase such as DdChlA (SEQ ID NO: 52) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 57 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the flavin reductase (EC 1.5.1.30) is a heterologous flavin reductase such as LaRebF (SEQ ID NO: 35) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 40 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the tryptophan decarboxylase (EC 4.1.1.105) is a heterologous tryptophan decarboxylase such as CrTDC (SEQ ID NO: 1) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 6 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, the indole N-methyltransferase (EC 2.1.1.49) is a heterologous indole N-methyltransferase such a OcINMT (SEQ ID NO: 36) or a functional variant thereof having at least 80% homology thereto, and encoded by a polynucleotide comprising or consisting of SEQ ID NO: 41 or a homologue thereof having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84% such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% homology thereto.
In some embodiments, one or more of the above polynucleotide(s) is/are codon-optimized for said cell.
In some embodiments, each of the nucleic acids encoding each of the present activities, i.e. a tryptophan halogenase, a tryptophan-2-halogenase, a tryptophan-5-halogenase, a tryptophan-6-halogenase or a tryptophan-7-halogenase, a flavin reductase, a tryptophan decarboxylase, and an indole N-methyltransferase, may be designed to be integrated within the genome of the cell or they may be within one or more vectors comprised within the cell.
In some embodiments, one or more of the nucleic acids encoding each of the present activities may be integrated in the genome of said cell. Methods for integrating a nucleic acid are well known in the art. Thus in some embodiments the activity of interest is encoded by introduction of a heterologous nucleic acid in the cell. The heterologous nucleic acid encoding said activity may be codon-optimized, or may comprise features that can help improve the activity. Such modifications include, but are not limited to, the introduction of localization signals, gain-of-function or loss-of-function mutations, fusion of the protein to a marker or a tag such as fluorescent tag, insertion of an inducible promoter, introduction of modifications conferring increased stability and/or half-life.
The introduction of the heterologous nucleic acid encoding the activity of interest can be performed by methods known in the art. The skilled person will recognize that such methods include, but are not limited to: cloning and homologous recombination-based methods. Cloning methods may involve the design and construction of a plasmid e.g. in an organism such as Escherichia coli. The plasmid may be an integrative or a non-integrative vector. Cloning-free methods comprise homologous recombination-based methods such as adaptamer-mediated PCR or gap repair. Such methods often result in integration of the heterologous nucleic acid in the genome of the cell.
The nucleic acids encoding the activities of interest may be present in high copy number.
In some embodiments, the nucleic acid construct further comprises or consists of one or more vectors, such as an integrative vector or a replicative vector. In some embodiments, the vector is a high copy replicative vector.
Each of the nucleic acid sequences comprised within the present nucleic acid constructs may be present in multiple copies. In some embodiments, at least one of the nucleic acid sequences is present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies. In some embodiments, all of the nucleic acid sequences are present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies.
The nucleic acid constructs may, in addition to the polynucleotides described above, also comprise additional polynucleotides useful for introducing additional modifications in the cell, to obtain cells as described in “Other modifications”. Designing such additional polynucleotides can be performed as is known in the art.
The nucleic acid constructs may be a PCR product or a synthetic DNA molecule.
Kit of Parts
Also provided herein is a kit of parts comprising a cell, and/or a nucleic acid construct as described herein, and instructions for use.
In some embodiments, the kit comprises a cell that can be used in the methods described herein. In other embodiments, the kit comprises a nucleic acid construct that can be used to engineer a cell useful for the methods described herein. In some embodiments, the kit comprises a cell and a nucleic acid construct as described herein.
In some embodiments, the kit comprises a cell capable of producing one or more mono-, di- and/or tri-halogenated tryptophans, wherein the cell expresses one or more tryptophan halogenases and optionally a flavin reductase. In some embodiments, the kit comprises a cell capable of producing one or more mono-, di-, and/or tri-halogenated tryptamines, wherein the cell expresses one or more tryptophan halogenases, a tryptophan decarboxylase and optionally a flavin reductase. In some embodiments, the kit comprises a cell capable of producing one or more mono-, di-, and/or tri-halogenated N-methyltryptamines, one or more mono-, di-, and/or tri-halogenated N,N-dimethyltryptamines, and/or one or more mono-, di-, and/or tri-halogenated N,N,N-trimethyltryptamines, wherein the cell expresses one or more tryptophan halogenases, a tryptophan decarboxylase, an indole N-methyltransferase and optionally a flavin reductase. The cell may be further modified as detailed in “Other modifications”.
In some embodiments, the kit comprises a nucleic construct comprising a polynucleotide encoding a tryptophan halogenase. In some embodiments, the kit comprises a nucleic construct comprising a polynucleotide encoding a tryptophan halogenase and a polynucleotide encoding a tryptophan decarboxylase. In some embodiments, the kit comprises a nucleic construct comprising a polynucleotide encoding a tryptophan halogenase, a polynucleotide encoding a tryptophan decarboxylase and a polynucleotide encoding an indole N-methyltransferase. Additionally, any of the previously cited may also comprise a polynucleotide encoding a flavin reductase.
In some embodiments, the kit comprises the nucleic acid constructs described herein and the cell to be modified. In some embodiments, the cell to be modified is a yeast cell. In a preferred embodiment, the cell to be modified is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
In some embodiments, the kit comprises the cell and a nucleic acid construct as described herein.
Methods and Cells for Production of Methylated Tryptamine
The present disclosure also relates to methods for producing methylated tryptamines, in particular N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine. The term “methylated tryptamine” herein refers to tryptamine substituted with one or more methyl groups, such as two or more, such as three or more methyl groups. Throughout the present disclosure, it will be understood that the cells can produce the compounds of interest listed herein when incubated in a cultivation medium under conditions that enable the cell to grow and produce the desired compound. From the description of the production host cells provided herein, and knowing the type of host cell used, the skilled person will not have difficulties in identifying suitable cultivation media and conditions to achieve production. In particular, the cultivation may be performed aerobically or anaerobically, at temperatures and at pH suitable for supporting growth of the cell. The cultivation medium should include the required nutrients, and may be supplemented with precursors as applicable. The time of cultivation will vary depending on which cell is used, but can easily be adapted by the skilled person.
Herein is also provided 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:
whereby the cell is capable of producing N-methyltryptamine, N,N-dimethyltryptamine, and/or N,N,N-trimethyltryptamine.
The cell may be as described herein above, in particular the cell may be a yeast cell for example a S. cerevisiae cell, or a cell as described in the section “Other organisms” above. Any of the modifications otherwise described herein, in particular in the section “Other modifications”, may also be applied to cells producing methylated tryptamines.
Such cells are useful in methods for producing methylated tryptamine. Also provided herein is 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:
Recovering the Methylated Tryptamines
Thus the present methods may comprise a further step of recovering the compounds obtained by the methods disclosed herein. Methods for recovering the products obtained by the present invention are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. Methods for recovering the products obtained by the present invention are known in the art, for example organic solvent extraction followed by lyophilisation and purification by preparative HPLC or similar column purification techniques. For example, the step of recovering the compound(s) may comprise separating the cell culture in a solid phase and in a liquid phase to obtain a supernatant. The supernatant can then be contacted with one or more adsorbent resins to which the compound(s) can bind, and the compound(s) can then be eluted as is known in the art. Alternatively, one or more ion exchange or reversed-phase chromatography columns can be used. Another option is to employ liquid-liquid extraction in an immiscible solvent, which may optionally be evaporated before precipitating the compound(s), or further liquid-liquid extraction may be employed.
The yeast cell is preferably as defined herein.
In some embodiments, the method is for production of N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine and further comprises a step of recovering the N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine.
Titers
The above cells can produce N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine with a titer of at least 20 mg/L, such as at least 30 mg/L, such as at least 40 mg/L, such as at least 50 mg/L, such as at least 60 mg/L, such as at least 70 mg/L, such as at least 80 mg/L, such as at least 90 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 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.
In some embodiments, the cell produces N-methyltryptamine with a titer of at least 20 mg/L, such as at least 30 mg/L, such as at least 40 mg/L, such as at least 50 mg/L, such as at least 60 mg/L, such as at least 70 mg/L, such as at least 80 mg/L, such as at least 90 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 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.
In some embodiments, the cell produces N,N-dimethyltryptamine with a titer of at least 20 mg/L, such as at least 30 mg/L, such as at least 40 mg/L, such as at least 50 mg/L, such as at least 60 mg/L, such as at least 70 mg/L, such as at least 80 mg/L, such as at least 90 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 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.
In some embodiments, the cell produces N,N,N-trimethyltryptamine with a titer of at least 20 mg/L, such as at least 30 mg/L, such as at least 40 mg/L, such as at least 50 mg/L, such as at least 60 mg/L, such as at least 70 mg/L, such as at least 80 mg/L, such as at least 90 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 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.
In some embodiments, the titer of methylated tryptamine, i.e. the sum of the titers of N-methyltryptamine, N,N-dimethyltryptamine and N,N,N-trimethyltryptamine produced by the cell, is at least 20 mg/L, such as at least 30 mg/L, such as at least 40 mg/L, such as at least 50 mg/L, such as at least 60 mg/L, such as at least 70 mg/L, such as at least 80 mg/L, such as at least 90 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 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.
Also provided herein is N-methyltryptamine, N,N-dimethyltryptamine and/or N,N-dimethyltryptamine obtainable by the methods disclosed herein.
Nucleic Acid Constructs
Also provided herein are nucleic acid constructs useful for engineering cells capable of producing methylated tryptamine.
Such constructs comprise:
The polynucleotide encoding the tryptophan decarboxylase may herein be referred to as the “first polynucleotide”. The polynucleotide encoding the indole N-methyltransferase may be referred to as the “seventh polynucleotide”. This does not imply that the construct comprises eight polynucleotides in total; in some embodiments the cell comprises only the first and the seventh polynucleotides.
The first polynucleotide is as described herein above.
The seventh polynucleotide is as described herein above.
In some embodiments, one or more of the first and seventh polynucleotide(s) is/are codon-optimised for said yeast cell.
In some embodiments, each of the nucleic acids encoding each of the present activities, i.e. a tryptophan decarboxylase or an indole N-methyltransferase, may be designed to be integrated within the genome of the yeast cell or they may be within one or more vectors comprised within the yeast cell.
In some embodiments, one or more of the nucleic acids encoding each of the present activities may be integrated in the genome of said yeast cell. Methods for integrating a nucleic acid are well known in the art. Thus in some embodiments the activity of interest is encoded by introduction of a heterologous nucleic acid in the yeast cell. The heterologous nucleic acid encoding said activity may be codon-optimised, or may comprise features that can help improve the activity. Such modifications include, but are not limited to, the introduction of localisation signals, gain-of-function or loss-of-function mutations, fusion of the protein to a marker or a tag such as fluorescent tag, insertion of an inducible promoter, introduction of modifications conferring increased stability and/or half-life.
The introduction of the heterologous nucleic acid encoding the activity of interest can be performed by methods known in the art. The skilled person will recognise that such methods include, but are not limited to: cloning and homologous recombination-based methods. Cloning methods may involve the design and construction of a plasmid e.g. in an organism such as Escherichia coli. The plasmid may be an integrative or a non-integrative vector. Cloning-free methods comprise homologous recombination-based methods such as adaptamer-mediated PCR or gap repair. Such methods often result in integration of the heterologous nucleic acid in the genome of the yeast cell.
The nucleic acids encoding the activities of interest may be present in high copy number.
In some embodiments, the nucleic acid construct further comprises or consists of one or more vectors, such as an integrative vector or a replicative vector. In some embodiments, the vector is a high copy replicative vector.
Each of the nucleic acid sequences comprised within the present nucleic acid constructs may be present in multiple copies. In some embodiments, at least one of the nucleic acid sequences is present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies. In some embodiments, all of the nucleic acid sequences are present in at least 2 copies, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 10 copies, such as at least 20 copies, such as at least 30 copies, such as at least 40 copies, such as at least 50 copies, such as at least 60 copies, such as at least 70 copies, such as at least 80 copies, such as at least 90 copies, such as at least 100 copies, such as at least 125 copies, such as at least 150 copies, such as at least 175 copies, such as at least 200 copies.
The nucleic acid constructs may, in addition to the first and seventh polynucleotides described above, also comprise additional polynucleotides useful for introducing additional modifications in the yeast cell, to obtain cells as described in “Other modifications”. Designing such additional polynucleotides can be performed as is known in the art.
The nucleic acid constructs may be a PCR product or a synthetic DNA molecule.
Kit of Parts
Also provided herein is a kit of parts comprising a cell, for example a yeast cell as described herein, or any other cell described herein, and/or a nucleic acid construct as described herein, and instructions for use.
In some embodiments, the kit comprises a yeast cell that can be used in the methods for producing methylated tryptamines described herein. In other embodiments, the kit comprises a nucleic acid construct that can be used to engineer a yeast cell useful for the methods for producing methylated tryptamines described herein. In some embodiments, the kit comprises a yeast cell and a nucleic acid construct as described herein.
In some embodiments, the kit comprises a yeast cell capable of producing methylated tryptamines, in particular N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine, wherein the yeast cell expresses a tryptophan decarboxylase and an indole N-methyltransferase. The yeast cell may be further modified as detailed in “Other modifications”.
In some embodiments, the kit comprises a nucleic construct comprising a first polynucleotide encoding a tryptophan decarboxylase and a seventh polynucleotide encoding an indole N-methyltransferase.
In some embodiments, the kit comprises the nucleic acid construct as described herein and the yeast cell to be modified. In some embodiments, the yeast cell to be modified is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell.
In some embodiments, the kit comprises the yeast cell and a nucleic acid construct as described herein.
Advanced Microbiome Therapy
Many diseases relate to a condition referred to as dysbiosis, a microbial imbalance in the gut associated with a bloom of pathobionts, loss of commensals, and loss of diversity within the gut. Given the implications of the gut microbiome in disease, means for modulating the gut microbiome have been explored with the goal of lowering disease prevalence. Among the approaches tested is the administration of faecal microbial transplants for the treatment of diseases ranging from irritable bowel syndrome to chronic fatigue syndrome. However, such faecal microbial transplants have recently been linked to multiple cases of serious and fatal adverse events due to the transfer of drug-resistant bacteria. In view of concerns as to the safety and efficacy of this treatment, alternative methods for modulating the gut microbiome are needed.
One such method is through the use of prebiotics, which are non-digestible substrates associated with an increased density of health-promoting microorganisms. As such, prebiotics serve as a less invasive and short-term mechanism for modulating the gut microbiome for health benefits. Overall, consumption of prebiotics such as inulin, fructo-oligosaccharides, and galacto-oligosaccharides increases the density of beneficial microorganisms, in particular bacteria such as Bifidobacterium and Lactobacilli species.
Probiotics, living organisms that are beneficial to health, have also been shown to help modulate the gut microbiome in order to improve health. As with prebiotics, probiotics are specifically used to alter the gut environment. However, instead of seeking to up-regulate beneficial bacteria by providing prebiotic substrates, such as dietary fibers, probiotics are used to directly introduce beneficial strains. Probiotics function by either directly interacting with the host via chemical and physical signals or by affecting the make-up of the gut microbial community. Previously, probiotics have been useful in treating obesity, diabetes, inflammation, cancer, allergies, and many other ailments.
The success of probiotics in benefitting the gut environment, as well as human health as a whole, has led to a new generation of probiotics engineered to augment the innate benefits of probiotics through a wide range of mechanisms such as the production of therapeutics. These next generation probiotics are often referred to as smart probiotics, living therapeutics, or advanced microbial therapeutics. In such systems, microbial production of therapeutics allows for a continuous and inexpensive supply of molecules such as hormones, interleukins, and antibodies. With a potential for secreting a range of molecules, these living therapeutics have a wide scope of possibilities stretching far beyond the already important role of gut microbes. As some therapeutics are unstable or require high doses, utilizing engineered microbials may be a superior alternative to traditional drug delivery as the microbe-produced therapeutic avoids exposure to the harsh acidic conditions of the upper gastrointestinal tract. Additionally, with an ever-expanding toolbox of sensors, killswitches, memory circuits, etc., these microorganisms can be fine-tuned to better secrete therapeutics, sense signals within the gut environment, and respond to physiological changes.
Accordingly, the present disclosure also provides for the yeast cells disclosed herein to be used as probiotics. These yeast cells have been engineered to produce one or more compounds as described above, where the one or more compounds is selected from the group consisting of: a halogenated tryptophan, a halogenated tryptamine, a halogenated, di-halogenated or tri-halogenated N-methyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N-dimethyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N,N-trimethyltryptamine, N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine, norbaeocystin, baeocystin, norpsilocin, psilocybin, psilocin, aeruginascin, dephosphorylated aeruginascin, N-acetyl-4-hydroxytryptamine, and derivatives thereof. Any of the yeast cells described herein above may be used as probiotic, particularly in the context of advance microbiome therapy.
The yeast cell may be provided as a composition comprising the yeast cell, such as a pharmaceutical composition comprising the yeast cell.
In some embodiments, the genus of said yeast is selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In some embodiments, the genus of said yeast is Saccharomyces or Yarrowia.
The yeast cell may be selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica. In preferred embodiments, the yeast cell is a Saccharomyces cerevisiae cell, a Saccharomyces boulardii cell or a Yarrowia lipolytica cell. Most preferably, the yeast cell is a Saccharomyces boulardii cell.
Any of the yeast cells of the present disclosure, or any composition or pharmaceutical composition comprising said yeast cells, may thus be used as a probiotic. Without being bound by theory, it is envisaged that upon administration to the subject, the yeast cells release the one or more compounds produced by the yeast cell, in particular one or more of a halogenated tryptophan, a halogenated tryptamine, a halogenated, di-halogenated or tri-halogenated N-methyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N-dimethyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N,N-trimethyltryptamine, N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine, norbaeocystin, baeocystin, norpsilocin, psilocybin, psilocin, aeruginascin, dephosphorylated aeruginascin, N-acetyl-4-hydroxytryptamine, and derivatives thereof. Yeast cells producing said compounds have been described in detail herein above.
In addition to the modifications described herein, the yeast cell may be further engineered to allow for biocontainment of the yeast cells. Thus in some embodiments, the yeast cell further comprises means for biocontainment. Such means may include a conditional suicide system or a genetic switch system, which trigger inactivation or destruction of the yeast cell upon activation, for example through engineered auxotrophy. Other means for biocontainment include kill switches, where death of the yeast cell can be induced by the presence of an inducer. Alternatively, interruption of administration can also be used in order to prevent the microorganisms from settling in the intestinal tract in the long term.
Preferably, the yeast cell, the composition or the pharmaceutical composition comprising the yeast cell, are for oral administration. The skilled person will know how to formulate the yeast cell, the composition or the pharmaceutical composition for such administration.
The yeast cells and compositions may be useful for treating or preventing a disorder or a disease, particularly disorders and diseases where it is envisioned that one of the above compounds, i.e. a halogenated tryptophan, a halogenated tryptamine, a halogenated, di-halogenated or tri-halogenated N-methyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N-dimethyltryptamine, a halogenated, di-halogenated or tri-halogenated N,N,N-trimethyltryptamine, N-methyltryptamine, N,N-dimethyltryptamine and/or N,N,N-trimethyltryptamine, norbaeocystin, baeocystin, norpsilocin, psilocybin, psilocin, aeruginascin, dephosphorylated aeruginascin, N-acetyl-4-hydroxytryptamine, and derivatives thereof, is expected to have a therapeutic effect. In preferred embodiments, the compound is psilocybin or psilocin.
Relevant diseases and disorders include depression, such as major depressive disorder or treatment-resistant depression, anxiety disorders, obsessive-compulsive disorder, post-traumatic stress disorder, substance addiction or dependence such as alcohol or tobacco addiction or dependence, migraine and headache, preferably chronic migraines and chronic headaches.
The yeast cell, the composition or the pharmaceutical composition, are to be administered to a subject in need thereof. Subjects in need thereof include subjects suffering of, suspected of suffering of, or at risk of suffering of any one of the above listed diseases and disorders. The subject may be a mammal, such as a human, a farm animal such as a pig, a cow, a sheep, poultry, or a pet, in particular mammalian pets such as cats and dogs.
The present yeast cells and compositions are also expected to be useful in methods for increasing empathy and/or creativity of the subject to which they are administered.
Thus is also provided herein a method for increasing empathy and/or creativity of a subject, comprising administering to the subject any of the yeast cells described herein, or a composition or pharmaceutical composition comprising any of the yeast cells described herein.
The yeast cell, the composition or the pharmaceutical composition are preferably administered in such a dosage that only minute amounts of the one or more compounds are released in the intestinal tract of the subject. In some embodiments, the one or more compounds is delivered to the subject in an amount in the range of 1 ng to 1 mg, such as between 1 ng and 750 μg, such as between 5 ng and 500 μg, such as between 10 ng and 250 μg, such as between 25 ng and 100 μg, such as between 50 ng and 75 μg, such as between 75 ng and 50 μg, such as between 50 ng and 25 μg, such as between 75 ng and 10 μg, such as between 100 ng and 7.5 μg, such as between 250 ng and 5 μg, such as between 500 ng and 2.5 μg, such as between 750 ng and 1 μg.
The yeast cell, the composition or the pharmaceutical composition may thus be administered one to five times a day, such as once daily, twice daily, thrice daily, four times daily or five times daily, or every second day, every third day, once a week, every second week, or once a month.
Strains Media and Maintenance
All S. cerevisiae strains used in this study (Table 1) were derived from the CEN.PK strain family background. Frozen stocks of E. coli and S. cerevisiae were prepared by addition of glycerol (30% (v/v)) to exponentially growing cells and aseptically storing 1 mL aliquots at −80° C. Cultures were grown in synthetic medium according to the following recipes. Synthetic medium was prepared with 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO47H2O and appropriate growth factors. Synthetic complete media minus uracil supplementation (SC-Ura) was prepared with 3 g/L synthetic complete minus uracil powder and 5 g/L (NH4)2SO4. YP medium was prepared with 10 g/L yeast extract and 20 g/L peptone. In all cases unless stated otherwise, 20 g/L glucose was added. Synthetic feed-in-time (FIT) media was prepared by adding 60 g/L EnPump 200 substrate (polysaccharide) and 0.3% reagent A (hydrolyzing enzyme) to synthetic media. Media was supplemented with 200 mg/L G418 and 100 mg/L nourseothricin when required. E. coli strains were grown in Luria-Bertani (LB) media and supplemented with 100 mg/L ampicillin when required. Agar plates were prepared as described above but with the addition of 20 g/L agar.
Plasmid and Strain Construction
E. coli DH5 was used for all plasmid cloning and propagation. Single integration plasmids were constructed using the EasyClone-MarkerFree system (Jessop-Fabre et al., 2016), and multiple integration plasmids were constructed using a modified version of the EasyCloneMulti system (Maury et al., 2016) using a backbone plasmid were multiple integration was achieved using a Kluyveromyces lactis URA3 gene (KIURA3) under control of a truncated 10 bp KIURA3 promoter. Heterologous genes were codon-optimized for expression in S. cerevisiae using the JCat algorithm (Grote et al., 2005) and ordered as synthetic gene strings (GeneArt). DNA was transformed into S. cerevisiae using the LiAc method according to (Gietz and Woods, 2002).
Cultivation and Analysis
E. coli cells were cultured at 37° C. with shaking at 300 rpm. S. cerevisiae cells were cultured at 30° C. with shaking at 300 rpm. For micro-titer plate (MTP) cultivation of psilocybin producing S. cerevisiae strains, cells were inoculated from a 400 μL synthetic media pre-culture into 500 μL synthetic FIT media in a 96-deep well microtiter plate with air-penetrable lid (EnzyScreen, NL) and incubated for 72 h. When required, uracil was added at a final concentration of 200 mg/L.
Extraction of extracellular metabolites for analysis was performed as follows; Cell culture broth was supplemented with 100% acetonitrile at a ratio of 1:1, vortexed thoroughly then centrifuged at 3000 g for 5 min. The resulting supernatant was further diluted in 50% acetonitrile if required and analyzed using LC-MS with the following conditions; High resolution LC-MS measurements were carried out on a Dionex UltiMate 3000 UHPLC, connected to an Orbitrap Fusion Mass Spectrometer. The UHPLC was equipped with a zic-Hilic column, 15 cm×2.1 mm, 3 μm column. The temperature was 35.0 and the flow rate 0.5 mL/min. The system was running an isocratic gradient with a mobile phase consisting of 20% 10 mM ammonium formate (pH 3) and 80% acetonitrile, with 0.1% formic acid. The samples were passed on to the MS equipped with a heated electrospray ionization source (HESI) in positive-ion mode. The scan range was 100 to 1000 Da. Psilocybin, psilocin, tryptophan and tryptamine authentic analytical standards were used to quantify production in engineered strains.
Heterologous genes encoding the catalytic enzymes for psilocybin biosynthesis were introduced into S. cerevisiae strain ST8251 (CEN.PK113-5D+Cas9) (as outlined in
The basic heterologous pathway was introduced into S. cerevisiae (ST9327), then, using an Orbitrap Fusion Mass Spectrometer and authentic analytical standards, successful production of psilocybin, as well as the pathway intermediate tryptamine, and the spontaneous degradation product psilocin was confirmed in micro-titer plate cultivation (
While psilocybin was successfully produced in yeast, the initial titers were low. Furthermore, analysis revealed the extracellular accumulation of tryptamine (90 mg/L) indicating a limitation in the conversion of tryptamine to 4-Hydroxytryptamine encoded by the cytochrome P450 enzyme PcPsiH (
Functional implementation of PcCpr resulted in a significant increase in psilocybin and psilocin titers, and furthermore, significantly reduced extracellular accumulation of the first product in the heterologous pathway, tryptamine (11.7 mg/L,
To boost psilocybin precursor supply we introduced a series of modifications including overexpression of genes in the shikimate pathway (ARO1, ARO2), overexpression of feedback insensitive mutant genes in the shikimate pathway (ARO4K229L, TRP2S65R, S76L) (Graf et al., 1993; Luttik et al., 2008), and deletion of genes involved in regulation of the shikimate pathway (RIC1) (Suástegui et al., 2017). Iterative introduction of these modifications led to a modest yet significant increase in psilocybin and psilocin titer with ST9318 producing 155 mg/L psilocybin and 113 mg/L psilocin (
Finally, in an attempt to expand the utility of these engineered strains, we investigated whether S. cerevisiae could be engineered to produce natural and new-to-nature tryptamine derivatives. While enzymes typically display strict substrate specificity, others display relaxed substrate specificity and can accept multiple substrates with various affinities. LC-MS analysis of the psilocybin producing strain ST9328 detected the presence of norbaeocystin (non N-methylated) (
We then investigated whether this enzyme could catalyze a third iterative N-methylation to produce the tri-methylated derivative aeruginascin (Jensen et al., 2006). Interestingly, while we could not observe a peak matching the expected m/z of aeruginascin, a peak matching the expected m/z of the dephosphorylated version of aeruginascin was detected in ST9328. Furthermore, introduction of multiple copies of PcpsiM using a Ty integrative vector (ST9335) led to a 10-fold increase in the dephosphorylated aeruginascin peak area (
To investigate whether introduction of a P. cubensis cytochrome b5 could improve psilocybin titers, efforts to identify a cytochrome b5-encoding gene in the P. cubensis genome were undertaken. A gene encoding a putative P. cubensis cytochrome b5 (PcCYB5) was identified. To test its function and effect on psilocybin titers, PcCYB5 was expressed in ST9328 which carries the fully psilocybin biosynthetic pathway plus the P. cubensis cytochrome P450 reductase (PcCPR), resulting in strain ST9740. Strains were cultivated in media containing 5 g/L tryptophan in order to drive as much flux as possible through the pathway in order to distinguish whether expression of PcCYB5 could facilitate a higher conversion of tryptamine to 4-hydroxytryptamine.
LC-MS analysis (
We speculated that N-methylation catalyzed by PcPsiM was a rate-limiting step in the psilocybin pathway, perhaps due to insufficient availability of SAM, which acts as the methyl donor in the reaction. Accordingly, we set out to investigate strategies for increasing the availability of SAM in S. cerevisiae.
Hence, we performed single gene knock-outs of SPE2 and ERG4, respectively, in the high psilocybin producing strain ST9316, yielding strains ST9733 and ST9734.
According to LC-MS analysis, both ST9733 and ST9734 produced higher titers of psilocybin compared to the control ST9316 (
The 4-hydroxylation step in the psilocybin biosynthetic pathway where tryptamine is converted to 4-HT by PcPsiH consumes NADPH. We hypothesized that the supply of NADPH might be a limiting factor for this catalytic reaction, hence we explored different strategies in attempts to increase NADPH supply and thereby psilocybin production, namely overexpression of the native S. cerevisiae gene POS5 in the psilocybin producing strain ST9328. POS5 encodes a mitochondrial NADH kinase, responsible for NADPH generation in the mitochondria. Overexpression of POS5 in ST9328 led to a two-fold increase in psilocybin titers (
All tryptamine derivatives including psilocybin are produced from the common intermediate tryptophan which itself is produced from the amino acids serine and glutamine. While metabolic engineering to increase flux through the shikimate pathway (e.g. by overexpression of ARO1 and ARO2) had a positive effect on psilocybin titers, boosting the shikimate pathway flux only considers the carbon skeleton of tryptophan and not the two nitrogen groups present on the molecule. We therefore hypothesized that increasing flux towards the nitrogen groups of tryptophan would have a positive effect. To test this, ST9328 was cultivated in different media containing 5 g/L glutamine. LC-MS analysis showed that strains cultivated in glutamine containing media produced significantly higher amounts of psilocybin and psilocin with a 2-fold increase in titer observed.
Human and rabbit (Oryctolagus cuniculus) INMTs have both been cloned and heterologously expressed in COS-1 cells (immortalized Chlorocebus aethiops kidney cells). The O. cuniculus derived INMT (OcINMT) showed higher affinity in vitro for tryptamine than the human derived INMT (Km of 0.27 and 2.92, respectively), and was selected for production of DMT. Multiple copies of the OcINMT gene were integrated into the genome of strain ST9337, which expresses CrTDC and produces approximately 70 mg/L tryptamine. This yielded strain ST9647 that when cultivated produced metabolites with masses and fragmentation patterns corresponding to N-methyltryptamine (NMT), N,N-dimethyltryptamine (DMT) and N,N,N-trimethyltryptamine (TMT) as shown in
In nature, halogenated compounds are produced by haloperoxidases, perhydrolases and flavin-dependent halogenases. While haloperoxidases and perhydrolases in general lack regioselectivity, flavin-dependent halogenases display a high degree of substrate specificity and regioselectivity, which could make them more amenable for applications in biotechnological production. In addition to oxygen and halide ions, flavin-dependent halogenases require input of FADH2 provided by a partner flavin-reductase that performs NADH-driven reduction of FAD to FADH2.
To attempt production of halogenated tryptophan, integrations of Tryptophan 5-halogenase (SrPyrH), tryptophan 6-halogenase (SttH) and tryptophan 7-halogenase (LaRebH) into a wildtype S. cerevisiae strain (ST7574) were carried out with and without co-integration of the partner flavin reductase LaRebF. The same integrations were additionally performed in strain ST9336 expressing CrTDC in order to attempt production of halogenated tryptamine. Transformants were cultivated in synthetic minimal media supplemented with 25 mM KCl or 25 mM KBr. For extraction of intracellular products, the cultivation broths were subjected to cell lysis, which was carried out by adding a small aliquot of acid washed glass beads (212-300 p, Sigma) and running the samples for two cycles of 20 sec. at 5500 rpm on a Precellys 24 Homogenizer. The lysed cell broths were centrifuged at 17000 g for 1 min. and the supernatants were analyzed by LC-MS. Chloro- and bromo-tryptophan were present in all wild-type (ST7574) strains expressing a tryptophan halogenase, even in absence of LaRebF (
This observation was surprising. Flavin-dependent halogenases are speculated to use free FADH2 supplied by the flavin reductase, rather than forming a complex with the flavin reductase. FADH2 is produced in the citric acid cycle in S. cerevisiae, but as this takes place in the mitochondria it is unlikely that the FADH2 generated this way could be responsible for the observed halogenation. However, the observed chlorination and bromination in absence of LaRebF indicates that FADH2 was present in the cytosol of S. cerevisiae. However, the levels of halogenated tryptophan were approximately 100-fold higher in the presence of LaRebF, clearly demonstrating that FADH2 was a limiting factor of the chlorination and that expression of LaRebF increases cytosolic FADH2. Chloro- and bromotryptamine was produced by strains expressing CrTDC and a halogenase. Since none of these halogenases have been shown to chlorinate tryptamine, this observation indicates that CrTDC expressed in S. cerevisiae can accept chlorinated and brominated tryptophan as a substrate. In order to determine the efficiency of production of these halogenated derivatives the production of bromotryptamine was compared with an authentic analytical standard of 5-bromotryptamine. Comparison revealed that these strains produced approximately 0.13-0.29 mg/L of bromotryptamine.
By expressing different combinations of halogenases in a single strain, the production of di-halo and di-bromo tryptophan was observed (
In order to evaluate the substrate specificity of tryptophan halogenases, their ability to directly halogenate a tryptophan derivative like tryptamine was tested. Strains expressing individual tryptophan halogenases together with the partner flavin reductase LaRebF were cultivated in synthetic minimal media supplemented with 25 mM KCl or 25 mM KBr and 1 mM tryptamine. Cultivation samples were subjected to the intracellular extraction method and the supernatants were analyzed by LC-MS. The production of halogenated tryptamine was observed in strains expressing SttH and LaRebH when tryptamine was supplemented to the media (Table 3).
Psilocybin has been found to bind various receptors of serotonin, an important neurotransmitter relevant to various psychological and neurological afflictions, e.g. depression and migraine. Serotonin receptors are widely expressed in the gut, in fact it has been estimated that around 95% of serotonin receptors are found in the GI tract. It is becoming increasingly clear that the neural networks of the brain (central) and gut (enteric) are tightly linked, and that the bidirectional regulatory signals exchanged between these organs can be a significant contributor or mediator of disease. The complexities of the brain-gut connection have yet to be fully unraveled, however the recent recognition of the importance of the gut microbiome to the physiological function of the gut and brain, confirms the possibility of manipulating neurological function via the gut. This presents an intriguing opportunity to engineer probiotic organisms, e.g. Saccharomyces boulardii for the in vivo delivery of various beneficial compounds. Auxotrophic S. boulardii strains (ΔURA3 ΔHIS3 ΔTRP1 ΔLEU2) are generated from Saccharomyces cerevisiae Meyen ex E. C. Hansen (ATCC MYA-796) by introducing a stop codon near the beginning of the genes, thereby disrupting their expression. The Cas9 plasmid (pCfB2312) is introduced using the LiAc method according to (Gietz and Woods, 2002), and maintained on 200 mg/L G418. Using the EasyClone-MarkerFree system, integration plasmids containing SEQ ID NO: 6-7, 8, and 9-10 are constructed and integrated into the S. boulardii genome at the X-3, X-4, and XII-5 integration sites, respectively. The gRNA is maintained via URA auxotrophy or nourseothricin sensitivity (50 mg/L; S. boulardii exhibits heightened nourseothricin sensitivity compared to S. cerevisiae). The production of psilocybin-related tryptamine derivatives is achieved via the introduction of any SEQ ID NO: 6-31; 37-41; 43; 45; 47; 49; 55-59 DNA sequences, or other sequences encoding the necessary enzymes. In order to increase production under GI conditions, the yeast can be subjected to various optimisations, e.g. medium optimization (e.g. growth on alternative carbon sources) or by modification of the biosynthesis pathway (e.g. introducing, reducing or modifying alternative anaerobic biosynthesis steps).
Sequences
Catharanthus roseus,
Psilocybe cubensis,
Psilocybe cubensis
Psilocybe cubensis,
Psilocybe cubensis,
Catharanthus roseus
Psilocybe cubensis
Psilocybe cubensis
Psilocybe cubensis
Psilocybe cubensis
Bos taurus
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Bos taurus
Saccharomyces cerevisiae
Streptomyces rugosporus tryptophan-5-
Streptomyces toxytricini tryptophan 6-
Lechevalieria aerocolonigenes Tryptophan 7-
Lechevalieria aerocolonigenes flavin
Oryctolagus cuniculus (Uniprot Seq. ID:
Psilocybe cubensis putative Cytochrome b5
S. cerevisiae Sterol reductase (Uniprot ID:
S. cerevisiae SAM decarboxylase (Uniprot
Chondromyces crocatus
Pseudomonas fluorescens Tryptophan 7-
Streptomyces albogriseolus Tryptophan 6-
Dictyostelium discoideum Tryptophan
Kutzneria sp. 744 Tryptophan 7-halogenase
Kutzneria sp. 744 Tryptophan 6-halogenase
Item List 1
Item list 2
| Number | Date | Country | Kind |
|---|---|---|---|
| 19197432.8 | Sep 2019 | EP | regional |
| 20164388.9 | Mar 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/075823 | 9/16/2020 | WO |
