Patent Application
20230242919
The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
The present invention generally relates to the production of substituted indoles, e.g. N-methyl-L-tryptophan (NMTP), N,N-dimethyl-L-tryptophan (DMTP), and N,N,N-trimethyl-L-tryptophan (TMTP), and related tryptamines, e.g. N-methyltryptamine (NMT), N,N-dimethyltryptamine (DMT), and N,N,N-trimethyltryptamine (TMT), in a modified heterologous microorganism.
Mental health problems, which may also be referred to as mental illness or psychiatric disorder, are behavioral or mental patterns which impair the functioning of individuals across the world. Such mental health disorders include: personality disorders, anxiety disorders, major depressions, and various addictions. Indolic and tryptamine-based compounds similar in structure to the endogenous neurotransmitter serotonin have been increasingly evaluated for treating mental health problems. In contrast to anxiolytic medicines, usage of substituted indoles and methylated tryptamines, such as N,N-dimethyltryptamine does not lead to physical dependence.
The chemical synthesis of hydroxy, methoxy, phosphorylated, prenylated, and halogenated substituted tryptamines and indoles typically involve tedious techniques of organic chemistry. Often, reproducibility is elusive and the solvents used during the syntheses of substituted tryptamines are environmentally toxic. Decarboxylations and selective methylations can be difficult to obtain via the techniques of organic chemistry. Further, the yields and purity of the intermediates for obtaining the target molecules can be low, where, for example, the starting molecule is L-tryptophan and the target molecule is N,N-dimethyltryptophan (DMTP), bufotenine, 5-MeO-dimethyltryptamine (5-MeO-DMT), 7-dimethylallyltryptophan, psilocybin, aeruginascin, among others.
The present invention provides for producing substituted tryptamines and indoles in recombinant microorganisms, providing for a more environmentally benign and higher yielding processes for production of those compounds.
In some embodiments, provided is a non-naturally occurring nucleic acid comprising a sequence encoding an enzyme or regulatory protein in tryptamine metabolism, where the enzyme or regulatory protein is an N-methyltransferase (INMT, PsiM, TrpM), a tryptophan decarboxylase (AADC), a tryptophan hydroxylase (TPH), a tryptamine 4′ hydroxylase (T4H), a tryptamine 5′ hydroxylase (T5H), a truncated cytochrome p450 reductase (T4H-CPR, T5H-CPR), an hydroxytryptamine O-methyltransferase (IOMT or CaffOMT), an N-acetyltransferase (NAT), a deacetylase (DAC), a hydroxyl tryptamine kinase (PsiK), a tryptophan synthase (TrpS), a toluene monooxygenase (TMO), an aminotransferase/methyltransferase fusion (ATMT), a phosphatase, an oxidase, a dimethylallyltryptophan synthase (DMAT), an isopentenyl-diphosphate isomerase (IDII), a tryptophan halogenase (TrpHalo), an aspartate oxidase/quinolinic acid synthase fusion (AOQS), a tryptophan importer (TAT2), a methionine importer (MUP1), or a SAMe importer (SAM3).
Also provided is an expression cassette comprising any of the above nucleic acids with a promoter functional in a recombinant microorganism.
Additionally provided is a recombinant microorganism comprising the above expression cassette, that expresses the enzyme or regulatory protein encoded therein.
Further provided is a non-naturally occurring enzyme or regulatory protein comprising an amino acid sequence encoded by any of the above-identified nucleic acids.
To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:
Conservative amino acid substitutions: As used herein, when referring to mutations in a protein, “conservative amino acid substitutions” are those in which at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gln for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gln; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.
Functional variant: The term “functional variant,” as used herein, refers to a recombinant enzyme such as an INMTenzyme that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent protein and that is still capable of performing an enzymatic function (e.g., synthesis of DMT) of the parent enzyme. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent enzyme may cause desirable changes in reaction parameters without altering fundamental enzymatic function encoded by the nucleotide sequence or containing the amino acid sequence. The functional variant may have conservative change including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids. Also envisioned is the use of amino acid analogs, e.g. amino acids not DNA or RNA encoded in biological systems, and labels such as fluorescent dyes, radioactive elements, electron dense agents, or any other protein modification, now known or later discovered.
Recombinant nucleic acid and recombinant protein: As used herein, a recombinant nucleic acid or protein is a nucleic acid or protein produced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012).
Polypeptide, protein, and peptide: The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.
The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).
Primer, probe and oligonucleotide: The terms “primer,” “probe,” and “oligonucleotide” may be used herein interchangeably to refer to a relatively short nucleic acid fragment or sequence. They can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands that can be separated apart by denaturation. In certain aspects, they are of a length of from about 8 nucleotides to about 200 nucleotides. In other aspects, they are from about 12 nucleotides to about 100 nucleotides. In additional aspects, they are about 18 to about 50 nucleotides. They can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications.
Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Various vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
Linker: The term “linker” refers to a short amino acid sequence that separates multiple domains of a polypeptide. In some embodiments, the linker prohibits energetically or structurally unfavorable interactions between the discrete domains.
Codon optimized: As used herein, a recombinant gene is “codon optimized” when its nucleotide sequence is modified to accommodate codon bias of the host organism to improve gene expression and increase translational efficiency of the gene.
Expression cassette: As used herein, an “expression cassette” is a nucleic acid that comprises a gene and a regulatory sequence operatively coupled to the gene such that the promoter drives the expression of the gene in a cell. An example is a gene for an enzyme with a promoter functional in yeast, where the promoter is situated such that the promoter drives the expression of the enzyme in a yeast cell.
Compounds and abbreviations in use of or contained within systems and methods herein are provided in Table 1.
Enzymes and regulatory proteins, and abbreviations, in use of or contained within systems and methods herein are provided in Table 2.
The present invention is directed to biosynthetic production of molecules that are analogs of indoles, tryptophans, and tryptamines, which can also serve as precursors to larger tryptamine alkaloids, such as tryptamines and tryptophans modified by hydroxylation, halogenation, methylation, phosphorylation, prenylation, and halogenation in recombinant organisms.
By engineering various enzymes and regulatory proteins into a microorganism, tryptophan, tryptamine and other substituted indoles can be modified into a large array of useful compounds, which can be harvested from cultures of the microorganisms.
As depicted in
Thus, in some embodiments, provided is a non-naturally occurring nucleic acid comprising a sequence encoding an enzyme or regulatory protein in tryptamine metabolism, where the enzyme or regulatory protein is an N-methyltransferase (INMT, PsiM, TrpM), a tryptophan decarboxylase (AADC), a tryptophan hydroxylase (TPH), a tryptamine 4′ hydroxylase (T4H), a tryptamine 5′ hydroxylase (T5H), a truncated cytochrome p450 reductase (T4H-CPR, T5H-CPR), an hydroxytryptamine O-methyltransferase (IOMT or CaffOMT), an N-acetyltransferase (NAT), a deacetylase (DAC), a hydroxyl tryptamine kinase (PsiK), a tryptophan synthase (TrpS), a toluene monooxygenase (TMO), an aminotransferase/methyltransferase fusion (ATMT), a phosphatase, an oxidase, a dimethylallyltryptophan synthase (DMAT or DMATS), an isopentenyl-diphosphate isomerase (IDI1), a tryptophan halogenase (TrpHalo), an aspartate oxidase/quinolinic acid synthase fusion (AOQS), a tryptophan importer (TAT2), a methionine importer (MUP1), or a SAMe importer (SAM3).
These enzymes and regulatory proteins are further characterized as follows.
Indolethylamine N-methyltransferase (INMT) catalyzes the alkylation (i.e., adding a methyl (CH3) group) of the primary amine on a tryptamine substrate. The methylation reaction uses up the methyl donor cofactor, SAMe (see
Indole-O-methyltransferase (IOMTor CaffOMT) catalyzes the alkylation of the primary amine on the 5-hydroxy moiety on an indole ring. The methylation reaction uses up the methyl donor cofactor, SAMe (
Tryptamine 5′ hydroxylase (T5H) is a p450 tryptamine hydroxylase which prefers hydroxylation at the 5′ position of the indole ring, such as generating serotonin from tryptamine (
Tryptophan methyltransferase (TrpM) catalyzes the alkylation of the primary amine of L-tryptophan to produce N-methyltryptophan (NMTP, also called L-abrine), the mono-methylated product; N,N-dimethyltryptophan (DMTP), the di-methylated product; and N,N,N-trimethyltryptophan (TMTP), the tri-methylated product. See
Psilocybin synthase (PsiM) is an N-methyltransferase that prefers a substituted tryptamine, such as the phosphorylated tryptamine, norbaeocystin. Novel chimeric PsiMs, were generated to remove potentially deleterious regulatory regions of the enzymes by swapping PsiM domains with the related small rRNA methyltransferases from Ascomycota, the phylum of S. cerevisiae.
Aromatic amino acid decarboxylase or tryptophan decarboxylase (AADC) catalyzes the decarboxylation of an aliphatic carboxylic acid (i.e., releases carbon dioxide) from compounds such as L-tryptophan to create tryptamine, 5-HTP to create serotonin; 5-OH-DMTP to create bufotenine; and 5-MeO-DMTP to create 5-MeO-DMT, as depicted in
Tryptophan hydroxylase (TPH), adds a hydroxy group to the 5-carbon of L-tryptophan. The L-tryptophan hydroxylase can catalyze the OH addition to the 5-carbon with the cofactor BH4 and oxygen (Biotechnol J. 2016 May;11(5):717-24) (
Tryptamine 4′ hydroxylase (T4H) is a p450 tryptamine hydroxylase which prefers hydroxylation at the 4′ position of the indole ring, in conjunction with the cofactors NAD(P)H, FMN, and FAD+. When derived from psychedelic mushrooms, these are also called PsiH. The T4H enzyme can convert tryptamine to 4-OH-tryptamine, which is a part of the psilocybin pathway. P450s such as the T4Hs are generally membrane-associated, with the N-termini imparting an effect on the efficiency of the p450 enzymatic function, including a p450′s interaction with an associated CPR, which assists with electron transfer.
From psychedelic a mushroom derived PsiH and CPR, we generated chimeric p450s and CPRs to better match a heterologous host (SEQ ID NO:162, 179-180, and 451, 468-469), where the N termini of a yeast p450 and CPR replaced the N terminus. Due to the enhancing action of CPRs on p450 enzymatic activity, we determined an optimal fusion between T4H and T4H CPR, where the T4H_CPR listed are truncated at the N termini and replaced with a linker region. In some embodiments, the T4H nucleic acids have, at the 3′ end, an optimized nucleic acid encoding a T4H_CPR, e.g., having SEQ ID NOs:171-180, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:460-469 fused at the C terminus of the enzyme polypeptide, generating recombinant T4H-CPR fusion polypeptides.
Similar to the T4H CPR fusions, we generated T5H CPR fusions to enhance the hydroxylation activity. In those embodiments, the T5H nucleic acids have, at the 3′ end, an optimized nucleic acid encoding a T5H-CPR, e.g., having SEQ ID NOs:181-192, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:470-481 fused at the C terminus of the enzyme polypeptide, generating recombinant T5H-CPR fusion polypeptides.
Examples of the utilization of the T4H-CPR and T5H-CPR in recombinant cells are shown in
Localizing O-methyltransferase activity to hydroxylation can be beneficial for generating methoxytryptamines, such as 5-MT, 5-MeO-DMT, and melatonin. In some embodiments, the T5H nucleic acids have, at the 3′ end, an optimized nucleic acid encoding an IOMT e.g., having SEQ ID NOs:99-130, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:388-419 fused at the C terminus of the enzyme polypeptide, generating recombinant T5H-IOMT fusion polypeptides.
In all of the fusions described herein, the N-terminal coding sequence has any STOP codon removed, if present, before fusion to a C-terminal coding sequence. If the N-terminal coding sequence does not have a START (ATG) codon, a START codon is added.
N-acetyltransferase (NAT) adds an acetyl group from acetyl-CoA to the terminal amino group of e.g., a tryptamine such as serotonin (
Deacetylase (DAC) removes an acetyl group from the terminal amino group of a tryptamine such as melatonin. As an example of DAC activity, DAC can act on melatonin to create 5-MeO-tryptamine, which in turn can be acted on by an INMT to generate 5-MeO-DMT (
Hydroxy tryptamine kinase (PsiK) phosphorylates a hydroxy-indole, in conjunction with ATP. For example, PsiK can act on 4-OH tryptamine to generate norbaeocystin as part of the psilocybin pathway. PsiKs are found in certain mushrooms and parasitic fungi. For psychedelic mushroom derived PsiKs, we generated chimeric PsiKs based on yeast choline kinase to better match a heterologous host.
Non-natural tryptamine analogs can be created with the addition of a synthetic precursor to the fermentation of a recombinant host expressing enzymes capable of utilizing the substrate. For example, the addition of an alpha-methylated amino acid such as alpha-methyl tryptophan to a fermentation where an organism expresses a an indole-N-methyltransferase (INMT) leads to the generation of alpha-methylated DMT (e.g.,
For certain indole ring modifications, such as non-natural indoles, bacterial tryptophan synthases (TrpS) can be used to combine an indole with L-serine or L-threonine to create variants of tryptophan and beta-methyl tryptophan, respectively (
Alternatively to T4H, T5H, and TPH enzymes, hydroxylation of the indole ring of tryptamines and related indole-like compounds can be carried out by complexes known as toluenemonooxygenases (TMO) typically found in bacteria within the genus Pseudomonas. The polypeptides that form this complex can be expressed in a modified host as an alternative to P450-based hydroxylation for compounds such as psilocybin and aeruginascin, whose biosynthetic pathway involves 4′OH hydroxylation. Other non-P450 monooxygenases from genuses of Pseudomonas and Burkeholderia can be optimized and expressed in a modified host for hydroxylation of different indole positions, such as the 3′ carbon of the indole ring. TMO complexes are made up of several subunits. For efficient expression of TMOs in a recombinant heterologous host, we generated fusion polypeptide pairs of the four core subunits.
Beta-methylated tryptamine analogs are created by combined expression of a recombinant aminotransferase-methyltransferase (ATMT) fusion polypeptide and an aromatic amino acid decarboxylase (AADC) (
In some embodiments, recombinant phosphatases and oxidases are used to generate hydroxylated tryptamine dimers such as one psilocin or bufotenine molecule conjugated to another psilocin or bufotenine molecule (
Dimethylallyl tryptophan synthase (DMATS or DMAT) generates prenylated tryptophans and tryptamines. DMATS is a prenyltransferase that prefers the dimethylallyl diphosphate (DMAPP) prenyl donor to prenylate tryptophan and tryptamine compounds.
Localizing DMAPP generation to the DMATS enzyme can be beneficial for generating prenylated tryptophans, such as 7-dimethylallyltryptophan. In yeast, IDI1 is the enzyme which generates DMAPP as part of the mevalonate pathway. In some embodiments, the DMATS nucleic acids have, at the 3′ end, an optimized nucleic acid encoding IDI1 e.g., having SEQ ID NO:67, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NO:356 fused at the C terminus of the enzyme polypeptide, generating recombinant DMATS-IDI1 fusion polypeptides (
Tryptophan halogenase (TrpHalo) is a flavin-associated halogenase that adds fluorine (F), chlorine (Cl), bromine (Br), and/or iodine (I) to various indoles and biogenic amines (
In other embodiments, TrpHalo nucleic acids have, at the 5′end, a nucleic acid encoding a secretion tag with or without a 6xHIS tag for purification, e.g., having SEQ ID NO:1, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NO:290 fused at the N terminus of the enzyme polypeptide, generating recombinant fusion polypeptides. In one embodiment, TrpHalo is also coexpressed with the yeast fluoride exporter, Fex1, SEQ ID NO:66,355, to limit halide toxicity on the heterologous host.
To improve the yield of tryptophan and tryptamine variants discussed herein, modifying the heterologous host which expresses these genes and enzymes, in various combinatorial ways, to prevent tryptophan and tryptamine compound degradation is beneficial. Replacing the yeast pathway which degrades certain tryptamine and tryptophan compounds for de novo NAD+ production, which is an important source of cofactors for cell viability, with an alternative route to NAD+ production can preserve tryptophan as a precursor and increase product yields. In some embodiments, a new de novo pathway is expressed in a heterologous host, where the pathway is composed of a fusion protein containing the two enzymatic functions required to convert the amino acid aspartate into quinolinic acid (AOQS), SEQ ID NO: 26-27,315-316, which replaces the endogenous use of tryptophan for generating quinolinic acid in the pathway for NAD+.
In some embodiments, the nucleic acids have, at the 5′ end, a nucleic acid encoding codon optimized cofolding peptides to create a fusion protein, e.g., having SEQ ID NOs:256-269, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:554-558 fused at the N terminus of the enzyme polypeptide, generating recombinant fusion polypeptides.
In some embodiments, the nucleic acids have, at the 5′ end, a nucleic acid encoding a secretion signal, creating a secreted protein, e.g., having SEQ ID NOs:282-286, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:571-575 fused at the N terminus of the enzyme polypeptide, generating recombinant fusion polypeptides.
In some embodiments, the nucleic acids have, at the 5′ or 3′ end, an optimized nucleic acid encoding a localization scaffold composed of multiple domains where proteins tagged with affibodies can bind and colocalize together (for example,
In some embodiments, the nucleic acids have, at the 5′ or 3′ end, an optimized nucleic acid encoding an affibody tag that can bind one of the domains of the localization scaffold, thereby colocalizing multiple enzymes and creating protein scaffold fusion, e.g., having SEQ ID NOs:259-264, joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NOs:548-553 fused at the N or C terminus of the enzyme polypeptide, generating recombinant fusion polypeptides.
The initial substrates for DMTP, DMT, and related compound production are L-tryptophan and S-Adenosyl-L-methionine (SAMe). The initial substrate can be produced endogenously in a recombinant host as described and/or provided exogenously to a fermentation involving a recombinant host, whereby the host uptakes the starting substrates to feed into the biosynthetic pathway for indoles and tryptamines. The recombinant hosts herein described that are expressing all, one, or multiple combinations of the engineered INMT, AADC, TPH, T4H, T5H, T4H-CPR, T5H-CPR, IOMT, NAT, DAC, PsiK, TrpS, TMO, ATMT, DMATS, IDI1, and TrpHalo genes can produce tryptamine, NMTP, DMTP, TMTP, NMT, DMT, TMT, psilocybin, bufotenine, 5-MeO DMT, 4-bromo-tryptamine, 4-dimethylallyl tryptamine, alpha-methylated DMTP, beta-methylated DMTP, melatonin, etc.
As depicted in
The methylation occurs selectively at the primary amine of L-tryptophan and tryptamine in the presence of TrpM and INMT enzymes. The nitrogen in the heterocycle and hydroxyl group in the carboxylic acid of L-tryptophan are also sites of alkylation, as SAMe is a highly reactive methylating agent. The TrpM enzyme directs methylation such that di-methylation of the primary amine occurs. Using the traditional techniques of organic chemistry where robust methylating agents, such as methyl iodide, trimethyl sulfonium iodide, and dimethyl sulfate, are employed, a mixture of products is formed. The mixture of products may include: mono, di, and tri-methylation of the amine; O-methylation of the carboxylic acid (i.e., the methyl ester), and N-methylation of the indole ring. Separation of these products are tedious and reduces the yield of a desired product. Additionally, SAMe has a primary amine group which may readily undergo intramolecular methylation at the amine. The systems and methods herein in the recombinant host with TrpM and INMT enzymes maintain the structure of SAMe without methylation of the amine of the SAMe prior to methylating the amine of L-tryptophan and tryptamine.
Heterologous pathway enzymes that are expressed to produce substituted indole and tryptamine compounds such as DMTP and DMT use L-tryptophan as a directing molecule. Tryptophan production in cells is normally tightly regulated. Tryptophan accumulation in a recombinant host is increased by: (a) overexpressing feedback-resistant versions of the endogenous tryptophan-producing enzymes; (b) knocking out off-pathway tryptophan-consuming genes and enzymes; and (c) overexpressing a recombinant L-tryptophan transporter. This allows for exogenous tryptophan to be fed to the cells and transported in the recombinant host. These modifications, genes, and methods are disclosed in U.S. Pat. Publication 2021/0147888, incorporated by reference.
On-pathway genes and enzymes can be overexpressed for L-tryptophan accumulation. The immediate precursors for L-tryptophan include chorismate, L-serine, and L-glutamine. To increase the on-pathway flux to L-tryptophan and the substituted indole and tryptamine pathway, off-pathway genes which consume L-tryptophan are deleted. The genes that encode the enzymes, Pdc5 and Aro10 are deleted to reduce pathway flux through the pathways that produce aromatic alcohols. The gene encoding the Aro7 enzyme is deleted to reduce production of tyrosine and phenylalanine from L-tryptophan. The genes that encode the enzymes Pdz1 and Pdz2 are also deleted to reduce pathway flux through the pABA production pathway. The gene encoding the enzyme Bna2 is deleted to reduce consumption of L-tryptophan by the kynurenine pathway.
In some embodiments, a recombinant host is modified to increase the accumulation of the methyl donor, SAMe, which is used by the recombinant TrpM and INMT enzymes to methylate indole and tryptamine molecules, such as L-tryptophan and NMT. SAMe accumulation in the recombinant host cell is increased by: (a) overexpressing enzymes to promote conversion of L-methionine to SAMe; (b) deleting off-pathway genes which encode for enzymes that deplete SAMe for unwanted side products; and (c) overexpressing a permease. This enables exogenous L-methionine to be fed to and transported into the cells.
The TrpM and INMT methyltransferase reactions consume one equivalent of adenosine triphosphate (ATP) and of SAMe. SAMe is a robust methyl donor synthesized from methionine and ATP via the L-methionine adenosyltransferase enzyme, Sam2. In various embodiments, Sam2 is overexpressed in a recombinant host to increase the conversion of L-methionine to SAMe. In other embodiments, to support the increased pathway flux and generate more ATP, the adenylate kinase enzyme, Adk1, is overexpressed. In additional embodiments, to increase the uptake of exogenous L-methionine fed into the SAMe pathway, recombinant Mup1 is overexpressed, which is a methionine transporter. SAMe is a precursor molecule for spermidine production and glycogen biosynthesis. To keep SAMe levels high in the pathways of the recombinant host and decrease off-pathway usage of SAMe, the SPE2 gene can be deleted in the recombinant host, thereby blocking the conversion of SAMe to spermidine. Glycogen biosynthesis consumes ATP, which is required for the conversion of L-methionine to SAMe. The gene encoding the enzyme Glc3 can be deleted in the recombinant host, thereby reducing production of glycogen, maintaining higher levels of ATP in the host cell, and increasing on-pathway flux of SAMe for methyltransferase activity.
As depicted in
As depicted in
Heterologous pathway enzymes that are expressed to produce substituted indole and tryptamine compounds such as DMTP and DMT use L-tryptophan as a directing molecule. Tryptophan production in cells is normally tightly regulated. Tryptophan accumulation in a recombinant host is increased by: (a) overexpressing feedback-resistant versions of the endogenous tryptophan-producing enzymes; (b) knocking out off-pathway tryptophan-consuming genes and enzymes; and (c) overexpressing a recombinant L-tryptophan transporter. This allows for exogenous tryptophan to be fed to the cells and transported in the recombinant host. See also U.S. Pat. Publication 2021/0147888.
On-pathway genes and enzymes can be overexpressed for L-tryptophan accumulation. The immediate precursors for L-tryptophan include chorismate, L-serine, and L-glutamine. To increase the on-pathway flux to L-tryptophan and the substituted indole and tryptamine pathway, off-pathway genes which consume L-tryptophan may be deleted. In some embodiments, the genes that encode the enzymes Pdc5 and Aro10 are deleted to reduce pathway flux through the pathways that produce aromatic alcohols. In other embodiments, the gene encoding the Aro7 enzyme is deleted to reduce production of tyrosine and phenylalanine from L-tryptophan. In additional embodiments, the genes that encode the enzymes Pdz1 and Pdz2 are also deleted to reduce pathway flux through the pABA production pathway. In further embodiments, the gene encoding the enzyme Bna2 is deleted to reduce consumption of L-tryptophan by the kynurenine pathway.
In some embodiments, the nucleic acids described herewith encode a polypeptide or oligopeptide having an amino acid sequence that is naturally occurring. In other embodiments, the nucleic acids encode a polypeptide or oligopeptide having an amino acid sequence that is not naturally occurring. The encoded polypeptides or oligopeptides that are not naturally occurring can vary from a naturally occurring polypeptide or oligopeptide, or portion thereof, by a small amount (e.g., one conservative amino acid substitution or a histidine tag) or extensively (e.g., further comprising a fusion peptide, a substituted or added domain from another protein, a scaffold, etc.).
The nucleic acids can be derived from a naturally occurring gene from any source, e.g., any microorganism, protist, plant, or animal.
In some embodiments, the gene for the enzyme or regulatory protein is derived from a bacterium. It is envisioned that an enzyme or regulatory protein derived from any bacterium now known or later discovered can be utilized in the present invention. For example, the bacterium can be from phylum Abditibacteriota, including class Abditibacteria, including order Abditibacteriales; phylum Abyssubacteria or Acidobacteria, including class Acidobacteriia, Blastocatellia, Holophagae, Thermoanaerobaculia, or Vicinamibacteria, including order Acidobacteriales, Bryobacterales, Blastocatellales, Acanthopleuribacterales, Holophagales, Thermotomaculales, Thermoanaerobaculales, or Vicinamibacteraceae; phylum Actinobacteria, including class Acidimicrobiia, Actinobacteria, Actinomarinidae, Coriobacteriia, Nitriliruptoria, Rubrobacteria, or Thermoleophilia, including orders Acidimicrobiales, Acidothermales, Actinomycetales, Actinopolysporales, Bifidobacteriales, Nanopelagicales, Catenulisporales, Corunebacteriales, Cryptosporangiales, Frankiales, Geodermatophilales, Glycomycetales, Jiangellales, Micrococcales, Micromonosporales, Nakamurellales, Propionibacteriales, Pseudonocardiales, Sporichthyales, Streptomycetales, Streptosporangiales, Actinomarinales, Coriobacteriales, Eggerthellales, Egibacterales, Egicoccales, Euzebyales, Nitriliruptorales, Gaiellales, Rubrobacterales, Solirubrobacterales, or Thermoleophilales; phylum Aquificae, including class Aquificae, including order Aquificales or Desulfurobacteriales; phylum Armatimonadetes, including class Armatimonadia, including order Armatimonadales, Capsulimonadales, Chthonomonadetes, Chthonomonadales, Fimbriimonadia, or Fimbriimonadales; phylum Aureabacteria or Bacteroidetes, including class Armatimonadia, Bacteroidia, Chitinophagia, Cytophagia, Flavobacteria, Saprospiria or Sphingobacteriia, including order Bacteroidales, Marinilabiliales, Chitinophagales, Cytophagales, Flavobacteriales, Saprospirales, or Sphingopacteriales; phylum Balneolaeota, Caldiserica, Calditrichaeota, or Chlamydiae, including class Balneolia, Caldisericia, Calditrichae, or Chlamydia, including order Balneolales, Caldisericales, Calditrichales, Anoxychlamydiales, Chlamydiales, or Parachlamydiales; phylum Chlorobi or Chloroflexi, including class Chlorobia, Anaerolineae, Ardenticatenia, Caldilineae, Thermofonsia, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Tepidiformia, Thermoflexia, Thermomicrobia, or Sphaerobacteridae, including order Chlorobiales, Anaerolineales, Ardenticatenales, Caldilineales, Chloroflexales, Herpetosiphonales, Kallotenuales, Dehalococcoidales, Dehalogenimonas, Ktedonobacterales, Thermogemmatisporales, Tepidiformales, Thermoflexales, Thermomicrobiales, or Sphaerobacterales; phylum Chrysiogenetes, Cloacimonetes, Coprothermobacterota, Cryosericota, or Cyanobacteria, including class Chrysiogenetes, Coprothermobacteria, Gloeobacteria, or Oscillatoriophycideae, including order Chrysiogenales, Coprothermobacterales, Chroococcidiopsidales, Gloeoemargaritales, Nostocales, Pleurocapsales, Spirulinales, Synechococcales, Gloeobacterales, Chroococcales, or Oscillatoriales; phyla: Eferribacteres, Deinococcus-thermus, Dictyoglomi, Dormibacteraeota, Elusimicrobia, Eremiobacteraeota, Fermentibacteria, or Fibrobacteres, including class Deferribacteres, Deinococci, Dictyoglomia, Elusimicrobia, Endomicrobia, Chitinispirillia, Chitinivibrionia, or Fibrobacteria, including order Deferribacterales, Deinococcales, Thermales, Dictyoglomales, Elusimicrobiales, Endomicrobiales, Chitinspirillales, Chitinvibrionales, Fibrobacterales, or Fibromonadales; phylum Firmicutes, Fusobacteria, Gemmatimonadetes, or Hydrogenedentes, including class Bacilli, Clostridia, Erysipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, Tissierellia, Fusobacteriia, Gemmatimonadetes, Longimicrobia, including order Bacillales, Lactobacillales, Borkfalkiales, Clostridiales, Halanaerobiales, Natranaerobiales, Thermoanaerobacterales, Erysipelotrichales, Limnochordales, Acidaminococcales, Selenomonadales, Veillonellales, Thermolithobacterales, Tissierellales, Fusobacteriales, Gemmatimonadales, or Longimicrobia; phylum Hydrogenedentes, Ignavibacteriae, Kapabacteria, Kiritimatiellaeota, Krumholzibacteriota, Kryptonia, Latescibacteria, LCP-89, Lentisphaerae, Margulisbacteria, Marinimicrobia, Melainabacteria, Nitrospinae, or Omnitrophica, including class Ignavibacteria, Kiritimatiellae, Krumholzibacteria, Lentisphaeria, Oligosphaeria, or Nitrospinae, including order Ignavibacteriales, Kiritimatiellales, Krumholzibacteriales, Lentisphaerales, Victivallales, Oligosphaerales, or Nitrospinia; phylum Omnitrophica or Planctomycetes, including class Brocadiae, Phycisphaerae, Planctomycetia, or Phycisphaerales, including order Sedimentisphaerales, Tepidisphaerales, Gemmatales, Isosphaerales, Pirellulales, or Planctomycetales; phylum Proteobacteria including class Acidithiobacillia, Alphaproteobacteria, Betaproteobacteria, Lambdaproteobacteria, Muproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Hydrogenophilalia, Oligoflexia, or Zetaproteobacteria, including order Acidithiobacillales, Caulobacterales, Emcibacterales, Holosporales, Iodidimonadales, Kiloniellales, Kopriimonadales, Kordiimonadales, Magnetococcales, Micropepsales, Minwuiales, Parvularculales, Pelagibacterales, Rhizobiales, Rhodobacterales, Rhodospirillales, Rhodothalassiales, Rickettsiales, Sneathiellales, Sphingomonadales, Burkholderiales, Ferritrophicales, Ferrovales, Neisseriales, Nitrosomonadales, Procabacteriales, Rhodocyclales, Bradymonadales, Acidulodesulfobacterales, Desulfarculales, Desulfobacterales, Desulfovibrionales, Desulfurellales, Desulfuromonadales, Myxococcales, Syntrophobacterales, Campylobacterales, Nautiliales, Acidiferrobacterales, Aeromonadales, Alteromonadales, Arenicellales, Cardiobacteriales, Cellvibrionales, Chromatiales, Enterobacterales, Immundisolibacterales, Legionellales, Methylococcales, Nevskiales, Oceanospirillales, Orbales, Pasteurellales Pseudomonadales, Salinisphaerales, Thiotrichales, Vibrionales, Xanthomonadales, Hydrogenophilales, Bacteriovoracales, Bdellovibrionales, Oligoflexales, Silvanigrellales, or Mariprofundales; phylum Rhodothermaeota, Saganbacteria, Sericytochromatia, Spirochaetes, Synergistetes, Tectomicrobia, or Tenericutes, including class Rhodothermia, Spirochaetia, Synergistia, Izimaplasma, or Mollicutes, including order Rhodothermales, Brachyspirales, Brevinematales, Leptospirales, Spirochaetales, Synergistales, Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, or Mycoplasmatales; phylum Thermodesulfobacteria, Thermotogae, Verrucomicrobia, or Zixibacteria, including class Thermodesulfobacteria, Thermotogae, Methylacidiphilae, Opitutae, Spartobacteria, or Verrucomicrobiae, including order Thermodesulfobacteriales, Kosmotogales, Mesoaciditogales, Petrotogales, Thermotogales, Methylacidiphilales, Opitutales, Puniceicoccales, Xiphinematobacter, Chthoniobacterales, Terrimicrobium, or Verrucomicrobiales.
In other embodiments, the gene for the enzyme or regulatory protein is derived from an archaeon. It is envisioned that an enzyme or regulatory protein derived from any archaeon now known or later discovered can be utilized in the present invention. For example, the archaeon can be from phylum Euryarchaeota, including class Archaeoglobi, Hadesarchaea, Halobacteria, Methanobacteria, Methanococci, Methanofastidiosa, Methanomicrobia, Methanopyri, Nanohaloarchaea, Theionarchaea, Thermococci, or Thermoplasmata, including order Archaeoglobales, Hadesarchaeales, Halobacteriales, Methanobacteriales, Methanococcales, Methanocellales, Methanomicrobiales, Methanophagales, Methanosarcinales, Methanopyrales, Thermococcales, Methanomassiliicoccales, Thermoplasmatales, or Nanoarchaeales; DPANN superphylum, including subphyla Aenigmarcheota, Altiarchaeota, Diapherotrites, Micrarchaeota, Nanoarchaeota, Pacearchaeota, Parvarchaeota, or Woesearchaeota; TACK superphylum, including subphylum Korarchaeota, Crenarchaeota, Aigarchaeota, Geoarchaeota, Thaumarchaeota, or Bathyarchaeota; Asgard superphylum including subphylium Odinarchaeota, Thorarchaeota, Lokiarchaeota, Helarchaeota, or Heimdallarchaeota.
In additional embodiments, the gene for the enzyme or regulatory protein is derived from a fungus. It is envisioned that an enzyme or regulatory protein derived from any fungus now known or later discovered can be utilized in the present invention. This includes but is not limited to the phyla Chytridiomycota, Basidiomycota, Ascomycota, Blastocladiomycota, Ascomycota, Microsporidia, Basidiomycota, Glomeromycota, Symbiomycota, and Neocallimastigomycota. For example, the fungus can be from the phylum Ascomycota, including classes and orders Pezizomycotina, Arthoniomycetes, Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Geoglossomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, Orbiliomycetes, Pezizomycetes, Sordariomycetes, Xylonomycetes, Lahmiales, Itchiclahmadion, Triblidiales, Saccharomycotina, Saccharomycetes, Taphrinomycotina, Archaeorhizomyces, Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes, Taphrinomycetes; phylum Basidiomycota including subphyla or classes Pucciniomycotina, Ustilaginomycotina, Wallemiomycetes, and Entorrhizomycetes; subphylum Agaricomycotina including classes Tremellomycetes, Dacrymycetes, and Agaricomycetes; phylum Symbiomycota, including class Entorrhizomycota; subphylum Ustilaginomycotina including classes Ustilaginomycetes and Exobasidiomycetes; phylum Glomeromycota including classes Archaeosporomycetes, Glomeromycetes, and Paraglomeromycetes; subphylum Pucciniomycotina including orders and classes: Pucciniomycotina, Cystobasidiomycetes, Agaricostilbomycetes, Microbotryomycetes, Atractiellomycetes, Classiculomycetes, Mixiomycetes, and Cryptomycocolacomycetes; subphylum incertae sedis Mucoromyceta including orders Calcarisporiellomycota and Mucoromycota; phylum Mortierellomyceta including class Mortierellomycota; subphylum incertae sedis Entomophthoromycotina including order Entomophthorales; phylum Zoopagomyceta including classes Basidiobolomycota, Entomophthoromycota, Kickxellomycota, and Zoopagomycotina; subphylum incertae sedis Mucoromycotina including orders Mucorales, Endogonales, and Mortierellales; phylum Neocallimastigomycota including class Neocallimastigomycetes; phylum Blastocladiomycota including classes Physodermatomycetes and Blastocladiomycetes; phylum Rozellomyceta including classes Rozellomycota and Microsporidia; phylum Aphelidiomyceta including class Aphelidiomycota; Chytridiomyceta including classes Chytridiomycetes and Monoblepharidomycetes; and phylum Oomycota including classes or orders Leptomitales, Myzocytiopsidales, Olpidiopsidales, Peronosporales, Pythiales, Rhipidiales, Salilagenidiales, Saprolegniales, Sclerosporales, Anisolpidiales, Lagenismatales, Rozellopsidales, and Haptoglossales.
In additional embodiments, the gene for the enzyme or regulatory protein is derived from the organism below. This includes but is not limited to: Acanthurus tractus, Aplysina aerophoba, Bos Taurus, Bufo bufo, Bufotes viridis, Chrysochloris asiatica, Fukomys damarensis, Homo sapiens, Rattus norvegicus, Rhinella marina, Rhinella spinulosa, Schistosoma mansoni, Xenopus laevis, Xenopus tropicalis, Acacia koa, Arabidopsis thaliana, Brassica oleracea, Citrus sinensis, Hordeum vulgare, Juglans cinereal, Lophophora williamsii, Nymphaea colorata, Oryza sativa, Ricinus communis, Solanum lycopersicum, Sorghum bicolor, Theobroma cacao, and Triticum aestivum.
In some embodiments, the nucleic acids are codon optimized to improve expression, e.g., using techniques as disclosed in U.S. Pat. No. 10,435,727. More specifically, optimized nucleotide sequences are generated based on a number of considerations: (1) For each amino acid of the recombinant polypeptide to be expressed, a codon (triplet of nucleotide bases) is selected based on the frequency of each codon in the Saccharomyces cerevisiae genome; the codon can be chosen to be the most frequent codon or can be selected probabilistically based on the frequencies of all possible codons. (2) In order to prevent DNA cleavage due to a restriction enzyme, certain restriction sites are removed by changing codons that cover those sites. (3) To prevent lowcomplexity regions, long repeats (sequences of any single base longer than five bases) are modified. (2) and (3) are performed recursively to ensure that codon modification does not lead to additional undesirable sequences. (4) A ribosome binding site is added to the N-terminus. (5) A stop codon is added. (6) A localization signal is removed or replaced.
In some of the above embodiments, the nucleic acid provided herein comprises the sequence of any one of SEQ ID NOs:1-289.
In various embodiments, the nucleic acids further comprise additional nucleic acids encoding amino acids that are not part of the included enzymes or regulatory proteins herein. In some of these embodiments, the additional sequences encode additional amino acids present when the nucleic acid is translated, encoding, for example, a cofolding peptide, as previously discussed, or an additional protein domain, with or without a linker sequence, creating a fusion protein. Other examples are localization sequences, i.e., signals directing the localization of the folded protein to a specific subcellular compartment or membrane. Additional nonlimiting examples are an affibody tag, a localization scaffold, a vacuolar localization tag, a secretion signal, and a 6xhis tag.
In some embodiments, the nucleic acid comprises additional nucleotide sequences that are not translated. Nonlimiting examples include promoters, terminators, barcodes, Kozak sequences, targeting sequences, and enhancer elements. Particularly useful here are promoters that are functional in yeast.
Expression of a gene encoding an enzyme or regulatory protein is determined by the promoter controlling the gene. In order for a gene to be expressed, a promoter must be present within 1,000 nucleotides upstream of the gene. A gene is generally cloned under the control of a desired promoter. The promoter regulates the amount of enzyme expressed in the cell and also the timing of expression, or expression in response to external factors such as sugar source.
Any promoter now known or later discovered can be utilized to drive the expression of the enzymes and regulatory proteins described herein. See e.g. http://parts.igem.org/Yeast for a listing of various yeast promoters. Exemplary promoters listed in Table 3 below drive strong expression, constant gene expression, medium or weak gene expression, or inducible gene expression. Inducible or repressible gene expression is dependent on the presence or absence of a certain molecule. For example, the GAL1, GAL7, and GAL10 promoters are activated by the presence of the sugar galactose and repressed by the presence of the sugar glucose. The HO promoter is active and drives gene expression only in the presence of the alpha factor peptide. The HXT1 promoter is activated by the presence of glucose while the ADH2 promoter is repressed by the presence of glucose.
In various embodiments, the nucleic acid is in an expression cassette, e.g., a yeast expression cassette. Any yeast expression cassette capable of expressing the enzyme in a yeast cell can be utilized.
Additional regulatory elements can also be present in the expression cassette, including restriction enzyme cleavage sites, antibiotic resistance genes, integration sites, auxotrophic selection markers, origins of replication, and degrons.
The expression cassette can be present in a vector that, when transformed into a host cell, either integrates into chromosomal DNA or remains episomal in the host cell. Such vectors are well-known in the art. See e.g. http://parts.igem.org/Yeast for a listing of various yeast vectors.
A nonlimiting example of a yeast vector is a yeast episomal plasmid (YEp) that contains the pBluescript II SK(+) phagemid backbone, an auxotrophic selectable marker, yeast and bacterial origins of replication and multiple cloning sites enabling gene cloning under a suitable promoter (see Table 3). Other exemplary vectors include pRS series plasmids.
The present invention is also directed to genetically engineered host cells that comprise the above-described nucleic acids. Such cells may be, e.g., any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules, intermediate molecules, or cannabinoid molecules. Host cells may also be any species of bacteria, including but not limited to Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or Lactobacillus.
In some embodiments, the genetically engineered host cell is a yeast cell, which may comprise any of the above-described expression cassettes, and capable of expressing the recombinant enzyme encoded therein.
Any yeast cell capable of being genetically engineered can be utilized in these embodiments. Nonlimiting examples of such yeast cells include species of Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, or Yarrowia.
These cells can achieve gene expression controlled by inducible promoter systems; natural or induced mutagenesis, recombination, and/or shuffling of genes, pathways, and whole cells performed sequentially or in cycles; overexpression and/or deletion of single or multiple genes and reducing or eliminating parasitic side pathways that reduce precursor concentration.
The host cells of the recombinant organism may also be engineered to produce any or all precursor molecules necessary for the biosynthesis of substituted indoles, tryptophans and tryptamines.
Construction of Saccharomyces cerevisiae strains expressing the enzymes and regulatory proteins provided herein is carried out via expression of a gene which encodes for the enzyme. The gene encoding the enzyme can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the gene encoding the enzyme may be inserted into the recombinant host genome. Integration may be achieved by a single or double cross-over insertion event of a plasmid, or by nuclease-based genome editing methods, as are known in the art e.g. CRISPR, TALEN and ZFR. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing. See, e.g., Green and Sambrook (2012).
In some embodiments, the recombinant microorganism expresses TPH, TrpM, and AADC, where the recombinant microorganism produces at least one hydroxy substituted tryptamine compound, e.g., bufotenine, 5-OH-NMT, or 5-OH-TMT (
In other embodiments, the recombinant microorganism expresses TPH, TrpM, AADC, and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT (
In additional embodiments, the recombinant microorganism expresses AADC, T5H or T5H-CPR and INMT, where the recombinant microorganism produces at least one hydroxy substituted tryptamine compound, e.g., bufotenine, 5-OH-NMT, or 5-OH-TMT (
In further embodiments, the recombinant microorganism expresses AADC, T5H or T5H-CPR, INMT, and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT (
In other embodiments, the recombinant microorganism expresses TrpM and TPH, where the recombinant microorganism produces at least one hydroxy substituted tryptophan compound, e.g., 5-HTP, 5-OH-NMTP, 5-OH-DMTP or 5-OH-TMTP.
In additional embodiments, the recombinant microorganism expresses TrpM, TPH and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptophan compound, e.g., 5-MeO-NMTP, 5-MeO-DMTP or 5-MeO-TMTP.
In further embodiments, the recombinant microorganism expresses INMT and T5H, where the recombinant microorganism produces at least one hydroxy substituted tryptamine compound, e.g., bufotenine, 5-OH-NMT, or 5-OH-TMT.
In other embodiments, the recombinant microorganism expresses INMT, T5H and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT.
In additional embodiments, the recombinant microorganism expresses INMT, where the recombinant microorganism produces at least one hydroxy substituted tryptophan compound, e.g., 5-OH-NMTP, 5-OH-DMTP or 5-OH-TMTP.
In further embodiments, the recombinant microorganism expresses INMT and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptophan compound, e.g., 5-MeO-NMTP, 5-MeO-DMTP or 5-MeO-TMTP.
In other embodiments, the recombinant microorganism expresses INMT and AADC, where the recombinant microorganism produces at least one hydroxy substituted tryptamine compound, e.g., bufotenine, 5-OH-NMT, or 5-OH-TMT.
In additional embodiments, the recombinant microorganism expresses INMT, AADC and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT.
In further embodiments, the recombinant microorganism expresses INMT, where the recombinant microorganism produces at least one hydroxy substituted tryptamine compound, e.g., bufotenine, 5-OH-NMT, or 5-OH-TMT.
In other embodiments, the recombinant microorganism expresses INMT and IOMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT.
In additional embodiments, the recombinant microorganism expresses INMT, where the recombinant microorganism produces at least one methoxy substituted tryptamine compound, e.g., 5-MeO-NMT, 5-MeO-DMT, or 5-MeO-TMT.
As depicted in
As depicted in
In accordance with the present invention, a recombinant host may also be modified to increase the accumulation of the methyl donor, SAMe, which is used by the recombinant TrpM and INMT enzymes to methylate indole and tryptamine molecules such as L-tryptophan and NMT. SAMe accumulation in the recombinant host cell may be increased by: (d) overexpressing enzymes to promote conversion of L-methionine to SAMe; (e) deleting off-pathway genes that encode for enzymes that deplete SAMe for unwanted side products; and (f) overexpressing a permease, which enables exogenous L-methionine to be fed to and transported into the cells.
The TrpM and INMT methyltransferase reactions consume one equivalent of adenosine triphosphate (ATP) and of SAMe. SAMe is a robust methyl donor synthesized from methionine and ATP via the L-methionine adenosyltransferase enzyme, Sam2. Sam2 may be overexpressed in a recombinant host to increase the conversion of L-methionine to SAMe. To support the increased pathway flux and generate more ATP, the adenylate kinase enzyme, Adk1, may also be overexpressed. To increase the uptake of exogenous L-methionine to feed into the SAMe pathway, recombinant Mup1, which is a methionine transporter, may be overexpressed.
SAMe is a precursor molecule for spermidine production and glycogen biosynthesis. To keep SAMe levels high in the pathways of the recombinant host and decrease off-pathway usage of SAMe, the SPE2 gene may be deleted in the recombinant host, thereby blocking the conversion of SAMe to spermidine. Glycogen biosynthesis consumes ATP, which is required for the conversion of L-methionine to SAMe. The gene encoding the enzyme Glc3 may be deleted in the recombinant host, thereby reducing production of glycogen, maintaining higher levels of ATP in the host cell, and increasing on-pathway flux of SAMe for methyltransferase activity.
The present invention is also directed to a non-naturally occurring enzyme or regulatory protein comprising an amino acid sequence encoded by any of the nucleic acids described above. In some embodiments, the amino acid sequence is 85%, 90%, 95%, 98%, or 100% identical to any one of SEQ ID NO:290-578. In these embodiments, the enzyme or regulatory protein can be isolated in vitro and used in vitro to provide enzyme activity. Alternatively, as discussed above, the enzyme can be expressed in a recombinant organism, e.g., a microorganism or a plant. In some of these embodiments, the recombinant microorganism is a bacterium, for example an E. coli. In other embodiments, the recombinant microorganism is a yeast cell, e.g., a species of Saccharomyces (for example S. cerevisiae), Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, Aspergillus or Yarrowia.
The systems and methods herein include: (i) growing modified recombinant host cells and thereby yielding a recombinant host organism; (ii) expressing engineered indole and tryptamine biosynthesis genes and enzymes in the recombinant host organism; (iii) producing or synthesizing substituted indoles and tryptamines in the recombinant host organism; (iv) fermenting the recombinant host organism; and (v) isolating the substituted indoles and tryptamines from the recombinant host organism. Endogenous pathways of the recombinant host can be modified by the systems and methods herein to produce high purity substituted indoles and tryptamines.
To produce the desired substituted indole, the nucleic acid encoding the enzymes and/or regulatory proteins are introduced into a host cell using standard cell (e.g., yeast) transformation techniques (Green and Sambrook, 2012). Cells are subjected to fermentation under conditions that activate the promoter controlling the synthesis of the enzyme and/or regulatory protein. The broth may be subsequently subjected to HPLC analysis to determine the presence or yield of the desired substituted indole, as in
In various embodiments, the host cells are provided with various feedstocks to drive production of the desired substituted indole, e.g., glucose, fructose, sucrose, ethanol, fatty acids, glycerol, molasses, corn steep liquor, dairy, fish waste, etc. for example as discussed in U.S. Pat. Application 17/078636.
In some embodiments, for recombinant enzyme purification, the gene encoding the enzyme and/or regulatory protein is cloned into an expression vector such as the pET expression vectors from Novagen, transformed into a protease deficient strain of E. coli such as BL21 and expressed by induction with IPTG. The protein of interest may be tagged with an affinity tag to facilitate purification, e.g. hexahistidine, GST, calmodulin, TAP, AP, CAT, HA, FLAG, MBP etc. Coexpression of a bacterial chaperone such as dnaK, GroES/GroEL or SecY may help facilitate protein folding. See Green and Sambrook (2012).
Any of the enzymes and/or regulatory proteins described above can also be produced in transgenic plants, using techniques known in the art (see, e.g., Keshavareddy et al., 2018). In these embodiments, the above-described nucleic acid encoding the enzyme and/or regulatory protein further comprises a promoter functional in a plant. In various embodiments, the nucleic acid is in a plant expression cassette. Any plant capable of being transformed with the nucleic acid can be utilized here. In some embodiments, the plant is a tobacco or cannabis.
Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
In the examples below, genetically engineered host cells may be any species of yeast herein, including but not limited to any species of Saccharomyces, Candida, Schizosaccharomyces, Yarrowia, etc., which have been genetically altered to produce precursor molecules, intermediate molecules, and psilocybin molecules. Additionally, genetically engineered host cells may be any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules such as L-tryptophan and substituted indole and tryptamine molecules. Some of the species of yeast herein for the recombinant host organism include but are not limited to: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica.
In these examples, the gene sequences from gene source organisms are codon optimized to improve expression using techniques disclosed in U.S. Pat.10,435,727.
DNA sequences are synthesized and cloned using techniques known in the art. Gene expression can be controlled by inducible or constitutive promoter systems using the appropriate expression vectors. Genes are transformed into an organism using standard yeast or fungus transformation methods to generate modified host strains (i.e., the recombinant host organism). The modified strains express genes for: (i) producing L-tryptophan, SAMe and precursor molecules to L-tryptophan and SAMe; (ii) increasing an output of L-tryptophan molecules and precursor molecules to L-tryptophan and SAMe molecules; (iii) increasing the import of exogenous L-tryptophan, L-methionine, SAMe and TMG into the host strain; and (iv) the genes for biosynthetic pathways that generate DMT, DMTP, bufotenine, 5-MeO-DMT and all intermediate indole and tryptamine compounds synthesized and described herein. In the presence or absence of exogenous L-tryptophan, L-methionine, SAMe, TMG, 5-HTP, melatonin, and serotonin, fermentations are run to determine if the cell will convert the fed precursors into tryptamine, serotonin, methylated versions of serotonin, melatonin, or methylated versions of melatonin. The L-tryptophan, SAMe, hydroxylation, decarboxylation, and methylation pathway genes herein can be integrated into the genome of the cell or maintained as an episomal plasmid. Samples are: (i) prepared and extracted using a combination of fermentation, dissolution, and purification steps; and (ii) analyzed by HPLC for the presence of directing molecules (e.g., SAMe and L-tryptophan), precursor molecules, intermediate molecules, and target molecules such as bufotenine and 5-MeO-DMT.
Using the systems and methods herein, the genes which can be expressed to encode for a corresponding enzyme or other type of proteins include but are not limited to: ENO2, TAL1, ARO1, ADK1, MUP1, SAM2, MHT1, SAM4, SAM3, TAT2, AADC, TRPM, INMT, TPH, genes encoding enzymes for the BH4 biosynthesis pathway, genes encoding enzymes for the BH4 regeneration pathway, T5H, IOMT, caffOMT, NAT, DAC, T4H, PsiK, oxidase, phosphatase, TrpHalo, DMAT, T4H-CPR, T5H-CPR, TrpS, and ATMT. For example, the AADC gene is expressed, or overexpressed, to encode for the aromatic amino decarboxylase enzyme; the TRPM gene is expressed to encode for the TrpM enzyme; and so forth. Gene sequences can be determined using standard techniques known in the art, e.g., the techniques disclosed in U.S. Pat. 10,671,632.
The construction of Saccharomyces cerevisiae platform strains with elevated metabolic flux towards L-tryptophan is carried out by overexpressing five optimized enzymes in or upstream of the shikimate pathway to make the aromatic compound intermediate, chorismate, and one optimized enzyme in the tryptophan pathway to make L-tryptophan. Further, tryptophan levels in the cell are enhanced with the expression of TAT2, a tryptophan importer, and L-tryptophan supplementation in the media up to 1% mass to volume. Finally, five enzymes are deleted in the cell to decrease off-pathway consumption of the L-tryptophan. The genetically modified host described herein can be the same host used for production of psilocybin and DMT as both production pathways use the precursor, L-tryptophan. A specific description of the strain with elevated L-tryptophan is disclosed in U.S. Pat. Publication 2021/0147888.
Construction of Saccharomyces cerevisiae platform strains with elevated SAMe production is carried out via expression of SAM2, a SAMe synthetase gene. The SAM2 gene is cloned from Saccharomyces cerevisiae using techniques known in the art. The gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the SAM2 gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of Saccharomyces cerevisiae platform strains with elevated SAMe production via expression of the ADK1, adenylate kinase gene. The ADK1 gene is cloned from Saccharomyces cerevisiae using techniques known in the art. The gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the ADK1 gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Further SAM accumulation for methyl donor availability is achieved herein by engineering the homocysteine to methionine side of the methylation pathway. SAH is generated after methylation of serotonin and other intermediates to produce bufotenine and other compounds described herein. SAH is recycled back to methionine after methyl donation by TMG (trimethylglycine) or betaine. TMG is fed to the cells up to 1% (v/v) in the growth media. Two Saccharomyces cerevisiae genes, MHT1 and SAM4 encode the enzymes, Mht1 and Sam4, that are responsible for homocysteine re-methylation using TMG as a methyl donor. MHT1 and SAM4 are overexpressed from a high copy vector with a strong promoter.
Construction of Saccharomyces cerevisiae platform strains with elevated SAMe production is carried out via expression of MUP1, the methionine permease gene. The MUP1 gene is cloned from Saccharomyces cerevisiae using techniques known in the art. The gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the MUP1 gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Herein we describe a strategy to increase the SAM accumulation by increasing transport of exogenous SAM into the cell. SAM levels are increased by overexpressing the gene, SAM3. SAM3 encodes for the Sam3 protein, the predominant Saccharomyces cerevisiae transporter that is responsible for SAM import. SAM3 is expressed from a high-copy vector with a strong promoter and media is supplemented with 0.5 - 1.0 mM SAMe.
Construction of Saccharomyces cerevisiae platform strains with elevated metabolic flux towards SAMe is carried out via deletion of SPE2 to reduce SAMe decarboxylation. Deletion of SPE2 is performed by replacement of the SPE2 gene with the URA3 cassette in the recombinant host. The SPE2 URA3 knockout fragment, carrying the marker cassette, URA3, and homologous sequence to the targeted gene, SPE2, can be generated by bipartite PCR amplification. The PCR product is transformed into a recombinant host and transformants can be selected on synthetic URA drop-out media. Further verification of the modification in said strain can be carried out by genome sequencing, then analyzed by the techniques disclosed in U.S. Pat. 10,671,632.
Saccharomyces cerevisiae platform strains are constructed with elevated metabolic flux towards SAMe via deletion of GLC3 to reduce ATP consumption. Deletion of GLC3 is performed by replacement of the GLC3 gene with the URA3 cassette in the recombinant host. The GLC3 URA3 knockout fragment, carrying the marker cassette, URA3, and homologous sequence to the targeted gene, GLC3, can be generated by bipartite PCR amplification. The PCR product is transformed into a recombinant host and transformants can be selected on synthetic URA drop-out media. Further verification of the modification in said strain can be carried out by genome sequencing and analyzed by the techniques disclosed in U.S. Pat. 10,671,632.
Saccharomyces cerevisiae platform strains with accumulation of tryptophan are generated by deletion of BNA2. Bna2 is an enzyme necessary for de novo NAD+ production from tryptophan. Deletion of BNA2 is performed by replacement of the BNA2 gene with the URA3 cassette in the recombinant host. The BNA2 URA3 knockout fragment, carrying the marker cassette, URA3, and homologous sequence to the targeted gene, BNA2, can be generated by bipartite PCR amplification. The PCR product is transformed into a recombinant host and transformants can be selected on synthetic URA drop-out media. Further verification of the modification in said strain can be carried out by genome sequencing and analyzed by the techniques disclosed in U.S. Pat. 10,671,632.
Construction of Saccharomyces cerevisiae NMTP, DMTP, and TMTP production strains is carried out via expression of the TrpM methyltransferase gene. The optimized TrpM gene is synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized TrpM gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of Saccharomyces cerevisiae tryptamine production strains is carried out via expression of the AADC gene which encodes the enzyme that converts L-tryptophan to tryptamine. AACD also encodes the enzyme that converts 5HTP to serotonin. This specific conversion may be carried out by the same enzyme encoded by the AADC gene that converts L-tryptophan to tryptamine. It also may be carried out by the gene product of a novel AADC described herein. The optimized AADC gene is synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized AADC gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of the Saccharomyces cerevisiae 5-HTP production strains is carried out via expression of the gene that encodes tryptophan hydroxylase. 5-HTP is a precursor compound for production of serotonin and variants described herein. Tryptophan hydroxylase activity is dependent on the availability of the BH4 cofactor. The optimized TPH, BH4 biosynthesis and BH4 regeneration genes are synthesized using DNA synthesis techniques known in the art. The optimized genes can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized TPH, BH4 biosynthesis and BH4 regeneration genes are inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of the Saccharomyces cerevisiae serotonin production strains is carried out via expression of the gene that encodes tryptamine 5-hydroxylase (T5H). 5-HT or serotonin is a precursor compound for production of bufotenine and variants described herein. T5H activity is dependent on the availability of the intermediate indole compound, tryptamine, production of which is disclosed in U.S. Pat. Publication 2021/0147888 and further described herein.
T5H, as a cytochrome p450-containing monooxygenase, is also dependent on the cytochrome p450 reductase enzyme (CPR) for full activity. The CPR facilitates electron transfer from the NAD(P)H. The optimized T5H and CPR genes are synthesized using DNA synthesis techniques known in the art. The optimized genes can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized T5H and CPR genes are inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of Saccharomyces cerevisiae DMT production strains is carried out via expression of the INMT gene which encodes the enzyme that methylates tryptamine to DMT. INMT also encodes the enzyme that converts serotonin to bufotenine. Finally, INMT encodes the enzyme that converts 5-MeO-tryptamine to 5-Meo-DMT. These unique conversions may be carried out by the same enzyme encoded by the INMT gene that converts tryptamine to DMT. It also may be carried out by the gene product of a novel INMT described herein. The optimized INMT gene is synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized INMT gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of Saccharomyces cerevisiae 5-MeO-DMT production strains is carried out via expression of the IOMT gene which encodes the enzyme that methylates the 5-OH in bufotenine, an intermediate derived from the INMT conversion of serotonin, described herein. The IOMT gene also encodes for the enzyme that converts serotonin to 5-MeO-tryptamine in the first intermediate to make melatonin. The IOMT enzyme also methylates the 5-OH of N-acetyl-serotonin to generate melatonin as an intermediate to make 5-MeO-tryptamine and further, 5-MeO-DMT. Alternatively, the enzyme that converts serotonin to 5-MeO-tryptamine can be carried out with a CaffOMT enzyme, an enzyme shared with the phenylpropanoid biosynthesis pathway. This same CaffOMT enzyme can also methylate N-acetyl-serotonin to generate melatonin. The optimized IOMT or CaffOMT gene is synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized IOMT or CaffOMT gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Construction of Saccharomyces cerevisiae 5-MeO-DMT production strains can alternatively be carried out via expression of the two more enzymes, NAT and NAD. NAT acetylation of serotonin produces the intermediate N-acetyl-serotonin or NAS. NAS is converted to melatonin with the IOMT (or CaffOMT) enzyme described herein. DAC deacetylases melatonin to 5-MeO-tryptamine which is converted to 5-MeO-DMT via the INMT enzyme described herein. The optimized NAT and DAC genes are synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized NAT and DAC genes are inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene(s) can be screened by rescue of auxotrophy and genome sequencing.
Serotonin is the precursor molecule for both bufotenine and 5-MeO-DMT. Construction of a Saccharomyces cerevisiae serotonin strain is carried out by expression of AADC and TPH or AADC and T5H genes described herein for the enzymatic conversion of L-tryptophan to serotonin. Exogenous serotonin is also fed to the strains to increase precursor levels at concentrations of 0.5 mM to 2 mM. Exogenous 5-HTP with expression of the AADC gene is fed to the cells as a mechanism to increase the serotonin precursor.
In order to accumulate serotonin in the cell and prevent off pathway conversion of serotonin to unwanted products, the endogenous Saccharomyces cerevisiae gene, PAA1 (YDR071C) is deleted. PAA1 is a polyamine acetyltransferase that would acetylate serotonin and use up valuable acetyl-CoA.
Modified host cells that yield substituted indoles and tryptamine compounds, such as a bufotenine-producing strain herein, express engineered bufotenine biosynthesis genes and enzymes. More specifically, the bufotenine-producing strain herein is grown in a minimal, complete culture media containing yeast nitrogen base, amino acids, vitamins, ammonium sulfate, and a carbon source of glucose and galactose. The recombinant host cells are grown in 24-well plates or shake flasks in a volume range of 2 mL to 100 mL of media starting from an inoculation density of OD600nm=1. Exogenous serotonin, melatonin, tryptamine, 5HTP, SAMe and TMG can be added to media to supplement the precursor pool for final compound production or support methyl donor accumulation.
Herein we describe a strategy for 5-methoxy-tryptamine (5-MT) production by recombinant expression and secretion of the melatonin deacetylase, DAC in BL21(DE3)pLysS E. coli. The DAC enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG) 2) an N-terminal secretory signal peptide [MKKTAIAIAVALAGFATVAQA (SEQ ID NO:286,575)] and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the NAD-expression vector are grown in M9 minimal media with 1% glucose for 18h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG. After a 3 h induction at 18° C. and 300 rpm shaking, melatonin is added to the media at a final concentration of 1-2 mM. Cells are grown at room temperature for 48 h, shaking at 300 rpm. Media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compound, 5-methoxy-tryptamine (5-MT) by recombinant expression and secretion of the melatonin deacetylase, DAC in Saccharomyces cerevisiae. The DAC enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the sugar, galactose 2) an N-terminal alpha factor secretion leader sequence, [MEGVSLEKREAEA (SEQ ID NO:574] and 3) c-terminal fusion to a HIS tag for purification. Saccharomyces cerevisiae cells harboring the DAC-expression vector are grown in CM minimal media with 2% glucose for 18 h at 30° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh CM minimal media with 2% galactose. After 24 h of induction at 30° C. and 300 rpm shaking, melatonin is added to the media at a final concentration of 1-2 mM. Cells are grown at 30° C. and 300 rpm shaking for 48 h. Media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compound, 5-methoxy-tryptamine (5-MT) by recombinant expression and secretion of the melatonin deacetylase (DAC) in Komagataella phaffii (Pichia pastoris). The DAC enzyme is cloned into a high-copy vector with key features that allow 1) induction by methanol with the AOX1 promoter and 2) a secretion signal consisting of the α-factor pro region. K. phaffii cells harboring the DAC enzyme are inoculated into 5 mL of YPD in a 15-mL culture tube. After a day of incubation at 30° C. with shaking at 220 rpm, an aliquot of the culture is diluted to an OD600=0.2 in 5 mL of BMG (buffered minimal glycerol media) in a 15-mL culture tube. This tube is incubated under the same conditions as before. The following day, the culture is centrifuged at 3000 rpm (2000×g) for 5 min and resuspended in 25 mL BMM (buffered minimal methanol media) to attain an OD600 = 1.0. 25 mL of this culture is placed in a 250-mL baffled flask, and during this induction phase, the cells are incubated at 25° C. with shaking at 150 rpm to reduce loss of methanol. After 1 day of induction, an additional dose of 125 µL methanol is added (yielding a final concentration of 0.5%), melatonin is added to the media at a final concentration of 1-2 mM, and the incubation is continued for another day. After 48 h of induction, media is collected at at 24 h and 48 h and analyzed by HPLC as described herein.
Herein we describe a strategy for 5-MeO-NMT, 5-MeO-DMT, and 5-MeO-TMT production by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in in BL21(DE3)pLysS E. coli. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG) 2) an N-terminal secretory signal peptide [MKKTAIAIAVALAGFATVAQA (SEQ ID NO:574)] and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the INMT-expression vector are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG. After a 3 h induction at 18° C. and 300 rpm shaking, melatonin is added to the media at a final concentration of 1-2 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at room temperature for 48 h, shaking at 300 rpm. Media is collected at 24h and 48h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, 5-MeO-NMT, 5-MeO-DMT, and 5-MeO-TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Saccharomyces cerevisiae. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the sugar, galactose 2) an N-terminal alpha factor secretion leader sequence, [MEGVSLEKREAEA (SEQ ID NO:574)] and 3) c-terminal fusion to a HIS tag for purification. Saccharomyces cerevisiae cells harboring the INMT-expression vector are grown in CM minimal media with 2% glucose for 18 h at 30° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh CM minimal media with 2% galactose. After 24 h of induction at 30° C. and 300 rpm shaking, melatonin is added to the media at a final concentration of 1-2 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at 30° C. and 300 rpm shaking for 48h. Media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, 5-MeO-NMT, 5-MeO-DMT, and 5-MeO-TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Komagataella phaffii. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) induction by methanol with the AOX1 promoter and 2) a secretion signal consisting of the α-factor pro region. K. phaffii cells harboring the DAC enzyme are inoculated into 5 mL of YPD in a 15-mL culture tube. After a day of incubation at 30° C. with shaking at 220 rpm, an aliquot of the culture is diluted to an OD600=0.2 in 5 mL of BMG (buffered minimal glycerol media) in a 15-mL culture tube. This tube is incubated under the same conditions as before. The following day, the culture is centrifuged at 3000 rpm (2000×g) for 5 min and resuspended in 25 mL BMM (buffered minimal methanol media) to attain an OD600 = 1.0. 25 mL of this culture is placed in a 250-mL baffled flask, and during this induction phase, the cells are incubated at 25° C. with shaking at 150 rpm to reduce loss of methanol. After 1 day of induction, an additional dose of 125 µL methanol is added (yielding a final concentration of 0.5%), melatonin is added to the media at a final concentration of 1-2 mM, SAMe is added to the media at a final concentration of 1-2 mM, and the incubation is continued for another day. After 48 h of induction, media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Herein we describe a strategy for NMT, DMT, and TMT production by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in BL21(DE3)pLysS E. coli. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG) 2) an N-terminal secretory signal peptide [MKKTAIAIAVALAGFATVAQA (SEQ ID NO:574)] and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the INMT-expression vector are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG. After a 3h induction at 18° C. and 300 rpm shaking, tryptamine is added to the media at a final concentration of 1-2 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at room temperature for 48 h, shaking at 300 rpm. Media is collected at 24 h and 48h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, NMT, DMT, and TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Saccharomyces cerevisiae. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the sugar, galactose 2) an N-terminal alpha factor secretion leader sequence, [MEGVSLEKREAEA (SEQ ID NO:574)] and 3) c-terminal fusion to a HIS tag for purification. Saccharomyces cerevisiae cells harboring the INMT-expression vector are grown in CM minimal media with 2% glucose for 18 h at 30° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh CM minimal media with 2% galactose. After 24h of induction at 30° C. and 300 rpm shaking, tryptamine is added to the media at a final concentration of 1-2 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at 30° C. and 300 rpm shaking for 48 h. Media is collected at 24h and 48h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, NMT, DMT, and TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Komagataella phaffii. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) induction by methanol with the AOX1 promoter and 2) a secretion signal consisting of the α-factor pro region. K. phaffii cells harboring the DAC enzyme are inoculated into 5 mL of YPD in a 15-mL culture tube. After a day of incubation at 30° C. with shaking at 220 rpm, an aliquot of the culture is diluted to an OD600=0.2 in 5 mL of BMG (buffered minimal glycerol media) in a 15-mL culture tube. This tube is incubated under the same conditions as before. The following day, the culture is centrifuged at 3000 rpm (2000×g) for 5 min and resuspended in 25 mL BMM (buffered minimal methanol media) to attain an OD600 = 1.0. 25 mL of this culture is placed in a 250-mL baffled flask, and during this induction phase, the cells are incubated at 25° C. with shaking at 150 rpm to reduce loss of methanol. After 1 day of induction, an additional dose of 125 µL methanol is added (yielding a final concentration of 0.5%), tryptamine is added to the media at a final concentration of 1-2 mM, SAMe is added to the media at a final concentration of 1-2 mM, and the incubation is continued for another day. After 48 h of induction, media is collected at at 24 h and 48 h and analyzed by HPLC as described herein.
Herein we describe a strategy for 5-OH-NMT, 5-OH-DMT, and 5-OH-TMT production by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in BL21(DE3)pLysS E. coli. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG) 2) an N-terminal secretory signal peptide [MKKTAIAIAVALAGFATVAQA (SEQ ID NO:574)] and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the INMT-expression vector are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG. After a 3 h induction at 18° C. and 300 rpm shaking, serotonin is added to the media at a final concentration of 5 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at room temperature for 48 h, shaking at 300 rpm. Media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, 5-OH-NMT, 5-OH-DMT, and 5-OH-TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Saccharomyces cerevisiae. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the sugar, galactose 2) an N-terminal alpha factor secretion leader sequence, [MEGVSLEKREAEA (SEQ ID NO:574)] and 3) c-terminal fusion to a HIS tag for purification. Saccharomyces cerevisiae cells harboring the INMT-expression vector are grown in CM minimal media with 2% glucose for 18h at 30° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh CM minimal media with 2% galactose. After 24 h of induction at 30° C. and 300 rpm shaking, serotonin is added to the media at a final concentration of 5 mM and SAMe is added to the media at a final concentration of 1-2 mM. Cells are grown at 30° C. and 300 rpm shaking for 48 h. Media is collected at 24 h and 48 h and analyzed by HPLC as described herein.
Alternatively, we describe a strategy for production of the compounds, 5-OH-NMT, 5-OH-DMT, and 5-OH-TMT by recombinant expression and secretion of the indolethylamine-N-methyltransferase (INMT) in Komagataella phaffii. The INMT enzyme is cloned into a high-copy vector with key features that allow 1) induction by methanol with the AOX1 promoter and 2) a secretion signal consisting of the α-factor pro region. K. phaffii cells harboring the DAC enzyme are inoculated into 5 mL of YPD in a 15-mL culture tube. After a day of incubation at 30° C. with shaking at 220 rpm, an aliquot of the culture is diluted to an OD600=0.2 in 5 mL of BMG (buffered minimal glycerol media) in a 15-mL culture tube. This tube is incubated under the same conditions as before. The following day, the culture is centrifuged at 3000 rpm (2000×g) for 5 min and resuspended in 25 mL BMM (buffered minimal methanol media) to attain an OD600 = 1.0. 25 mL of this culture is placed in a 250-mL baffled flask, and during this induction phase, the cells are incubated at 25° C. with shaking at 150 rpm to reduce loss of methanol. After 1 day of induction, an additional dose of 125 µl methanol is added (yielding a final concentration of 0.5%), serotonin is added to the media at a final concentration of 5 mM, SAMe is added to the media at a final concentration of 1-2 mM and the incubation is continued for another day. After 48 h of induction, media is collected at 24 h and 48 h and analyzed by HPLC as herein.
The INMT enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG) 2) an N-terminal secretory signal peptide [MKKTAIAIAVALAGFATVAQA (SEQ ID NO:574] and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the INMT-expression vector are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG and grown for 48 h.
The supernatant containing the recombinant proteins is equilibrated in binding buffer (50 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 10% glycerol, 10 mM 2-mercaptoethanol, 1 mM PMSF, Complete EDTA-free (1 tablet/100 ml), 20 mM 1-phenyl-2- thio-urea; pH 7.4) and centrifuged at 2,500 g for 5 min to remove insoluble matter. Then the supernatant is filtered through a 0.45 µm filter (Millipore, MA, USA) and applied onto a HisTrap HP column (GE Healthcare Bioscience). The recombinant proteins are eluted with a step gradient of imidazole (concentrations of 5, 20, 40 and 300 mM). Fractions are analyzed by SDS-PAGE and stored at -80° C. before use.
Purified INMT protein is resuspended in activity buffer [100 mM sodium phosphate buffer, pH 6.55, PMSF (1 mM), EDTA-free protease inhibitor] cocktail at working concentration (Roche, Meylan, France) for use in in vitro assays. 0.1 mg/mL of INMT protein is added to a tube with a final volume of 600 uL per sample and added to 100 mM sodium phosphate buffer (pH 7.5), 2 mM tryptamine, serotonin, or melatonin, 2 mM S-adenosylmethionine, and 5 mM MgCl2.
Alternatively, 0.1 mg/mL BSA protein-equivalent of INMT lysate is used in the same reaction. INMT lysate is derived from E. coli cells harboring the INMT-expression vector. They are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600 =1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG and grown for 48 h. Cell pellets are resuspended in 100 mM sodium phosphate buffer at pH 7.5 and lysed using sonication. After lysis, samples are pelleted by centrifugation (16,000 g, 4° C., 20 min) and supernatant containing INMT is harvested.
Modified host cells that yield substituted indoles and tryptamine compounds, such as the DMTP-producing strain herein, express engineered DMTP biosynthesis genes and enzymes. More specifically, the DMTP-producing strain herein is grown in a minimal, complete culture media containing yeast nitrogen base, amino acids, vitamins, ammonium sulfate, and a carbon source of glucose and galactose. The recombinant host cells are grown in 24-well plates or shake flasks in a volume range of 2 mL to 100 mL of media starting from an inoculation density of OD600nm=1. Exogenous L-tryptophan and L-methionine up to 1% can be added to media to supplement the precursor pool for DMTP production. Exogenous L-tryptophan can be taken up by strains expressing the TAT2 L-tryptophan importer protein. Exogenous L-methionine can be taken up by strains expressing the MUP1 L-methionine permease protein. The strains herein can be harvested during a fermentation period ranging from 12 hours onward from the start of pathway enzyme induction.
To identify fermentation-derived tryptamine, DMTP, NMT, DMT, and all other products of a recombinant host expressing an engineered biosynthetic pathway for substituted indoles (see
As another example,
In some instances, it may be preferable, for reasons of either cost or product quality, to utilize recombinant host pathways to accomplish the first part of a substituted indole synthesis and complete the remaining steps synthetically. The tryptamine, as obtained from the recombinant organism, is of a particular grade such that methylations with robust methylating agents selectively leads to mono- or di-methylation. One of ordinary skill in the art would appreciate this as improvement when a primary amine subjected to robust methylating agents as a mixture of alkylation products are not obtained, while obviating the need for tedious chromatography.
One example would be the production of tryptamine via fermentation of a recombinant host organism, followed by N,N-methylation via methylation chemistry to yield DMT. In one embodiment, the reaction of tryptamine would proceed with a 30-fold molar excess of dimethyl carbonate (DMC) under an inert atmosphere, utilizing a Y-type zeolite catalyst (see
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U.S. Pat. No. 10,671,632.
U.S. Pat. Application 16/553103.
U.S. Pat. Application 16/553120.
U.S. Pat. Application 17/068636.
U.S. Provisional Pat. Application 63/035692.
U.S. Provisional Pat. Application 63/053539.
U.S. Provisional Pat. Application 63/141486.
U.S. Provisional Pat. Application 63/164126.
U.S. Pat.Application Publication 2020/0063170.
U.S. Pat. Application Publication 2020/0063171.
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In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification, including but not limited to patent publications and non-patent literature, and references cited therein, are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure.
That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements can optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.
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This application is a 371 National Stage entry of PCT Application Serial No. PCT/US21/36031, filed Jun. 4, 2021, which claims the benefit of U.S. Provisional Application No. 63/035,692, filed Jun. 6, 2020, and incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/036031 | 6/4/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63035692 | Jun 2020 | US |
