Patent Application
20160340704
N.A.
The present invention relates to a method of making a benzylisoquinoline alkaloid (BIA) metabolite. More specifically, the present invention is concerned with an improved method of making a (BIA) metabolite (e.g., reticuline) in a recombinant host cell.
Plant secondary metabolites are a rich source of bioactive molecules. The chemical diversity of these compounds derives from enzymes that have diversified to perform an array of stereo- and enantioselective modifications. Coupling these reactions by chemical synthesis to reach high yields can be difficult if not impossible. While plants remain the main source of these valuable natural compounds, the use of microbial platform has emerged as an attractive alternative.
Many pharmaceutical drugs are isolated directly from plants or are semisynthetic derivatives of natural products1,2. Information from New Drug Applications and clinical trials is evidence that the pharmaceutical industry continues to use natural products as a source of new drug leads3. However, the pipeline of drug discovery is difficult to sustain, due to technical challenges in isolating new compounds with diverse structures and complex chemistries in sufficient quantities for screening4.
Next generation DNA sequencing technology has provided rapid access to the genetic diversity underpinning the immense biosynthetic capacity of plants and microbes5,6. Although Saccharomyces cerevisiae has traditionally been used for the biosynthesis of simple molecules derived from central metabolism7, advances in recombinant DNA technology streamlining the cloning of large DNA sequences makes this microbe an attractive platform for functional characterization of enzymes as well as the reconstitution of complex metabolic pathways8,9. When combined with genetic information on an ever-increasing number of species, microbial hosts provide new opportunities for the discovery and production of diverse and complex natural products. A recent example demonstrating the power of these combined technologies is the high-level production of the artemisinin antimalarial drug precursor artemisinic acid in yeast10.
Benzylisoquinoline alkaloids (BIAs) are a diverse class of plant secondary metabolites including such pharmaceuticals as the antitussive codeine and its derivatives, the analgesic morphine and its derivatives, the antitussive and anticancer drug noscapine11 and the antibacterial and potential antineoplastic drugs berberine and sanguinarine12. Their complex molecular backbone and the presence of multiple stereocenters make the complete chemical synthesis of most BIAs commercially unfeasible13-15. Consequentially, plant extraction is the only commercial source of BIAs, which limits the diversity of BIA structures available for drug discovery due to their low abundance16. The pharmaceutical value of BIAs and advances made in the elucidation of their biosynthesis in plants have made these compounds high-value candidates for production using microbial hosts17.
Despite their structural diversity, BIAs share many common biosynthetic steps and intermediates (
Sanguinarine is a BIA with recognized antimicrobial activities and potential as an antineoplastic drug23,24. The last steps in sanguinarine biosynthesis were recently elucidated, laying the groundwork for complete synthesis of this molecule in a heterologous host25-27. In the present invention, the applicants combine gene discovery with multi-gene heterologous expression in S. cerevisiae to reconstitute a 10-gene BIA pathway for the biosynthesis of dihydrosanguinarine and sanguinarine from the commercial precursor (R,S)-norlaudanosoline. The applicants also demonstrate the activity of tetrahydroprotoberberine cis-N-methyltransferase (TNMT) towards scoulerine and cheilanthifoline and synthesize N-methylscoulerine and N-methylcheilanthifoline in yeast and show that the pathway for reticuline synthesis from norlaudanosoline is enantioselective for (S)-reticuline. The applicants also identify novel Ring A and Ring B closers able to convert scoulerine, nandinine and/or cheilanthifoline into BIA metabolites. The reconstitution of a complex pathway for BIA synthesis in S. cerevisiae represents an important advance towards the production of a broader class of alkaloids in a microbial host.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
Next-generation sequencing technology and the accelerated discovery of genes combined with advances in synthetic biology are opening up new opportunities for the reconstitution of plant natural product biosynthetic pathways in microbes46. In the last five years, there has been a growing trend in the complexity and diversity of the chemical structures achieved by these pathways. For example microbes have been engineered for the synthesis of terpenoids such as artemisinin acid10 and taxa-di-ene47, BIAs such as magnoflorine20 and canadine22 and glucosinates such as indolylglucosinolate48. Cytochrome P450s are required for the synthesis of a wide range of plant natural products and the efficient recombinant expression of this class of enzyme can be difficult. Of all pathways reconstituted in microbes thus far, only those of the mammalian hydrocortisone and plant dihydrosanguinarine pathways require the heterologous expression of four cytochrome P450s. The dihydrosanguinarine pathway described herein represents the most complex plant alkaloid biosynthetic pathway ever reconstituted in yeast and provides a glimpse into the potential of engineering microbes for the synthesis of ever more complex plant natural products.
In an aspect, the applicants reconstituted a multiple-gene plant pathway in Saccharomyces cerevisiae that allows for the production of various metabolites (e.g., reticuline, stylopine and dihydrosanguinarine and its oxidized derivative sanguinarine) from (R,S)-norlaudanosoline. Synthesis of dihydrosanguinarine also yields the side-products N-methylscoulerine and N-methylcheilanthifoline, the latter of which had not been detected in plants before then. The present invention provides the longest reconstituted alkaloid pathway ever assembled in yeast and demonstrates the feasibility of the production of high-value alkaloids in microbial systems.
More specifically, in accordance with an aspect of the present invention, there is provided a method of preparing a benzylisoquinoline alkaloid (BIA) metabolite comprising: (a) culturing a host cell under conditions suitable for protein production, including a pH of between about 7 and about 10 said host cell comprising: a. a first heterologous coding sequence encoding a first enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; b. a second heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; c. a third heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; (b) adding (R,S)-norlaudanosoline to the cell culture; and (c) recovering the metabolite from the cell culture.
More specifically, in accordance with an aspect of the present invention, there is provided a method of preparing a benzylisoquinoline alkaloid (BIA) metabolite comprising: (a) culturing a host cell under conditions suitable for protein production, said host cell comprising: a. a first heterologous coding sequence encoding a first enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; b. a second heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; c. a third heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; (b) adding (R,S)-norlaudanosoline to the cell culture; and (c) recovering the metabolite from the cell culture. In a specific embodiment, the conditions include a first pH (i.e. first fermentation at a pH of) between about 7 and about 10. This may be useful for a synthesis comprising the full or a part of the sequence of enzymes of blocks 1 and 2 (see e.g.,
in accordance with another aspect of the present invention, there is provided a method of preparing a benzylisoquinoline alkaloid (BIA) metabolite comprising: (a) culturing a host cell under conditions suitable for protein production, including a first fermentation at a pH of between about 7 and about 10, and, optionnaly followed by a second fermentation at a pH between about 3 and about 6, said host cell comprising: a. a first heterologous coding sequence encoding a first enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; b. a second heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; c. a third heterologous coding sequence encoding a second enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite; (b) adding (R,S)-norlaudanosoline to the cell culture; and (c) recovering the metabolite from the cell culture.
In a specific embodiment of these methods, the host cell is a yeast cell. In another specific embodiment, the yeast is Saccharomyces. In another specific embodiment, the Sacharomyces is Sacharomyces cerevisiae.
In a specific embodiment, the metabolite is (S)-reticuline. In another specific embodiment, the first enzyme is 6-O-methyltransferase (6OMT); the second enzyme is coclaurine N-methyltransferase (CNMT); and/or the third enzyme is 4′-O-methyltransferase 2 (4′OMT2).
In another specific embodiment, the 6OMT is as set forth in any one of the sequences as depicted in
In another specific embodiment, 6OMT is from Papaver somniferum; CNMT is from Papaver somniferum; and/or 4′OMT2 is from Papaver somniferum.
In another specific embodiment, Ps6OMT is as set forth in SEQ ID NO: 34 (
In another specific embodiment, the cell further comprises a fourth heterologous coding sequence encoding a fourth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is (S)-scoulerine. In another specific embodiment, the fourth enzyme is berberine bridge enzyme (BBE). In another specific embodiment, the BBE is as set forth in any one of the sequences as depicted in
In another specific embodiment, the cell further comprises a fifth heterologous coding sequence encoding a fifth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is nandinine or (S)-cheilanthifoline. In another specific embodiment, the fifth enzyme is a Ring B closer able to transform scoulerine into cheilanthifoline. In another specific embodiment, the Ring B closer is further able to transform nandinine in stylopine. In another specific embodiment, the Ring B closer is as set forth in any one of the sequences depicted in
In another specific embodiment, the cell further comprises a sixth heterologous coding sequence encoding a sixth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is (S)-stylopine. In another specific embodiment, the sixth enzyme is a Ring A closer able to transform cheilanthifoline in (S)-stylopine. In another specific embodiment, the Ring A closer is further able to transform scoulerine in nandinine. In another specific embodiment, the Ring A closer is as set forth in any one of the sequences depicted in
In another specific embodiment, the method comprises the second fermentation and wherein the cell further comprises a seventh heterologous coding sequence encoding a seventh enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is (S)—N-cis-methylstylopine. In another specific embodiment, the seventh enzyme is tetrahydroprotoberberine cis-N-methyltransferase (TNMT). In another specific embodiment, the TNMT is as set forth in any one of the sequences as depicted in
In another specific embodiment, the cell further comprises a eight heterologous coding sequence encoding a eight enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is protopine. In another specific embodiment, the eighth enzyme is (S)-cis-N-methylstylopine 14-hydroxylase (MSH). In another specific embodiment, the MSH is as set forth in any one of the sequences as depicted in
In another specific embodiment, wherein the cell further comprises a ninth heterologous coding sequence encoding a ninth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, In another specific embodiment, the metabolite is 6-hydroxyprotopine. In another specific embodiment, the ninth enzyme is protopine 6-hydroxylase (P6H). In another specific embodiment, the P6H is as set forth in any one of the sequences as depicted in
In another specific embodiment, the cell further comprises a tenth heterologous coding sequence encoding a tenth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the tenth enzyme is cytochrome P450 reductase (CPR). In another specific embodiment, the CPR is as set forth in any one of the sequences as depicted in
In another specific embodiment, 6OMT, CNMT and 4′OMT2 are expressed from a plasmid. In another specific embodiment, BBE and CPR are expressed from a plasmid and CFS, SPS, TNMT, MSH and P6H are expressed from a chromosome.
In another specific embodiment, the metabolite is (S)-stylopine. In another specific embodiment, the first enzyme is berberine bridge enzyme (BBE); the second enzyme is cheilanthifoline synthase (CFS) or a Ring B closer able to transform scoulerine into cheilanthifoline; the third enzyme is stylopine syntase (SPS) or a Ring A closer able to transform cheilanthifoline in (S)-stylopine; and/or the fourth enzyme is cytochrome P450 reductase (CPR).
In another specific embodiment, the BBE is as set forth in any one of the sequences as depicted in
In another specific embodiment, BBE is from Papaver somniferum; CFS is from Papaver somniferum; SPS is from Papaver somniferum; and/or CPR is from Papaver somniferum.
In another specific embodiment, PsBBE is as set forth in SEQ ID NO: 48 (
In another specific embodiment, the method comprises the second fermentation and the cell further comprises a fifth heterologous coding sequence encoding a fifth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is (S)—N-cis-methylstylopine. In another specific embodiment, the fifth enzyme is tetrahydroprotoberberine cis-N-methyltransferase (TNMT). In another specific embodiment, the TNMT is as set forth in any one of the sequences as depicted in
In another specific embodiment, the cell further comprises a sixth heterologous coding sequence encoding a sixth enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is protopine. In another specific embodiment, the sixth enzyme is (S)-cis-N-methylstylopine 14-hydroxylase (MSH). In another specific embodiment, the MSH is as set forth in any one of the sequences as depicted in
In another specific embodiment, the cell further comprises a seventh heterologous coding sequence encoding a seventh enzyme involved in a metabolite pathway that converts (R,S)-norlaudanosoline into the metabolite. In another specific embodiment, the metabolite is 6-hydroxyprotopine. In another specific embodiment, the seventh enzyme is protopine 6-hydroxylase (P6H). In another specific embodiment, the P6H is as set forth in any one of the sequences as depicted in
In another specific embodiment, the BBE, CFS, SPS and CPR are expressed from plasmid(s). In another specific embodiment, the TNMT, MSH and P6H are are expressed from plasmid(s). In another specific embodiment, the BBE, CFS and SPS are expressed from chromosome.
In another specific embodiment, the method comprises the second fermentation and wherein the metabolite is (S)-dihydrosanguinarine.
In another specific embodiment, the first enzyme is tetrahydroprotoberberine cis-N-methyltransferase (TNMT); the second enzyme is (S)-cis-N-methylstylopine 14-hydroxylase (MSH); the third enzyme is protopine 6-hydroxylase (P6H); and/or the fourth enzyme is cytochrome P450 reductase (CPR).
In another specific embodiment, the TNMT is as set forth in any one of the sequences as depicted in
In another specific embodiment, TNMT is from Papaver somniferum; MSH is from Papaver somniferum; P6H is from Eschscholzia californica; and/or CPR is from Papaver somniferum.
In another specific embodiment, PsTNMT is as set forth in SEQ ID NO: 58 (
In another specific embodiment, the TNMT, MSH and P6H are expressed from a plasmid.
In another specific embodiment, the host cell further expresses a cytochrome b5 (Cytb5). In another specific embodiment, the Cytb5 is as set forth in any one of the sequences as depicted in
In accordance with another aspect of the present invention, there is provided a plasmid comprising nucleic acid encoding: (a) the 6OMT, CNMT and 4′OMT2 enzymes as defined in the present invention; (b) the (i) BBE, (ii) (a) CFS or (b) Ring B closer, and (iii) (a) SPS or (b) Ring A closer enzymes as defined in the present invention; (c) the TNMT, MSH and P6H enzymes as defined in the present invention; (c) the CPR enzyme as defined in the present invention; or (d) the BBE enzyme as defined in the present invention.
In a specific embodiment, the plasmid further comprises a terminator and/or a promoter. In another specific embodiment, the plasmid is as set forth in: SEQ ID NO: 7 (
In accordance with another aspect of the present invention, there is provided a host cell expressing (a) the 6OMT, CNMT and 4′OMT2 enzymes as defined in the present invention; (b) the (i) BBE, (ii) (a) CFS or (b) Ring B closer, and (iii) (a) SPS or (b) Ring A closer enzymes as defined in the present invention; (c) the TNMT, MSH and P6H enzymes as defined in the present invention, and the CPR enzyme as defined in the present invention; (d) the enzymes of (a) and (b); or (b) and (c); (e) the enzymes of (a), (b) and (c); or (f) one or more of the plasmids as defined in the present invention.
In a specific embodiment, the host cell expresses the enzymes of (a) in a plasmid. In another specific embodiment, the host cell expresses the enzymes of (b) in a plasmid. In another specific embodiment, the host cell expresses the enzymes of (b) in a chromosome. In another specific embodiment, the host cell expresses the enzymes of (c) in a plasmid. In another specific embodiment, the host cell expresses the enzymes of (b) and (c) in a chromosome. In another specific embodiment, the host cell expresses in a plasmid the enzymes of (a) and BBE; and in a chromosome, the enzymes of (b) and (c).
In another specific embodiment, the host cell further expresses cytochrome b5.
In accordance with another aspect of the present invention, there is provided a CYP719 polypeptide that is any one of EX45-48 (SEQ ID NOs: 324-327), EX53-58 (SEQ ID NOs: 332-337), EX65-76 (SEQ ID NOs: 344-355), EX78-80 (SEQ ID NOs: 357-359), EX82 (SEQ ID NO: 361), EX86-93 (SEQ ID NOs: 365-372), EX95-101 (SEQ ID NOs: 374-380) and EX104-105 (SEQ ID NOs: 383-384).
In accordance with another aspect of the present invention, there is provided a method of preparing a benzylisoquinoline alkaloid (BIA) metabolite comprising contacting (a) a CYP719 polypeptide of the present invention; or (b) A CYP719 polypeptide that is any one of EX43-44 (SEQ ID NOs: 322-323), EX49 (SEQ ID NO:328), EX51-52 (SEQ ID NOs: 330-331), EX63-64 (SEQ ID NOs: 342-343), EX77 (SEQ ID NO: 356) or EX103 (SEQ ID NO: 382), with scoulerine, nandinine and/or cheilanthifoline.
In accordance with another aspect of the present invention, there is provided a method of producing (i) N-methylcheilanthifoline; or (ii) N-methylcoulerine, comprising contacting cheilanthifoline or scoulerine, respectively, with tetrahydroprotoberberine cis-N-methyltransferase (TNMT), whereby (i) N-methylcheilanthifoline; or (ii) N-methylcoulerine are produced.
In accordance with another aspect of the present invention, there is provided a method of producing nandinine comprising contacting scoulerine with a Ring B closer as set forth in SEQ ID NO: 483, SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 324, SEQ ID NO: 353, SEQ ID NO: 320, SEQ ID NO: 363, SEQ ID NO: 338, SEQ ID NO: 378, SEQ ID NO: 333, SEQ ID NO: 377, SEQ ID NO: 344, or SEQ ID NO: 374.
In another specific embodiment, the Ring B closer as set forth in SEQ ID NO: 484, SEQ ID NO: 485, SEQ ID NO: 324, SEQ ID NO: 333 or SEQ ID NO: 377
Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.
In the present description, a number of terms are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.
Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
As used herein, the term “consists of” or “consisting of” means including only the elements, steps, or ingredients specifically recited in the particular claimed embodiment or claim.
The present invention relates to enzymes involved in a BIA synthetic pathway encoded by plasmids or chromosomes in a host cell and improved methods of use thereof to produce various BIA metabolites.
Without being so limited, enzymes encompassed by the present invention include: native or synthetic 6-O-methyltransferase (6OMT); coclaurine-N-methyltransferase (CNMT); 4′-O-methyltransferase 2 (4′OMT2); berberine bridge enzyme (BBE); cheilanthifoline synthase (CFS); stylopine synthase (SPS); protoberberine Ring A closer (e.g., able to convert scoulerine into nandinine and/or cheilanthifoline into stylopine); Ring A closer able to promote production of N-methylcanadine by a high affinity to tetrahydrocolumbamine (e.g., noscapine pathway), protoberberine Ring B closer (e.g., able to convert scoulerine into cheilanthifoline and/or nandinine into stylopine); tetrahydroprotoberberine cis-N-methyltransferase (TNMT); (S)-cis-N-methylstylopine 14-hydroxylases (MSH); protopine 6-hydroxylase (P6H); cytochrome P450 reductase (CPR); cytochrome b5 and dihydrobenzophenanthridine oxidase (DBOX). Useful enzymes for the present invention may be isolated from Papaver somniferum, Eschscholzia californica, other Papaveraceae (e.g., Papaver bracteatum, Sanguinaria canadensis, Chelidonium majus, Stylophorum diphyllum, Glaucium flavum, Argemone mexicana and Corydalis cheilanthifolia), Ranunculaceae (e.g., Thalictrum flavum, Aquilegia Formosa, Hydrastis canadensis, Nigella sativa, Xanthorhiza simplicissima and Coptis japonica), Berberidaceae (e.g., Berberis thunbergii, Mahonia aquifolium, Jeffersonia diphylla, and Nandina domestica), or Menispermaceae (e.g., Menispermum canadense, Cissampelos mucronata, Tinospora cordifolia, and Cocculus trilobus), etc. The truncated (e.g., devoid of transmembrane domains) and full amino acid sequences of illustrative examples of these enzymes are presented in Figs. herein (e.g.,
Consensuses derived from the alignments of certain of these orthologues are also presented in
In other specific embodiments of the enzymes as used in the present invention (e.g., consensuses in
Hence enzymes in accordance with the present invention include enzymes having the specific nucleotide or amino acid sequences described in
In a more specific embodiment, the enzymes are from Papaver somniferum, Eschscholzia californica, Argemone mexicana, Aquilegia formosa, Corydalis cheilanthifolia, Coptis chinensis, Coptis japonica, Chelidonium majus, Cissampelos mucronata, Glaucium flavum, Hydrastis canadensis, Mahonia aquifolium, Menispermum canadense, Nandina domestica, Nelumbo nucifera, Papaver bracteatum, Podophyllum peltatum, Sanguinaria canadensis, Stylophorum diphyllum, Sinopodophyllum hexandrum, Thalictrum flavum or Xanthorhiza simplicissima. In a more specific embodiment, the P6H, when present is from Eschscholzia californica; and/or 6OMT, CNMT, 4′OMT2, BBE, CFS, SPS, TNMT, MSH, P6H, CPR and/or cytochrome b5, when present are from Papaver somniferum and/or the protoberberine Ring A closer (e.g., able to convert scoulerine into nandinine and/or cheilanthifoline into stylopine) are from Eschscholzia californica, Argemone mexicana, Aquilegia formosa, Corydalis cheilanthifolia, Coptis japonica, Chelidonium majus, Glaucium flavum, Mahonia aquifolium, Nandina domestica, Sanguinaria canadensis, Stylophorum diphyllum, Thalictrum flavum or Xanthorhiza simplicissima and/or the protoberberine Ring B closer (e.g. able to convert scoulerine into cheilanthifoline and/or nandinine into stylopine) are from Papaver somniferum, Eschscholzia californica, Argemone mexicana, Corydalis cheilanthifolia, Chelidonium majus, Glaucium flavum, Nandina domestica, Sanguinaria canadensis, or Stylophorum diphyllum.
For example, the enzymes may be as described in
Percent identities between amino acid sequences of certain enzymes of the present invention are also presented (see e.g.,
Relatedness of enzymes of the present invention are also presented by way of phylogenetic trees (see e.g.,
The enzymes could also be modified for better expression/stability/yield in the host cell (e.g., replacing the native N-terminal membrane-spanning domain by the N-terminal membrane-spanning domain from another plant or yeast gene (e.g., Lactuca sativa (lettuce) germacrene A oxidase) or from a yeast ER bound protein (e.g., erg1 or erg8); codon optimization for expression in the heterologous host; use of different combinations of promoter/terminators for optimal coexpression of multiple enzymes; spatial colocalization of sequential enzymes using a linker system or organelle-specific membrane domain. In a more specific embodiment, useful enzymes are as shown in
A substantially identical sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a mutant peptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and mutant sequences would be considered “substantially identical” polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; a conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g., size, charge, or polarity).
In a non-limiting example, a conservative mutation may be an amino acid substitution. Such a conservative amino acid substitution may be a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term “basic amino acid” it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term “neutral amino acid” (also “polar amino acid”), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term “hydrophobic amino acid” (also “non-polar amino acid”) is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). “Acidic amino acid” refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).
Sequence identity is used to evaluate the similarity of two sequences; it is determined by calculating the percent of residues that are the same when the two sequences are aligned for maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software is available to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as NCBI BLAST2, BLAST-P, BLAST-N, COBALT or FASTA-N, or any other appropriate software/tool that is known in the art (Johnson M, et al. (2008) Nucleic Acids Res. 36:W5-W9; Papadopoulos J S and Agarwala R (2007) Bioinformatics 23:1073-79).
The substantially identical sequences of the present invention may be at least 75% identical; in another example, the substantially identical sequences may be at least 80, 85, 90, 95, 96, 97, 98 or 99% identical at the amino acid level to sequences described herein. The substantially identical sequences retain substantially the activity and specificity of the reference sequence.
The present invention also relates to nucleic acids comprising nucleotide sequences encoding the above-mentioned enzymes. The nucleic acid may be codon-optimized. The nucleic acid can be a DNA or an RNA. The nucleic acid sequence can be deduced by the skilled artisan on the basis of the disclosed amino acid sequences. In a specific embodiment, the nucleic acid encodes one of the amino acid sequences as presented in any one of
The present invention also encompasses vectors (plasmids) comprising the above-mentioned nucleic acids. The vectors can be of any type suitable, e.g., for expression of said polypeptides or propagation of genes encoding said polypeptides in a particular organism. The organism may be of eukaryotic or prokaryotic origin (e.g., yeast). The specific choice of vector depends on the host organism and is known to a person skilled in the art. In an embodiment, the vector comprises transcriptional regulatory sequences or a promoter operably-linked to a nucleic acid comprising a sequence encoding an enzyme involved in the BIA pathway of the invention. A first nucleic acid sequence is “operably-linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since for example enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. “Transcriptional regulatory sequences” or “transcriptional regulatory elements” are generic terms that refer to DNA sequences, such as initiation and termination signals (terminators), enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably-linked.
Plasmids useful to express the enzymes of the present invention include the modified centromeric plasmids pGREG503 (
Plasmids including enzymes in accordance with specific embodiments of the present invention include pGC1189 (CPR); pGC1190 (CPRb-CFS); pGC1191(CPRb-SPSΔNb); pGC1062 (Block 1) (
Promoters useful to express the enzymes of the present invention include the constitutive promoters from the following S. cerevisiae CEN.PK2-1 D genes: glyceraldehyde-3-phosphate dehydrogenase 3 (PTDH3) (
Terminators useful for the present invention include terminators from the following S. cerevisiae CEN.PK2_1 D genes: cytochrome C1 (TCYC1) (
The term “heterologous coding sequence” refers herein to a nucleic acid molecule that is not normally produced by the host cell in nature.
The terms “benzylisoquinoline alkaloid metabolite” or “BIA metabolite” as used herein refer to any BIA metabolite produced by the host cells of the present invention when fed the relevant substrate. Such BIA metabolites include plant native (e.g., reticuline) and non-native metabolites (e.g., N-methylscoulerine and N-methylcheilanthifoline). Without being so limited, it includes (R,S)-6-O-methyl-norlaudanosoline, (R,S)-3′-hydroxy-N-methylcoclaurine, (R,S)-reticuline, (R)-reticuline, (S)-reticuline, (S)-scoulerine, (S)-cheilanthifoline, (S)-stylopine, (S)—N-cis-methylstylopine, protopine, 6-hydroxyprotopine, dihydrosanguinarine, sanguinarine, N-methylscoulerine, N-methylcheilanthifoline, racemic mixtures of any of these compounds and stereoisomers of any of these compounds.
A recombinant expression vector (plasmid) comprising a nucleic acid sequence of the present invention may be introduced into a cell, e.g., a host cell, which may include a living cell capable of expressing the protein coding region from the defined recombinant expression vector. Accordingly, the present invention also relates to cells (host cells) comprising the nucleic acid and/or vector as described above. The suitable host cell may be any cell of eukaryotic (e.g., yeast) or prokaryotic (bacterial) origin that is suitable, e.g., for expression of the enzymes or propagation of genes/nucleic acids encoding said enzyme. The eukaryotic cell line may be of mammalian, of yeast, or invertebrate origin. The specific choice of cell line is known to a person skilled in the art. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny(ies) may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. Vectors can be introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection and viral-mediated transfection. Suitable methods for transforming or transfecting host cells can for example be found in Sambrook et al. (supra), Sambrook and Russell (supra) and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known, and may be used to deliver the vector DNA of the invention to a subject for gene therapy.
In a specific embodiment, the host cells can be a yeast or a bacteria (E. coli). In a more specific embodiment, it can be a Saccharomycetaceae such as a Saccharomyces, Pichia or Zygosaccharomyces. In a more specific embodiment, it can be a Saccharomyces. In a more specific embodiment, it can be a Saccharomyces cerevisiae (S. cerevisiae). Yeast is advantageous in that cytochrome P450 proteins, involved in certain steps in the BIA pathways, are able to fold properly into the endoplasmic reticulum membrane so that activity is maintained, as opposed to bacterial cells which lack such intracellular compartments. The present invention encompasses the use of yeast strains that are aploid, and contain auxotropies for selection that facilitate the manipulation with plasmid. Yeast strains that can be used in the invention include, but are not limited to, CEN.PK, S288C, W303, A363A and YPH499, strains derived from S288C (FY4, DBY12020, DBY12021, XJ24-249) and strains isogenic to S288C (FY1679, AB972, DC5). In specific examples, the yeast strain is any of CEN.PK2-1D (MATalpha ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8C; SUC2) or CEN.PK2-1C (MATa ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8c; SUC2) or any of their single, double or triple auxotrophs derivatives. In a more specific embodiment, the yeast strain is any of the yeast strains listed in Table 1 or Table 2. In another specific embodiment, the particular strain of yeast cell is S288C (MATalpha SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1), which is commercially available. In another specific embodiment, the particular strain of yeast cell is W303.alpha (MAT.alpha.; his3-11,15 trp1-1 leu2-3 ura3-1 ade2-1), which is commercially available. The identity and genotype of additional examples of yeast strains can be found at EUROSCARF, available through the World Wide Web at web.uni-frankfurt.deffb15/mikro/euroscarf/col_index.html or through the Saccharomyces Genome Database (www.yeastgenome.org).
The above-mentioned nucleic acid or vector may be delivered to cells in vivo (to induce the expression of the enzymes and generates BIA metabolites in accordance with the present invention) using methods well known in the art such as direct injection of DNA, receptor-mediated DNA uptake, viral-mediated transfection or non-viral transfection and lipid based transfection, all of which may involve the use of gene therapy vectors. Direct injection has been used to introduce naked DNA into cells in vivo. A delivery apparatus (e.g., a “gene gun”) for injecting DNA into cells in vivo may be used. Such an apparatus may be commercially available (e.g., from BioRad). Naked DNA may also be introduced into cells by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor. Binding of the DNA-ligand complex to the receptor may facilitate uptake of the DNA by receptor-mediated endocytosis. A DNA-ligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, may be used to avoid degradation of the complex by intracellular lysosomes.
The present invention encompasses a method of using a host cell as described above expressing enzymes in accordance with the present invention for generating a significant yield of benzylisoquinoline alkaloid. The applicants have surprisingly discovered that by using first buffering conditions enabling the maintenance of a useful pH of over about 7, and, optionally, second buffering conditions between about 3 and about 6, the host cells of the present invention produced a significantly improved yield of BIA metabolite.
The present invention therefore provide a method of using a host cell as described above expressing enzymes in accordance with the present invention for generating a significant yield of benzylisoquinoline alkaloid using a first useful pH. As used herein, the terms “first useful pH” refer to a pH used for a first fermentation and refer to a pH of over about 7 (over about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, etc.), more preferably between about 7 (or about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, etc.) and about 10 (or about 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10), more preferably, about 7 (or about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8 or 7.9, etc.) to about 9 (or about 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9); about 7 to about 9.9; about 7 to about 9.8; about 7 to about 9.7; about 7 to about 9.6; about 7 to about 9.5; about 7 to about 9.4; about 7 to about 9.3; about 7 to about 9.2; about 7 to about 9.1; about 7.1 to about 8.9; about 7.1 to about 8.8; about 7.1 to about 8.7; about 7.1 to about 8.6; about 7.1 to about 8.5; about 7.1 to about 8.4; about 7.1 to about 8.3; about 7.1 to about 8.2; about 7.1 to about 8.1; about 7.2 to about 9.9; about 7.2 to about 9.8; about 7.2 to about 9.7; about 7.2 to about 9.6; about 7.2 to about 9.5; about 7.2 to about 9.4; about 7.2 to about 9.3; about 7.2 to about 9.2; about 7.2 to about 9.1; about 7.2 to about 8.9; about 7.2 to about 8.8; about 7.2 to about 8.7; about 7.2 to about 8.6; about 7.2 to about 8.5; about 7.2 to about 8.4; about 7.2 to about 8.3; about 7.2 to about 8.2; about 7.2 to about 8.1; about 7.2 to about 8.6; about 7.2 to about 8.5; about 7.2 to about 8.4; about 7.2 to about 8.3; about 7.2 to about 8.2; about 7.2 to about 8.1; about 7.2 to about 9.9; about 7.2 to about 9.8; about 7.2 to about 9.7; about 7.2 to about 9.6; about 7.2 to about 9.5; about 7.2 to about 9.4; about 7.2 to about 9.3; about 7.2 to about 9.2; about 7.2 to about 9.1; about 7.2 to about 8.9; about 7.2 to about 8.8; about 7.2 to about 8.7; about 7.2 to about 8.6; about 7.2 to about 8.5; about 7.2 to about 8.4; about 7.2 to about 8.3; about 7.2 to about 8.2; about 7.2 to about 8.1; about 7.3 to about 8.6; about 7.3 to about 8.5; about 7.3 to about 8.4; about 7.3 to about 8.3; about 7.3 to about 8.2; about 7.3 to about 8.1; about 7.3 to about 9.9; about 7.3 to about 9.8; about 7.3 to about 9.7; about 7.3 to about 9.6; about 7.3 to about 9.5; about 7.3 to about 9.4; about 7.3 to about 9.3; about 7.3 to about 9.2; about 7.3 to about 9.1; about 7.3 to about 8.9; about 7.3 to about 8.8; about 7.3 to about 8.7; about 7.3 to about 8.6; about 7.3 to about 8.5; about 7.3 to about 8.4; about 7.3 to about 8.3; about 7.3 to about 8.2; about 7.3 to about 8.1; about 7.4 to about 8.6; about 7.4 to about 8.5; about 7.4 to about 8.4; about 7.4 to about 8.3; about 7.4 to about 8.2; about 7.4 to about 8.1; about 7.4 to about 9.9; about 7.4 to about 9.8; about 7.4 to about 9.7; about 7.4 to about 9.6; about 7.4 to about 9.5; about 7.4 to about 9.4; about 7.4 to about 9.3; about 7.4 to about 9.2; about 7.4 to about 9.1; about 7.4 to about 8.9; about 7.4 to about 8.8; about 7.4 to about 8.7; about 7.4 to about 8.6; about 7.4 to about 8.5; about 7.4 to about 8.4; about 7.4 to about 8.3; about 7.4 to about 8.2; about 7.4 to about 8.1; about 7.5 to about 8.6; about 7.5 to about 8.5; about 7.5 to about 8.4; about 7.5 to about 8.3; about 7.5 to about 8.2; about 7.5 to about 8.1; about 7.5 to about 9.9; about 7.5 to about 9.8; about 7.5 to about 9.7; about 7.5 to about 9.6; about 7.5 to about 9.5; about 7.5 to about 9.4; about 7.5 to about 9.3; about 7.5 to about 9.2; about 7.5 to about 9.1; about 7.5 to about 8.9; about 7.5 to about 8.8; about 7.5 to about 8.7; about 7.5 to about 8.6; about 7.5 to about 8.5; about 7.5 to about 8.4; about 7.5 to about 8.3; about 7.5 to about 8.2; about 7.5 to about 8.1; about 7.6 to about 8.6; about 7.6 to about 8.5; about 7.6 to about 8.4; about 7.6 to about 8.3; about 7.6 to about 8.2; about 7.6 to about 8.1; about 7.6 to about 9.9; about 7.6 to about 9.8; about 7.6 to about 9.7; about 7.6 to about 9.6; about 7.6 to about 9.5; about 7.6 to about 9.4; about 7.6 to about 9.3; about 7.6 to about 9.2; about 7.6 to about 9.1; about 7.6 to about 8.9; about 7.6 to about 8.8; about 7.6 to about 8.7; about 7.6 to about 8.6; about 7.6 to about 8.5; about 7.6 to about 8.4; about 7.6 to about 8.3; about 7.6 to about 8.2; about 7.6 to about 8.1; about 7.7 to about 8.6; about 7.7 to about 8.5; about 7.7 to about 8.4; about 7.7 to about 8.3; about 7.7 to about 8.2; about 7.7 to about 8.1; about 7.7 to about 9.9; about 7.7 to about 9.8; about 7.7 to about 9.7; about 7.7 to about 9.6; about 7.7 to about 9.5; about 7.7 to about 9.4; about 7.7 to about 9.3; about 7.7 to about 9.2; about 7.7 to about 9.1; about 7.7 to about 8.9; about 7.7 to about 8.8; about 7.7 to about 8.7; about 7.7 to about 8.6; about 7.7 to about 8.5; about 7.7 to about 8.4; about 7.7 to about 8.3; about 7.7 to about 8.2; about 7.7 to about 8.1; about 7.8 to about 8.6; about 7.8 to about 8.5; about 7.8 to about 8.4; about 7.8 to about 8.3; about 7.8 to about 8.2; about 7.8 to about 8.1; about 7.8 to about 9.9; about 7.8 to about 9.8; about 7.8 to about 9.7; about 7.8 to about 9.6; about 7.8 to about 9.5; about 7.8 to about 9.4; about 7.8 to about 9.3; about 7.8 to about 9.2; about 7.8 to about 9.1; about 7.8 to about 8.9; about 7.8 to about 8.8; about 7.8 to about 8.7; about 7.8 to about 8.6; about 7.8 to about 8.5; about 7.8 to about 8.4; about 7.8 to about 8.3; about 7.8 to about 8.2; about 7.8 to about 8.1; about 7.9 to about 8.6; about 7.9 to about 8.5; about 7.9 to about 8.4; about 7.9 to about 8.3; about 7.9 to about 8.2; about 7.9 to about 8.1; about 7.9 to about 9.9; about 7.9 to about 9.8; about 7.9 to about 9.7; about 7.9 to about 9.6; about 7.9 to about 9.5; about 7.9 to about 9.4; about 7.9 to about 9.3; about 7.9 to about 9.2; about 7.9 to about 9.1; about 7.9 to about 8.9; about 7.9 to about 8.8; about 7.9 to about 8.7; about 7.9 to about 8.6; about 7.9 to about 8.5; about 7.9 to about 8.4; about 7.9 to about 8.3; about 7.9 to about 8.2; about 7.9 to about 8.1. As used herein, the terms “second useful pH” refer to the pH used for the optional second fermentation and refer to a pH of between about 2.7 and about 6.3 (e.g., YNB conditions), between about 2.8 and about 6.2, between about 2.9 and about 6.1, between about 3 and about 6.0, between about 3 and about 5.9, between about 3 and about 5.8, between about 3 and about 5.7, between about 3 and about 5.6, between about 3 and about 5.5.
Without being so limited, useful buffering conditions capable of maintaining a pH of about 7 to about 10 include: a buffer or mixture of buffers such as Tris; yeast growing medium (e.g., yeast nitrogen broth, synthetic dropout supplement, 2% α-D-glucose and amino acids) (YNB); YNB and a sufficient concentration of Tris; YNB and HEPES; Tris; and Tris and EDTA. Additional examples of such buffers are PBS, PIPES, MOPS, and taurine. A more exhaustive list can be found online at http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html. In a specific embodiment, such conditions include using about 5 mM to about 150 mM of Tris or Tris and EDTA. In a more specific embodiment, the range is of about 10 to 150 mM; 10 to 140 mM; 10 to 130 mM; 10 to 120 mM; 10 to 110 mM; 10 to 100 mM; 10 to 90 mM; 10 to 80 mM; 10 to 70 mM; 10 to 60 mM; 10 to 55 mM; 10 to 50 mM; 20 to 150 mM; 20 to 140 mM; 20 to 130 mM; 20 to 120 mM; 20 to 110 mM; 20 to 100 mM; 20 to 90 mM; 20 to 80 mM; 20 to 70 mM; 20 to 60 mM; 20 to 55 mM; 20 to 50 mM; 30 to 150 mM; 30 to 140 mM; 30 to 130 mM; 30 to 120 mM; 30 to 110 mM; 30 to 100 mM; 30 to 90 mM; 30 to 80 mM; 30 to 70 mM; 30 to 60 mM; 30 to 55 mM; 30 to 50 mM; 40 to 150 mM; 40 to 140 mM; 40 to 130 mM; 40 to 120 mM; 40 to 110 mM; 40 to 100 mM; 40 to 90 mM; 40 to 80 mM; 40 to 70 mM; 40 to 60 mM; 40 to 55 mM; 40 to 50 mM; 45 to 150 mM; 45 to 140 mM; 45 to 130 mM; 45 to 120 mM; 45 to 110 mM; 45 to 100 mM; 45 to 90 mM; 45 to 80 mM; 45 to 70 mM; 45 to 60 mM; 45 to 55 mM; or 45 to 50 mM.
In one embodiment, the method comprising incubating (R,S)-norlaudanosoline (fed substrate) with a host cell expressing 6OMT, CNMT and 4′OMT2 in buffering conditions enabling a useful pH (namely in that case a pH of about 8) yielded about 20% of (S)-reticuline. As used herein the yield may be defined as the ratio of the end product (metabolite) produced to the fed substrate. Hence 20% of the total fed (R,S)-norlaudanosoline was converted to (S)-reticuline in the host cell combined supernatant and cell extract. In another embodiment, the method comprising incubating (S)-scoulerine (fed substrate) with a host cell expressing BBE, CFS, SPS and CPR in buffering conditions enabling a useful pH (namely in that case a pH of about 8) yielded about 19% of (S)-stylopine. In another embodiment, the method comprising incubating (S)-stylopine (fed substrate) with a host cell expressing TNMT, MSH, P6H and CPR in buffering conditions enabling a useful pH (namely in that case a pH of about 8) yielded about 57% of dihydrosanguinarine. In another embodiment, the method comprising incubating (S)-scoulerine (fed substrate) with a host cell expressing BBE, CFS, SPS, TNMT, MSH, P6H and CPR in buffering conditions enabling a useful pH (namely in that case a pH of about 8) yielded about 7.5% of dihydrosanguinarine. In another embodiment, the method comprising incubating (R,S)-norlaudanosoline (fed substrate) with a host cell expressing 6OMT, CNMT, 4′OMT2, BBE, CFS, SPS, TNMT, MSH, P6H and CPR in buffering conditions enabling a useful pH (namely in that case a pH of about 8) yielded about 1.5% of dihydrosanguinarine. As used herein, a significant yield of BIA metabolite includes about 1.5% or more. In another embodiment, the method comprising incubating scoulerine (fed substrate) with a host expressing a Ring A closer and A Ring B closer (see
The present invention is illustrated in further details by the following non-limiting examples.
(S)-Reticuline was a gift from Johnson & Johnson. (R,S)-Norlaudanosoline was purchased from Enamine Ltd. (Kiev, Ukraine), (S)-scoulerine and (S)-stylopine from ChromaDex (Irvine, Calif., USA), protopine from TRC Inc. (North York, Ontario, Canada) and sanguinarine from Sigma. Dihydrosanguinarine was prepared by NaBH4 reduction of sanguinarine50. Antibiotics, growth media and α-D-glucose were purchased from Sigma-Aldrich. Restriction enzymes, T4 DNA polymerase, and T4 DNA ligase were from New England Biolabs (NEB). Polymerase chain reactions (PCRs) for the assembly of expression cassettes were performed using Phusion™ High-Fidelity DNA polymerase (NEB/Thermo Scientific). Taq polymerase (Fermentas/Thermo Scientific) was used in PCRs confirming DNA assembly or chromosomal integration. PCR-amplified products were gel purified using the QIAquick™ purification kit (Qiagen). Plasmid extractions were done using the GeneJET™ plasmid mini-prep kit (Thermo Scientific). Genomic DNA preparations were done using the DNeasy™ blood and tissue kit (Qiagen). HPLC-grade water was purchased from Fluke. HPLC-grade methanol and acetonitrile were purchased from Fischer Scientific.
RNA extraction from root or stems of the Papaver somniferum (opium poppy) cultivar Bea's Choice, cDNA library construction, Illumine sequencing, sequence assembly and annotation, and gene expression analysis were performed as described previously6.
The pESC-CPR vector encoding opium poppy cytochrome P450 reductase (PsCPR) fused to a c-Myc tag was used for the heterologous expression of plant proteins in S. cerevisiae25. The native PsCPR sequence and that optimized for the host are shown in
Reconstitution of the Sanguinarine Pathway in S. cerevisiae
For liquid cultures, S. cerevisiae was grown in yeast nitrogen broth, synthetic dropout supplement, 2% α-D-glucose and amino acids as appropriate (YNB-DO-GLU) at 30° C. and 200 rpm. For solid media, selection for plasmid transformation was on YNB-DO-GLU/agar, while selection for chromosomal integration was on YPD/agar (yeast extract peptone-dextrose) with the appropriate antibiotic. Lithium acetate transformation was performed according to Gietz and Schiestl53, electroporation was performed according to Shao et al9.
Coding sequences for 6OMT, CNMT, 4′OMT2, BBE, CFS, SPS, TNMT, MSH AND CPR are from P. somniferum and that of P6H is from Eschscholzia californica. Coding sequences for protoberberine Ring A and Ring B closers are from various species as listed in Table 6. Synthetic sequences of Ps6OMT (GenBank KF554144), PsCNMT (GenBank KF661326), Ps4′OMT2 (GenBank KF661327), PsSPSΔN and PsP450R(CPR) (GenBank KF661328) were codon-optimized by DNA2.0 for optimal expression in yeast (See
a All coding sequences are from Papaver somniferum except PH6 which is from Eschscholzia californica
bSynthetic gene. Codon-optimized sequence for expression in Saccharomyces cerevisiae.
C6-H1-C1-PPDC1-BBEΔN-TADH1-C6-H2-C1-PPMA1-
C6-H1-C1-PTDH3-TNMT-TADH1-C6
C1- PPDC1-CNMTb-TPGI1-C6,
a All coding sequences are from Papaver somniferum except PH6 which is from Eschscholzia californica.
bSynthetic gene. Codon-optimized sequence for expression in Saccharomyces cerevisiae.
c Linkers used for cloning purposes are in bold.
Blocks of enzymes were designed to independently express sequential enzymes of the dihydrosanguinarine pathway. Enzyme blocks were cloned into the pGREG series of E. coli-S. cerevisiae shuttle vectors55. Vectors pGREG503, 504, 505 and 506, harbouring the HIS3, TRP1, LEU2 and URA3 auxotrophic markers, respectively, were modified by site-directed mutagenesis to contain a unique KpnI site at the 3′ end of a stuffer cassette in the multiple cloning site using the PCR primers reported in Supplemental Table 3. Gene expression cassettes were inserted by homologous recombination into pGREG vectors previously linearized with AscI/KpnI. Empty pGREG control plasmids were created by intra-molecular ligation of the linearized pGREG made blunt with T4 DNA polymerase.
The DNA assembler technique, which takes advantage of in vivo homologous recombination in yeast9, was used for the assembly of the sanguinarine pathway. Promoters, genes, and terminators were assembled by incorporating a ˜50-bp homologous region between the segments. Expression cassettes were joined to each other and to the vector backbone using DNA linkers (C6-H(n)-C1 linkers in Table 4), with the exception of some components of Block 3. DNA linkers were added to promoters and terminators by PCR using the primers listed in Table 4 and CEN.PK genomic DNA as template. In addition, a NotI site was introduced in the 3′ linker primer containing homology to pGREG backbones, allowing the excision of enzyme blocks by AscI/NotI double digest. PsBBEΔN was also independently cloned into the 2μ, high copy vector pYES2. For DNA assembly, the pYES2 backbone was amplified by PCR using primers pYES2 for and pYES2 rev described in Table 4. Transformation of DNA fragments in yeast for homologous recombination was accomplished by electroporation. Assembled plasmids were transferred to E. coli and sequenced-verified. All the plasmids used in examples presented herein are described in Table 1 and Table 2.
Integration of Enzyme Blocks into the Genome
Integration of enzyme blocks into the genome of S. cerevisiae was achieved through targeted homologous recombination to integration sites shown to support relatively high levels of gene expression35. Enzyme blocks were integrated into the genome using upstream and downstream homology regions, selection cassettes, and gene cassettes, as parts for chromosomal DNA assembly9. Selection cassettes HygR and G418R (for Blocks 2 and 3, respectively), were amplified from pZC3 and pUG6, while genomic homology regions (site 18 on chromosome XV and site 20 on chromosome XVI for Blocks 2 and 3, respectively) were amplified from CEN.PK genomic DNA using primers described in Table 5. Gene cassettes of Blocks 2 and 3 were excised from their plasmids by AscI//NotI-HF/XbaI digestion. Parts for assembly were transformed into S. cerevisiae by the lithium acetate method and integrants were selected on solid media. Successful integration was verified by PCR using genomic DNA as template.
Whole cell substrate feeding assays were used to test the function of each enzyme block individually and in combinations. To prepare the cells for the feeding assays, a colony of S. cerevisiae was inoculated in YNB-DO-GLU and incubated for 24 hours. Cultures were diluted to an OD600 of 0.8 into 6 ml of fresh YNB-DO-GLU and incubated for an additional 7 hours. Cells were harvested by centrifugation at 2000×g for 2 min. Supernatants were decanted and cells were suspended in 2 ml of Tris-EDTA (10 mM Tris-HCl, 1 mM EDTA, pH 8), containing 10 μM of one of the following feeding substrates: (R,S)-norlaudanosoline, (S)-reticuline, (S)-scoulerine or (S)-stylopine. Cells were incubated for 16 hours then harvested by centrifugation at 15000×g for 1 min. For BIA extraction from cells, the cell pellet was suspended in 500 μl methanol with ˜50 μl acid-washed glass beads and vortexed for 30 min. Cell extracts were clarified by centrifugation at 15000×g for 1 min and used directly for LC-MS analysis.
Analysis of enzyme assays was performed using an Agilent™ 1200 liquid chromatography system equipped with a 6410 triple-quadrupole mass spectrometer (Agilent Technologies; Santa Clara, Calif.). Ten microliters of the reaction mixtures were separated as described previously25 and the eluate was applied to the mass analyser using the following parameters: capillary voltage, 4000 V; fragmentor voltage, 125 V; source temperature, 350° C.; nebulizer pressure, 50 psi; gas flow, 10 L min−1. Scoulerine, cheilanthifoline, and stylopine were detected in multiple reaction monitoring (MRM) mode using a collision energy of 25 eV and monitored transitions of m/z 328→178, 326→178 and 324→176, respectively.
Detection of alkaloids in the sanguinarine biosynthetic pathway was performed by FT-MS using a 7T-LTQ FT ICR instrument (Thermo Scientific, Bremen, Germany). Alkaloids were separated by reverse phase HPLC (Perkin Elmer SERIES 200 Micropump, Norfolk, Conn.) using an Agilent Zorbax™ Rapid Resolution HT C18 2.1*30 mm, 1.8 micron column. Solvent A (0.1% acetic acid) and solvent B (100% acetonitrile) were used in a gradient elution to separate the metabolites of interest as follows: 0-1 min at 100% A, 1-6 min 0 to 95% B (linear gradient), 7-8 min 95% B, 8-8.2 min 100% A, followed by a 1 min equilibration at 100% A. Three microliters of either cell extract or supernatant fraction were loaded on the HPLC column run at a flow rate of 0.25 ml/min. Dilutions in methanol were performed to keep alkaloid concentrations within the range of standard curve values and avoid saturating FT signals. Following LC separation, metabolites were injected into the LTQ-FT-MS (ESI source in positive ion mode) using the following parameters: resolution, 100000; scanning range, 250 to 450 AMU; capillary voltage, 5 kV; source temperature, 350° C.; AGC target setting for full MS were set at 5×105 ions. Identification of alkaloids was done using retention time and exact mass (<2 ppm) of the monoisotopic mass of the protonated molecular ion [M+H]+. LC-FT-MS data were processed using the freely available program Maven56. When available authentic standards were used to confirm the identity of the BIA intermediates (using HPLC retention times and exact masses) and to quantify sanguinarine alkaloids. When unavailable, we assumed equal ionization efficiency between an intermediate and the closest available quantifiable alkaloid (m/z 302 and m/z=316: reticuline; cheilanthifoline: stylopine).
Sanguinarine biosynthesis from (S)-scoulerine proceeds with the formation of two methylenedioxy bridges catalysed by the P450s cheilanthifoline synthase (CFS) and stylopine synthase (SPS), to yield cheilanthifoline and stylopine respectively (
Constructs for the heterologous expression of PsCFS and PsSPS in S. cerevisiae were assembled in a vector harbouring PsCPR (Table 1 above). As PsSPS was poorly expressed (data not shown), the native N-terminal membrane-spanning domain was swapped with that of lettuce germacrene A oxidase, generating recombinant PsSPSΔN. Western blot analysis confirmed expression of the recombinant proteins in yeast (
The synthesis of sanguinarine from norlaudanosoline requires ten enzymatic reactions (
To confirm functional expression of enzymes, plasmids expressing each of the three blocks were individually transformed into S. cerevisiae and cultures of the strains were supplemented with either (R,S)-norlaudanosoline, (S)-reticuline, (S)-scoulerine or (S)-stylopine. Functional expression was verified by detection of the expected end products. As negative controls, yeast strains lacking enzyme blocks were incubated with each of the pathway intermediate to evaluate substrate recovery and to assess the relative proportion recovered in cellular extracts versus culture supernatants.
Block 1 contains the P. somniferum enzymes 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), and 4′-O-methyltransferase 2 (4′OMT2), which catalyse three methylation reactions to convert (R,S)-norlaudanosoline to (S)-reticuline. The committed step of BIA synthesis in plants is the condensation of the L-tyrosine derivatives L-dopamine and 4-hydroxyphenylacetaldehyde to produce (S)-norcoclaurine, catalysed by the enzyme (S)-norcoclaurine synthase (NCS) (
Strain GCY1086, expressing Block 1 enzymes from a plasmid, was incubated with (R,S)-norlaudanosoline. The end product reticuline was produced with a yield of 20% (
To investigate the possibility that the reticuline produced from (R,S)-norlaudanosoline by the three opium poppy MTs was not racemic, chiral analysis by HPLC was used to reveal the presence or absence of reticuline enantiomers.
Separation of the (R)- and (S)-enantiomers of reticuline was performed using the chiral column CHIRALCEL OD-H (4.6×250 mm, Daicel Chemical Industries) and the solvent system hexane:2-propanol:diethylamine (78:22:0.01) at a flow rate of 0.55 ml min−1 60. Following LC separation, metabolites were injected into an LTQ ion trap mass spectrometer (Thermo Electron, San Jose, Calif.) and detected by selected reaction monitoring (SRM). SRM transitions of m/z 288→164.0 (CID@35) and 330→192 (CID@30) were used to detect reticuline. Retention times for reticuline obtained in samples matched retention times observed with authentic standards.
Chiral analysis of enantio-pure standards of (R)- and (S)-reticuline and of racemic (R,S)-reticuline was first performed to confirm the separation of the two enantiomers (
Block 2 contains the P. somniferum enzymes berberine bridge enzyme (BBE), CFS and SPS. In plants, the flavoprotein oxidase BBE stereoselectively converts (S)-reticuline to (S)-scoulerine33. A truncated version of BBE (PsBBEΔN) was cloned with CFS and recombinant PsSPSΔN into plasmid pGC994 (Table 1). When cells expressing enzymes of Block 2 and CPR (strain GCY1090) were incubated with (S)-reticuline, no scoulerine, cheilanthifoline, or stylopine were detected (
The third block encodes the three enzymes catalysing the conversion of stylopine to dihydrosanguinarine. Stylopine is N-methylated to (S)-cis-N-methylstylopine by tetrahydroprotoberberine cis-N-methyltransferase (TNMT)34. The next two biosynthetic steps are catalysed by the P450 hydroxylases (S)-cis-N-methylstylopine 14-hydroxylase (MSH) and protopine 6-hydroxylase (P6H). Both enzymes were recently identified and characterized from P. somniferum and Eschscholzia californica respectively (CYP82N4 and CYP82N2v2)25,27. 6-Hydroxyprotopine spontaneously rearranges to dihydrosanguinarine. When cells expressing Block 3 and CPR (strain GCY1094) were incubated with (S)-stylopine, the majority of the stylopine was consumed, resulting in 57% conversion to dihydrosanguinarine (
Approximately 55% of the pathway intermediate N-methylstylopine (N-st) was found in the culture supernatant as opposed to stylopine (Sty) and dihydrosanguinarine (DHS), which were mostly found in the cellular extract fraction (
The vectors harbouring Blocks 1, 2, 3, and CPR are closely related and share several of the same promoters and terminators. A loss of function was occasionally observed from strains harbouring multiple plasmids, which was attributed to recombination. To address this problem, Blocks 2 and 3 were integrated into the genome of S. cerevisiae (Table 1 and Table 2). Block 2 was integrated into YORWΔ17(ChrXV). Block 3 was integrated into YPRCΔ15(ChrXVI). The plasmids were single copy.
Block 2 was integrated into S. cerevisiae and the strain was transformed with CPR (strain GCY1101). When incubated with scoulerine, 14% of the substrate was converted to stylopine, which is comparable to what was obtained by expressing Block 2 from a centromeric plasmid (single copy) (strain GCY1090)(
Block 3 was integrated into the Block 2 strain, generating a Block 2-Block 3 double integrant, which was subsequently transformed with CPR (strain GCY1104). Incubation of this strain with (S)-scoulerine resulted in 7.5% conversion to dihydrosanguinarine (
When cells of the Block 2-Block 3 double integrant were incubated with (S)-scoulerine, compounds with exact masses of 342.1710 m/z and 340.1548 m/z were detected in addition to expected sanguinarine pathway intermediates. These exact masses, and their CIDs, correspond to N-methylscoulerine and N-methylcheilanthifoline, respectively36. The compounds had been previously identified in opium poppy cell culture and were predicted to be a product of TNMT activity, an enzyme that had also been shown to methylate (S)-canadine34. To determine whether TNMT was responsible for the N-methylation of cheilanthifoline and scoulerine, the Block 2 integrant strain harbouring CPR on a plasmid (strain GCY1101) was compared with the Block 2 integrant strain harbouring both CPR and TNMT on a plasmid (strain GCY1127). The resulting chromatograms and CIDs (
Because the applicants lacked standards to quantify the N-methylated products and since the three compounds are similar in structure, they assumed equal ionization efficiency and estimated their relative proportions by peak area: when strain GCY1127 is fed (S)-scoulerine, the expected product N-methylstylopine is 33%, while N-methylscoulerine is 7% and N-methylcheilanthifoline is 60% (
The three functional blocks were combined to assemble a complete dihydrosanguinarine pathway in yeast. The Block 2-Block 3 integrant served as a background strain for the transformation of Block 1 and CPR (strain GCY1108). When this strain was incubated with (R,S)-norlaudanosoline, trace levels of dihydrosanguinarine were observed (
The applicants co-transformed a high-copy vector expressing psBBEΔN along with Block 1 and CPR plasmids into the double integrant (strain GCY1125). When strain GCY1125 was incubated with (R,S)-norlaudanosoline, conversion to dihydrosanguinarine improved from trace to 1.5% (
Finally, the applicants independently incubated GCY1125 cells with (R,S)-norlaudanosoline, (S)-reticuline, (S)-scoulerine, or (S)-stylopine to assess the efficiency of substrate conversion at different points in the complete pathway (
Block 2_Block 3 enzymes were tested in whole cell substrate (GCY1104) feeding assays for the production of dihydrosanguinarine from scoulerine using different buffering conditions (
A colony of S. cerevisiae was inoculated in YNB-DO-GLU and incubated for 24 hours. Cultures were diluted to an OD600 of 0.8 into 6 ml of fresh YNB-DO-GLU and incubated for an additional 7 hours. Cells were harvested by centrifugation at 2000×g for 2 min. Supernatants were decanted and cells were suspended in 2 ml of each of the following media containing 10 μM of of (S)-scoulerine:
Cells were incubated for 16 hours and at the end of the feeding the pHs were verified. In sample 1 the pH dropped from 5 to 3 as expected. In sample 2 the pH dropped from 8 to 5, indicating that the buffer strength was not enough to maintain the pH at 8. All other buffers maintained the pH at 8. Extraction of alkaloids and analysis were performed as described above.
Results shown in
Additional pHs of 3, 6, 7, 8, 9 were tested on the individual Blocks 1, 2 and 3 and on the three Blocks 1-2-3 combined in GCY1125 in order to evaluate the effect of pH on sanguinarine pathway BIA synthesis and recovery.
A variety of media was used: the yeast media YNB, which has a starting pH of about 5.5 but decreases to about pH 3 upon fermentation; 10 mM Sørenson's phosphate buffer, pH 6.0; TE buffer pH 7.0 (10 mM Tris, 1 mM EDTA), TE buffer pH 8.0 (10 mM Tris, 1 mM EDTA), and TE buffer pH 9.0 (10 mM Tris, 1 mM EDTA). Yeast expressing no heterologous BIA pathway enzymes were inoculated in YNB with appropriate supplementation overnight, back-diluted 1:10 in 96-well deep-well plates, allowed to grow for 7 h and concentrated 3× in incubation media containing 5 uM of either (R,S)-norlaudanosoline, (S)-scoulerine, or (S)-stylopine. After 16 hours, supernatant was recovered and diluted 1:2 in MeOH before analysis by HPLC-FT-MS. In addition, cell pellets were resuspended in MeOH and vortexed for 30 minutes before analysis by HPLC-FT-MS.
Total recovery of BIAs was not consistent across all pHs, nor was it consistent across all BIAs. (R,S)-norlaudanosoline recovery was highest in YNB, and dropped as pH increased, until at pH 9, no norlaudanosoline was recovered (
BIAs were not recovered equally from supernatant and cell extract, nor was recovery consistent across the pH range from 3-9. While for (R,S)-norlaudanosoline recovery in cell extract remained within 3-5% across all pHs, (S)-scoulerine and (S)-stylopine recovery in cell extract increased with pH (
In contrast to recovery studies, activity assays tended to favour higher pHs. Yeast expressing Block 1 (GCY1086), Block 2 and PsCPR (GCY1090), or Block 3 and PsCPR (GCY1094) were inoculated overnight, back-diluted 1:10 in 96-well deep-well plates, allowed to grow for 7 h and concentrated 3× in incubation media containing 5 uM of (R,S)-norlaudanosoline (
Poor recovery of (R,S)-norlaudanosoline at pHs greater or equal to 6 suggests degradation, perhaps by oxidation as hypothesized by Kim et al 201371. They observed higher conversion of fermented (S)-norlaudanosoline to (S)-reticuline at pH 6 than pH 8. While the Applicant observed that norlaudanosoline is highly affected by higher pHs, conversion of norlaudanosoline towards downstream products is also higher at these pHs, thus preventing oxidation. Kim et al.71 was supplementing dopamine, which must be enzymatically condensed with its derivative 3,4-dHPAA to form norlaudanosoline; it is possible that this enzymatic step provided an extra bottleneck which led to increased oxidation.
The effect of higher pHs on Block 1 and Block 2 conversion was remarkably similar: 0% activity in YNB, 10-25% activity in pHs 6 and 7, and activity levelling off at ˜40% at pHs 8 and 9. In contrast, Block 3 was most active in YNB, where 75% of stylopine was converted into dihydrosanguinarine and sanguinarine. This suggests that a two-step fermentation could be performed, with a higher pH to promote conversion to stylopine, followed by a lower pH to promote conversion to dihydrosanguinarine and sanguinarine.
The present invention encompasses increasing the activity of SPS and CFS, and thus the flux of alkaloids towards sanguinarine. Orthologous genes from different plants can have varying kinetic constants and expression efficiency in yeast.
There are 14 enzyme families and 300 genes in the BIA gene order. The cheilanthifoline and stylopine synthases belong to the CYP719 family.
The Applicant purchased all published CYP719s (including those without published activities). Published CYP719 protein sequences were used as queries for a tblastx™ search of the PhytoMetaSyn™ transcriptome database. The interface for BLAST was on PhytoMetaSyn™'s website. Protein sequences with percent similarity of 55% or greater to published CYP719s were saved for downstream analysis.
In parallel with the BLAST approach, PhytoMetaSyn™'s transcriptome data (RNA) was downloaded and converted into predicted ORFs (protein) using the OrfPredictor algorithm developed in Dr. Tsang's laboratory at Concordia University63. Two motifs were used to search the database of predicted PhytoMetaSyn™ ORFs. The first was the highly conserved heme-binding motif FXXGXRXC (SEQ ID NO: 481). The second was a common N-terminal hydrophobic region downstream of the membrane-anchor sequence, found to be conserved amongst published CYP719s: P(hydrophobic)(hydrophobic)GN64. Protein sequences containing both motifs of interest were saved for downstream analysis.
Predicted ORFs identified through the tblastx™ search and/or motif searches described above were sorted into CYP families by percent sequence identity using the program BLAST-CLUST™ (http://toolkit.tuebingen.mpg.de/blastclust). BLAST-CLUST™ requires two inputs: “sequence length to be covered” and “percent identity threshold”. Sequence length was set to 95% to allow for variability in identity and length of membrane-anchor sequences. Percent identity was set to 40% because the CYP nomenclature committee defines CYP families as CYPs with 40% identity or more64. All published CYP719s cluster together using these settings. Predicted ORFs that clustered with published CYP719s were selected for further analysis. Additional outliers were discarded using Clustal Omega™'s multiple sequence alignment, and phylogenetic trees were generated using the program MEGA6™65 (See
The Applicant ordered 42 CYP719s from the PhytoMetaSyn™ database, along with 19 published CYP719s, from the DNA synthesis company Gen9 (referred to herein as “purchased CYP719”). A phylogenetic tree of the ordered sequences is presented in
The pBOT vector system is modular and flexible, and can be used to synthesize an unlimited number and type of vector backbones. Each vector feature is amplified individually, flanked by 40 bp linkers such that features can be combined via cloning methods relying on homologous regions of DNA. Any number of features can be used, depending on the nature of linkers used. Features used in the pBOT-TRP vector were: 1) E. coli antibiotic resistance and origin of replication; 2) yeast origin of replication; 3) yeast antibiotic resistance; 4) yeast auxotrophy; and 5) expression cassette.
The four basic pBOT vectors contain unique promoter and terminator combinations, allowing for cassette assembly via cloning methods relying on homologous regions of DNA. Genes were directionally cloned into pBOT expression cassettes as GFP fusion proteins via the type II restriction enzyme SapI. Protein expression can be measured indirectly via GFP fluorescence. GFP can be removed by digestion with KasI followed by dilution and religation, resulting in a functional expression cassette with a two amino acid scar (glycine-alanine) at the C terminus of the gene. The four pBOT versions available contain a different auxotrophy (LEU, URA, HIS or TRP) and different promoter-terminator pairs associated with each auxotrophy. Any gene of interest can be cloned by SapI restriction digestion and ligation. Target genes are PCR amplified using primers that add a SapI site at the 5′ and at the 3′ as follows: 5′-GCTCTTCTACA (SEQ ID NO: 565)-GENE-GGCTGAAGAGC-3′ (SEQ ID NO: 566). Digestion of vector generates 5′ overhangs on vector (TGT and GGC) which complement designed 5′ overhangs on digested gene sequences (ACA and CCG). Ligation of SapI digested plasmid and target gene will reconstitute a functional Kozak sequence at the 5′ of the gene (AAACA (SEQ ID NO: 567) followed by the ATG first codon and no extra UTRs region added as described. A linker of 36 nucleotides (12 amino acids) between the gene and the GFP in present.
To broadly identify CYP719 activities on BIAs, a substrate affinity test was performed with the protoberberine BIA scoulerine. CYP719s have been described to form methylenedioxy bridges on BIAs from an alcohol group and a methyl group on adjacent carbons. Two different rings can be made on scoulerine, which can be called “Ring A” and “Ring B”, with the BIA products being called “nandinine” and “cheilanthifoline”, respectively (see
Plasmids harboring CYP719s were transformed into either GC1333 containing an integrated PsCPR (
Argemone mexicana
Argemone mexicana
Aquilegia formosa
Aquilegia formosa
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Coptis chinensis
Coptis japonica
Coptis japonica
Coptis japonica
Chelidonium majus
Chelidonium majus
Chelidonium majus
Chelidonium majus
Cissampelos mucronata
Cissampelos mucronata
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Glaucium flavum
Glaucium flavum
Glaucium flavum
Hydrastis canadensis
Mahonia aquifolium
Menispermum canadense
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nelumbo nucifera
Papaver bracteatum
Papaver bracteatum
Papaver bracteatum
Podophyllum peltatum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Sanguinaria canadensis
Sanguinaria canadensis
Sanguinaria canadensis
Stylophorum diphyllum
Stylophorum diphyllum
Stylophorum diphyllum
Stylophorum diphyllum
Sinopodophyllum hexandrum
Thalictrum flavum
Thalictrum flavum
Xanthorhiza simplicissima
Argemone
mexicana
Argemone
mexicana
Aquilegia
formosa
Aquilegia
formosa
Coptis chinensis
Coptis japonica
Coptis japonica
Coptis japonica
Eschscholzia
californica
Eschscholzia
californica
Eschscholzia
californica
Eschscholzia
californica
Eschscholzia
californica
Eschscholzia
californica
Nelumbo nucifera
Podophyllum
peltatum
Papaver
somniferum
Papaver
somniferum
Papaver
somniferum
Papaver
somniferum
Sinopodophyllum
hexandrum
Thalictrum
flavum
The resulting CYP719-harboring strains (strains SF41-105) were supplemented with scoulerine. After 16 hours, BIAs were extracted and the molar ratio of scoulerine to downstream BIAs was calculated (
10 of the 61 assayed CYP719s were observed to convert scoulerine into cheilanthifoline (Ring B closers) (EX41, EX45, EX54, EX59, EX65, EX74, EX84, EX95, EX98 and EX99). All 10 converted over 95% of scoulerine to the Ring B product cheilanthifoline. 23 of the 61 assayed CYP719s converted at least 5% of supplemented scoulerine to the Ring A product nandinine. 10 of 23 converted over 95% of scoulerine to nandinine. This could indicate that while scoulerine was accepted, it was not a preferred substrate for the other 13 Ring A-closing CYP719s.
The 10 CYP719s capable of Ring A closure of >95% of scoulerine (EX42, EX50, EX56, EX60, EX67, EX69, EX72, EX76, EX96 and EX101), along with several other purchased CYP719s with a range of Ring A-closing activity on scoulerine from 0% to 89% (EX44, EX46, EX47, EX48, EX58, EX61, EX66, EX103 and EX105) were introduced to the Block 2 pathway and tested for affinity for cheilanthifoline. Plasmids harboring individual CYP719s were transformed into strain GC1316, which harbored PsCPR and PsCFS integrated into the genome. Strains were supplemented with scoulerine and after 16 hours total BIAs were extracted and the molar ratio was compared (
Because nandinine was not a preferred substrate of PsCFS, most stylopine was generated from cheilanthifoline. Therefore, the ratio of nandinine:cheilanthifoline:stylopine was affected by two factors. The first factor was the acceptance of cheilanthifoline by Ring A-closing CYP719s. Of the 10 CYP719s capable of 95% Ring A closure of scoulerine, residual cheilanthifoline was detected in three (EX50, EX60, EX72). Conversely, EX46 was capable of just 66% Ring A closure of scoulerine (see
The second factor affecting the ratio of nandinine:cheilanthifoline:stylopine was the relative rate of activity of Ring A and Ring B closure. Scoulerine was a substrate for both Ring A closure and Ring B closure. If Ring A closure occurred at a greater rate than Ring B closure, nandinine was produced, which accumulated. If Ring B closure occurred at a greater rate than Ring A closure, cheilanthifoline was produced, which either accumulated or was converted to stylopine depending on the specificity of the Ring A-closing CYP719. Ring B closure has previously been observed to occur at a higher rate than Ring A closure in the CYP719s of Argemone mexicana (EX41 vs. EX42)28. It is this difference in Ring A and Ring B closure rates that results in cheilanthifoline accumulation in vivo, which is then a substrate for TNMT to generate the undesirable side product N-methylcheilanthifoline.
To optimize the turnover of scoulerine to stylopine, there were two options considered. First, a Ring A-closing CYP719 that did not synthesize nandinine could be identified, avoiding the nandinine side-product. However, most CYP719s predicted to close Ring A of various protoberberines were able to accept scoulerine, limiting the number of Ring A-closers available to compare relative rates of activity in vivo. Alternatively, a Ring B-closing CYP719 could be identified which could accept both scoulerine and nandinine. Consequently, the Ring A product nandinine would no longer be a dead-end but an intermediate. As a result, any CYP719 capable of closing Ring A on scoulerine and/or cheilanthifoline would be a potential candidate for pathway optimization.
The 10 CYP719s with 95% activity on Ring A of scoulerine and the 10 CYP719s with activity on Ring B of scoulerine were tested for activity on cheilanthifoline and nandinine, respectively, in order to generate a branched stylopine synthesis pathway. As the Applicant did not have pure cheilanthifoline or nandinine, it generated these compounds through in vivo conversion of scoulerine. CYP719s PsCFS and EX101, capable of converting >98% of scoulerine to cheilanthifoline and nandinine, respectively, were incubated with scoulerine. After 16 h, cells were pelleted and the supernatant fraction was collected. The supernatant was then applied to fresh yeast strains in order to supplement them with either TE containing nandinine or cheilanthifoline as necessary.
7 of 10 CYP719s with >95% activity on Ring A of scoulerine converted >98% of cheilanthifoline to stylopine: EX42, EX50, EX56, EX67, EX76, EX96, EX101 (
Argemone mexicana
Argemone mexicana
Aquilegia formosa
Aquilegia formosa
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Corydalis cheilanthifolia
Coptis chinensis
Coptis japonica
Coptis japonica
Coptis japonica
Chelidonium majus
Chelidonium majus
Chelidonium majus
Chelidonium majus
Cissampelos mucronata
Cissampelos mucronata
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Eschscholzia californica
Glaucium flavum
Glaucium flavum
Glaucium flavum
Hydrastis canadensis
Mahonia aquifolium
Menispermum canadense
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nandina domestica
Nelumbo nucifera
Papaver bracteatum
Papaver bracteatum
Papaver bracteatum
Podophyllum peltatum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Papaver somniferum
Sanguinaria canadensis
Sanguinaria canadensis
Sanguinaria canadensis
Stylophorum diphyllum
Stylophorum diphyllum
Stylophorum diphyllum
Stylophorum diphyllum
Sinopodophyllum hexandrum
Thalictrum flavum
Thalictrum flavum
Xanthorhiza simplicissima
All CYP719s having been ordered pre-cloned into the same expression vector: pBOT-TRP, plasmids harboring CYP719s could not be co-transformed until some enzymes were expressed from a different auxotrophies. The pBOT expression cassettes were designed to be excisable via the restriction enzymes AscI and NotI. Therefore, pBOT-LEU was digested with AscI and NotI, and the expression cassettes of P1E6 and P2A2 were liberated from pBOT-TRP backbones via AscI and NotI digestion. Religation and transformation resulted in P1E6 and P2A2 expressed from pBOT-LEU. As a result, all CYP719s were ready for combinatorial testing.
CYP719s capable of closing Ring A and Ring B were co-transformed into either GC1333, harbouring PsCPR integrated into the genome (
When expressed individually, all Ring A-closing CYP719s converted >98% of scoulerine to nandinine, and all Ring B-closing CYP719s converted >98% of scoulerine to cheilanthifoline (
When individual CYP719s with Ring B-closing activity were expressed in combination with TNMT, >98% of scoulerine was converted to N-methylcheilanthifoline (
The side-product N-methylcheilanthifoline was not observed (<2% of extracted BIAs) when scoulerine was supplemented to yeast expressing various combinations of CYP719s with Ring A-closing and Ring B-closing activity in the presence of TNMT. Several combinations of CYP719s (e.g., EX54 and EX98 against EX42, EX50, EX67, EX76 and EX101) are expressed in the presence of Block 3 (TNMT, MSH, P6H) and supplemented with scoulerine in order to observe downstream products in the presence of a larger number of heterologous enzymes. Yields are expected to increase when these genes are combined with the rest of the sanguinarine pathway as described herein.
The screens of purchased CYP719s herein have been focused on the synthesis of N-methylstylopine for the purpose of optimization of dihydrosanguinarine yields. In the process, combinations of TNMT and purchased CYP719s were used to efficiently generate a variety of N-methylated and unmethylated protoberberines: cheilanthifoline, nandinine, stylopine, N-methylscoulerine, N-methylnandinine, N-methylcheilanthifoline, and N-methylstylopine.
Other activities of CYP719s on protoberberines have previously been published, such as the Ring A closure of scoulerine-derived tetrahydrocolumbamine to produce canadine. Scoulerine is methylated by scoulerine O-methyl transferase (SOMT) to generate tetrahydrocolumbamin72. CYP719-catalyzed Ring A-closing of tetrahydrocolumbamine produces canadine, which can be methylated by TNMT to generate N-methylcanadine, a precursor to noscapine. The presence of both a Ring A-closing CYP719 and TNMT in this pathway will also require CYP719 optimization for efficient yields of noscapine in a microbial host.
The major route for the synthesis of (R)-reticuline in P. somniferum is considered to be epimerization from (S)-reticuline, which was proposed to proceed via dehydrogenation of (S)-reticuline to 1,2-dehydroreticuline and subsequent enantioselective reduction to (R)-reticuline. However, the genes encoding these enzymes have never been cloned and those reaction never fully characterized60-61. It should however be noted that (R)-reticuline is not the only (R)-BIA intermediate found in Ranunculales62. This suggests the possibility of an alternative pathway for the synthesis of (R)-intermediates, possibly the existence of enzymes selective for the (R)-enantiomers from the very beginning of the reticuline synthesis pathway. For example, both (S)- and (R)—N-methylcoclaurine were isolated in Berberis stolonifera. These two enantiomers of N-methylcoclaurine are required by the cytochrome P450 berbamunine synthase for the synthesis of berbamunine in Berberis stolonifera62.
While P. somniferum does not make (R,S)-norlaudanosoline, results presented herein indicate that only (S)-reticuline is produced from racemic norlaudanosoline using opium poppy's native methyltransferases. Some evidence for the enantioselectivity of MTs involved in BIA synthesis can be found in the literature. For example, a study reporting on the activity of Coptis japonica MTs for the production of reticuline from racemic norlaudanosoline in engineered E. coli reported a prevalent synthesis of (S)-reticuline over (R)-reticuline18. This data clearly indicated that some of the C. japonica MTs have a preference for the (S)-enantiomer with limited activity on the (R)-enantiomer. It is therefore possible that MTs strictly enantioselective for the (R)-enantiomer exist and the epimerization to the (R)-enantiomers happens upstream reticuline.
Cytochrome b5 has been reported to enhance activity of certain cytochrome P450s48. Tuning expression of the four P450s, CPR and cognate cytochrome b5 could increase pathway efficiency. The impact of cytochrome b5 on yield is tested by expressing b5 in a plasmid or integrated in a chromosome in host cells expressing block(s) 1, 1-2, 2-3 or 1-2-3.
The high number of P450s expressed the cells may be affecting yields of dihydrosanguinarine. (S)-Scoulerine fed to Block 2-Block 3 integrant strains expressing four P450s yielded the same conversion to dihydrosanguinarine whether or not Block 1 and BBEΔN-2μ were expressed (7.5% vs. 7.7;
Synthesis of the side products N-methylcheilanthifoline and N-methylscoulerine by TNMT was shown to be a major limiting factor in the reconstituted pathway. Promiscuity is a common theme among enzymes involved in plant specialized metabolism and is one of the factors contributing to the great chemodiversity of plant secondary metabolites41. While broad substrate specificity of PsTNMT had been previously described34, the applicants present the first experimental evidence of its acceptance of scoulerine and cheilanthifoline as substrates. The present invention encompasses the use of orthologous plant TNMT enzymes with narrower substrate specificity, enzyme engineering42, mutagenesis, substrate channeling and/or spatio-temporal sequestration of the reactions43,44. TNMT orthologues as shown in
While N-methylscoulerine and N-methylcheilanthifoline are undesirable side-products, they may themselves be end products of interest. Both compounds are quaternary benzylisoquinoline alkaloids like sanguinarine and berberine. N-methylscoulerine (cyclanoline) can be extracted from several plants of the genus Stephania and has been described as an acetylcholinesterase inhibitor45, but to the best of the applicants' knowledge N-methylcheilanthifoline has never been detected in plants. The promiscuity of TNMT could also be further explored to generate other quaternary benzylisoquinoline alkaloids. Synthesis of N-methylcheilanthifoline, although serendipitous, highlights the potential of combinatorial biology in S. cerevisiae, through which libraries of alkaloids can be generated independent of their abundance in nature.
Synthesis of (S)-reticuline from glucose and glycerol has been reported in E. coli18,19 but not in S. cerevisiae. Thus, supplemented (R,S)-norlaudanosoline was provided to measure the efficiency of the reconstituted BIA pathway. The applicants observed that just 10% of fed norlaudanosoline was detected in the cell extract after 16 hours of incubation with the negative control yeast cells (
Other activities of CYP719s on protoberberines have previously been published, such as the Ring A closure of scoulerine-derived tetrahydrocolumbamine (THC) to produce canadine. In particular, CYP719s catalyzing the Ring A closure of THC to produce canadine have been described in P. somniferum, C. japonica, A. mexicana, and E. californica68. Scoulerine is methylated by scoulerine-O-methyltransferase (SOMT) to generate tetrahydrocolumbamine. CYP719A13 from Argemone mexicana was shown to catalyze the Ring A closure of both cheilanthifoline and tetrahydrocolumbamine28 strongly indicating that the CYP719 library in
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
This application is a PCT application Serial No. PCT/CA2015/0* filed on Jan. 13, 2015 and published in English under PCT Article 21(2), which itself claims benefit of U.S. provisional application Ser. No. 61/926,648, filed on Jan. 13, 2014. All documents above are incorporated herein in their entirety by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2015/050021 | 1/13/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61926648 | Jan 2014 | US |
