Methods and cell lines for producing phytocannabinoids and phytocannabinoid analogs in yeast
阅读说明：本技术 用于在酵母中生产植物***素和植物***素类似物的方法和细胞系 (Methods and cell lines for producing phytocannabinoids and phytocannabinoid analogs in yeast ) 是由 肖哈姆·慕克吉 亚历山大·詹姆斯·坎贝尔 扎卡里·道格拉斯·威尔特希尔 凯文·约翰·陈 于 2018-02-19 设计创作，主要内容包括：用于在酵母中生产植物大麻素和植物大麻素类似物的方法和细胞系。该方法应用,以及该细胞系包括,用聚酮化合物合成酶CDS和胞质异戊烯基转移酶CDS转化的酵母细胞。聚酮化合物合成酶催化橄榄醇或甲基橄榄醇的合成,并且可包括大麻橄榄醇酸合成酶或盘基网柄菌聚酮化合物合成酶(“DiPKS”)。可修饰酵母细胞,以包括磷酸泛酰巯基乙胺基转移酶,用于增加DiPKS的活性。可修饰酵母细胞,以减轻线粒体乙醛分解代谢,用于增加合成橄榄醇或甲基橄榄醇可用的丙二酰基辅酶A。异戊烯基转移酶催化大麻萜酚或大麻萜酚类似物的合成,并且可包括来自链霉菌属CL190的αββα胞质异戊烯基转移酶。可修饰酵母细胞,以减轻焦磷酸香叶酯的消耗,用于增加用于异戊烯化可用的焦磷酸香叶酯。(Methods and cell lines for the production of phytocannabinoids and phytocannabinoid analogs in yeast. The method uses, and the cell line comprises, yeast cells transformed with polyketide synthase CDS and cytoplasmic prenyltransferase CDS. Polyketide synthases catalyze the synthesis of olivine or methylolivine and may include cannabinol synthase or dictyostelium discodermatum polyketide synthase ("dicks"). Yeast cells can be modified to include a phosphopantetheinyl transferase for increasing the activity of dicks. Yeast cells can be modified to reduce mitochondrial acetaldehyde catabolism for increased malonyl-coa available for synthesis of olivetol or methyl olivetol. The prenyltransferase catalyzes the synthesis of cannabigerol or cannabigerol analogs, and may include an α β α cytosolic prenyltransferase from streptomyces CL 190. Yeast cells can be modified to mitigate geranyl pyrophosphate consumption for increasing the available geranyl pyrophosphate for prenylation.)
1. A method of producing a phytocannabinoid or a phytocannabinoid analog, the method comprising:
providing a yeast cell comprising a first polynucleotide encoding a polyketide synthase and a second polynucleotide encoding a cytosolic prenyltransferase, wherein:
the polyketide synthase is for producing at least one precursor chemical from malonyl-coa, the precursor chemical having structure I:
wherein, in structure I, R1 is an alkyl group having a chain length of 1, 2, 3, 4, or 5 carbons, R2 is H, carboxy, or methyl, and R3 is H, carboxy, or methyl; and is
The cytosolic prenyltransferase is used to prenylate the at least one precursor chemical to provide at least one species of phytocannabinoid or phytocannabinoid analog; and
propagating the yeast cells for providing a yeast cell culture.
2. The method of claim 1, wherein:
the yeast cell comprises a third polynucleotide encoding a hexanoyl synthase;
the polyketide synthase comprises an OAS enzyme from cannabis; and is
Propagating the yeast cell comprises propagating the yeast cell in a nutritional formulation comprising hexanoic acid.
3. The method of claim 2, wherein the yeast cells do not comprise cannabis polyketide cyclase and the at least one species of phytocannabinoid or phytocannabinoid analog comprises a decarboxylated phytocannabinoid or phytocannabinoid analog.
4. The method of any one of claims 2 or 3, wherein the first polynucleotide comprises a coding sequence for the OAS enzyme from Cannabis, the OAS enzyme having a primary structure with between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 3841 to 4995 of SEQ ID NO: 45.
5. The method of claim 4, wherein the first polynucleotide has between 80% and 100% base sequence homology with bases 3841 to 4995 of SEQ ID NO 45.
6. The method of any one of claims 1 to 5, wherein R1 is alkyl having a chain length of 3 carbons, R2 is H, and R3 is H.
7. The method of any one of claims 1 to 5, wherein R1 is alkyl having a chain length of 3 carbons, R2 is carboxyl, and R3 is H.
8. The method of any one of claims 1 to 5, wherein R1 is alkyl having a chain length of 3 carbons, R2 is methyl, and R3 is H.
9. The method of any one of claims 1 to 5, wherein R1 is an alkyl group having a chain length of 3 carbons, R2 is a carboxyl group, and R3 is a methyl group.
10. The method of claim 1, wherein the polyketide synthase comprises a DiPKS polyketide synthase from Dictyotanus teres.
11. The method of claim 10, wherein the first polynucleotide comprises a coding sequence for the DiPKS polyketide synthase having a primary structure with between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 535 to 9978 of SEQ ID NO 46.
12. The method of claim 11, wherein said first polynucleotide has between 80% and 100% base sequence homology with bases 535 to 9978 of SEQ ID No. 46.
13. The method according to any one of claims 10 to 12, wherein the at least one precursor chemical comprises a methyl group at R2 and the at least one species of phytocannabinoid or phytocannabinoid analog comprises a methylated phytocannabinoid analog.
14. The method of claim 10, wherein:
the DiPKS polyketide synthase includes a mutation that affects an active site of a C-Met domain for mitigating methylation of the at least one precursor chemical, such that the at least one precursor chemical includes a first precursor chemical where R2 is methyl and R3 is H, and a second precursor chemical where R2 is H and R3 is H; and is
The at least one species of phytocannabinoid or phytocannabinoid analogue comprises a methylated phytocannabinoid analogue and an unmethylated phytocannabinoid.
15. The method of claim 14, wherein the DiPKS polyketide synthase comprises DiPKSG1516D ；G1518APolyketide synthase.
16. The method of claim 15, wherein the first polynucleotide is comprised for the DiPKSG1516D；G1518ACoding sequence of polyketide synthase, said DiPKSG1516D；G1518AThe primary structure of the polyketide synthase has between 80% and 100% amino acid residue sequence homology with the protein encoded by the reading frame defined by bases 523 to 9966 of SEQ ID NO 37.
17. The method of claim 16, wherein the first polynucleotide has between 80% and 100% base sequence homology with bases 523 to 9966 of SEQ ID No. 37.
18. The method of claim 14, wherein the DiPKS polyketide synthase comprises DiPKSG1516RPolyketide synthase.
19. The method of claim 18, wherein the first polynucleotide comprises a polynucleotide for the DiPKSG1516RCoding sequence of polyketide synthase, said DiPKSG1516RThe primary structure of the polyketide synthase has between 80% and 100% amino acid residue sequence homology with the protein encoded by the reading frame defined by bases 523 to 9966 of SEQ ID NO 38.
20. The method of claim 19, wherein said first polynucleotide has between 80% and 100% base sequence homology with bases 523 to 9966 of SEQ ID No. 38.
21. The method of claim 10, wherein:
the DiPKS polyketide synthase comprises a mutation that reduces activity at an active site of the C-Met domain of the DiPKS polyketide synthase for preventing methylation of the at least one precursor chemical such that the at least one precursor chemical has a hydrogen R2 group and a hydrogen R3 group; and is
The at least one species of phytocannabinoid or phytocannabinoid analogue comprises a decarboxylated phytocannabinoid or phytocannabinoid analogue.
22. The method of any one of claims 10-21, wherein the yeast cell comprises a third polynucleotide encoding a phosphopantetheinyl transferase for increasing activity of a DiPKS.
23. The method of claim 22, wherein said phosphopantetheinyl transferase comprises an NpgA phosphopantetheinyl transferase from aspergillus nidulans.
24. The method according to claim 23, wherein the third polynucleotide comprises a coding sequence for the NpgA phosphopantetheinyl transferase from aspergillus nidulans having a primary structure with between 80% and 100% amino acid residue sequence homology to the protein encoded by the reading frame defined by bases 1170 to 2201 of SEQ ID No. 10.
25. The method of claim 24, wherein the third polynucleotide has between 80% and 100% base sequence homology with bases 1170 to 2201 of SEQ ID No. 10.
26. The method of any one of claims 1 to 25, wherein the polyketide synthase includes an active site for synthesis of the at least one precursor chemical from malonyl coa without requiring longer chain carbonyl coa.
27. The method of claim 26, wherein the at least one precursor chemical comprises a pentyl group at R1 and the at least one species of phytocannabinoid or phytocannabinoid analog comprises a pentyl phytocannabinoid or a methylated pentyl phytocannabinoid analog.
28. The method of claim 27, wherein the at least one precursor chemical comprises at least one of olivinic acid, methyl olivinic acid, or methyl olivinic acid, and the at least one species of phytocannabinoid or phytocannabinoid analog comprises at least one of CBG, CBGa, meCBG, or meCBGa.
29. The method of any one of claims 1-28, wherein said cytosolic prenyltransferase comprises an NphB prenyltransferase from streptomyces CL 190.
30. The method of claim 29, wherein the second polynucleotide comprises a coding sequence for an NphB prenyltransferase from streptomyces CL190 having a primary structure with between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 987 to 1913 of SEQ ID No. 44.
31. The method of claim 30, wherein said second polynucleotide has between 80% and 100% base sequence homology with bases 987 to 1913 of SEQ ID No. 44.
32. The method of any one of claims 1 to 31, wherein R1 is alkyl having a chain length of 5 carbons, R2 is H, and R3 is H.
33. The method of any one of claims 1 to 31, wherein R1 is alkyl having a chain length of 5 carbons, R2 is carboxyl, and R3 is H.
34. The method of any one of claims 1 to 31, wherein R1 is alkyl having a chain length of 5 carbons, R2 is methyl, and R3 is H.
35. The method of any one of claims 1 to 31, wherein R1 is an alkyl group with a chain length of 5 carbons, R2 is a carboxyl group, and R3 is a methyl group.
36. The method of any one of claims 1-35, wherein the yeast cell comprises a genetic modification that increases the availability of geranyl pyrophosphate.
37. The method of claim 36, wherein the genetic modification comprises inactivation of the Erg20 enzyme.
38. The method of claim 37, wherein the yeast cell comprises a third polynucleotide comprising a polynucleotide for Erg20K197EThe coding sequence of Erg20K197EHas a sequence homology of between 80% and 100% amino acid residues with the protein encoded by the reading frame defined by SEQ ID NO 3.
39. The method of claim 38, wherein the third polynucleotide has between 80% and 100% base sequence homology with SEQ ID No. 3.
40. The method of any one of claims 1 to 39, wherein the yeast cell comprises a genetic modification that increases available malonyl-coa.
41. The method of claim 40, wherein the genetic modification comprises increased expression of Maf 1.
42. The method of claim 41, wherein the yeast cell comprises a third polynucleotide comprising a coding sequence for Maf1, the primary structure of Maf1 having between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 936 to 2123 of SEQ ID NO 8.
43. The method of claim 42, wherein said third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to SEQ ID NO 8.
44. The method of claim 40, wherein the genetic modification comprises a modification to increase cytosolic expression of aldehyde dehydrogenase and acetyl-CoA synthetase.
45. The method of claim 44, wherein the yeast cell comprises a third polynucleotide comprising Acs for Salmonella entericaL641PAnd the coding sequence for Ald6 from Saccharomyces cerevisiae, said AcsL641PHas between 80% and 100% amino acid residue sequence homology with the protein encoded by the reading frame defined by bases 3938 to 5893 of SEQ ID No. 4, and said primary structure of Ald6 has between 80% and 100% amino acid residue sequence homology with the protein encoded by the reading frame defined by bases 1494 to 2999 of SEQ ID No. 4.
46. The method of claim 45, wherein the third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to bases 51 to 7114 of SEQ ID NO. 4.
47. The method of claim 40, wherein the genetic modification comprises a modification to increase malonyl-coa synthetase activity.
48. The method of claim 47, wherein the yeast cell comprises a third polynucleotide comprising Acc1 for Saccharomyces cerevisiaeS659A；S1167AThe coding sequence of (a).
49. The method of claim 48, wherein said third polynucleotide comprises a polynucleotide for said Acc1S659A；S1167ACoding sequence of an enzyme, Acc1S659A；S1167AThe primary structure of a part of the enzyme is shown by SEQ ID NO 7Acc1S659A；S1167AThe reading frame defined by bases 9 to 1716 encodes a protein portion having between 80% and 100% amino acid residue sequence homology.
50. The method of claim 49, wherein said third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to SEQ ID NO. 7.
51. The method of claim 47, wherein the yeast cell comprises a third polynucleotide comprising a coding sequence for Acc1 from Saccharomyces cerevisiae that is regulated by a constitutive promoter.
52. The method of claim 51, wherein said constitutive promoter comprises PGK1 promoter from Saccharomyces cerevisiae.
53. The method of claim 52, wherein said PGK1 promoter has between 80% and 100% nucleotide homology with bases 7 to 750 of SEQ ID NO 6.
54. The method of claim 40, wherein the genetic modification comprises increased expression of an activator for sterol biosynthesis.
55. The method of claim 54, wherein the yeast cell comprises a third polynucleotide comprising Upc2 for use from Saccharomyces cerevisiaeE888DThe coding sequence of Upc2E888DHas a sequence homology of between 80% and 100% amino acid residues with the protein encoded by the reading frame defined by bases 975 to 3701 of SEQ ID NO 9.
56. The method according to claim 55, wherein the third polynucleotide further comprises a promoter sequence, a terminator sequence and an integration sequence and has between 80% and 100% base sequence homology to SEQ ID NO 9.
57. The method of any one of claims 1 to 28 or 31 to 56, wherein the second polynucleotide comprises a coding sequence for a cytoplasmic prenyltransferase having a primary structure with between 80% and 100% amino acid residue sequence homology to any one of: 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.
58. The method of any one of claims 1-57, further comprising extracting the at least one species of phytocannabinoid or phytocannabinoid analog from the yeast cell culture.
59. A yeast cell for producing a phytocannabinoid or a phytocannabinoid analog, the yeast cell comprising:
a first polynucleotide encoding a polyketide synthase; and
a second polynucleotide encoding a cytosolic prenyltransferase.
60. The yeast cell of claim 59, further comprising the features of one or more of the yeast cell, the first polynucleotide, or the second polynucleotide as claimed in the yeast cell as provided in connection with any one of method claims 1 to 57.
61. A method of transforming a yeast cell for the production of a phytocannabinoid or a phytocannabinoid analog, the method comprising:
introducing a first polynucleotide encoding a polyketide synthase into a yeast cell line; and
introducing a second polynucleotide encoding a cytosolic prenyltransferase into the yeast.
62. The method according to claim 61, further comprising one or more of the features of the yeast cell, the first polynucleotide or the second polynucleotide as claimed in conjunction with the yeast cell provided in any one of method claims 1 to 57.
63. A phytocannabinoid analog having the following structure II:
wherein, in structure II, R1 is an alkyl group having a chain length of 1, 2, 3, 4, or 5 carbons;
r2 is methyl; and is
R3 is H, carboxy or methyl.
64. The phytocannabinoid analog according to claim 63, wherein R1 has a chain length of 5 carbons and R3 is H.
65. The phytocannabinoid analog of claim 63, wherein the phytocannabinoid analog is produced by biosynthesis in yeast.
66. A phytocannabinoid analog having the following structure III:
wherein, in structure III, R1 is pentyl;
r2 is methyl; and is
R3 is H.
Technical Field
The present disclosure relates generally to the production of phyto-cannabinoids and analogues of phyto-cannabinoids in yeast.
Background
Phytocannabinoids are naturally produced in Cannabis sativa (Cannabis sativa), other plants and some fungi. Over 105 phytocannabinoids are known to be either biosynthesized in cannabis or derived from the thermal or other decomposition of phytocannabinoids biosynthesized in cannabis. While cannabis plants are also a valuable source of grain, fiber and other materials, planting cannabis for the production of phytocannabinoids, particularly indoor planting, is energy and labor intensive. Subsequent extraction, purification and fractionation of phytocannabinoids from the cannabis plant is also labor and energy intensive.
Phytocannabinoids are pharmacologically active molecules that contribute to the medicinal and psychotherapeutic effects of cannabis. The biosynthesis of phytocannabinoids in the cannabis plant is similar in scale to other agricultural projects. As with other agricultural projects, the large-scale production of phytocannabinoids by planting cannabis requires various inputs (e.g. nutrients, light, pest control, CO)2Etc.). The investment required for the cultivation of hemp must be provided. Furthermore, where permitted, the breeding of cannabis is currently subject to strict regulations, taxes and strict quality controls, further increasing costs, where products prepared from the plant are used for commercial purposes. Phytocannabinoid analogs are pharmacologically active molecules structurally similar to phytocannabinoids. Plant cannabinoid analogs are typically chemically synthesized, which can be labor intensive and expensive. Thus, it may be economical to produce phytocannabinoids and phytocannabinoid analogs in robust and scalable fermentable organisms. Saccharomyces cerevisiae (Saccharomyces cerevisiae) is an example of a fermentable organism that has been used to produce similar molecules on an industrial scale.
The time, energy and labor involved in planting cannabis for the production of naturally occurring phytocannabinoids provides an incentive to prepare transgenic cell lines for the production of phytocannabinoids in yeast. An example of such an effort is provided in U.S. patent application publication No. US 2016/0010126 to Poulos and Farnia.
Disclosure of Invention
It is an object of the present disclosure to obviate or mitigate at least one disadvantage of previous methods of producing phytocannabinoid in yeast and previous methods of producing phytocannabinoid analogs. Many of the 105 phytocannabinoids found in cannabis may be synthesized in yeast and it may be desirable to improve yeast-based production. Similarly, methods that allow the production of phytocannabinoid analogs without the need for labor-intensive synthesis would be desirable.
The methods and cell lines provided herein are applicable and include transgenic s.cerevisiae that has been transformed with a gene encoding NphB prenyltransferase ("AltPT") from Streptomyces coelicolor ("CL 190"). AltPT is an α β β α ("ABBA") type prenyltransferase. AltPT catalyzes the synthesis of cannabigerolic acid ("CBGa") from olivinic acid and geranyl pyrophosphate ("GPP"). AltPT also catalyzes the synthesis of cannabigerol ("CBG") from olivine and GPP. In cannabis, prenyltransferases catalyze the synthesis of CBGa from olive alcohol and GPP. Cannabis prenyltransferases are membrane bound, complicating expression in s.cerevisiae. In contrast, AltPT is cytosolic and is expressed at higher levels in saccharomyces cerevisiae than cannabiprenyltransferase. AltPT, when expressed in saccharomyces cerevisiae to catalyze the synthesis of CBGa from olivinic acid and GPP or CBG from olivine and GPP, may offer advantages over membrane-bound cannabiprenyltransferase. S. cerevisiae may have one or more mutations in Erg20, Maf1, or UPC2, or other genes that support enzymes or other proteins that deplete the metabolic pathways of GPP, that serve to increase available GPP. Alternatively, other species of yeast may be used, including Yarrowia lipolytica (Yarrowia lipolytica), Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces lactis (Kluyveromyces lactis), Rhodosporidium toruloides (Rhodosporidium toruloides), Cryptococcus curvatus (Cryptococcus curvatus), Trichosporon pullulans (Trichosporon), and Lipomyces oleosus (Lipomyces lipofect).
In some of the methods and cell lines provided herein, the transgenic saccharomyces cerevisiae includes genes for cannabis polyketide synthase (also known as olivine synthase or "OAS"). OAS catalyzes the synthesis of olivine from malonyl-coa and hexanoyl-coa. The reaction has a stoichiometric ratio of malonyl-coa to hexanoyl-coa to olivetol of 2:1: 1. In cannabis, olivine is carboxylated to olivine acid in the presence of olivine acid cyclase ("OAC") or another polyketide cyclase, and olivine acid is sent to the CBGa anabolic pathway catalyzed by membrane-bound isopentenyl transferases in cannabis as described above in connection with AltPT and other cytosolic isopentenyl transferases. The OAC enzyme from cannabis may be excluded from transgenic saccharomyces cerevisiae to facilitate the synthesis of CBG instead of CBGa by AltPT.
In some of the methods and cell lines provided herein, the transgenic saccharomyces cerevisiae includes genes for Dictyostelium discodermium polyketide synthase ("DiPKS"). The DiPKS is a fusion protein composed of a type I fatty acid synthase ("FAS") and a polyketide synthase, and is referred to as a hybrid "FAS-PKS" protein. DiPKS catalyzes the synthesis of methyl olivetol from malonyl-coa. The reaction was carried out with a stoichiometric ratio of malonyl-coa to methylolivil of 6: 1. AltPT catalyses the synthesis of methyl cannabigerol ("meCBG") from methyl olivetol, similar to the synthesis of CBG from olivetol described above. Caproic acid is toxic to Saccharomyces cerevisiae. When OAS is used, hexanoyl-CoA is an essential precursor for the synthesis of olivetol. The absence of hexanoic acid in the growth medium may lead to increased growth of the Saccharomyces cerevisiae culture and higher production of mecBG compared to the CBG production when OAS is used.
For some applications meCBG and methylated downstream phytocannabinoid analogs that can be synthesized from meCBG (similar to downstream phytocannabinoids synthesized from CBGa in cannabis) are of value. In other cases, phytocannabinoids that are structurally identical to the decarboxylated form of the naturally occurring phytocannabinoids may be more desirable. To produce phytocannabinoids that are structurally identical to the decarboxylated form of the naturally occurring phytocannabinoid, the DiPKS may be modified relative to the wild-type DiPKS to reduce methylation of olive alcohol, such that CBG is synthesised rather than meCBG. S.cerevisiae may include a cofactor loading enzyme that increases the activity of the DiPKS.
Synthesis of olivetol and methyl olivetol is promoted by increasing the level of malonyl-coa in the cytoplasm. Saccharomyces cerevisiae may have overexpression of native acetaldehyde dehydrogenase and expression of mutant acetyl-CoA synthetases or other genes, the mutations resulting in reduced mitochondrial acetaldehyde catabolism. Reducing mitochondrial acetaldehyde catabolism by transferring acetaldehyde to acetyl-coa production increases the malonyl-coa available for synthesis of olive alcohol. Acc1 is a natural yeast malonyl-coenzyme A synthetase. S.cerevisiae may have overexpression of Acc1 or modification of Acc1 for increased activity and increased available malonyl-coa. Saccharomyces cerevisiae may include modified expression of Maf1 or other modulators of tRNA biosynthesis. Overexpression of native Maf1 has been shown to reduce the loss of isopentyl pyrophosphate ("IPP") of tRNA biosynthesis, thereby increasing monoterpene production in yeast. IPP is an intermediate of the mevalonate pathway. Upc2 is an activator of sterol biosynthesis in Saccharomyces cerevisiae and the Glu888Asp mutation of Upc2 increases monoterpene production in yeast.
In a first aspect, provided herein are methods and cell lines for the production of phytocannabinoids and phytocannabinoid analogs in yeast. The method applies and the cell line comprises a yeast cell transformed with a polyketide synthase CDS and a cytoplasmic prenyltransferase CDS. Polyketide synthases catalyze the synthesis of olivine or methylolivine and may include cannabinol synthase or dictyostelium discodermatum polyketide synthase ("dicks"). Yeast cells can be modified to include a phosphopantetheinyl transferase for increasing the activity of DiPKS. Yeast cells can be modified to reduce mitochondrial acetaldehyde catabolism for increasing malonyl-coa available for synthesis of olivetol or methyl olivetol. The prenyltransferase catalyzes the synthesis of cannabigerol or cannabigerol analogs, and may include an α β α cytosolic prenyltransferase from streptomyces CL 190. Yeast cells can be modified to mitigate geranyl pyrophosphate consumption for increasing prenylation available geranyl pyrophosphate.
In a further aspect, provided herein is a method of producing a phytocannabinoid or a phytocannabinoid analog, the method comprising: providing a yeast cell comprising a first polynucleotide encoding a polyketide synthase and a second polynucleotide encoding a cytosolic prenyltransferase, and propagating the yeast cell for providing a yeast cell culture. The polyketide synthase is for producing at least one precursor chemical from malonyl-coa, the precursor chemical having structure I:
In some embodiments, the yeast cell comprises a third polynucleotide encoding a hexanoyl synthase; polyketide synthases include OAS enzymes from cannabis; and propagating the yeast cell comprises propagating the yeast cell in a nutritional formulation comprising hexanoic acid. In some embodiments, the yeast cell does not comprise a cannabis polyketide cyclase and the at least one class of phytocannabinoids or phytocannabinoid analogs comprises a decarboxylated phytocannabinoid or phytocannabinoid analog. In some embodiments, the first polynucleotide comprises a coding sequence for an OAS enzyme from cannabis, wherein the primary structure of the OAS enzyme has between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 3841 to 4995 of SEQ ID NO: 45. In some embodiments, the first polynucleotide has between 80% and 100% base sequence homology with bases 3841 to 4995 of SEQ ID NO. 45. In some embodiments, the first polynucleotide has between 80% and 100% base sequence homology with bases 3841 to 4995 of SEQ ID NO. 45.
In some embodiments, R1 is an alkyl group with a chain length of 3 carbons, R2 is H, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 3 carbons, R2 is a carboxyl group, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 3 carbons, R2 is methyl, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 3 carbons, R2 is a carboxyl group, and R3 is a methyl group.
In some embodiments, the polyketide synthase comprises a DiPKS polyketide synthase from dictyostelium discodermatum. In some embodiments, the first polynucleotide comprises a coding sequence for a DiPKS polyketide synthase, wherein the DiPKS polyketide synthase has a primary structure with between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 535 to 9978 of SEQ ID No. 46. In some embodiments, the first polynucleotide has between 80% and 100% base sequence homology with bases 535 to 9978 of SEQ ID NO. 46. In some embodiments, the at least one precursor chemical comprises a methyl group at R2, and the at least one species of phytocannabinoid or phytocannabinoid analog comprises a methylated phytocannabinoid analog. In some embodiments, the DiPKS polyketide synthase includes a mutation that affects an active site of the C-Met domain for mitigating methylation of at least one precursor chemical, such that the at least one precursor chemical includes a first precursor chemical where R2 is methyl and R3 is H, and a second precursor chemical where R2 is H and R3 is H; and the at least one species of phytocannabinoid or phytocannabinoid analogue comprises a methylated phytocannabinoid analogue and an unmethylated phytocannabinoid. In some embodiments, the DiPKS polyketide synthase comprises DiPKSG 1516D; G1518A polyketide synthase. In some embodiments, the first polynucleotide comprises the polynucleotide for DiPKSG 1516D; G1518A polyketide synthase, wherein DiPKSG 1516D; the primary structure of the G1518A polyketide synthase has between 80% and 100% amino acid residue sequence homology with the protein encoded by the reading frame defined by bases 523 to 9966 of SEQ ID NO: 37. In some embodiments, the first polynucleotide has between 80% and 100% base sequence homology with bases 523 to 9966 of SEQ ID NO: 37. In some embodiments, the DiPKS polyketide synthase comprises a DiPKSG1516R polyketide synthase. In some embodiments, the first polynucleotide comprises a coding sequence for a dipsg 1516R polyketide synthase having a primary structure with between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 523 to 9966 of SEQ ID NO: 38. In some embodiments, the first polynucleotide has between 80% and 100% base sequence homology with bases 523 to 9966 of SEQ ID NO. 38. In some embodiments, the DiPKS polyketide synthase comprises a mutation that reduces activity at an active site of a C-Met domain of the DiPKS polyketide synthase for preventing methylation of at least one precursor chemical such that the at least one precursor chemical has a hydrogen R2 group and a hydrogen R3 group; and at least one class of phytocannabinoids or phytocannabinoid analogues comprises decarboxylated phytocannabinoids or phytocannabinoid analogues. In some embodiments, the yeast cell comprises a third polynucleotide encoding a phosphopantetheinyl transferase, for increasing activity of a DiPKS. In some embodiments, the phosphopantetheinyl transferase comprises an NpgA phosphopantetheinyl transferase from aspergillus nidulans (a. nidulans). In some embodiments, the third polynucleotide comprises a coding sequence for an NpgA phosphopantetheinyl transferase from aspergillus nidulans, wherein the primary structure of the NpgA phosphopantetheinyl transferase has between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 1170 to 2201 of SEQ ID NO: 10. In some embodiments, the third polynucleotide has between 80% and 100% base sequence homology with bases 1170 to 2201 of SEQ ID NO. 10.
In some embodiments, the polyketide synthase includes an active site for the synthesis of at least one precursor chemical from malonyl-coa without the need for longer chain carbonyl-coa. In some embodiments, the at least one precursor chemical comprises a pentyl group at R1, and the at least one species of phytocannabinoid or phytocannabinoid analog comprises a pentyl phytocannabinoid or a methylated pentyl phytocannabinoid analog. In some embodiments, the at least one precursor chemical comprises at least one of olivetol, methyl olivetol or methyl olivetol acid, and the at least one species of phytocannabinoid or phytocannabinoid analog comprises at least one of CBG, CBGa, meCBG or meCBGa.
In some embodiments, the cytosolic prenyltransferase comprises an NphB prenyltransferase from streptomyces CL 190. In some embodiments, the second polynucleotide comprises a coding sequence for an NphB prenyltransferase from streptomyces CL190, wherein the primary structure of the NphB prenyltransferase has between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 987 to 1913 of SEQ ID No. 44. In some embodiments, the second polynucleotide has between 80% and 100% base sequence homology with bases 987 to 1913 of SEQ ID NO. 44.
In some embodiments, R1 is an alkyl group with a chain length of 5 carbons, R2 is H, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 5 carbons, R2 is a carboxyl group, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 5 carbons, R2 is methyl, and R3 is H.
In some embodiments, R1 is an alkyl group with a chain length of 5 carbons, R2 is a carboxyl group, and R3 is a methyl group.
In some embodiments, the yeast cell comprises a genetic modification that increases the availability of geranyl pyrophosphate. In some embodiments, the genetic modification comprises inactivation of the Erg20 enzyme. In some embodiments, the yeast cell comprises a third polynucleotide comprising a coding sequence for Erg20K197E, wherein the primary structure of Erg20K197E has between 80% and 100% amino acid residue sequence homology to the protein encoded by the reading frame defined by seq id No. 3. In some embodiments, the third polynucleotide has between 80% and 100% base sequence homology with SEQ ID NO. 3.
In some embodiments, the yeast cell comprises a genetic modification that increases available malonyl-coa. In some embodiments, the genetic modification comprises increased expression of Mafl. In some embodiments, the yeast cell comprises a third polynucleotide comprising a coding sequence for Maf1, wherein the primary structure of Maf1 has between 80% and 100% amino acid residue sequence homology to a protein encoded by the reading frame defined by bases 936 to 2123 of SEQ ID NO: 8. In some embodiments, the third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to SEQ ID No. 8. In some embodiments, the genetic modification comprises a modification to increase cytosolic expression of aldehyde dehydrogenase and acetyl-coa synthetase. In some embodiments, the yeast cell comprises a third polynucleotide comprising a coding sequence for AcsL641P from salmonella enterica and a coding sequence for Ald6 from saccharomyces cerevisiae, wherein the primary structure of AcsL641P has between 80% and 100% amino acid residue sequence homology to the protein encoded by the reading frame defined by bases 3938 to 5893 of SEQ ID NO:4, and wherein the primary structure of Ald6 has between 80% and 100% amino acid residue sequence homology to the protein encoded by the reading frame defined by bases 1494 to 2999 of SEQ ID NO: 4. In some embodiments, the third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to bases 51 to 7114 of SEQ ID No. 4. In some embodiments, the genetic modification comprises a modification to increase malonyl-coa synthetase activity. In some embodiments, the yeast cell comprises a third polynucleotide comprising Acc1S659A for use from saccharomyces cerevisiae; S1167A. In some embodiments, the third polynucleotide comprises the polynucleotide for Acc1S 659A; a coding sequence for an S1167A enzyme, wherein Acc1S 659A; the primary structure of a portion of the S1167A enzyme has between 80% and 100% amino acid residue sequence homology with the portion of the protein encoded by the reading frame defined by bases 9 to 1716 of SEQ ID NO. 7. In some embodiments, the third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to SEQ ID No. 7. In some embodiments, the yeast cell comprises a third polynucleotide comprising a coding sequence for Acc1 from saccharomyces cerevisiae that is regulated by a constitutive promoter. In some embodiments, the constitutive promoter comprises the PGK1 promoter from saccharomyces cerevisiae. In some embodiments, the PGK1 promoter has between 80% and 100% nucleotide homology with bases 7 to 750 of SEQ ID NO. 6. In some embodiments, the genetic modification comprises increased expression of an activator for sterol biosynthesis. In some embodiments, the yeast cell comprises a third polynucleotide comprising a coding sequence for Upc2E888D from saccharomyces cerevisiae, wherein the primary structure of Upc2E888D has between 80% and 100% amino acid residue sequence homology to the protein encoded by the reading frame defined by bases 975 to 3701 of SEQ ID No. 9. In some embodiments, the third polynucleotide further comprises a promoter sequence, a terminator sequence, and an integration sequence, and has between 80% and 100% base sequence homology to SEQ ID No. 9.
In some embodiments, the second polynucleotide comprises a coding sequence for a cytosolic prenyltransferase, wherein the cytosolic prenyltransferase has a primary structure with between 80% and 100% amino acid residue sequence homology to any one of: 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36.
In some embodiments, the methods comprise extracting at least one species of phytocannabinoid or phytocannabinoid analog from a yeast cell culture.
In a further aspect, provided herein is a yeast cell for producing a phytocannabinoid or a phytocannabinoid analog, the yeast cell comprising: a first polynucleotide encoding a polyketide synthase; and a second polynucleotide encoding a cytosolic prenyltransferase.
In some embodiments, the features of one or more of the yeast cells, first polynucleotides, or second polynucleotides described herein are included in the yeast cells.
In a further aspect, provided herein is a method of transforming a yeast cell for the production of phytocannabinoids or phytocannabinoids. The method comprises the following steps: introducing a first polynucleotide encoding a polyketide synthase into a yeast cell line; and introducing into the yeast a second polynucleotide encoding a cytosolic prenyltransferase.
In some embodiments, the features of one or more of the yeast cell, the first polynucleotide, or the second polynucleotide described herein are applied to a transformed yeast cell.
In a further aspect, provided herein are phytocannabinoid analogs having the following structure II:in structure II, R1 is an alkyl group with a chain length of 1, 2, 3, 4, or 5 carbons. R2 is methyl. R3 is H, carboxy or methyl.
In some embodiments, R1 has a chain length of 5 carbons and R3 is H. In some embodiments, the phytocannabinoid analog is produced by biosynthesis in yeast.
In a further aspect, provided herein are phytocannabinoid analogs having the following structure III:
Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
Drawings
Embodiments of the present disclosure will now be described, by way of example only, with reference to the accompanying drawings.
FIG. 1 is a schematic representation of the biosynthesis of olivinic acid and related compounds with different alkyl chain lengths in cannabis;
FIG. 2 is a schematic representation of the biosynthesis of CBGa from hexanoic acid, malonyl-coa and geranyl pyrophosphate in cannabis;
FIG. 3 is a schematic representation of the biosynthesis of acid forms of downstream phyto-cannabinoids from CBGa in cannabis;
FIG. 4 is a schematic of CBG biosynthesis by OAS and AltPT in transformed yeast cells;
FIG. 5 is a schematic of the biosynthesis of downstream phytocannabinoids from CBGs in transformed yeast cells;
FIG. 6 is a schematic representation of the biosynthesis of mecBG by DiPKS and AltPT in transformed yeast cells;
FIG. 7 is a schematic representation of the biosynthesis of downstream methylated phytocannabinoid analogs from mecBG in transformed yeast cells;
FIG. 8 is a schematic representation of the biosynthesis of downstream methylated phytocannabinoid analogs from mecBG in transformed yeast cells;
FIG. 9 is a schematic of functional domains in DiPKS with a mutation in C-methyltransferase for reducing methylation of olive alcohol;
FIG. 10 is a graph of the expression by DiPKS in transformed yeast cellsG1516D；G1518AAnd a schematic representation of AltPT biosynthesis of meCBG and CBG;
FIG. 11 is a graph of the expression by DiPKS in transformed yeast cellsG1516RAnd a schematic of the biosynthesis of CBG by AltPT;
FIG. 12 shows the growth of Saccharomyces cerevisiae at different concentrations of hexanoic acid;
FIG. 13 shows Saccharomyces cerevisiae growth and olive alcohol production before and after introduction of hexanoic acid;
FIG. 14 shows yeast growth and CBG production before and after introduction of hexanoic acid;
FIG. 15 shows yeast growth and hexanoic acid consumption in Saccharomyces cerevisiae before and after introduction of hexanoic acid;
FIG. 16 shows cytoplasmic expression of cannabis membrane-bound prenyltransferase and AltPT in Saccharomyces cerevisiae.
FIG. 17 shows CBG production with cannabis OAS and AltPT and mecBG production with DiPKS and AltPT in Saccharomyces cerevisiae;
FIG. 18 shows the production of methylolivil by DiPKS, and by DiPKSG1516D；G1518AProducing both methyl olivetol and olivetol;
FIG. 19 shows the production of methylolivil by DiPKS in two separate strains of s.cerevisiae;
FIG. 20 shows the production of methylolivil by DiPKS in two separate strains of s.cerevisiae;
FIG. 21 shows the production of mecBG by AltPT in two separate strains of s.cerevisiae;
FIG. 22 shows the production of methylolivil by DiPKS and by DiPKS in two separate strains of s.cerevisiaeG1516RProducing both methyl olivetol and olivetol;
FIG. 23 shows the passage of DiPKS in three separate strains of s.cerevisiaeG1516RProducing the olive alcohol; and
FIG. 24 shows CBG production by Cannabis OAS and AltPT, mecBG production by DiPKS and AltPT, and DiPKS production by DiPKS in three Saccharomyces strainsG1516RAnd AltPT produces CBG.
Detailed Description
In general, the present disclosure provides methods and yeast cell lines for producing phytocannabinoids that are naturally biosynthetic in cannabis plants and methylated phytocannabinoid analogs that are biosynthetic from methylolivetol. Phyto-cannabinoids and phyto-cannabinoid analogs are produced in transgenic yeast. The methods and cell lines provided herein include the use of genes for enzymes not present in cannabis plants. The application of genes in cannabis plants outside the complete genome that encode enzymes that give phytocannabinoids in the biosynthetic pathway may provide one or more benefits, including the biosynthesis of decarboxylated phytocannabinoids, the biosynthesis of methylated phytocannabinoid analogs, and the biosynthesis of phytocannabinoids without the input of caproic acid, which is toxic to saccharomyces cerevisiae and other species of yeast.
The modifier "decarboxylated" as used herein refers to a form of phytocannabinoid or phytocannabinoid analog that lacks an acid group, for example at
FIG. 1 shows the biosynthesis of olivetol from the polyketide condensation product of malonyl-coenzyme A and hexanoyl-coenzyme A as occurs in cannabis. Olivonic acid is a metabolic precursor of CBGa. As described in further detail below, CBGa is a precursor to a number of downstream phytocannabinoids. In most varieties of cannabis, the majority of phytocannabinoids are amylcannabinoids that are biosynthesized from olivinic acid, which is synthesized from malonyl-coa and hexanoyl-coa in a 2:1 stoichiometric ratio. Some propyl cannabinoids are observed and are often identified with a "v" suffix in the widely used three-letter abbreviations (e.g., tetrahydrocannabivarin is commonly referred to as "THCv", cannabidiol is commonly referred to as "CBDv", etc.). Figure 1 also shows the biosynthesis of diphenolic acid (divarinolic acid) by condensation of malonyl-coa with n-butyl-coa, which will provide the downstream propyl phytocannabinoid.
Figure 1 also shows the biosynthesis of orcinol from the condensation of malonyl-coa with acetyl-coa, which provides downstream methyl phytocannabinoid. In this context, the term "methyl phytocannabinoids" means that their alkyl side chains are methyl, wherein most phytocannabinoids have a pentyl group on the alkyl side chain and phenolic acid (varinic) phytocannabinoids have a propyl group on the alkyl side chain. The context in which meCBG and other methylated phytocannabinoid analogs are referred to as "methylated" is different and parallel to the use of "methyl phytocannabinoid" as a prefix and "methyl" in figure 1. Similarly, methyl olivetol refers to methylation on the ring rather than to the shorter side chain, since olivetol has a side chain of defined length (which would otherwise be one of the other three polyketides shown in fig. 1 rather than "olivetol").
Figure 1 also shows the biosynthesis of 2, 4-diol-6-propylbenzoic acid by condensation of malonyl-coa with valeryl-coa, which will provide the downstream butyl phytocannabinoid.
FIG. 2 shows the biosynthesis of CBGa from hexanoic acid, malonyl-coa and geranyl pyrophosphate ("GPP") in cannabis, which includes the olivine acid biosynthesis steps shown in FIG. 1. Hexanoic acid is activated with coenzyme A by hexanoyl coenzyme A synthase ("
Figure 3 shows the biosynthesis of the downstream acid form of phyto-cannabinoids from CBGa in cannabis. CBGa is oxidatively cyclized to Δ 9-tetrahydrocannabinolic acid ("THCa") by THCa synthase. Oxidative cyclization of CBGa to cannabidiolic acid ("CBDa") by CBDa synthetase. Other phytocannabinoids, such as cannabichromenic acid ("CBCa"), cannabinoidic acid (cannabiisoic acid) ("CBEa"), iso-tetrahydrocannabinolic acid ("iso-THCa"), cannabicyclamic acid ("CBLa") or cannabiceric acid ("CBTa") are also synthesized in cannabis by other synthetases or by altering conditions in plant cells in a way that affects the enzymatic activity against the phytocannabinoid structures produced. The acid form of each of these general phytocannabinoid types is shown in fig. 3, where the general "R" group shows the alkyl side chain, which would be the 5 carbon chain in the case of the synthesis of olivinic acid from hexanoyl-coa and malonyl-coa. In some cases, the carboxyl group may alternatively be present at a ring position opposite to the position of the R group shown in fig. 3 (e.g., the 4 position of THC instead of the 2 position shown in fig. 3, etc.). The decarboxylated forms of the phytocannabinoids shown in figure 3 are THC, cannabidiol ("CBD"), cannabichromene ("CBC"), cannabinoids ("CBE"), iso-tetrahydrocannabinol ("iso-THC"), cannabiconol ("CBL") or cannabichromene ("CBT"), respectively.
U.S. publication No. 2016/0010126 to Poulos et al describes the expression of five natural hemp genes in Saccharomyces cerevisiae and Kluyveromyces marxianus. Expression of genes from the natural cannabis pathway in yeast for the production of phytocannabinoids may present disadvantages. Cannabis OAS uses hexanoyl-coa as a polyketide substrate. Caproic acid is toxic to s.cerevisiae and some other yeast strains. Furthermore, synthesis of CBGa from olivinic acid requires a membrane-bound cannabiprenyltransferase, which is poorly expressed in fungi.
The methods and yeast cells provided herein for the production of phytocannabinoids and phytocannabinoid analogs may be applied and include saccharomyces cerevisiae transformed with a gene for the prenyltransferase NphB from streptomyces CL 190. Streptomyces CL190NphB prenyltransferase provides an alternative to cannabis prenyltransferase and is hereinafter referred to as "AltPT". AltPT is an α β β α ("ABBA") type prenyltransferase. AltPT is highly promiscuous, accepting most polyketides as substrates for prenylation. AltPT is specific to GPP as a terpenoid donor. AltPT is a cytoplasmic enzyme expressed in streptomyces CL190 (gram positive bacteria), in contrast to membrane-bound prenyltransferase expressed in cannabis (plant). Bacterial cytosolic enzymes are expressed at higher levels in yeast than plant membrane-associated enzymes. AltPT prenylated olivinic acid to CBGa, similar to the reaction catalyzed by membrane-bound prenyltransferases in hemp. AltPT also prenylated olivol to cannabigerol ("CBG") or methyl olivol to methyl cannabigerol ("meCBG"). The synthetic sequence of AltPT that is codon optimized for yeast is included herein in
FIG. 4 shows the biosynthetic pathway for CBG production from hexanoic acid, malonyl-coa and GPP in transgenic yeast. Yeast strains provided herein for the production of CBG as shown in fig. 4 may include genes encoding streptomyces CL190AltPT, cannabis Hex1, and cannabis OAS. Examples of such yeast strains are provided as "HB 37" and "HB 88," each of which is described in table 7.
FIG. 5 shows the biosynthesis of downstream phyto-cannabinoids by CBG. CBG is oxidatively cyclized to THC, CBD, CBC, CBE, iso-THC, CBL or CBT. The decarboxylated form of each of these general phytocannabinoid types is shown in fig. 5, where the general "R" group shows an alkyl side chain, which in phytocannabinoids biosynthesized from olive alcohol would be a 5 carbon chain.
Figure 4 shows the production of hexanoyl-coa from hexanoic acid by
The pathway shown in fig. 4 includes cannabis HEx1 and cannabis OAS. The pathway shown in figure 4 does not include cannabis OAC. The transgenic yeast cells used to implement the pathway of fig. 4 will accordingly include genes for OAS, but not for cannabis OAC. During biosynthesis of olivinic acid, cannabis OAC carboxylates olivine alcohol to olivinic acid. In the presence of OAS and in the absence of OAC or other polyketide cyclase, olive alcohol is produced rather than olive alcohol acid produced in cannabis. As a result, the AltPT catalyzed reaction yields CBG instead of CBGa. The downstream reactions for producing phytocannabinoids will then correspondingly produce decarboxylated species of phytocannabinoids, including
The phytocannabinoids of figure 5, while in the presence of OAC or another polyketide cyclase (such as in cannabis), will produce phytocannabinoids in acid form, including those of figure 3.
Conversion of hexanoyl-coa to olivetol catalyzed by OAS in
FIG. 6 shows the biosynthetic pathway for the production of mecBG from malonyl-coa and GPP in transgenic yeast. Yeast strains for CBG production provided herein as shown in figure 6 may include genes for AltPT and genes for DiPKS that support polyketide production from malonyl-coa only, without the need for hexanoic acid in the culture medium. The DiPKS includes functional domains similar to those found in fatty acid synthetase, a methyltransferase domain, and a Pks III domain (see fig. 9). Examples of yeast strains comprising codon-optimized synthetic sequences encoding wild-type DiPKS genes are provided as "HB 84", "
FIG. 6 shows the production of methyl olivetol by malonyl-coa catalysed by DiPKS (
Figures 7 and 8 show downstream methylated phytocannabinoid analogs corresponding to methyl-tetrahydrocannabinol ("medhc"), methyl-cannabidiol ("meCBD"), methyl-cannabichromene ("meCBC"), methyl-cannabiisolone ("meCBE"), iso-methyl-tetrahydrocannabinol ("iso-meTHC"), methyl-cannabiconol ("meCBL"), or methyl-cannabisica ("meCBT"), which are methylated analogs of THC, CBD, CBC, CBE, iso-THC, CBL, and CBT, respectively, which may be prepared when methyl olive alcohol is provided as the precursor chemical instead of olive alcohol or olive alcohol. The decarboxylated form of each of these methylated phytocannabinoid analogs is shown in fig. 7 and 8, where the general "R" group shows an alkyl side chain, which in the case of synthesis enabled by hexanoyl-coa and malonyl-coa or malonyl-coa alone is a 5 carbon chain.
In addition to meCBD, a portion of the structure of each of the downstream phytocannabinoid analogs shown in fig. 7 and 8 includes a rotation-limiting group bonded to an aromatic ring. As a result, each of the downstream phytocannabinoid analogs shown in figures 7 and 8, except meCBD, can be synthesized from meCBG in one of two rotamers. Depending on the rotamer of meCBG during synthesis, the methyl group in the resulting cyclized methylated phytocannabinoid analogue may be at the position shown for the isomer of meTHC, meCBC, meCBE, iso-meTHC, meCBL or meCBT in figure 7, or the position shown for the isomer of meTHC, meCBC, meCBE, iso-meTHC, meCBL or meCBT in figure 8. References herein to meTHC, meCBC, meCBE, iso-meTHC, meCBL or meCBT include either or both of the isomers shown in figures 7 and 8.
The DiPKS comprises a C-methyltransferase domain that methylates olivetol at the 4-position on an aromatic ring. As a result, AltPT prenylated methyl olivil to give meCBG, a phytocannabinoid analogue, rather than CBGa, which is known to be synthesized in cannabis. Any downstream reactions that might produce phyto-cannabinoids should produce the unmethylated acid form of the phyto-cannabinoids in cannabis (as in figure 3) using CBGa or CBG as the input would produce the decarboxylated species of methylated phyto-cannabinoid analogs shown in figures 7 and 8 accordingly. If OAC or another polyketide cyclase is included, methyl olivil may be converted to meCBGa by OAC or other polyketide cyclase, as the methylated and carboxylated carbons may be in different positions. For example, meTHC synthesized from meCBG may be methylated at
Figure 9 is a schematic of the functional domains of the DiPKS showing β -ketoacyl-synthetase ("KS"), acyl transacetylase ("AT"), dehydratase ("DH"), C-methyltransferase ("C-Met"), enoyl reductase ("ER"), ketoreductase ("KR"), and acyl carrier protein ("ACP"). The "TYPE III" domain is a
The C-Met domain is delineated in fig. 9, schematically illustrating modifications to the DiPKS protein that inactivate the C-Met domain and reduce or eliminate methylation function. The TYPE III domain catalyzes iterative polyketide extension and cyclization of a thioester of hexanoic acid that transfers from ACP to TYPE III domain.
FIG. 10 shows the biosynthetic pathway for the production of both mecBG and CBG from malonyl-coa and GPP in transgenic yeast. As shown in fig. 10, yeast strains provided herein for the production of both CBG and meCBG may comprise genes for AltPT and genes for mutant DiPKS with reduced activity at the C-Met domain as schematically shown in fig. 9. The C-Met domain of the DiPKS protein includes amino acid residues 1510 to 1633 of DiPKS. The C-Met domain includes three motifs. The first motif includes residues 1510 to 1518. The second motif includes residues 1596 to 1603. The third motif includes residues 1623 to 1633. Disruption of one or more of these three motifs may result in reduced activity at the C-Met domain.
Expression of modified DiPK with reduced activity in the C-Met domainAn example of a yeast strain for S is provided in example V below as "HB 80A". HB80A includes modifications of the yeast-codon optimized gene encoding the wild-type DiPKS protein. HB80A includes modifications of the dicks gene such that the dicks protein is modified in the first motif of the C-Met domain. Due to these modifications to the DiPKS gene, the DiPKS protein has substitutions of Gly1516Asp and Gly1518 Ala. HB80A includes DiPKS onlyG1516D；G1518ABut not AltPT, resulting in catalysis of
FIG. 10 shows both the production of methyl olivetol from malonyl-coa (
FIG. 11 shows the biosynthetic pathway for the production of only CBG from malonyl-coa and GPP in transgenic yeast. As shown in fig. 11, yeast strains provided herein for the production of CBG only may include a gene for AltPT and a mutant DiPKS gene for reduced activity at the C-Met domain as schematically shown in fig. 9.
Examples of yeast strains expressing modified DiPKS that are essentially inactive in the C-Met domain are provided in examples VIII, IX and X below as "HB 135", "HB 137", "HB 138" and "HB 139". HB135, HB137, HB138 and HB139 each comprise a modification of a yeast-codon optimized gene encoding a wild-type DiPKS protein. HB135, HB137, HB138 and HB139 each include a modification of the dicks gene such that the dicks protein is modified in the first motif of the C-Met domain. Due to this modification of the DiPKS gene, the DiPKS protein has a substitution of Gly1516 Arg.
DiPKSG1516RCatalyzing reaction 1 in fig. 11. Gly1516Arg substitution at the active site of C-Met domain and reduction of DiPKSG1516RCatalysis of methylation at the 4-position of the olivine ring. Malonyl-coa added is catalyzed according to
Increasing availability of biosynthetic precursors
The biosynthetic pathways shown in figures 4, 6, 10 and 11 each require malonyl-coa and GPP to produce CBGa, CBG or meCBG, respectively. In addition to introducing polyketide synthase (such as OAS or DiPKS) and in addition to introducing cytosolic prenyltransferase (such as AltPT), the yeast cell can be mutated, genes from other species can be introduced, genes can be up-or down-regulated, or the yeast cell can be otherwise genetically modified to increase the availability of malonyl-coa A, GPP or other input metabolites needed to support the biosynthetic pathway of any of FIGS. 4, 6, 10, and 11.
Yeast cells can be modified to increase available GPP. S.cerevisiae may have one or more additional mutations in Erg20 or other genes that support enzymes that consume the metabolic pathways of GPP. Erg20 catalyzes GPP production in yeast cells. Erg20 also adds a subunit of 3-isopentyl pyrophosphate ("IPP") to GPP to give farnesyl pyrophosphate ("FPP"), a metabolite for the biosynthesis of downstream sesquiterpenes and sterols. It has been demonstrated that Erg20Some of the mutations in (a) reduce the conversion of GPP to FPP, increasing the available GPP in the cell. The substitution mutation Lys197Glu in Erg20 reduced the conversion of GPP to FPP by
Yeast strains can be modified for increased availability of malonyl-coa. The reduced mitochondrial acetaldehyde catabolism shifts acetaldehyde from ethanol catabolism to acetyl-coa production, which in turn drives the production of malonyl-coa and downstream polyketides and terpenoids. Saccharomyces cerevisiae may be modified to express acetyl-CoA synthetase ("Acs") from Salmonella enterica (Salmonella enterica) with a leucine to proline substitution at residue 641L641P") and aldehyde dehydrogenase 6 from Saccharomyces cerevisiae (" Ald6 "). The Leu641 Pro mutation removes the downstream regulation of Acs, as compared to wild-type AcsL641PThe mutant has higher activity. Together, cytoplasmic expression of these two enzymes increases the concentration of acetyl-coa in the cytoplasm. Greater acetyl-coa concentrations in the cytosol reduce mitochondrial catabolism, bypassing mitochondrial pyruvate dehydrogenase ("PDH"), providing a PDH bypass. As a result, more acetyl-CoA is available for malonyl-CoA production.
Another method used to increase cytoplasmic malonyl-coa is to up-regulate Acc1, Acc1 is the native yeast malonyl-coa synthetase. The promoter sequence of Acc1 gene was replaced by the constitutive yeast promoter of PGK1 gene. The promoter from the PGK1 gene allows multiple copies of Acc1 to be present in the cell. The native Acc1 promoter only allows a single copy of the protein to be present in the cell at a time. The native promoter region is shown in
In addition to upregulating the expression of Acc1, saccharomyces cerevisiae may also include one or more modifications of Acc1 to increase Acc1 activity and cytosolic acetyl-coa concentration. Two mutations in the regulatory sequences were identified in the literature that abrogated the inhibition of Acc1, resulting in higher Acc1 expression and higher malonyl-coa production. SEQ ID NO 7 is a polynucleotide of Acc1 gene native to the s.cerevisiae genome that can be used to modify by homologous recombination. SEQ ID NO 7 includes a portion of the coding sequence for the Acc1 gene with Ser659Ala and Ser1167Ala modifications. As a result, Saccharomyces cerevisiae transformed with this sequence will express Acc1S659A；S1167A. For example, similar results can be achieved by integrating the sequences with the Tef1 promoter, Acc1 with Ser659Ala and Ser1167Ala modifications, and the Prm9 terminator at any suitable site. The end result will be Tef1, Acc1S659A；S1167AAnd Prm9 is flanked by genomic DNA sequences for facilitating integration into the saccharomyces cerevisiae genome. This was attempted at
Saccharomyces cerevisiae may include modified expression of Maf1 or other tRNA biosynthesis regulators. It has been shown that over-expressing native Maf1 can reduce the loss of IPP for tRNA biosynthesis, thereby increasing monoterpene production in yeast. IPP is an intermediate of the mevalonate pathway. SEQ ID NO 8 is a polynucleotide integrated into the Saccharomyces cerevisiae genome at
Upc2 is an activator for sterol biosynthesis in Saccharomyces cerevisiae. The Glu888Asp mutation of Upc2 increased monoterpene production in yeast. SEQ ID NO 9 is integrable into the genome to provide expression of Upc2 under the Tef1 promoterE888DThe polynucleotide of (1). SEQ ID NO 9 includes Tef1 promoter, Upc2E888DGene and Prm9 terminator. Tef1, Upc2E888DAnd Prm9 are flanked together by genomic DNA sequences for facilitating integration into the saccharomyces cerevisiae genome.
The above gene, Erg20K197E、AcsL641P、Ald6、Maf1、Acc1S659A；S1167AOr Upc2E888DEither of which may be expressed from a plasmid or integrated into the genome of s.cerevisiae. Genomic integration can be by homologous recombination, including CRISPR recombination, or any suitable method. The promoter of Acc1 may be similarly modified by recombination. The coding sequence and regulatory sequences in each of
Increased DiPKS functionality
As shown in fig. 9, the DiPKS includes an ACP domain. The ACP domain of DiPKS requires a phosphopantetheinyl group as a cofactor. NpgA is a 4' -phosphopantetheinyl transferase from Aspergillus nidulans (Aspergillus nidulans). Copies of codon-optimized NpgA for saccharomyces cerevisiae can be introduced into and converted into saccharomyces cerevisiae, including by homologous recombination. The NpgA gene cassette was integrated into the genome of saccharomyces cerevisiae at the Flagfeldt site 14 to produce
Expression of NpgA provides a greater catalytic effect of aspergillus nidulans phosphopantetheinyl transferase for loading phosphopantetheinyl groups onto the ACP domain of DiPKS. As a result, the reaction catalyzed by DiPKS (
Other prenyltransferases
ABBA prenyltransferase structure was searched based on DELTA BLAST to define NphB variants. The list is redefined by looking for binding pockets that are appropriate for GPP rather than IPP, dimethylallyl pyrophosphate, or other isopentenyl groups. SEQ ID NO 12 through 33 provide the primary structural amino acid residue sequences of fungal and bacterial cytoplasmic prenyltransferases located in the DELTA BLAST search. DELTA BLAST searches of the cannabis genome were also performed, and membrane-bound prenyltransferases were located in these searches. Some cannabis membrane-bound prenyltransferases are poorly expressed in some yeast species and are not introduced into the yeast strains provided herein for the production of phytocannabinoids or phytocannabinoid analogs.
SEQ ID NO 33 to SEQ ID NO 36 provide the primary structural amino acid residue sequences of fungal and bacterial cytoplasmic prenyltransferases located in manual literature searches. 33 to 36 are the primary structural amino acid residue sequences of cytoplasmic prenyltransferases named FNQ26, FNQ28, FUR7 and NAPT9, respectively.
Any of SEQ ID NO 11 through SEQ ID NO 36 can be applied to the yeast strains described herein as a cytoplasmic prenyltransferase. Each of these prenyltransferases is summarized in table 1.
Table 1: prenyltransferases
Modification of DiPKS
DiPKS can be modified to reduce or eliminate the activity of C-Met.
SEQ ID NO 37 is a modified form of the synthetic sequence of DIPKS optimized for yeast codons, where DiPKS includes a Gly1516Asp substitution and a Gly1518Ala substitution, which together disrupt the activity of the C-met domain. The following example IV for the inclusion of strain HB80A provides DiPKS in Saccharomyces cerevisiae culturesG1516D，G1518AThe results of the expression. Other modifications may be introduced into the DiPKS to destroy or eliminate all of the active sites of C-Met or all of C-Met. Each of these modified DiPKS enzymes can be introduced into saccharomyces cerevisiae as described for wild-type DiPKS.
38 is a modified form of the synthetic sequence of DIPKS optimized for yeast codons, where DiPKS includes a Gly1516Arg substitution that disrupts the activity of the C-met domain. The following combination of example VIII, including strain HB135, and example IX, including strains HB135, HB137, and HB138 provides DiPKS in Saccharomyces cerevisiae culturesG1516RThe results of the expression.
Except in particular for DiPKSG1516D，G1518AAnd DiPKSG1516RIn addition, other modifications were introduced into the DiPKS to destroy or eliminate all active sites of C-Met or all C-Met: (a) by
Transformation and growth of yeast cells
Details of specific examples of methods and yeast cells prepared according to the present description are provided below as examples I through X. Similar methods were used in each of these 10 embodiments to construct plasmids, transform yeast, quantify strain growth, and quantify intracellular metabolites. These common features in the 10 embodiments are described below, followed by results and other details relating to one or more of the 10 embodiments.
Plasmid construction
The plasmids assembled for use in applying and preparing the embodiments of the methods and yeast cells provided herein are shown in table 2. In table 2, for expression plasmids pYES and pYES2,
Table 2: plasmids and cassettes for the preparation of yeast strains
Plasmids for introduction into s.cerevisiae were amplified by polymerase chain reaction ("PCR") using primers from Operon Eurofins and Phusion HF polymerase (ThermoFisheRF-530S) according to the manufacturer' S recommended protocol using an Eppendorf Mastercycler ep Gradient 5341.
All plasmids were assembled in s.cerevisiae using overlapping DNA portions and transformation-assisted recombination. Plasmids were transformed into s.cerevisiae using a lithium acetate heat shock method as described by Gietz, r.d. and Schiestl, r.h. "High-efficiency yeast transformation using the LiAc/SScarrier DNA/PEG method" nat. protoc.2, 31-34 (2007). The basic yeast strains used to assemble the plasmids are shown in table 3:
bacterial strains
Background
Decoration
Note
HB24
-LEU
Is free of
Unmodified yeast with leucine auxotrophy for assembling plasmids
HB25
-URA
Is free of
Uracil for assembling plasmidsAuxotrophic, unmodified yeast
Table 3: basal yeast strains
The pAltPT plasmid was assembled in the HB24 leucine auxotroph. The pNPGA, pDiPKSm1, pDiPKSm2, pGFP, ppptgfp, pAPTGFP, pH1OAS, pDiPKS, pCRISPR and pdh plasmids were assembled in the HB25 uracil auxotroph. Transformed s.cerevisiae cells were selected by auxotrophic selection on agar plates. Colonies recovered from the petri dish were grown in liquid selective medium at 30 ℃ for 16 hours while shaking at 250 RPM.
After growth in liquid selective medium, transformed Saccharomyces cerevisiae cells were collected and plasmid DNA was extracted. The extracted plasmid DNA was transformed into E.coli (Escherichia coli). Transformed E.coli was selected by growth on agar plates containing ampicillin. Coli was cultured to amplify the plasmid. Plasmids grown in E.coli were extracted and sequenced using Sanger dideoxy sequencing to verify the exact construction. The sequence-verified plasmids were then used for genome modification or stable transformation of s.cerevisiae.
Genome modification of Saccharomyces cerevisiae
The Saccharomyces cerevisiae strains described herein may be prepared by stable transformation of plasmids or by genomic modification. Genome modification can be achieved by homologous recombination, including by methods utilizing CRISPR.
The method of using CRISPR is employed to delete DNA from the saccharomyces cerevisiae genome and introduce heterologous DNA into the saccharomyces cerevisiae genome. Guide RNA ("gRNA") sequences for targeting Cas9 endonuclease to desired locations on the saccharomyces cerevisiae genome were designed with bench top online DNA editing software. DNA splicing by overlap extension ("SOEing") and PCR is applied to assemble gRNA sequences and amplify DNA sequences that include functional gRNA cassettes.
Functional gRNA cassettes, Cas9 expressing gene cassettes and the pYes2(URA) plasmid were assembled into pCRISPR plasmid and transformed into saccharomyces cerevisiae for facilitating targeted DNA double strand cleavage. The resulting DNA cleavage was repaired by adding a linear fragment of the targeted DNA.
Erg20 shown in SEQ ID NO. 3 by homologous recombinationK197EThe CDS of the protein is integrated into the genome of HB13, and an HB42 basic strain is obtained.
Bases 51 to 7114 of SEQ ID No. 4 were integrated into HB42 strain by CRISPR to provide HB82 base strain with the PDH bypass gene in saccharomyces cerevisiae. After assembly in s.cerevisiae, the sequence of the pPDH plasmid was verified. The sequence-verified pPDH plasmid was grown in E.coli, purified, and digested with BciV1 restriction enzyme. Digestion with BciV1 provided, as in Table 2, including Ald6 and SeAcsL641PThe gene of (a), a promoter, a terminator and an integration site homology sequence for integration into the saccharomyces cerevisiae genome at PDH-site 19 next to Cas 9. The resulting linear PDH bypass donor polynucleotide was purified by gel separation as shown in bases 51 to 7114 of SEQ ID NO. 4.
In both PDH bypass genes (Ald6 and Acs)L641P) A PDH bypass donor polynucleotide is co-transformed into saccharomyces cerevisiae with pCRISPR, with the PDH bypass polynucleotide on top of the single PDH bypass polynucleotide. The transformation was performed by lithium acetate heat shock method described by Gietz. The pCRISPR plasmid expresses Cas9, which is targeted to a selected location of the saccharomyces cerevisiae genome by a gRNA molecule. At this position, the Cas9 protein causes a double strand break in the DNA. A PDH bypass donor polynucleotide is used as the donor polynucleotide in the CRISPR reaction. Including Ald6, AcsL641PThe PDH bypass donor polynucleotide of the promoter and terminator was integrated by homologous recombination into the genome at the break site, site 19, resulting in strain HB 82.
The NpgA donor polynucleotide shown in SEQ ID NO. 10 was prepared and amplified. DNA SOEing was used to generate a single donor DNA fragment from 3 polynucleotides for NpgA integration. The first polynucleotide is a 5' region of genomic homology that allows donor recombination at a specific locus in the genome. The second polynucleotide encodes an NpgA gene cassette. The NpgA gene cassette includes a Tef1 promoter, an NpgA coding sequence, and a Prm9 terminator. The third polynucleotide includes a 3' region of genomic homology to facilitate targeted integration into the s.cerevisiae genome.
The NpgA donor polynucleotide was co-transformed with pCRISPR plasmid into strain HB 82. The pCRISPR plasmid is expressed and the endonuclease Cas9 is targeted by the gRNA molecule to a location on the saccharomyces cerevisiae genome. At this position, the Cas9 protein creates a double strand break in the DNA, and integrates the NpgA donor polynucleotide into the genome at the break by homologous recombination to provide the HB100 base strain.
The Maf1 donor polynucleotide shown in SEQ ID NO. 8 was prepared and amplified. DNA SOEing was used to generate a single donor DNA fragment from 3 polynucleotides for Maf1 integration. The first polynucleotide is a 5' region of genomic homology that allows donor recombination at a specific locus in the genome. The second polynucleotide encodes a Maf1 gene cassette. The Maf1 gene cassette includes a Tef1 promoter, a Maf1 coding sequence, and a Prm9 terminator. The third polynucleotide includes a 3' region of genomic homology to facilitate targeted integration into the s.cerevisiae genome.
The Maf1 donor polynucleotide was co-transformed with pCRISPR plasmid into
Acc1-PGK1p donor polynucleotide shown in SEQ ID NO 6 was prepared and amplified. DNA SOEing was used to generate a single donor DNA fragment from 3 polynucleotides for Acc1-PGK1 integration. The first polynucleotide is a 5' region of genomic homology that allows donor recombination at a specific locus in the genome. The second polynucleotide encodes the PGK1 promoter region. The third polynucleotide includes a 3' region of genomic homology to facilitate targeted integration into the s.cerevisiae genome.
Acc1-PGK1 donor polynucleotide was co-transformed with pCRISPR plasmid. The pCRISPR plasmid is expressed and the endonuclease Cas9 is targeted by the gRNA molecule to a location on the saccharomyces cerevisiae genome. At this position, the Cas9 protein creates a double-strand break in the DNA, and the Acc1-PGK1 donor polynucleotide is integrated by homologous recombination at the break in the genome. Stable transformation of donor polynucleotides into HB100 strain an HB110 base strain with Acc1 regulated by the PGK1 promoter is provided.
Table 4 provides a summary of the base strains prepared by genomic modification of saccharomyces cerevisiae. Each of the basic strains shown in table 4 is leucine and uracil auxotroph, and none of them includes a plasmid.
Table 4: base-transformed strain for confirming protein expression and phytocannabinoid production
Stable transformation for strain construction
Plasmids were transformed into s.cerevisiae using a lithium acetate heat shock method as described by Gietz.
As shown in Table 5 below, transgenic Saccharomyces cerevisiae strains HB1, HB6, and HB7 were prepared from the HB25 base strain by introducing the plasmids in Table 2 into
Bacterial strains
Basic strain
Plasmids
HB1
HB25
pGFP
HB6
HB25
pPTGFP
HB7
HB25
pAPTGFP
HB13
HB25
pEV
Table 5: transformed yeast strains prepared to confirm protein expression and phytocannabinoid production including expression plasmids
As shown in Table 6 below, by transforming HB42 with the expression plasmids, transgenic Saccharomyces cerevisiae HB80, HB80A, HB98, HB102, HB135, HB137 and HB138 were prepared from HB42, HB100, HB106 and HB110 base strains, and HB80A was prepared by transforming
Table 6: strain comprising plasmid expressing polyketide synthase
Transgenic saccharomyces cerevisiae HB37, HB84, HB88, HB90, HB105 and HB130 were prepared from the base strains shown in table 7 by transforming the base strains with the expression plasmids shown in table 7 below. HB37 and HB88 include and express AltPT and OAS, respectively. HB80, HB90 and HB105 each include and express AltPT and DiPKS. HB139 includes and expresses AltPT and DiPKSG1516R。
Bacterial strains
Plasmid 1
HB37
HB42
pAltPT
pH1OAS
HB84
HB42
pAltPT
pDiPKS
HB88
HB82
pAltPT
pH1OAS
HB90
HB82
pAltPT
pDiPKS
HB105
HB100
pAltPT
pDIPKS
HB139
HB106
pAltPT
pDIPKSm2
Table 7: strain comprising plasmid expressing cytosolic prenyltransferase
Yeast growth and feeding conditions
Yeast cultures were grown in overnight culture with selective media to provide starter cultures. The resulting initial culture was then used to inoculate triplicate 50ml cultures to an optical density of absorbance at 600nm ("A)600") is 0.1. Table 6 shows details of the medium used to grow each strain.
Bacterial strains
Growth medium
HB13-HA
YNB + 2% glucose +1.6g/L4DO +0.5mM hexanoic acid
HB13-No
YNB + 2% raffinose + 2% galactose +1.6g/L4DO
HB37-HA
YNB + 2% glucose +1.6g/L4DO +0.5mM hexanoic acid
HB84-No
YNB + 2% raffinose + 2% galactose +1.6g/L4DO
Table 8: growth medium for yeast
In table 8, "4 DO" refers to a supplement to the yeast synthetic deletion medium lacking leucine and uracil. With respect to strain HB13, "HB 13-HaA" refers to HB13 grown in the presence of 0.5mM hexanoic acid, and "HB 13-No" refers to HB13 grown in the absence of hexanoic acid. In table 8, "YNB" is a nutrient broth comprising the chemicals listed in the first two columns of table 9. The chemicals listed in
Table 9: YNB nutrient broth and 4DO supplement
Quantification of metabolites
Intracellular metabolites were extracted from saccharomyces cerevisiae cells using methanol extraction. 1mL of the liquid culture was centrifuged at 12,000 Xg for 3 minutes. 250 μ l of the resulting supernatant was used for extracellular metabolite quantification. The resulting cell pellet was suspended in 200. mu.l of 80% methanol at-40 ℃. The mixture was swirled and cooled on ice for 10 minutes. After cooling on ice for 10 minutes, the mixture was spun at 15,000 Xg for 14 minutes at 4 ℃. The resulting supernatant was collected. An additional 200. mu.l of-40 ℃ 80% methanol was added to the cell debris pellet and the mixture was vortexed and cooled on ice for 10 minutes. After cooling on ice for 10 minutes, the mixture was centrifuged at 15,000 Xg for 14 minutes at 4 ℃. The resulting 200. mu.l supernatant was added to the previously collected 200. mu.l supernatant, providing a total of 400. mu.l of 80% methanol and intracellular metabolites.
Intracellular metabolites were quantified using high performance liquid chromatography ("HPLC") and mass spectrometry ("MS") methods. An Agilent 1260 autosampler and HPLC system connected to a thermo finnigan LTQ mass spectrometer was used. The HPLC system included a Zorbax eclipseC 182.1 μm by 5.6mm by 100mm column.
Metabolites were injected into 10. mu.l samples using an autosampler and separated on HPLC using a flow rate of 1 ml/min. An HPLC separation protocol for a total of 20 minutes, wherein (a)0-2 minutes, 98% solvent a and 2% solvent B; (b) reaching 98 percent of solvent B after 2 to 15 minutes; (c) 98% solvent B in 15-16.5 min; (d) reaching 98 percent A in 16.5-17.5 minutes; and (e) final equilibration for 2.5 minutes at 98% solvent a. Solvent a was acetonitrile + 0.1% formic acid in MS water, and solvent B was 0.1% formic acid in MS water.
After HPLC separation, the samples were injected into the mass spectrometer by electrospray ionization and analyzed in positive mode. The capillary temperature was maintained at 380 ℃. The tube lens voltage was 30V, the capillary voltage was 0V, and the ejection voltage was 5 kV. After HPLC-MS/MS, CBG analyzed as the parent ion at 317.2 and the daughter ion at 193.1, while mecBG analyzed as the parent ion at 331.2. Similarly, after HPLC-MS/MS, olivetol was analyzed as the parent ion at 181.2 and the daughter ion at 111, while methyl olivetol was analyzed as the parent ion at 193.2 and the daughter ion at 125.
Different concentrations of known standards were injected to generate a linear standard curve. Standards for CBG and meCBG were purchased from Toronto Research Chemicals. meCBG was customized on demand because Toronto research chemicals did not synthesize the chemical before being asked for the standard. Olivetol and methyl olivetol standards were purchased from Sigma Aldrich.
Effect of hexanoic acid on growth of Saccharomyces cerevisiae
The genes encoding enzymes required for hexanoic acid biosynthesis were not introduced into s.cerevisiae. Rather, in yeast cells that include OAS genes, such as HB37, hexanoic acid is included in the growth medium.
FIG. 12 shows the effect of caproic acid supplementation on growth of Saccharomyces cerevisiae. HB13 was cultured in YNB + 2% glucose +1.6g/L4DO +0.5mM hexanoic acid. Hexanoic acid was added at 36 hours of incubation. Caproic acid was added to the culture samples at concentrations of 0, 0.5, 1.0 and 3.0mM, respectively. Caproic acid is toxic to Saccharomyces cerevisiae. A reduction in growth was observed in the presence of hexanoic acid. The extent of the decrease in growth of Saccharomyces cerevisiae corresponds to the concentration of hexanoic acid in the growth medium. Culture suspension A600Values quantify the growth rate, which are shown in fig. 12 as hexanoic acid concentrations of 0, 0.5, 1.0, and 3.0 mM.
HB13 and HB37 were grown in the presence of 0.5mM hexanoic acid for 96 hours, with samples taken at the points of 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, and 96 hours. In the absence of hexanoic acid, HB13 and HB84 were grown and a single time point was taken at 72 hours. HB13 was used as a control in both experiments. Growth media are described above in connection with tables 8 and 9.
Point in time
HB13-HA
HB13-No
HB37-HA
HB84-No
24 hours
5.33
(without data)
3.33
(without data)
36 hours
5.80
(without data)
3.43
(without data)
48 hours
4.67
(without data)
3.33
(without data)
60 hours
6.07
(without data)
3.53
(without data)
72 hours
8.96
10.7
4.48
6.9
84 hours
7.23
(without data)
4.13
(without data)
96 hours
8.28
(without data)
4.33
(without data)
Table 10: HB13 and HB37(0.5mM hexanoic acid) and HB13 and HB84 (without hexanoic acid) growth
As shown in table 10, HB84 grew faster than HB 37. Furthermore, HB84 does not require hexanoic acid to produce meCBG, whereas HB37 requires hexanoic acid to produce CBG. Similarly, HB13 showed better growth at 72 hours in the absence of hexanoic acid compared to the presence of 0.5mM hexanoic acid, consistent with the data shown in fig. 12.
FIGS. 13 to 15 each show A of HB37 cultures listed in Table 10600Value (dashed line with triangle data points). In addition, each of fig. 13 to 15 shows another solid line with circular data pointsA series of data.
FIG. 13 shows the olive alcohol yield (μ g olive alcohol/L medium) as a solid line with circular data points.
FIG. 14 shows CBG production (μ g CBG/L medium) as a solid line with circular data points.
FIG. 15 shows hexanoic acid present in the culture (mg hexanoic acid/L medium) as a solid line with circular data points.
Figures 13 to 15 together are consistent with a second transition (dioxicshift) occurring between 50 and 60 hours. Secondary transitions include metabolic transitions from glucose catabolism to acetate and ethanol catabolism. With the second shift, many secondary metabolic pathways become more active, and AltPT and OAS activities similarly increase.
Figures 12 to 15 and table 10 show data consistent with: production of phytocannabinoids by consumption of hexanoic acid does not appear to alleviate hexanoic acid toxicity to a great extent until the hexanoic acid level drops between 50 and 60 hours and then continues to drop. As shown in fig. 12 and 13, olivetol and CBG were produced starting from the introduction of hexanoic acid. However, when CBG is produced and caproic acid is converted to olivine, A of the culture600With the production of olivine alcohol and CBG not increasing significantly. As shown in FIG. 15, A only after between 50 and 60 hours of initial hexanoic acid depletion600Is increased. Depletion is due at least in part to olive alcohol production. However, no culture A was observed during the production of olivetol and CBG after 36 hours of caproic acid introduction600Increase significantly until the hexanoic acid concentration is exhausted.
Expression of cytosolic and membrane-bound prenyltransferases
Cannabis prenyltransferases are membrane-bound plant proteins, whereas AltPT is a cytoplasmic bacterial protein. The use of AltPT in saccharomyces cerevisiae, rather than cannabiprenyltransferase, provides higher protein expression levels in yeast cells. HB1, HB6, HB7 and HB13 shown in table 5 were each grown overnight in YNB, 2% glucose and 1.6g/L4 DO. After overnight growth, the resulting cultures were normalized to 1.0A600Then, in YNB, 2% glucose and 1.6g/L4DO in 4 hours growth. Measurement using a BD Acuri C6 flow cytometerFluorescence of each culture suspension.
HB1 expresses green fluorescent protein ("GFP"). HB6 and HB7 each express a GFP-isopentenyl transferase fusion protein. Neither HB6 nor HB7 included genes from pDiPKS or pH1OAS plasmids. Accordingly, neither HB6 nor HB7 expressed polyketide synthase genes or included all enzymes that implement the biosynthetic pathway in any of fig. 4, 6 or 9.
Fig. 16 shows the mean fluorescence levels of cell culture samples from HB13 ("negative"), HB1 ("positive"), HB6 ("isopentenyl transferase _ cannabis") and HB7 ("isopentenyl transferase _ Alt"). Fluorescence levels corresponded to protein expression levels, showing relative expression levels of cannabis prenyltransferase of HB6 and AltPT of HB 7. The common membrane-bound cannabis prenyltransferases have low expression in the cytoplasm of s.cerevisiae. Cytoplasmic AltPT is expressed in the cytoplasm of saccharomyces cerevisiae at higher levels than the common membrane-bound cannabiprenyltransferase.
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