Jay KEASLING, et al.
Yeast Biosynthesis of Cannabinoids
Yeast Biosynthesis of Cannabinoids
Related : THC Synthesis Patents
https://onezero.medium.com/
Scientists are making THC and CBD without Marijuana
by Tim McDonnell
New research paves the way for cannabinoids without cannabis
by Tim McDonnell
New research paves the way for cannabinoids without cannabis
...In a paper published today (Complete Biosynthesis of Cannabinoids and their Unnatural Analogues in Yeast) in the peer-reviewed journal Nature, biochemists at the University of California, Berkeley report what some cannabis industry experts are describing as a breakthrough in biosynthetic cannabinoid production.
By using genetically modified yeast, the Berkeley scientists were able to convert simple sugars into the active chemical compounds in marijuana...
The new research from the Berkeley scientists centers on the identification of a cannabis enzyme that can be transferred into yeast DNA so that it metabolizes sugar into cannabinoids instead of alcohol and carbon dioxide.
Jay Keasling, the biochemist at Berkeley who led the research, says his team tested dozens of options before finding the right combination of enzyme genes.
Yeast is a good host organism, Keasling says, because its DNA is thoroughly documented and because it's already in wide use for other commercial applications like beer-making and wine-making.
"It's as easy as brewing beer," says Keasling
"You feed the yeast sugar, it grows and replicates, produces the THC, and secretes it outside the cell so that it's floating in the sugar water that the yeast is growing in. It comes in high concentrations, and then we purify it away and you're left with a very pure white powder."
Keasling filed a patent for this method back in 2017, and has since been hammering down the science and working with a Bay Area biotech startup, Demetrix, to bring the process from the lab into commercial production.
Over the next few years, the company hopes to bring the cost of production below $1,000 per kilogram, far below the cost of chemically-synthesized cannabinoids (tens of thousands of dollars per kilogram) or cannabinoids extracted from a plant (more than $5,000 per kilogram), according to Jeff Ubersax, the company's CEO.
Biosynthesized cannabinoids aren't yet being produced commercially by any company.
And some of Demetrix's competitors remain skeptical that Keasling's approach is much different from what others are cooking up behind the scenes.
"They're showing how these pieces will all go together, which is cool, but I don't know that I would describe it as a breakthrough," says Kevin Chen, CEO of Montreal-based Hyasynth Bio, which recently received a $7.6 million investment from the Canadian cannabis distributor OrganiGram to speed up the rollout of its own biosynthetic cannabinoids.
"Everyone is in the research stage, so it's hard to say who will have the first product out."
Ronan Levy, chief strategic officer of another Demetrix competitor, New Mexico-based Trait Biosciences, says his company's preferred approach is to find ways to induce a cannabis plant to produce cannabinoids in every one of its cells, rather than only those in the "tricone" (better known as the "bud"), where it grows naturally.
"Instead of trying to find other organisms, why not figure out how to expand on what the plant can do?" he says. "Yeast is definitely interesting, we just don't think it makes the most sense."
"So you have a yeast that makes THC. Do you schedule this yeast?"
It's unclear whether biosynthesized cannabinoids would be subject to the same legal restrictions as plant-derived compounds, since federal law applies to the plant and not necessarily to the cannabinoids in it.
Cannabis researchers are subject to tight restrictions on where they can procure samples, and the Food and Drug Administration (FDA) has approved only one CBD-based drug, Epidiolex, for childhood epilepsy.
"So you have a yeast that makes THC. Do you schedule this yeast?" says Piomelli, referring to the DEA, Drug Enforcement Administration's drug classification system.
"We haven't seen the law at work enough to conclude, case by case, what will work. The legal landscape on cannabis is so confusing that almost anything goes."
Either way, biosynthesis is poised to change the way we think about getting high.
"We don't make insulin from animals anymore, and the plant won't be the way forward for industrial production of cannabinoids," says Banister.
"If big corporations want to move into the cannabis space, I think they will be moving in this direction."
https://www.bibliotecapleyades.net/archivos_pdf/complete-biosynthesis-cannabinoids.pdf
Complete
Synthesis of Cannabinoids... in Yeast
X. Luo, et al.
X. Luo, et al.
US2019078168
Generation of Water-Soluble Cannabinoid Compounds in Yeast and Plant Cell Suspension Cultures and Compositions of Matter
[ PDF ]
Generation of Water-Soluble Cannabinoid Compounds in Yeast and Plant Cell Suspension Cultures and Compositions of Matter
[ PDF ]
US9822384
Production of Tetrahydrocannabinolic Acid in Yeast
Production of Tetrahydrocannabinolic Acid in Yeast
[
PDF ]
Exemplary embodiments provided herein include genetically engineering microorganisms, such as yeast or bacteria, to produce cannabinoids by inserting genes that produce the appropriate enzymes for the metabolic production of a desired compound.
WO2019014490
PRODUCTION OF CANNABINOIDS IN YEAST
The
present disclosure relates to the production of cannabinoids in
yeast. In one aspect there is provided a genetically modified
yeast comprising: one or more GPP producing genes and
optionally, one or more GPP pathway genes; two or more
olivetolic acid producing genes; one or more cannabinoid
precursor or cannabinoid producing genes; one or more
Hexanoyl-CoA producing genes, and at least 5% dry weight of
fatty acids or fats.PRODUCTION OF CANNABINOIDS IN YEAST
WO2018200888
MICROORGANISMS AND METHODS FOR PRODUCING CANNABINOIDS AND CANNABINOID DERIVATIVES PRODUCTION OF CANNABINOIDS IN YEAST
MICROORGANISMS AND METHODS FOR PRODUCING CANNABINOIDS AND CANNABINOID DERIVATIVES PRODUCTION OF CANNABINOIDS IN YEAST
The present disclosure relates to the production of cannabinoids in yeast. In one aspect there is provided a genetically modified yeast comprising: one or more GPP producing genes and optionally, one or more GPP pathway genes; two or more olivetolic acid producing genes; one or more cannabinoid precursor or cannabinoid producing genes; one or more Hexanoyl-CoA producing genes, and at least 5% dry weight of fatty acids or fats...
[00133] The present disclosure provides methods, polypeptides, nucleic acids encoding said polypeptides, and genetically modified host cells for producing cannabinoids, cannabinoid precursors, cannabinoid derivatives (e.g., non-naturally occurring cannabinoids), or cannabinoid precursor derivatives (e.g., non-naturally occurring cannabinoid precursors). [00134] Geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT, Enzyme Commission Number 2.5.1.102) polypeptides play an important role in the biosynthesis of cannabinoids, but reconstituting their activity in a genetically modified host cell has proven challenging, hampering progress in the production of cannabinoids or cannabinoid derivatives. Herein, novel genes encoding polypeptides of the disclosure that catalyze production of cannabigerolic acid (CBGA) from GPP and olivetolic acid have been identified, isolated, and characterized. Surprisingly, these polypeptides of the present disclosure can catalyze production of CBGA from GPP and olivetolic acid in an amount at least ten times higher than previously discovered Cannabis polypeptides that catalyze production of CBGA from GPP and olivetolic acid (see, for example, U.S. PatentApplication Pub. No. US20120144523 and the GOT polypeptide, CsPT1, disclosed therein; SEQ ID NO:82 herein). The new polypeptides of the present disclosure that catalyze production of CBGA from GPP and olivetolic acid are GOT polypeptides (e.g., the CsPT4 polypeptide) and can generate cannabinoids and cannabinoid derivatives in vivo (e.g., within a genetically modified host cell) and in vitro (e.g., cell-free). These new GOT polypeptides, as well as nucleic acids encoding said GOT polypeptides, are useful in the methods and genetically modified host cells of the disclosure for producing cannabinoids or cannabinoid derivatives...
[00144] In Cannabis, cannabinoids are produced from the common metabolite precursors geranylpyrophosphate (GPP) and hexanoyl-CoA by the action of three polypeptides so far only identified in Cannabis. Hexanoyl-CoA and malonyl-CoA are combined to afford a 12-carbon tetraketide intermediate by a TKS polypeptide. This tetraketide intermediate is then cyclized by an OAC polypeptide to produce olivetolic acid. Olivetolic acid is then prenylated with the common isoprenoid precursor GPP by a GOT polypeptide (e.g., a CsPT4 polypeptide) to produce CBGA, the cannabinoid also known as the“mother cannabinoid.” Different synthase polypeptides then convert CBGA into other cannabinoids, e.g., a THCA synthase polypeptide produces THCA, a CBDA synthase polypeptide produces CBDA, etc. In the presence of heat or light, the acidic cannabinoids can undergo decarboxylation, e.g., THCA producing THC or CBDA producing CBD.
[00145] GPP and hexanoyl-CoA can be generated through several pathways (see FIGS.1 and 11). One or more nucleic acids encoding one or more polypeptides having at least one activity of a polypeptide present in these pathways can be useful in the methods and genetically modified host cells for the synthesis of cannabinoids, cannabinoid precursors, cannabinoid derivatives, or cannabinoid precursor derivatives. [00146] Polypeptides that generate GPP or are part of a biosynthetic pathway that generates GPP may be one or more polypeptides having at least one activity of a polypeptide present in the mevalonate (MEV) pathway. The term“mevalonate pathway” or“MEV pathway,” as used herein, may refer to the biosynthetic pathway that converts acetyl-CoA to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mevalonate pathway comprises polypeptides that catalyze the following steps: (a) condensing two molecules of acetyl-CoA to generate acetoacetyl-CoA (e.g., by action of an acetoacetyl-CoA thiolase polypeptide); (b) condensing acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) (e.g., by action of a HMG-CoA synthase (HMGS) polypeptide); (c) converting HMG-CoA to mevalonate (e.g., by action of a HMG- CoA reductase (HMGR) polypeptide); (d) phosphorylating mevalonate to mevalonate 5- phosphate (e.g., by action of a mevalonate kinase (MK) polypeptide); (e) converting mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., by action of a phosphomevalonate kinase (PMK) polypeptide); (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate (e.g., by action of a mevalonate pyrophosphate decarboxylase (MVD) polypeptide); and (g) converting isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP) (e.g., by action of an isopentenyl pyrophosphate isomerase (IDI) polypeptide) (FIGS.1 and 11). A geranyl diphosphate synthase (GPPS) polypeptide then acts on IPP and/or DMAPP to generate GPP. Additionally, polypeptides that generate GPP or are part of a biosynthetic pathway that generates GPP may be one or more polypeptides having at least one activity of a polypeptide present in the deoxyxylulose-5-phosphate (DXP) pathway, instead of those of the MEV pathway (FIG.1).
[00147] Polypeptides that generate hexanoyl-CoA may include polypeptides that generate acyl-CoA compounds or acyl-CoA compound derivatives (e.g., a hexanoyl-CoA synthase (HCS) polypeptide, an acyl-activating enzyme polypeptide, a fatty acyl-CoA synthetase polypeptide, or a fatty acyl-CoA ligase polypeptide). Hexanoyl-CoA may also be generated through pathways comprising one or more polypeptides that generate malonyl- CoA, such as an acetyl-CoA carboxylase (ACC) polypeptide. Additionally, hexanoyl-CoA may be generated with one or more polypeptides that are part of a biosynthetic pathway that produces hexanoyl-CoA, including, but not limited to: a malonyl CoA-acyl carrier protein transacylase (MCT1) polypeptide, a PaaH1 polypeptide, a Crt polypeptide, a Ter polypeptide, and a BktB polypeptide; a MCT1 polypeptide, a PhaB polypeptide, a PhaJ polypeptide, a Ter polypeptide, and a BktB polypeptide; a short chain fatty acyl-CoA thioesterase (SCFA-TE) polypeptide; or a fatty acid synthase (FAS) polypeptide (see FIGS. 1 and 11). Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such pathways and polypeptides.
[00148] GPP and hexanoyl-CoA may also be generated through pathways comprising polypeptides that condense two molecules of acetyl-CoA to generate acetoacetyl-CoA and pyruvate dehydrogenase complex polypeptides that generate acetyl-CoA from pyruvate (see FIGS.1 and 11). Hexanoyl CoA derivatives, acyl-CoA compounds, or acyl-CoA compound derivatives may also be formed via such pathways.
General Information
[00149] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature:“Molecular Cloning: A Laboratory Manual,” second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Animal Cell Culture” (R. I. Freshney, ed., 1987);“Methods in Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates);“PCR: The Polymerase Chain Reaction,” (Mullis et al., eds., 1994). Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y.1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y.1992), provide one skilled in the art with a general guide to many of the terms used in the present application.
[00150] “Cannabinoid” or“cannabinoid compound” as used herein may refer to a member of a class of unique meroterpenoids found until now only in Cannabis sativa.
Cannabinoids may include, but are not limited to, cannabichromene (CBC) type (e.g. cannabichromenic acid), cannabigerol (CBG) type (e.g. cannabigerolic acid), cannabidiol (CBD) type (e.g. cannabidiolic acid), ?<9>-trans-tetrahydrocannabinol (?<9>-THC) type (e.g. ?<9>- tetrahydrocannabinolic acid), ?<8>-trans-tetrahydrocannabinol (?<8>-THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabitriol (CBT) type, cannabigerolic acid (CBGA), cannabigerolic acid monomethylether (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCVA), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4(CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-C1), ?<9>–tetrahydrocannabinolic acid A (THCA-A), ?<9>–tetrahydrocannabinolic acid B (THCA-B), ?<9>–tetrahydrocannabinol (THC), ?<9>–tetrahydrocannabinolic acid-C4(THCA-C4), ?<9>–tetrahydrocannabinol-C4(THC-C4), ?<9>–tetrahydrocannabivarinic acid (THCVA), ?<9>–tetrahydrocannabivarin (THCV), ?<9>– tetrahydrocannabiorcolic acid (THCA-C1), ?<9>–tetrahydrocannabiorcol (THC-C1), ?<7>–cis- iso-tetrahydrocannabivarin, ?<8>–tetrahydrocannabinolic acid (?<8>–THCA), ?<8>–tetrahydrocannabinol (?<8>–THC), cannabicyclolic acid (CBLA), cannabicyclol (CBL), cannabicyclovarin (CBLV), cannabielsoic acid A (CBEA-A), cannabielsoic acid B (CBEA- B), cannabielsoin (CBE), cannabielsoinic acid, cannabicitranic acid, cannabinolic acid (CBNA), cannabinol (CBN), cannabinol methylether (CBNM), cannabinol-C4, (CBN-C4), cannabivarin (CBV), cannabinol-C2(CNB-C2), cannabiorcol (CBN-C1), cannabinodiol (CBND), cannabinodivarin (CBVD), cannabitriol (CBT), 10-ethyoxy-9-hydroxy-delta-6a- tetrahydrocannabinol, 8,9-dihydroxyl-delta-6a-tetrahydrocannabinol, cannabitriolvarin (CBTVE), dehydrocannabifuran (DCBF), cannabifuran (CBF), cannabichromanon (CBCN), cannabicitran (CBT), 10-oxo-delta-6a-tetrahydrocannabinol (OTHC), delta-9-cis- tetrahydrocannabinol (cis-THC), 3,4,5,6-tetrahydro-7-hydroxy-alpha-alpha-2-trimethyl-9-n- propyl-2,6-methano-2H-1-benzoxocin-5-methanol (OH-iso-HHCV), cannabiripsol (CBR), and trihydroxy-delta-9-tetrahydrocannabinol (triOH-THC).
[00151] “Cannabinoid precursor” as used herein may refer to any intermediate present in the cannabinoid biosynthetic pathway before the production of the“mother cannabinoid,” cannabigerolic acid (CBGA). Cannabinoid precursors may include, but are not limited to, GPP, olivetolic acid, hexanoyl-CoA, pyruvate, acetoacetyl-CoA, butyryl-CoA, acetyl-CoA, HMG-CoA, mevalonate, mevalonate-5-phosphate, mevalonate diphosphate, and malonyl- CoA...
[00169] As described herein, novel polypeptides for catalyzing production of cannabigerolic acid from GPP and olivetolic acid have been identified and characterized. Surprisingly, these new polypeptides of the present disclosure can catalyze production of cannabigerolic acid from GPP and olivetolic acid in an amount at least ten times higher than previously discovered Cannabis polypeptides that catalyze production of cannabigerolic acid from GPP and olivetolic acid (see, for example, U.S. Patent Application Pub. No. US20120144523 and the GOT polypeptide, CsPT1, disclosed therein; SEQ ID NO:82 herein)...
[00411] Exemplary GPPS heterologous nucleic acids disclosed herein may include nucleic acids that encode a GPPS polypeptide, such as, a full-length GPPS polypeptide, a fragment of a GPPS polypeptide, a variant of a GPPS polypeptide, a truncated GPPS polypeptide, or a fusion polypeptide that has at least one activity of a GPPS polypeptide...
[00707] Materials and methods suitable for the maintenance and growth of the recombinant cells of the disclosure are described herein, e.g., in the Examples section. Other materials and methods suitable for the maintenance and growth of cell (e.g. bacterial or yeast) cultures are well known in the art. Exemplary techniques can be found in International Publication No. WO2009/076676, U.S. Patent Application No.12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No.2010/0048964, WO 2009/132220, US Publ. No.2010/0003716, Manual of Methods for General Bacteriology Gerhardt et al, eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA.
[00708] Standard cell culture conditions can be used to culture the genetically modified host cells disclosed herein (see, for example, WO 2004/033646 and references cited therein).
[00709] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in International Publication No. WO 2009/076676, U.S. Patent Application No.12/335,071 (U.S. Publ. No.
2009/0203102), WO 2010/003007, US Publ. No.2010/0048964, WO 2009/132220, US Publ. No.2010/0003716, the contents of each of which are incorporated by reference herein in their entireties. Batch and Fed- Batch fermentations are common and well known in the art and examples can be found in Brock, Biotechnology: A Textbook of Industrial
Microbiology, Second Edition (1989) Sinauer Associates, Inc. Production and Recovery of Produced Cannabinoids, Cannabinoid Precursors,
Cannabinoid Derivatives or Cannabinoid Precursor Derivatives
SEQ ID SEQUENCE
SEQ ID NO:20 MIVKPMVRNNICLNAHPQGCKKGVEDQIEYTKKRITAEVKAGAKAPK Ter: trans-2-enoyl- NVLVLGCSNGYGLASRITAAFGYGAATIGVSFEKAGSETKYGTPGWY CoA reductase NNLAFDEAAKREGLYSVTIDGDAFSDEIKAQVIEEAKKKGIKFDLIVYS (Treponema sp.) LASPVRTDPDTGIMHKSVLKPFGKTFTGKTVDPFTGELKEISAEPANDE
EAAATVKVMGGEDWERWIKQLSKEGLLEEGCITLAYSYIGPEATQAL YRKGTIGKAKEHLEATAHRLNKENPSIRAFVSVNKGLVTRASAVIPVIP LYLASLFKVMKEKGNHEGCIEQITRLYAERLYRKDGTIPVDEENRIRID DWELEEDVQKAVSALMEKVTGENAESLTDLAGYRHDFLASNGFDVE GINYEAEVERFDRI SEQ ID NO:21 MTREVVVVSGVRTAIGTFGGSLKDVAPAELGALVVREALARAQVSGD BktB: beta- DVGHVVFGNVIQTEPRDMYLGRVAAVNGGVTINAPALTVNRLCGSGL ketothiolase QAIVSAAQTILLGDTDVAIGGGAESMSRAPYLAPAARWGARMGDAGL (Ralstonia sp.) VDMMLGALHDPFHRIHMGVTAENVAKEYDISRAQQDEAALESHRRAS
AAIKAGYFKDQIVPVVSKGRKGDVTFDTDEHVRHDATIDDMTKLRPV FVKENGTVTAGNASGLNDAAAAVVMMERAEAERRGLKPLARLVSYG HAGVDPKAMGIGPVPATKIALERAGLQVSDLDVIEANEAFAAQACAV TKALGLDPAKVNPNGSGISLGHPIGATGALITVKALHELNRVQGRYAL VTMCIGGGQGIAAIFERI SEQ ID NO:22 MKEVVMIDAARTPIGKYRGSLSPFTAVELGTLVTKGLLDKTKLKKDKI MvaE: acetyl-CoA DQVIFGNVLQAGNGQNVARQIALNSGLPVDVPAMTINEVCGSGMKAV acetyltransferase/HM ILARQLIQLGEAELVIAGGTESMSQAPMLKPYQSETNEYGEPISSMVND G-CoA reductase GLTDAFSNAHMGLTAEKVATQFSVSREEQDRYALSSQLKAAHAVEAG (Enterococcus sp.) VFSEEIIPVKISDEDVLSEDEAVRGNSTLEKLGTLRTVFSEEGTVTAGNA
SPLNDGASVVILASKEYAENNNLPYLATIKEVAEVGIDPSIMGIAPIKAI QKLTDRSGMNLSTIDLFEINEAFAASSIVVSQELQLDEEKVNIYGGAIAL GHPIGASGARILTTLAYGLLREQKRYGIASLCIGGGLGLAVLLEANMEQ THKDVQKKKFYQLTPSERRSQLIEKNVLTQETALIFQEQTLSEELSDHM IENQVSEVEIPMGIAQNFQINGKKKWIPMATEEPSVIAAASNGAKICGNI CAETPQRLMRGQIVLSGKSEYQAVINAVNHRKEELILCANESYPSIVKR GGGVQDISTREFMGSFHAYLSIDFLVDVKDAMGANMINSILESVANKL REWFPEEEILFSILSNFATESLASACCEIPFERLGRNKEIGEQIAKKIQQA GEYAKLDPYRAATHNKGIMNGIEAVVAATGNDTRAVSASIHAYAARN GLYQGLTDWQIKGDKLVGKLTVPLAVATVGGASNILPKAKASLAMLD IDSAKELAQVIAAVGLAQNLAALRALVTEGIQKGHMGLQARSLAISIG AIGEEIEQVAKKLREAEKMNQQTAIQILEKIREK SEQ ID NO:23 MKIGIDKLHFATSHLYVDMAELATARQAEPDKYLIGIGQSKMAVIPPS MvaS: HMG-CoA QDVVTLAANAAAPMLTATDIAAIDLLVVGTESGIDNSKASAIYVAKLL synthase GLSQRVRTIEMKEACYAATAGVQLAQDHVRVHPDKKALVIGSDVAR (Lactobacillus YGLNTPGEPTQGGGAVAMLISADPKVLVLGTESSLLSEDVMDFWRPL plantarum) YHTEALVDGKYSSNIYIDYFQDVFKNYLQTTQTSPDTLTALVFHLPYT
KMGLKALRSVLPLVDAEKQAQWLAHFEHARQLNRQVGNLYTGSLYL SLLSQLLTDPQLQPGNRLGLFSYGSGAEGEFYTGVIQPDYQTGLDHGLP QRLARRRRVSVAEYEALFSHQLQWRADDQSVSYADDPHRFVLTGQK NEQRQYLDQQV SEQ ID NO:24 MKLSTKLCWCGIKGRLRPQKQQQLHNTNLQMTELKKQKTAEQKTRP Erg13: HMG-CoA QNVGIKGIQIYIPTQCVNQSELEKFDGVSQGKYTIGLGQTNMSFVNDRE synthase DIYSMSLTVLSKLIKSYNIDTNKIGRLEVGTETLIDKSKSVKSVLMQLFG (Saccharomyces ENTDVEGIDTLNACYGGTNALFNSLNWIESNAWDGRDAIVVCGDIAIY cerevisiae) DKGAARPTGGAGTVAMWIGPDAPIVFDSVRASYMEHAYDFYKPDFTS
EYPYVDGHFSLTCYVKALDQVYKSYSKKAISKGLVSDPAGSDALNVL KYFDYNVFHVPTCKLVTKSYGRLLYNDFRANPQLFPEVDAELATRDY DESLTDKNIEKTFVNVAKPFHKERVAQSLIVPTNTGNMYTASVYAAFA SLLNYVGSDDLQGKRVGLFSYGSGLAASLYSCKIVGDVQHIIKELDITN
Recovery
[00925] Whole-cell broth from cultures comprising genetically modified host cells of the disclosure are extracted with a suitable organic solvent to afford cannabinoids, cannabinoid precursors, cannabinoid derivatives, or cannabinoid precursor derivatives. Suitable organic solvents include, but are not limited to, hexane, heptane, ethyl acetate, petroleum ether, and di-ethyl ether, chloroform, and ethyl acetate. The suitable organic solvent, such as hexane, is added to the whole-cell broth from fermentations comprising genetically modified host cells of the disclosure at a 10:1 ratio (10 parts whole-cell broth– 1 part organic solvent) and stirred for 30 minutes. The organic fraction is separated and extracted twice with an equal volume of acidic water (pH 2.5). The organic layer is then separated and dried in a concentrator (rotary evaporator or thin film evaporator under reduced pressure) to obtain crude cannabinoid, cannabinoid precursor, cannabinoid derivative, or cannabinoid precursor derivative crystals. The crude crystals may then be heated to 105 °C for 15 minutes followed by 145 °C for 55 minutes to decarboxylate a crude cannabinoid or cannabinoid derivative. The crude crystalline product is re-dissolved and recrystallized in a suitable solvent (e.g., n-pentane) and filtered through a 1 µm filter to remove any insoluble material. The solvent is then removed e.g. by rotary evaporation, to produce pure crystalline product. In vitro enzyme assay and cell-free production of cannabinoids or cannabinoid derivatives
[00926] In some embodiments, genetically modified host cells, e.g., genetically modified yeast cells, verified to comprise one or more heterologous nucleic acids encoding a GOT polypeptide that catalyzes production of cannabigerolic acid from geranyl pyrophosphate and olivetolic acid in an amount at least ten times higher than a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:82 or a polypeptide comprising an amino acid sequence having at least 65% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO:100 or SEQ ID NO:110, are cultured in 96-well microtiter plates containing 360 µL of YPD (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L dextrose (glucose)) and sealed with a breathable film seal. Cells are then cultured at 30 °C in a high capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 3 days until the cultures reach carbon exhaustion. The growth-saturated cultures are then subcultured into 200 mL of YPGAL media to an OD600 of 0.2 and incubated with shaking for 20 hours at 30 °C. Cells are then harvested by centrifugation at 3000 x g for 5 minutes at 4 °C. Harvested cells are then resuspended in 50 mL buffer (50 mM Tris-HCl, 1 mM EDTA, 0.1 M KCl, pH 7.4, 125 units Benzonase) and then lysed (Emulsiflex C3, Avestin, INC., 60 bar, 10 min). Cells debris is removed by centrifugation (10,000 × g, 10 min, 4 °C). Subsequently, the supernatant is then subjected to ultracentrifugation (150,000 × g, 1 h, 4 °C, Beckman Coulter L-90K, TI-70). The resulting membrane fractions of the GOT polypeptide that catalyzes production of cannabigerolic acid from geranyl pyrophosphate and olivetolic acid in an amount at least ten times higher than a polypeptide comprising an amino acid sequence set forth in SEQ ID NO:82 or the polypeptide comprising an amino acid sequence having at least 65% (e.g., at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%) sequence identity to SEQ ID NO:100 or SEQ ID NO:110 are then resuspended in 3.3 mL buffer (10 mM Tris-HCl, 10 mM MgCl2, pH 8.0, 10% glycerol) and solubilized with a tissue grinder. Then, 0.02% (v/v) of the respective membrane preparations are then dissolved in reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 8.5) and substrate (500 µM olivetolic acid, 500 µM GPP) to a total volume of 50 µL and incubated for 1 hour at 30 °C. Assays are then extracted by adding two reaction volumes of ethyl acetate followed by vortexing and centrifugation. The organic layer is evaporated for 30 minutes, resuspended in acetonitrile/H2O/formic acid (80:20:0.05%) and filtered with Ultrafree® -MC columns (0.22 µm pore size, PVDF membrane material). Cannabinoids or cannabinoid derivatives are then detected via LC-MS and/or recovered and purified. Yeast cultivation in a bioreactor
[00927] Single yeast colonies comprising genetically modified host cells disclosed herein are grown in 15 mL of Verduyn medium (originally described by Verduyn et al, Yeast 8(7): 501-17) with 50 mM succinate (pH 5.0) and 2% glucose in a 125 mL flask at 30 °C, with shaking at 200 rpm to an OD600 between 4 to 9. Glycerol is then added to the culture to a concentration of 20% and 1 mL vials of the genetically modified host cell suspension are stored at-80 °C. One to two vials of genetically modified host cells are thawed and grown in Verduyn medium with 50 mM succinate (pH 5.0) and 4% sucrose for 24 hours, then sub-cultured to an OD600 reading of 0.1 in the same media. After 24 hours of growth at 30 °C with shaking, 65 mL of culture is used to inoculate a 1.3-liter fermenter (Eppendorf DASGIP Bioreactor) with 585 mL of Verduyn fermentation media containing 20 g/L galactose supplemented with hexanoic acid (2 mM), a carboxylic acid other than hexanoic acid (2 mM), olivetolic acid (1 mM), or an olivetolic acid derivative (1 mM). A poly-alpha-olefin may be added to the fermenter as an extractive agent. The fermenter is maintained at 30 °C and pH 5.0 with addition of NH4OH. In an initial batch phase, the fermenter is aerated at 0.5 volume per volume per minute air (VVM) and agitation ramped to maintain 30% dissolved oxygen. After the initial sugar is consumed, the rise in dissolved oxygen triggers feeding of galactose + hexanoic acid (800 g galactose per liter + 9.28 g hexanoic acid per liter) at 10 g galactose per liter per hour in pulses of 10 g galactose per liter doses (alternatively, rather than feeding the genetically modified host cells disclosed herein hexanoic acid, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid is fed to the genetically modified host cells).
[00928] Between pulses, the feed rate is lowered to 5 g galactose per liter per hour. Upon a 10% rise in dissolved oxygen, the feed rate is resumed at 10 g L<-1>hour<-1>. As genetically modified host cell density increases, dissolved oxygen is allowed to reach 0%, and the pulse dose is increased to 50 g galacose per liter. Oxygen transfer rate is maintained at rates representative of full-scale conditions of 100 mM per liter per hour by adjusting agitation as volume increased. Feed rate is adjusted dynamically to meet demand using an algorithm that alternates between a high feed rate and low feed rate. During the low feed rate, genetically modified host cells should consume galactose and hexanoic acid, or, alternatively, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid, and any overflow metabolites accumulated during the high feed rate. A rise in dissolved oxygen triggers the high feed rate to resume. The length of time spent in the low feed rate reflects the extent to which genetically modified host cells are over- or under-fed in the prior high feed rate pulse; this information is then monitored and used to tune the high feed rate up or down, keeping the low feed rate within a defined range.
[00929] Over time, the feed rate matches sugar and hexanoic acid, or, alternatively, olivetolic acid, an olivetolic acid derivative, or a carboxylic acid other than hexanoic acid, demand from genetically modified host cells. This algorithm ensures minimal net accumulation of fermentation products other than cannabinoids, cannabinoid derivatives, cannabinoid precursors, or cannabinoid precursor derivatives; biomass; and CO2. In some embodiments, the process continues for 5 to 14 days. In certain such embodiments, accumulated broth is removed daily and assayed for biomass and cannabinoid, cannabinoid derivative, cannabinoid precursor, or cannabinoid precursor derivative concentration. A concentrated solution of NH4H2PO4, trace metals and vitamins are added periodically to maintain steady state concentrations. Example 1– Synthesis of Olivetolic Acid or Derivatives Thereof
[00930] The cannabinoid pathway is composed of four biosynthetic steps using the precursors hexanoyl-CoA, malonyl-CoA, and geranyl pyrophosphate (FIG.1, Box 4)...
Polypeptide Function Original host
[00937] Multiple polypeptides in pathway 1b require NADH as a co-factor. In order to maximize flux through pathway 1b, other biosynthetic pathways that compete for NADH supply are modified (FIG.1, Box 2). One target can be the ethanol pathway, mediated by various alcohol dehydrogenase polypeptides, but may also include other pathways that consume NADH, such as the glycerol biosynthesis pathway.
[00938] Another route conceived towards hexanoyl-CoA is described in pathway 1c: The alfatoxin biosynthetic gene cluster (iterative type I PKS) encodes a fatty acid synthase- based mechanism (FasA and FasB) for production of hexanoyl-CoA. In some embodiments, a heterologous nucleic acid encoding a thioesterase polypeptide and a heterologous nucleic acid encoding a CoA ligase polypeptide similar to a C6-tolerant thioesterase polypeptide (see BMC Biochem.2011 Aug 10;12:44. doi: 10.1186/1471-2091-12-44) and a heterologous nucleic acid encoding a HCS polypeptide are expressed to facilitate release of hexanoyl- ACP and activate free hexanoate to its acyl-CoA compound. Additionally, various type II PKS biosynthetic pathways (e.g. benastatin, R1128) contain a FabH-like KSIII (e.g. BenQ, ZhuH), AT and ACP component, which are crucial for providing and selecting the rare hexanoate PKS starter unit. Lastly, the type I PKS pathway for reveromycin biosynthesis encodes the fatty acyl-CoA ligase RevS polypeptide and the FabH-like KASIII component RevR polypeptide, which are suggested to provide hexanoyl-CoA via fatty acid degradation as well as de novo fatty acid biosynthesis. [00939] To avoid competitive consumption of hexanoyl-CoA via ß-oxidation, the fatty acid degradation pathway is engineered to have lowered activity. Alternatively, yeast are grown in presence of oleic acid to avoid competition for fatty acids as energy source.
[00940] The pathway of four genes encoding the NADH pathway for hexanoyl-CoA production, including polypeptides PaaH1, Crt, Ter, and BktB, was constructed under the control of Gal1, Gal10, Gal7, and TEF2 promoters, respectively. FIG.4. The whole cassette was inserted between the upstream and downstream homology region of ADE2 and was integrated into the genome of S. cerevisiae using CRISPR/Cas9 to generate yXL001 (using Construct 1/pXL044 as shown in FIG.4). The pathway of four genes encoding the NADPH pathway (including PhaB, PhaJ, Ter, and BktB polypeptides) was introduced into to S. cerevisiae in the same way to generate yXL002 (using Construct 1/pXL072 as shown in FIG.4). The MCT1 gene under the control of Gal1 promoter flanked by the 1622b homology region (Construct 2; FIG.4) was introduced into the genome of yXL001 and yXL002 using CRISPR/Cas9 to generate yXL003 and yXL004 (FIG.4).
[00941] A cassette encoding TKS and OAC genes under the control of Gal1 and Gal10 promoters flanked by ACC1 homology region (Construct 4; FIG.5) was introduced into the genome of yXL003 and yXL004 using CRISPR/Cas9 to generate yXL005 and yXL006. A heterologous nucleic acid encoding a TKS-OAC fusion polypeptide under the control of a Gal1 promoter (Construct 5; FIG.5) was introduced into yXL003 and yXL004 to generate yXL007 and yXL008. The resulting strains were inoculated into 10 mL YP medium supplemented with 2% dextrose. After an overnight culture at 30 °C and centrifugation at 3,000 × g for 5 mins, the pellet was resuspended into YP medium supplemented with 2% galactose. After two days expression, the culture supernatant was extracted with equal volume of ethyl acetate, and, after evaporation and filtration, the samples were analyzed by LC-MS, which showed the production of a significant amount of olivetolic acid (FIG.9 and FIG.10).
[00942] CsAAE (Construct 3; FIG.4), TKS, and OAC genes (Construct 4; FIG.5) were introduced into the genome of S. cerevisiae using CRISPR/Cas9 to generate yXL009, which can produce higher level of olivetolic acid in the presence of exogenously supplied hexanoate (FIG.11).
[00943] In addition, by supplementing the growth medium with various aliphatic acids, from C4-C10, various olivetolic acid derivatives can be produced from yXL009 (FIG. 11 and FIG.12). Some of the olivetolic acid derivatives can be further modified by biological or chemical means to covalently attach to other compounds. For example, click chemistry can be performed on the olivetolic derivative containing alkyne functional group. The olivetolic derivative is dissolved in biology grade dimethyl sulfoxide (DMSO) and treated with a DMSO solution of crosslinker containing an azide group (1.0 equiv.), TBTA (DMSO: tBuOH 1:1), CuSO45H2O, sodium ascorbate and HEPES-KOH pH: 7.0 (final HEPES-KOH˜250 mM). The reaction is placed on a water bath at 37 °C for 12 to 16 hours. Liquid chromatograph-mass spectrometry (LC-MS) analysis of the reaction mixture shows reaction completion after 16 hours to obtain the further modified olivetolic acid.
[00944] The GPPS large subunit (GPPSlsu) and small subunit (GPPSssu) genes from Cannabis sativa under the control of Gal1 and Gal10 promoters flanked by ADE1 homology region (Construct 10; FIG.7) were introduced into yXL008 and yXL009 to generate yXL010 and yXL011. A cassette encoding a NphB polypeptide and a THCAS polypeptide under the control of Gal1 and Gal10 promoters flanked by 1014a homology region
(Construct 12; FIG.8) was introduced into the genome of yXL010 and yXL011 to generate yXL012 and yXL013 using CRISPR/Cas9. The resulting strains were inoculated into 10 mL YP medium supplemented with 2% dextrose. After an overnight culture at 30 °C and centrifugation at 3,000 × g for 5 mins, the pellet was resuspended into YP medium supplemented with 2% galactose. After two days expression, the culture supernatant was extracted with equal volume of ethyl acetate, and, after evaporation and filtration, the samples were analyzed by LC-MS, which showed that the overexpression of NphB in yXL010 resulted in the production of cannabigerolic acid (FIGS.14 and 15). In the presence of a THCAS polypeptide, the cannabigerolic acid was transformed into THCA or into THC. With yXL013, C4-C10acids were added to the expression medium, resulting in the production of cannabigerolic acid derivatives, which were then modified by a THCAS polypeptide to produce THCA or THC derivatives. Those derivatives can then be further modified by chemical reactions (FIG.13). Example 3– Synthesis of Cannabinoid Precursors, Cannabinoids, or Derivatives of the Foregoing
[00945] To recreate cannabinoid production in microorganisms, chassis S. cerevisiae strains were developed containing metabolic pathways for the production of (1) GPP through the mevalonate (Mva) pathway, (2), olivetolic acid or derivatives, (3) CBGA or derivatives, and (4) different cannabinoids or cannabinoid derivatives produced by cannabinoid synthase polypeptides. Production of GPP
[00946] A GPP-overproducing strain, GTY23, was produced by overexpressing Mva pathway genes and introducing a repressible promoter on ERG9. A previously described ERG20 F96W-N127W mutant, ERG20mut, was added to provide a source of GPP precursor in the cell (FIG.16). This strain was used to screen GOT polypeptide candidates.
Production of Olivetolic Acid or Derivatives Thereof
[00947] Olivetolic acid was produced from sugar by introducing genes CsTKS and CsOAC, and pathways to produce hexanoyl-CoA. Pathways for the production of hexanoate and hexanoyl-CoA are known in the art (e.g., Gajewski et al,“Engineering fungal de novo fatty acid synthesis for short chain fatty acid production,” Nature Communications 2017). To produce olivetolic acid or its derivatives, rather than using hexanoyl-CoA pathways, a previously reported acyl-CoA ligase polypeptide, such as a CsAAE1 or CsAAE3 polypeptide, was introduced and exogenously fed cells hexanoate or a carboxylic acid other than hexanoate (FIGS.17-19). These pathways allow for the production of non-naturally occurring cannabinoids.
Production of CBGA
[00948] The mother cannabinoid CBGA, or derivatives thereof, was produced by a GOT polypeptide. A C. sativa GOT polypeptide was identified in the 1990s, yet no report was identified describing reconstituting GOT polypeptide activity in vivo. Twenty-five polypeptide variants were screened for in vivo production of CBGA in strains containing GPP pathways and exogenously fed olivetolic acid. These genes were all chromosomally integrated driven by GAL1 promoters and screened for activity in yeast extract peptone galactose (YPG) media. GC-MS and LC-MS analysis demonstrated in vivo production of CBGA from a CsPT4t polypeptide (FIGS 26A-C). The gene sequence of the CsPT4t polypeptide is referred to as a GOT polypeptide (FIG.20). yL444 was the strain used in the production of CBGA and expresses the following genotype: CEN.PK2-1D {1114a::GAL1p- CsPT4t-TDH1t; 308a::GAL1p-ERG20(F96W-N127W)-TDH1t; erg9::KanMX_CTR3p- ERG9; leu2-3,112::His3MX6_GAL1p-ERG19/GAL10p-ERG8; ura3-52::ura3/GAL1p- MvaS(A110G)/GAL10p-MvaE; his3_1::hphMX4_GAL1p-ERG12/GAL10p-IDI1; MATa} (FIGS.6 and 20). LC-MS was carried out as follows (FIGS 26A-C):
Column info: 2015 Kinetex XB-C182.1x100 mm RES6 method 10.6 min Method info:
0-5.6 mins, 45%-73%B, 0.2 mL/min
5.6-6.2 mins, 73%-97%B, 0.2 mL/min 6.2-11.3 mins, 97%B, 0.3 mL/min
11.3-12.7, 97-45%B, 0.3 mL/min
12.7-15.5, 45%B, 0.3 mL/min
A: H2O+0.05% TFA
Production of THCA and CBDA
[00949] Cannabinoid synthase genes have been identified from the Cannabis genome (including but not limited to THCA synthase (THCAS), CBDA synthase (CBDAS), JP450547, JP454863, JP471546, JP452622). To produce THCA and CBDA, the corresponding THCA synthase and CBDA synthase, respectively, were introduced into a strain producing CBGA containing a heterologous nucleic acid encoding a CsPT4t polypeptide. The synthases were introduced as N-terminal truncated polypeptides with polypeptide tags, e.g., ProA signal sequence (MIFDGTTMSIAIGLLSTLGIGAEA, from proteinase A with UniProt accession number F2QUG8) attached and the transcription of both synthases were under the control of GAL10 promoter. The final plasmid constructs were named as pESC-ProA-THCAS and pESC-ProA-CBDAS. Both plasmids were transformed individually into the above-mentioned strain, which has high CBGA production in the presence of olivetolic acid, to give strains yXL046 and yXL047 (FIGS.21-25).
[00950] After confirming the transformation by PCR of THCAS or CBDAS, two colonies from each culture were inoculated into a defined medium (SC-Leu + 2% Dextrose) and were incubated at 30 °C with shaking at 800 RPM. After two-day growth, the cultures were back-diluted 1:50 into inducing medium (SC-Leu + 2% galactose + 1 mM olivetolic acid + CuSO4) and incubated at 30 °C with shaking at 800 RPM for 4 days. After 4-day incubation, equal volume of ethyl acetate was added to the expression cultures and the mixtures were subjected to three rounds of bead beating. Then the mixtures were then spun down at 5000 RPM and the organic layers were sent for LC-MS analysis, which showed the production of THCA and CBGA from the corresponding cultures (FIGS.27 and 28). Example 4– Generation of a Base Yeast Strain Capable of High Flux to CBGA with Olivetolic Acid Feeding
[00951] CBGA production strains were created from wild-type Saccharomyces cerevisiae strain (CEN.PK2) by expressing genes of the mevalonate pathway polypeptides and a GOT polypeptide under control of the GAL1 or GAL10 promoter. The S21 strain comprised the following chromosomally integrated mevalonate pathway genes from S. cerevisiae: ERG10, ERG13, truncated HMG1 (tHMGR), ERG12, ERG8, ERG19, and IDI1. The S21 strain additionally comprised the chromosomally integrated pyruvate decarboxylase (PDC) from Zymomonas mobilis to increase flux from pyruvate towards acetyl-CoA.
[00952] To generate additionally strains, a mutant form of ERG20, ERG20mut, which preferentially generates GPP was added to the S21 strain with the following chromosomally integrated GOTs from C. sativa: CsPT1 (S164), a truncated CsPT1 (CsPT1_t75, S165), or CsPT4 (S29). Constructs used in S29, S164, and S165 are shown in Table 11.
[00953] Yeast colonies verified to contain the expected DNA assembly comprising one or more heterologous nucleic acids disclosed herein were picked into 96-well microtiter plates containing 360 µL of YPD (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L dextrose (glucose)) and sealed with a breathable film seal. Cells were cultured at 30 °C in a high capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 3 days until the cultures reached carbon exhaustion. The growth-saturated cultures were subcultured into fresh plates containing YPGAL and olivetolic acid (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L galactose, 1 g/L glucose and 1 mM olivetolic acid) by taking 14.4 µL from the saturated cultures and diluting into 360 µL of fresh media and sealed with a breathable film seal. Genetically modified host cells in the production media were cultured at 30 °C in a high capacity microtiter plate shaker at 1000 rpm and 80% humidity for an additional 3 days prior to extraction and analysis. Upon completion, 100 µL of whole cell broth was diluted into 900 µL of methanol, sealed with a foil seal, and shaken at 1500 rpm for 60 seconds to extract the cannabinoids. After shaking, the plate was centrifuged at 1000 x g for 60 seconds to remove any solids. After centrifugation, 12 µL of supernatant was transferred to a fresh assay plate containing 228 µL of methanol, sealed with a foil seal, shaken for 60 seconds at 900 rpm, and analyzed by LC-MS...