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Production of cannabinoids in yeast

A technology of cannabinoids and yeast, applied in the direction of enzymes, fermentation, biochemical equipment and methods, etc., can solve the problems of mixture purification, cannabinoid pollution, etc.

Pending Publication Date: 2020-03-17
BIOMEDICINES INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the structural similarity of many cannabinoids, it is difficult to purify these mixtures to high levels, resulting in cannabinoid contamination of the final product

Method used

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  • Production of cannabinoids in yeast
  • Production of cannabinoids in yeast
  • Production of cannabinoids in yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0261] Example 1: Vector Construction and Transformation

[0262] The Yarrowia lipolytica episomal plasmid contains a centromere, an origin, and a bacterial replicative backbone. Fragments of these regions were synthesized by TwistBioscience and cloned to make the episomal parental vector pBM-pa. Plasmids were constructed by Gibson assembly, Golden gate assembly, ligation or sequence and ligation independent cloning (SLIC). Genomic DNA was isolated from bacteria (E. coli) and yeast (Yarrowia lipolytica) using the Wizard Genomic DNA Purification Kit according to the manufacturer's protocol (Promega, USA). Synthetic genes were codon-optimized using GeneGenie or Genscript (USA) and assembled from gene fragments purchased from TwistBioscience. All engineered Yarrowia lipolytica strains were constructed by transformation of the corresponding plasmids. All gene expression cassettes were constructed using the TEF intronic promoter and a synthetic short terminator. Cloning of up t...

Embodiment 2

[0264] Embodiment 2: yeast culture condition

[0265] E. coli DH10B strain was used for cloning and plasmid propagation. DH10B were grown at 37°C with constant shaking in Luria-Bertani broth supplemented with 100 mg / L ampicillin for plasmid propagation. Yarrowia lipolytica strain W29 was used as the base strain for all experiments. Yarrowia lipolytica was cultured at 30°C under constant agitation. Yarrowia lipolytica cultures (2 ml) used in large-scale screens were grown in a shaking incubator at 250 rpm for 1 to 3 days, then larger culture volumes were shaken in 50 ml flasks or in bioreactors fermentation.

[0266] For colony selection and cell proliferation, Yarrowia lipolytica was grown on YPD liquid medium containing 10 g / L yeast extract, 20 g / L peptone and 20 g / L glucose or on YPD agar plates supplemented with 20 g / L agar. The medium is usually supplemented with 150 to 300 mg / L hygromycin B or 250 to 500 mg / L nourstacin as appropriate for selection. For cannabinoid-p...

Embodiment 3

[0267] Example 3: Cannabinoid Isolation

[0268] Yarrowia lipolytica cultures from shake flask experiments or bioreactors were pelleted and homogenized in acetonitrile followed by incubation on ice for 15 minutes. After centrifugation (13000 g, 4° C., 20 minutes), the supernatant was filtered (0.45 μm, Nylon) and analyzed by HPLC-DAD. The quantification of the product is based on the integrated peak area of ​​the UV chromatogram at 225nm. Standard curves were generated for CBGA and THCA. The identity of all compounds can be confirmed by Brukercompact using positive ionization mode TM Mass spectra and tandem mass spectra of each sample analyzed by ESI-Q-TOF were compared with co-eluting standards.

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Abstract

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, oneor 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% dryweight of fatty acids or fats.

Description

[0001] priority document [0002] This application claims priority to U.S. Provisional Patent Application No. 62531827, entitled "Production of terpenes including cannabinoids in yeast," filed on July 12, 2017, said U.S. The contents of the provisional patent application are hereby incorporated by reference in their entirety. technical field [0003] The present disclosure relates to the production of cannabinoids in yeast. Background technique [0004] Cannabinoids are a large class of chemicals that act on cannabinoid receptors and other target molecules to modulate a wide range of physiological behaviors, such as neurotransmitter release. Cannabinoids are produced naturally in humans (called endocannabinoids) and by several plant species, including Cannabis sativa (called phytocannabinoids). Cannabinoids have been shown to have several beneficial medical / therapeutic effects and are therefore an active area of ​​research by the pharmaceutical industry for use as medicina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/06C12P7/42
CPCC12P7/42C12P17/06C12N15/52C12N1/16C12Y205/01001C12N9/1085C12Y121/03007C12Y121/03008C12Y203/01206C12Y404/01026C12N9/0004C12N9/0006C12N9/1029C12N9/88C12N9/93C12Y101/01034C12Y203/0102C12Y604/01002
Inventor 马克西姆·米哈耶夫高第丰
Owner BIOMEDICINES INC
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