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Enzymes for cannabinoid synthesis and methods of making and using same

A kind of cannabinoid, cannabinoid phenolic acid technology, applied in the field of molecular biology

Pending Publication Date: 2022-07-08
杭州恩和生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many challenges remain in the production of cannabinoid compounds, which are traditionally extracted and purified from plants
Furthermore, it is difficult to produce multiple cannabinoid analogs using currently known methods

Method used

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  • Enzymes for cannabinoid synthesis and methods of making and using same
  • Enzymes for cannabinoid synthesis and methods of making and using same
  • Enzymes for cannabinoid synthesis and methods of making and using same

Examples

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example

[0145] Cloning, Expression and Purification of Prenyltransferases

[0146] In certain embodiments, gene candidates are expressed in E. coli, protein extracts are prepared, and purified enzymes are delivered for testing by affinity enzymatic purification using HIS tags to form CBGAs. In yet other exemplary embodiments, gene candidates are expressed in yeast. Expressed protein extracts were purified and used for testing. Illustrative examples demonstrating specific methods of cloning, expressing and purifying the enzymes of the present disclosure are described in the Examples herein.

[0147] Method for preparing cannabinoids

[0148] Cannabinoids can be produced using heterologous expression in microorganisms or yeast. Microorganisms can be genetically engineered to express cannabinoids or cannabinoid precursor molecules. Methods of heterologous expression of cannabinoid compounds are known in the art and are described, for example, in Carvalho et al., 2017, which is incorp...

example 1

[0151] Example 1: Cloning, Expression and Purification of Enzymes

[0152] The candidate gene was purchased from General Biosystems (Anhui, China) Corporation Limited as a gene block, and was located at the restriction endonuclease site NdeI ( CATATG) and XhoI (C TCGAG). All plasmids were transformed into BL21 (DE3) and named eCAN20005 to eCAN20060 (see Table 1).

[0153] 0.5 mL of the saturated culture in LB medium with 50 μg / mL kanamycin was inoculated into 50 mL of TB medium with 50 μg / mL kanamycin. Cultures were grown at 37°C to an OD600 of 0.5-0.8, induced with 0.5 mM IPTG, and then expressed at 25°C for 18 hours at 220 rpm.

[0154] Cells were harvested by centrifugation at 2500 x g, washed with lysis buffer (50 mM Tris-HCl, 500 mM NaCl, [pH 8.0], 10% (v / v) glycerol) and resuspended to OD550 in 10 ml of lysis buffer about 100. Cells were lysed using an ultrasonic cell disruptor for 10 min at 4°C. Lysates were clarified by centrifugation at 12,000 x g for 30 min at...

example 2

[0155] Example 2: In vitro enzyme assay

[0156] The reaction conditions for the enzyme assay consisted of the following: 50 mM HEPES (pH=7.5) with 5 mM MgCl2, 2 mM GPP, 2 mM olivetolic acid and 1 mg / ml of purified candidate enzyme in a final volume of 200 uL. After 18 hours of incubation at room temperature, the reaction mixture was extracted twice with 200 μL of ethyl acetate / formic acid (0.05% (v / v)). The organic extracts from each reaction were combined and solvent removed using a block heater. Samples were dissolved in 100 μL of resuspension (acetonitrile / H2O / formic acid (80% / 20% / 0.05% (v / v / v))) and filtered through a 0.22 μm PVDF membrane prior to LC-MS analysis.

[0157] figure 1 The enzymatic assay was validated by showing the results of titrating CBGA at four different concentrations. CBGA titration curve concentration: (A) 10ng / mL, (B) 100ng / mL, (C) 1ug / mL, (D) 10ug / mL. Table 2 also shows the LC-MS chromatogram of CBGA at m / z 359.5 with a retention time of 5.4...

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Abstract

Various enzymes for cannabinoid synthesis and methods of making and using the same are provided. Also provided are recombinant polypeptides having an isoprenyltransferase activity and / or geranyl pyrophosphate: oleylate geranyl transferase (GOT) activity, and engineered recombinant microorganisms for the production of CBGA, as well as methods of producing cannabinoid phenolic acid (CBGA) or analogs thereof, the method comprises the step of reacting a heterogeneously expressed isoprenyltransferase with a geranyl diphosphate ester (GPP) and an acid, such as oleylic acid (OA).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims US Provisional Patent Application Serial No. 62 / 909,227, filed on October 1, 2019, US Provisional Patent Application Serial No. 62 / 941,689, filed on November 27, 2019, and US Provisional Patent Application, filed on December 1, 2019 The benefit of and priority to Application Serial No. 62 / 942,198, which is incorporated herein by reference. [0003] Sequence Listing Reference [0004] This application contains a Sequence Listing in computer readable form, which is hereby incorporated by reference in its entirety. technical field [0005] This application relates to molecular biology, and more particularly to enzymes for cannabinoid synthesis. Background technique [0006] Cannabinoid compounds act on other targets of cannabinoid receptors and significantly affect the release of neurotransmitters in the brain. The use of cannabinoid compounds, such as cannabidiol for medical purposes, has expand...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22
CPCC12P17/06C12P7/22C12N15/70C12N15/81C12N15/815C12N9/1085C12Y205/01058C12Y205/01102C12P7/42
Inventor 吉约姆·科塔雷尔崔好曹佶聪
Owner 杭州恩和生物科技有限公司
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