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Expression vector for synthesizing cannabidiol, heterologous expression method and application

A technology of cannabidiol and expression vectors, which is applied in the field of synthetic cannabidiol expression vectors, can solve the problems of large-scale application, high oxygen consumption and energy consumption, and achieve the effect of reducing planting costs and efficient production

Inactive Publication Date: 2021-09-21
湖北碳元本草生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the synthetic pathway of CBD is analyzed, there is also the use of microbial heterologous host fermentation to produce CBD. Due to high oxygen consumption and energy consumption, this method cannot be applied on a large scale.

Method used

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  • Expression vector for synthesizing cannabidiol, heterologous expression method and application
  • Expression vector for synthesizing cannabidiol, heterologous expression method and application
  • Expression vector for synthesizing cannabidiol, heterologous expression method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Transient Transformation of Tobacco by Agrobacterium Engineering Bacteria to Produce Cannabidiol

[0068] 1. Pick the engineered Agrobacterium (bacterial strain EHA105) transformed with the expression plasmid in the LB medium containing the corresponding antibiotics, and carry out fermentation at 28±0.5°C for 24 hours.

[0069] 2. Add 100 μL of 0.5M MES and 2 μL of 100 mM AS to 5 mL of fresh LB containing the corresponding antibiotics, then inoculate 50 μL of Agrobacterium broth, and culture at 28° C. on a shaker at 250 rpm until OD600 = 1.0 (about 12-18 hours).

[0070] 3. Centrifuge at 4000rpm at room temperature for 10 minutes to collect the bacteria, resuspend with 10mM MgCl2 to OD600=1.0, add 100mM AS at a ratio of 2μL per ml of bacteria solution, and let stand for 3 hours. Specifically, the medium is selected from LB medium, TB medium or SOB medium, preferably TB medium.

[0071] 4. Take the tobacco (Nicotianabenthamiana) that is in the vigorous growth ...

Embodiment 2

[0073] The extraction of embodiment 2 cannabidiol

[0074] Tobacco leaves were taken 3-7 days after infection and analyzed for product. Tobacco leaves were taken and weighed to ensure that the wet weight of the leaves was the same for both the empty and the sample. Put the blade into the grinder, grind it with liquid nitrogen, grind it to a powder, add it to a 50mL centrifuge tube, and add about 10mL of 20% mass spectrometry grade methanol. Place the 50 mL centrifuge tube containing the sample on a shaker at 37 °C at 250 rpm for 1 hour. Then, at room temperature, using the method of dynamic immersion, put the centrifuge tube into the instrument and react for 45 minutes. Take out the centrifuge tube and centrifuge at 4500rpm for 10-15 minutes. Take the supernatant, filter it with a 0.45 μm organic phase filter membrane, use a rotary evaporator, and spin dry the sample.

[0075] Then dissolve the sample with about 100 μL of mass spectrometry grade methanol, take the reconsti...

Embodiment 3

[0076] Example 3 Exploration of Cannabidiol CBD Liquid Phase Conditions and Liquid Phase Conditions of Tobacco Biosynthesis Products

[0077] The direct transformation of the empty vector pC2300S (Kana resistance) into Agrobacterium EHA105 was used as a blank control. For the detection of positive clone results, see figure 2 As shown, wherein pAtUB10 is the promoter of gene AAE1, and tAdh is the terminator of gene AAE1. Both genes TKS and OAC use 35S promoter and 35S poly A as terminator. The genes CsPT4 and CBDAS use the NOS promoter and NOS terminator.

[0078] In addition, samples were taken from the engineering bacteria fermentation broth for HPLC analysis and mass spectrometry identification of LC-MS ( Figure 5-7 ) shows that the m / z value of the compound is 313.21730[M-H]-, and the secondary fragments are also the same as the standard cannabidiol, indicating that the engineering bacteria can produce cannabidiol from scratch.

[0079] Figure 5 Middle A-1 is the mas...

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Abstract

The invention discloses an expression vector for synthesizing cannabidiol, a heterologous expression method and application, five gene expression cassette vectors are constructed through an in-vitro de novo design and five enzymes of hexanoyl-CoA synthase, polyketide synthase, oleinic acid synthase, isopentenyl transferase and cannabidiol acid synthase, and a transient co-transformation experiment in tobacco shows that in this way, high yield of CBD can be achieved. According to the invention, efficient production of CBD can be realized by using a synthetic biological technical means, the synthetic product does not contain THC, and the measured CBD content is high; in addition, as a conventional commercial crop for agricultural production, the tobacco has advantages in planting technology, field management, yield of harvested leaves and the like, and planting cost can be greatly reduced.

Description

technical field [0001] The invention relates to the technical field of cannabidiol plant expression, in particular to an expression vector for synthesizing cannabidiol, a heterologous expression method and application. Background technique [0002] Cannabidiol (CBD) is the main active ingredient in industrial hemp. Industrial hemp, also known as hemp or hemp, has been used in China to make some textile products for the past 6,000 years. CBD is different from psychoactive tetrahydrocannabinol (THC). It is a multi-target drug with anti-inflammatory, analgesic, anti-anxiety, anti-cancer and other pharmacological activities. It is non-addictive and can counteract THC. The hallucinogenic effects, known as "anti-drug compounds." CBD is widely used. In cosmetics, it has the effects of repairing basal cells, anti-oxidation, and anti-aging; as a food additive, it can increase food nutrition; at the same time, CBD can also be used as one of the drugs for the treatment of epilepsy. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/54C12N15/60C12N15/53C12N5/10C12N1/21A01H5/12A01H6/82C12P7/22
CPCC12N15/8205C12N15/8243C12N15/52C12N9/1029C12N9/88C12N9/0004C12P7/22C12Y203/01199C12Y404/01026C12Y121/03008
Inventor 曹应龙虞沂邹小林
Owner 湖北碳元本草生物科技有限公司
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