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Synthesis method of tropinone

A synthesis method and tropinone technology are applied in the fields of synthetic biology and enzyme catalysis, which can solve the problems of insufficient stability and low expression, and achieve the effects of increasing yield and high application value.

Active Publication Date: 2022-04-08
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plant P450 is a membrane protein, which has defects such as low expression, insufficient stability, and difficulty in enzyme engineering

Method used

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  • Synthesis method of tropinone
  • Synthesis method of tropinone
  • Synthesis method of tropinone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Fermentative accumulation of engineering strain PL1-AaPKS 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid

[0030] Take out the engineering strain PL1-AaPKS from the -80°C refrigerator, inoculate it into 5mL YPD medium, culture it in a shaker at 30°C (rotating speed 200rpm) for 2-3 days, and inoculate it to 100mL YPD according to the inoculation amount of 10%-15%. The culture medium was cultured at 30°C for 5 days, and the fermentation supernatant was collected by centrifugation, freeze-dried and concentrated to the original volume of 5-10 mL.

Embodiment 2

[0031] Embodiment 2: the crude enzyme liquid freeze-dried powder preparation of monoamine oxidase

[0032] The synthesis of the monoamine oxidase gene sequence (shown in SEQ ID NO.2) and the construction of the monoamine oxidase expression plasmid pET28a-MAO-N were completed by Jinweizhi Biotechnology Co., Ltd. Take Escherichia coli competent cells E.coli BL21 out of the -80°C refrigerator and place them on ice, pipette 1 microliter of the constructed monoamine oxidase expression plasmid pET28a-MAO into the competent cells, mix gently, Ice-bathed for 30 minutes, then placed in a 42°C water bath for heat shock for 90 seconds, immediately placed on ice for 2 minutes, and added 1 mL of LB to the transformation system. After cultivating on a shaker at 37°C for 40-60 minutes, take out the low-speed centrifugation for 3 minutes, discard most of the supernatant, gently blow the resuspended bacteria with a pipette gun, and spread it on the LA plate containing 50 μg / mL kanamycin antibi...

Embodiment 3

[0035] Example 3: Biotransformation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid to produce tropinone

[0036] Add monoamine oxidase crude enzyme lyophilized powder to 200 μL of the fermented broth concentrated in Example 1 to a final concentration of 50 μg / mL, and at the same time add FAD to a final concentration of 1 mM, and peroxidase to a final concentration of 25 μg / mL; ~7 days, lyophilize the reaction solution, resuspend with 200 μL acetonitrile, centrifuge to get the supernatant and detect the formation of tropinone by LC-MS (MS detection results are as follows: Figure 4 ), the results showed that the conversion rate of this reaction was as high as 90%.

[0037] LC-MS detection conditions:

[0038] Instrument: SHIMADZU LCMS-2020UPLC-MS;

[0039] Analytical column: R227-32015-03Shim-pack Velox Biphenyl (2.7μm, 2.1x100 mm, Shimadzu Corporation), the flow rate is 0.4mL min -1 , the detected mobile phase is (A = deionized water, B = CH containing 0.1% formic acid ...

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Abstract

The invention discloses a synthesis method of tropinone, and belongs to the technical field of synthetic biology and enzyme catalysis. According to the present invention, the tropinone is synthesized by combining the engineering strain capable of generating the 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid and the monoamine oxidase capable of catalyzing the conversion of the 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid into the tropinone; the engineering strain contains a related gene for synthesizing the 4-(1-methyl-2-pyrrolidinyl)-3-oxobutyric acid, and the related gene is a gene for synthesizing the 4-(1-methyl-2-pyrrolidinyl)-3- The monoamine oxidase is a protein (namely MAO-N) with an amino acid sequence as shown in SEQ ID NO. 1. The invention provides a green and efficient new route for synthesis of tropinone, and has a better industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and enzyme catalysis, and more specifically relates to a synthesis method of tropinone. Background technique [0002] Tropinone (Tropinone) is a class of plant-derived tropane alkaloids containing a tertiary amine structure, which can be used to synthesize natural tropine drugs such as atropine. Its chemical name is 8-methyl-8azabicyclo[3.2 .1] Octan-3-one. At present, tropane drugs are mainly extracted from plants. Although chemists have realized the chemical total synthesis of tropinone (Robinson, R.LXIII.A synthesis of tropinone.Chem.Soc., Trans., 1917,111,762-768.) very early, it is difficult to control the follow-up The chiral structure of synthetic tropane drugs, environmental pollution and other problems have not been well applied. Therefore, researchers began to pay attention to the analysis of biosynthetic pathways and biosynthetic methods of tropane natural products, hoping ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/54C12N15/60C12N15/81C12N15/70C12P17/18
CPCY02P20/55
Inventor 瞿旭东林芝
Owner WUHAN UNIV
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