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Three FAD dependent enzymes in morus and application of three FAD dependent enzymes in synthesis of Diels-Alder adducts

A technology of oxidase and cyclization reaction, applied in the field of host cells, can solve problems such as limited sources, inability to realize biosynthesis, lack of selectivity in chemical synthesis, etc.

Pending Publication Date: 2021-10-29
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. The content of active Diels-Alder adducts in mulberry is low, the chemical synthesis lacks selectivity, and the source is limited;
[0007] 2. The key oxidase for diene formation in the biosynthesis of Diels-Alder adducts in mulberry and the cyclase for the intermolecular [4+2] cycloaddition reaction and its related genes have not been reported, and its biological synthesis

Method used

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  • Three FAD dependent enzymes in morus and application of three FAD dependent enzymes in synthesis of Diels-Alder adducts
  • Three FAD dependent enzymes in morus and application of three FAD dependent enzymes in synthesis of Diels-Alder adducts
  • Three FAD dependent enzymes in morus and application of three FAD dependent enzymes in synthesis of Diels-Alder adducts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Cloning of geneMaFAD1, geneMaFAD2, geneMaFAD3 genes

[0032] 1. Extraction of total RNA and synthesis of cDNA first strand

[0033] Select an appropriate amount of mulberry suspension cells as the material, quickly transfer them to a mortar pre-cooled with liquid nitrogen, and grind them thoroughly to a powder; transfer the powdered samples to a centrifuge tube, add 500 μL RCL buffer / 100 mg tissue (E.Z.N.A. TM Plant RNA kit), homogenate with a vortexer and pipette until mixed; incubate at 55°C for 3 minutes; centrifuge at 15,000×g for 5 minutes, transfer the supernatant to a filter column, and centrifuge at 14,000×g for 2 minutes. Add the filtered liquid to an equal volume of RCB buffer and invert up and down 5-10 times. Transfer the liquid to the RNA adsorption column and centrifuge at 10,000×g for 1 min; discard the filtrate, add 400 μL RWC elution buffer to the adsorption column, and centrifuge at 10,000×g for 1 min; dry the adsorption column at room t...

Embodiment 2

[0045] Example 2: Expression of geneMaFAD1, geneMaFAD2, geneMaFAD3 genes and separation and purification of recombinant proteins

[0046] 1. Construction of SMD1168-pPIC3.5K recombinant expression strain and induced expression of recombinant protein

[0047] According to the gene sequences on both sides of the multiple cloning site of the pPIC3.5K vector, primers containing homology arms of the vector were designed respectively, and homology arms were introduced into the N-terminal and C-terminal of the coding sequence of the target gene by PCR technology, and gel recovery PCR product. Homologous recombinase was used to connect the target gene with the linearized pPIC3.5K vector, construct the recombinant plasmid, transform Escherichia coli (Top10) cloning host, screen positive transformants by PCR, and send them for sequencing to verify the correctness of the reading frame. Transformants with correct sequencing were amplified and cultured, plasmids were extracted, linearized...

Embodiment 3

[0063] Example 3: Synthesis of chalcomoracin and kuwanon J adducts catalyzed by recombinant protein combination

[0064] The induced expression and isolation and purification of the recombinant protein are as described in Example 2. Moracin C is oxidized by dienoid oxidase MaFAD1 to form a dienoid, which undergoes Diels-Alder reaction with dienophile morachalcone A under the catalysis of cyclase MaFAD2 or MaFAD3. Through HPLC-MS analysis, it was found that MaFAD2 or MaFAD3 could efficiently catalyze the intermolecular [4+2] cyclization reaction to generate chalcomoracin. Morachalcone A can also be oxidized by the oxidase MaFAD1 to form a dienoid, and then undergo a Diels-Alder reaction with the dienophile morachalcone A under the enzymatic catalysis of the cyclase MaFAD2 or MaFAD3. It was found by HPLC-MS analysis ( Figure 1-2 ): MaFAD2 or MaFAD3 can efficiently catalyze the intermolecular [4+2] cyclization reaction to generate kuwanon J. The standard enzymatic catalytic r...

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Abstract

The invention belongs to the field of gene engineering, and relates to three FAD dependent enzymes in morus and application of the three FAD dependent enzymes in synthesis of Diels-Alder adducts, in particular to three FAD dependent enzymes MaFAD1, MaFAD2 and MaFAD3 in morus, nucleic acid molecules of encoded proteins of the three FAD dependent enzymes, expression vectors of the nucleic acid molecules, host cells containing the vectors, and a production method of the enzymes encoded by the nucleic acid molecules. The invention further relates to application of the MaFAD1 and the MaFAD2 in catalytic synthesis of Diels-Alder adducts such as chalcomracin and kuwanon J in morus, MaFAD1 is oxidase, and a diene body is synthesized through catalytic oxidation reaction; and maFAD2 and MaFAD3 are both cyclases, and a [4+2] cyclization reaction is catalyzed to synthesize the Diels-Alder adducts.

Description

technical field [0001] The present invention belongs to the field of genetic engineering, and specifically relates to a new nucleic acid molecule derived from three FAD-dependent enzymes MaFAD1, MaFAD2 and MaFAD3 derived from mulberry and its encoded protein, an expression vector containing the nucleic acid molecule, a host cell containing the vector, and a nucleic acid molecule composed of the nucleic acid molecule. The production method of molecularly encoded enzymes; it also relates to the application of these two types of enzymes in catalyzing the synthesis of Diels-Alder adducts in mulberry, wherein MaFAD1 is an oxidase that catalyzes the synthesis of dienes, and MaFAD2 and MaFAD3 are both cyclases that catalyze [4 +2] Cyclization reaction. Background technique [0002] Diels-Alder reaction, a typical [4+2] cyclization reaction, is a kind of reaction that is efficient and capable of constructing complex polycyclic compounds, and is widely used in organic synthetic chemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N9/02C12N15/53C12N15/54C12N15/81C12N1/19C12P17/04C12R1/84
CPCC12N9/1241C12N9/0083C12N15/815C12P17/04C12Y207/07002C12Y114/19001C12Y114/19006
Inventor 戴均贵苏聪解可波陈日道陈大伟刘继梅韩耀天
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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