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Biosynthesis method and application of N-acetyl-5-methoxytryptamine
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A technology of methoxytryptamine and its synthesis method, which is applied in the field of biosynthesis of N-acetyl-5-methoxytryptamine, can solve the problems of industrial application limitation, large energy consumption, pollution, etc., and achieve the reduction of production cost and condition Ease of control and productivity-enhancing effects
Pending Publication Date: 2022-06-28
河北维达康生物科技有限公司
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Problems solved by technology
Due to the low content of naturally occurring N-acetyl-5-methoxytryptamine in animals and plants, and the limited source of extraction raw materials, the extraction cost remains high, and the industrial application is subject to many restrictions
Chemical synthesis is currently a commonly used method in the industrial production of N-acetyl-5-methoxytryptamine. According to literature research, it is found that the synthesis methods are roughly divided into two categories: ring-building and ring-borrowing synthesis, which require pollution and are highly toxic. benzene ring-containing substances and catalysts, and the product separation and purification process is complicated, energy consumption is large, and the product purity is low.
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Embodiment 1
[0100] Example 1 Construction of 5-hydroxyβ-indolylalanine plasmid
[0101] Tandem expression of key enzyme gene TPH2 in β-indolylalanine hydroxylation pathway; key enzyme folE, PTPS and SPR in BH4 synthesis pathway; PCD and DHPR key enzyme in BH4 regeneration.
[0102] TPH2, folE, PTPS, SPR, PCD, DHPR genes were inserted between the NdeI and XhoI sites of pET28a(+) plasmid, respectively, to obtain pET28a-TPH2, pET28a-folE, pET28a-PTPS, pET28a-SPR, pET28a-PCD , pET28a-DHPR plasmid (such as Figure 1-6 shown).
[0103] Using F1 and R1 as primers and pET28a-TPH2 plasmid as template to clone the pET28a plasmid vector containing TPH2 gene; using F2 and R2 as primers and plasmid pET28a-folE as template to clone to obtain folE gene; using F3 and R3 as primers, plasmid pET28a - PTPS gene was cloned to obtain PTPS gene; F4 and R4 were used as primers, plasmid pET28a-SPR was used as template to obtain SPR gene; F5 and R5 were used as primers, and PCD gene was obtained by cloning plas...
Embodiment 2
[0108] Example 2 Construction of N-acetyl-5-hydroxytryptamine plasmid
[0109] Tandem expression of key enzyme genes AANAT and ACS in 5-hydroxyβ-indolylalanine-producing N-acetyl-5-hydroxytryptamine pathway.
[0110] The AANAT and ACS genes were inserted between the NcoI and AflII sites of the pACYCDuet plasmid to obtain the pACYCDuet-AANAT and pACYCDuet-ACS plasmids (such as Figure 8 , 9 shown).
[0111] Using F7 and R7 as primers, pACYCDuet-AANAT plasmid was used as template to clone to obtain pACYCDuet plasmid vector containing AANAT gene; using F8 and R8 as primers, plasmid pACYCDuet-ACS was used as template to clone to obtain ACS gene.
[0112] The above fragments were ligated together by a seamless cloning kit to form the plasmid pACYCDuet-AANAT-ACS (as Figure 10 ).
[0113] Table 2 Primer sequences 2
[0114]
Embodiment 3
[0115] Example 3 Construction of N-acetyl-5-methoxytryptamine plasmid
[0116] Tandem expression of key enzyme genes COMT and MAT in N-acetyl-5-hydroxytryptamine-producing N-acetyl-5-methoxytryptamine pathway.
[0117] The COMT and MAT genes were inserted between the NcoI and AflII sites of the plasmid pETDuet to obtain pETDuet-COMT and pETDuet-MAT plasmids (such as Figure 11 , 12 shown).
[0118] The pETDuet plasmid vector containing COMT gene was cloned with F9 and R9 as primers and pETDuet-COMT plasmid as template; MAT gene was cloned with F10 and R10 as primers and plasmid pETDuet-MAT as template.
[0119] The above fragments were ligated together by a seamless cloning kit to form plasmid pETDuet-CM (such as Figure 13 ).
[0120] Table 3 Primer sequences 3
[0121]
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Abstract
The invention provides a biological synthesis method of N-acetyl-5-methoxytryptamine and an application of the N-acetyl-5-methoxytryptamine. The N-acetyl-5-methoxytryptamine is synthesized by using all the protein coding genes for synthesizing the N-acetyl-5-methoxytryptamine or the recombinant genetic engineeringbacteria capable of expressing and synthesizing all the protein coding genes of the N-acetyl-5-methoxytryptamine. And separating the N-acetyl-5-methoxytryptamine from the system to obtain the N-acetyl-5-methoxytryptamine. The invention also discloses a recombinant vector containing a series of enzyme coding genes for synthesizing the N-acetyl-5-methoxytryptamine and a recombinant geneengineering bacterium. A series of enzyme coding genes for coding the synthesized N-acetyl-5-methoxytryptamine in a recombinant vector and a series of enzymes for synthesizing the N-acetyl-5-methoxytryptamine are expressed in a fermentation culture mode, the N-acetyl-5-methoxytryptamine is synthesized, and the yield of the N-acetyl-5-methoxytryptamine is high when the N-acetyl-5-methoxytryptamine is synthesized. The process is easy to implement, conditions are easy to control, and industrial production is suitable.
Description
technical field [0001] The invention relates to the technical fields of genetic engineering, metabolic engineering, enzymeengineering, bioinformatics and synthetic biology, in particular to a biosynthesis method of N-acetyl-5-methoxytryptamine and its application. Background technique [0002] N-acetyl-5-methoxytryptamine, a derivative of β-indolylalanine, is an amine hormone produced by the pineal gland of mammals after being stimulated by darkness. of antioxidants. Can improve animal sleep quality. With the increase of animal age, the secretion of N-acetyl-5-methoxytryptamine gradually decreased, thus affecting the sleep quality of animals. N-acetyl-5-methoxytryptamine was first discovered in the pineal gland of bovine and is considered to be an important neurohormone. Later studies found that N-acetyl-5-methoxytryptamine was found in various organs of the human body. It is distributed and plays different important functions. Now more and more studies have shown that N...
Claims
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Application Information
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