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Rice tryptophan decarboxylase and production method thereof

A technology of tryptophan decarboxylase and rice, which is applied in the field of bioengineering, can solve the problems of high price and achieve the effect of increasing the expression level and increasing the expression level

Active Publication Date: 2021-09-24
XINTAI JIAHE BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, my country's tryptamine products still mainly rely on imports (Chen Ning et al., 2017), and the price is quite expensive. It is urgent to develop a green, low-cost, and efficient new technology route for the biosynthesis of tryptamine

Method used

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  • Rice tryptophan decarboxylase and production method thereof
  • Rice tryptophan decarboxylase and production method thereof
  • Rice tryptophan decarboxylase and production method thereof

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1: Cloning and codon optimization of rice tryptophan decarboxylase (TDC) gene

[0031] The leaves of nutrient-deficient rice were selected, and the total RNA of the material was extracted by the CTAB method, according to the GenBank EST database ( https: / / www.ncbi.nlm.nih.gov / genbank / GenBank The complete cDNA ORF sequence information obtained in ) was used to design primers (TDCsense and TDCantisense), and the cDNA synthesized by reverse transcription was used as a template to amplify the full-length open reading frame by PCR.

[0032] TDCsense: 5'-GTAAGGTATAGTATCATC-3'; (SEQ ID NO.4)

[0033] TDCantisense: 5'-GTGAGTTAGCTAGGTTGGTGTG-3'. (SEQ ID NO.5)

[0034] The full-length cDNA of the rice tryptophan decarboxylase (TDC) gene includes a complete open reading frame of 1533bp, and the nucleotide sequence is shown in SEQ ID NO.3; it encodes a polypeptide of 507 amino acids, and its theoretical molecular weight is 56.21ku.

[0035] In order to make the gene ...

Embodiment 2

[0037] Embodiment 2: Construction of expression vector pGEX-4t-J:

[0038] Using the plasmid pGEX-4T-1 as the starting plasmid, first use pflmI to carry out a single enzyme cut at the 3250 position, then cut 2 pieces at the 3' end, cut 6 pieces at the 5' end, add A to the 3' end, and add CTAGT to the 5' end ;Reprotect; then use btgI to perform single enzyme digestion at the 4869 position, cut 4 pieces at the 5' end and then add AAGA to remove all protection; plus 5'-CTAGT...G-3' full-length 100bp Artificial chain, the omitted sequence in the middle of the artificial chain, as long as it does not contain the two enzyme cutting sites of SpeI and Bsc91I, the omitted sequence in the middle can be selected arbitrarily.

[0039] Plasmid pGEX-4t-J was double-digested with SpeI and Bsc91I and identified. After double-digestion, pEGX-4t-J appeared 3349 and 100, which proved that the expression vector pGEX-4t-J was successfully constructed.

Embodiment 3

[0040]Embodiment 3: Fermentative production of rice tryptophan decarboxylase

[0041] (1) Use SpeI and Bsc91I to double-digest the plasmid pGEX-4t-J, and then integrate the optimized TDC gene shown in SEQ ID NO.1 into the double-digested expression vector pGEX-4t by DNA ligase -J to obtain the recombinant expression vector pEGX-TDC; and then introduce the obtained recombinant expression vector into Escherichia coli B21 (DE3) to obtain the recombinant bacteria.

[0042] (2) Inoculate the recombinant bacteria into the culture medium for fermentation and cultivation. The conditions for fermentation and cultivation are: pH control at 7.0, tank pressure at 0.05MPa, temperature at 33°C, ventilation ratio 1:1, and fermentation until the fermentation medium is diluted 100 times. After OD 600 Value is 0.18-0.20, obtains fermentation medium;

[0043] Cool the fermentation culture liquid to 22°C, add the inducer IPTG to the fermentation culture liquid to make the final concentration of...

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Abstract

The invention discloses rice tryptophan decarboxylase and a production method thereof, and belongs to the technical field of bioengineering. The nucleotide sequence of gene TDC for coding the rice tryptophan decarboxylase is as shown in SEQ ID NO. 1. The production method comprises the following steps of connecting the gene TDC as shown in SEQ ID NO.1 into a plasmid pEGX-4t-J to obtain a recombinant expression vector; introducing the obtained recombinant expression vector into escherichia coli to obtain recombinant bacteria; inoculating the recombinant bacteria into a culture medium for fermentation culture to obtain a fermentation culture solution; adding an inducer into the fermentation culture solution for induced culture to obtain an induced culture; and separating and purifying the induced culture to obtain the rice tryptophan decarboxylase. The rice tryptophan decarboxylase produced by the method is high in expression level and good in catalytic activity.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a rice tryptophan decarboxylase and a production method thereof. Background technique [0002] Tryptophan decarboxylase (TDC) is a 5'-phosphate pyridoxal-5'-phosphate (PLP)-dependent decarboxylase, which can catalyze the decarboxylation of tryptophan to produce tryptamine (Wang Peng et al., 2014; López-Meyer et al., 1997). Tryptophan decarboxylase can be derived from plants, animals and microorganisms. In plants, tryptophan decarboxylase belongs to a class of proteins in the aromatic amino acid decarboxylase (Aromatic amino acid decarboxylases, AAADs) family of plants (Jiang Jingjie et al., 2019), and relies on the cofactor 5'-pyridoxal phosphate The function of catalyzing the decarboxylation of tryptophan to form tryptophan has been reported in many plants such as camptophyllum, periwinkle, brevity snakeroot, tomato, Indian ginseng, and beautiful capillary (Jadaun et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70
CPCC12N9/88C12N15/70C12Y401/01028C12N2800/22
Inventor 岳明瑞谢沛曹华杰郭永胜腾义卫
Owner XINTAI JIAHE BIOTECH CO LTD
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