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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: 2022-06-07
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

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

[0034] The full cDNA length of the rice tryptophan decarboxylase (TDC) gene contains a complete open reading frame of 1533 bp, the nucleotide sequence is shown in SEQ ID NO.3, and the polypeptide encoding 507 amino acids has a theoretical molecular mass of 56.21 ku.

[0035]In order to make the rice tryptophan decarboxy...

Embodiment 2

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

[0038] Taking the plasmid pGEX-4T-1 as the starting plasmid, pflmI was first used to perform single enzyme cleavage at the 3250 site, 2 more at the 3' end, 6 at the 5' end, A at the 3' end, and CTAGT at the 5' end; Reprotect; Then perform a single enzyme cleavage at the 4869 site with btgI., 4 at the 5' end and add AAGA to remove all protection; plus 5'-CTAGT... G-3' 100 bp of artificial chains, artificial chains omitted in the middle, as long as they do not contain speiI. and Bsc91I. two enzyme sites, the middle omitted sequence can be arbitrarily selected.

[0039] The plasmid pGEX-4t-J was double-digested and identified with SpeiI. and Bsc91I., and 3349 and 100 were present in pEGX-4t-J after double digestion, which proved that the expression vector pGEX-4t-J was successfully constructed.

Embodiment 3

[0040] Example 3: Fermentation production of rice tryptophan decarboxylase

[0041] (1) Double digestion of plasmid pGEX-4t-J using SpeiI. and Bsc91I., and then integrate the TDC gene shown in the optimized SEQ ID NO.1 into the expression vector pGEX-4t-J after double digestion by DNA ligase to obtain the recombinant expression vector pEGX-TDC; and then the obtained recombinant expression vector is introduced into E. coli B21 (DE3) to obtain recombinant bacteria.

[0042] (2) Inoculate recombinant bacteria into culture medium for fermentation culture, the conditions for fermentation culture are: pH controlled at 7.0, tank pressure 0.05MPa, temperature 33 °C, ventilation ratio 1:1, fermentation culture to OD after fermentation culture liquid dilution 100 times 600 The value is 0.18-0.20, and the fermentation culture liquid is obtained;

[0043]The fermentation culture medium was cooled to 22 °C, the inducer IPTG was added to the fermentation culture medium, so that the final concen...

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Abstract

The invention discloses a rice tryptophan decarboxylase and a production method thereof, belonging to the technical field of bioengineering. The nucleotide sequence of the gene TDC encoding rice tryptophan decarboxylase is shown in SEQ ID NO.1. Connect the gene TDC shown in SEQ ID NO.1 into the plasmid pEGX-4t-J to obtain a recombinant expression vector; then introduce the obtained recombinant expression vector into Escherichia coli to obtain recombinant bacteria; inoculate the recombinant bacteria into the medium for fermentation culturing to obtain a fermentation culture liquid; adding an inducer to the fermentation culture liquid for induction culture to obtain an induced culture; separating and purifying the induced culture to prepare rice tryptophan decarboxylase. The rice tryptophan decarboxylase produced by the invention has high expression level and good catalytic activity.

Description

Technical field [0001] The present invention relates to the field of bioengineering technology, specifically to a rice tryptophan decarboxylase and a production method thereof. Background [0002] Tryptophan decarboxylase (TDC) is a 5'-pyridoxal-5'-phosphphate(PLP)-dependent decarboxylase that catalyzes the production of trypamine after tryptophan decarboxylase (Wang et al., 2014; López-Meyer et al., 1997). Tryptophan decarboxylase can be derived from plants, animals, and microorganisms. In plants, tryptophan decarboxylase belongs to the class of aromatic amino aciddecarboxylases (AAADs) family of proteins in the plant category (Jiang Jingjie et al., 2019), relying on the action of cofactor 5'-pyridoxal phosphate to achieve the function of catalyzing tryptophan decarboxylation to form tryptophan, which has been reported in a variety of plants such as hibiscus, periwinkle, short snake root grass, tomato, Indian ginseng, and beautiful cap pillar tree ( Jadaun et al.,2017；Pang et al...

Claims

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Application Information

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Patent Type & Authority Patents(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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