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Method for biologically synthesizing indole-3-methyl glucosinolate I3M by using escherichia coli

A technology of methyl glucosinolate and Escherichia coli, applied in the biological field, can solve problems such as unreported, achieve the effect of broad prospects and improve synthesis efficiency

Pending Publication Date: 2022-02-08
KUNMING INST OF BOTANY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It has been reported in the literature that the synthesis pathway of aromatic glucosinolates was introduced into Escherichia coli, and the sulfur supply pathway was added, so that the desulfurized glucosinolate precursors could be sulfated under the action of sulfotransferase to form a complete aromatic glucosinolate. Glucosinolates (Petersen et al., 2019), indicating that the synthesis of glucosinolates using Escherichia coli as a biosynthetic engineering strain is feasible and has great application value, but the synthesis of indole glucosinolates in Escherichia coli Not yet reported

Method used

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  • Method for biologically synthesizing indole-3-methyl glucosinolate I3M by using escherichia coli
  • Method for biologically synthesizing indole-3-methyl glucosinolate I3M by using escherichia coli
  • Method for biologically synthesizing indole-3-methyl glucosinolate I3M by using escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Screening of Turnip Indole Glucosinolates (IGS) Synthetic Pathway Genes

[0051] Seven indole glucosinolate (I3M) biosynthetic genes (CYP79B2; CYP83B1; GSTF9; GGP1; SUR1; UGT74B1; ST5a) and BRAD ( http: / / brassicadb.org / brad / glucoGene.php) corresponding to the 7 families of Chinese cabbage (Brassica pekinensis) genes and the genes of corresponding families in cabbage (Brassica oleracea var. Seven gene family members corresponding to turnip (Brassicarapavar.rapa) were searched in genome database. Circus software (http: / / www.circos.ca / software / download / Circos / ) was used to generate the chromosomal map of the turnip genome sequence to determine the chromosomal location of the searched genes. Using the NeighborJoining method in the MEGA software to construct a phylogenetic tree, perform homology analysis on the genes screened from the turnip genome and seven genes in Arabidopsis thaliana, and select the gene with the highest homology to the corresponding gene . ...

Embodiment 2

[0052] Embodiment 2 sequence optimization

[0053] The codon optimization table of Escherichia coli was used to optimize the target gene. The sequence optimization and synthesis were completed by Shanghai Generay Biotech Co., Ltd. (Generay Biotech, Shanghai). The expression vector was pET-52b. The original vector plasmid was provided by Shanghai Generay Biotech Engineering Co., Ltd. (Generay Biotech, Shanghai) provided, considering the need for subsequent vector construction, restriction sites were added to the 5' and 3' ends of the partial gene sequence. The specific enzyme cutting sites are added as shown in Table 1 below:

[0054] Table 1 Restriction site addition

[0055]

[0056] Among them, GGTACC is the KpnI restriction site; GAATTC is the EcoRI restriction site.

Embodiment 3

[0057] Example 3 Vector construction and transformation of BL21(DE3)

[0058] The 7 target genes according to the attached figure 1 As shown, the construction of carrier A, carrier B and carrier C is completed respectively, and the specific construction methods are as follows:

[0059] (1) Construct vector A

[0060] 1. Using a high-fidelity enzyme ( Super-Fidelity DNA Polymerase, VazymeBiotech Co., Ltd) to amplify the target fragment, Table 2 is the list of primers used to construct the vector A amplified fragment (including the primers used to amplify the terminator):

[0061] Table 2 Primers used to construct vector A

[0062]

[0063] Note: The sequence of the terminator fragment is the same, but the linker sequence and restriction site are different due to the different connection position

[0064]2. Use primer pET-52b-P1-F / R to amplify the T7 promoter on the vector pET-52b together with the target gene CYP83B1 to obtain gene fragment P1 (T7+CYP83B1); use primer p...

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Abstract

The invention relates to a method for biologically synthesizing indole-3-methyl glucosinolate I3M by using escherichia coli, and belongs to the technical field of biology. According to the present invention, turnip genes CYP79B2, CYP83B1, GSTF9, GGP1, SUR1, UGT74B1 and ST5a are adopted to construct a carrier, and I3M is successfully synthesized in an escherichia coli strain BL21 (DE3) by using IPTG induction; quantitative analysis shows that the yield of indole-3-methyl glucosinolate I3M synthesized by the method reaches 1.89 mg / L, the synthesis efficiency of I3M is improved, the method can be applied to biosynthesis of other types of glucosinolate, certain theoretical support is provided for industrial synthesis of glucosinolate, and the prospect is very wide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for biosynthesizing indole-3-methylglucosinolate I3M by using Escherichia coli. Background technique [0002] Glucosinolates (GS for short), also known as glucosinolates, are a variety of plant secondary metabolites containing nitrogen and sulfur. In nature, glucosinolates are mainly distributed in Brassicaceae plants, most of which belong to the Brassica genus (Brassica), but there are also very small amounts of distribution in other families, some common Brassica edible vegetables Most contain glucosinolates. Glucosinolates and their degradation products have a variety of biological activities, and are the main source of the unique flavor of Brassica vegetables. The main degradation products, isothiocyanates, not only have anticancer activity, but also play a role in the defense of plants such as insect resistance and disease resistance. also play an important role in th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/29C12N15/54C12N15/53C12N15/55C12P19/64
CPCC12N15/70C12N15/52C12N9/0071C12N9/1088C12N9/485C12N9/1051C07K14/415C12P19/64C12Y205/01018
Inventor 胡越杨雅杨云强杨永平
Owner KUNMING INST OF BOTANY - CHINESE ACAD OF SCI