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Recombinant escherichia coli for heterologously synthesizing ambrein and construction method thereof

A technology for recombining Escherichia coli and ambroxol, which is applied in the biological field and can solve problems such as the inability to meet the requirements of industrial production, low yield, and no reports of ambroxol

Active Publication Date: 2018-03-23
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the chemical synthesis of ambroxol has made some progress, the chemical synthesis method is very complicated and the yield is very low, which is far from meeting the requirements of industrial production.
In recent years, some natural products have been heterologously synthesized using synthetic biology techniques, but the heterologous synthesis of ambroxol in microorganisms has not been reported

Method used

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  • Recombinant escherichia coli for heterologously synthesizing ambrein and construction method thereof
  • Recombinant escherichia coli for heterologously synthesizing ambrein and construction method thereof
  • Recombinant escherichia coli for heterologously synthesizing ambrein and construction method thereof

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Embodiment 1

[0051] The construction of the recombinant escherichia coli of embodiment 1 synthetic squalene

[0052] In order to construct the squalene synthetic metabolic pathway on the Escherichia coli genome, the Saccharomyces cerevisiae squalene synthase gene ERG9, which was truncated by 26 amino acid residues at the C-terminus, was first constructed downstream of the constitutive trc promoter as an operator module trcp-ERG9 .

[0053] The upstream and downstream homology arms of the Saccharomyces cerevisiae squalene synthase gene ERG9 truncated by 26 amino acid residues at the C-terminal were fused with the lacZ gene site of Escherichia coli ATCC.47076 (hereinafter referred to as E. coli ATCC.47076) as Donor DNA;

[0054] The expression plasmid for constructing the gRNA targeting the lacZ gene locus is plasmid 1.

[0055] The donor DNA and plasmid 1 were transformed into E. coli ATCC.47076, and the S. cerevisiae squalene synthase gene ERG9, which was truncated by 26 amino acid resid...

Embodiment 2

[0064] Example 2 Construction method of recombinant Escherichia coli for heterologous synthesis of ambroxol

[0065] 1. Construction of expression vectors for enzymes related to ambroxol synthesis

[0066] ① According to the amino acid residue sequence of Alicyclobacillus acidocaldarius squalene-hopene cyclase after the 377th amino acid residue was mutated to cysteine ​​residue, codon optimization for Escherichia coli was carried out, by Kings Rui Biological Technology Co., Ltd. designed and synthesized Alicyclobacillus acidocaldarius squalene-hopene cyclase gene D377C SHC (SEQ ID NO.2) after mutating the 377th amino acid residue to cysteine ​​residue .

[0067] The D377C SHC gene fragment was inserted into the SacI and BamHI restriction sites of the Escherichia coli expression plasmid p5C (Zhenquan Lin et al., Microbial Cell Factories. 2014, 13(1):104) to obtain plasmid 2.

[0068] The specific steps are: using the D377C SHC gene sequence (SEQ ID NO.2) as a template, and us...

Embodiment 3

[0080] Example 3 Construction Method of the Second Recombinant Escherichia coli for Heterologous Synthesis of Ambroxol

[0081]1. Construction of the expression vector of the BmeTC-Linker1-D377C SHC fusion protein for the synthesis of ambroxol

[0082] Design of fusion protein expressed by fusion of Alicyclobacillus acidocaldarius squalene-hopene cyclase and Bacillus megaterium tetraprenyl-β-curcumene cyclase after mutating the 377th amino acid residue to cysteine ​​residue Gene (BmeTC-Linker1-D377C SHC). The BmeTC-Linker1-D377C SHC gene (SEQ ID NO.4) was inserted into the EcoRI and KpnI restriction sites of the Escherichia coli expression plasmid p5C to obtain plasmid 5.

[0083] The design principle of the fusion protein is: delete the stop codon of the BmeTC gene, connect it with the amino acid sequence of the connecting peptide (Linker1), and then connect it with the D377C SHC gene fragment with the stop codon. Due to the absence of the stop codon of the BmeTC nucleotide...

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Abstract

The invention discloses recombinant escherichia coli for heterologously synthesizing ambrein and a construction method thereof. The construction method comprises the following steps of: (1) fusing saccharomyces-cerevisiae squalene synthase gene ERG9 with truncation of 26 amino acid residues at a C end and homologous arms at the upstream and downstream of a lacZ site of the escherichia coli into adonor DNA; constructing plasmid 1; transforming the donor DNA and the plasmid 1 together into the escherichia coli, eliminating the plasmid 1, obtaining the recombinant escherichia coli for synthesizing squalene, and naming the recombinant escherichia coli as a strain 1; (2) connecting alicyclobacillus-acidocardarius squalene-hopene cyclase gene D377C SHC with mutation from the 377th amino acid residue into cysteine residue and bacillus-megaterium cyclase gene BmeTC into one segment, then inserting the segment into escherichia-coli expression plasmid p5C to obtain plasmid 4; (3) transforming the plasmid 4 into the strain 1 to obtain the recombinant escherichia coli for heterologously synthesizing the ambrein, and naming the recombinant escherichia coli as a strain 4. Proved by experiments,the recombinant escherichia coli for heterologously synthesizing the ambrein is fermented to obtain the ambrein.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant Escherichia coli for heterologously synthesizing ambroxol and a construction method thereof. Background technique [0002] Ambrein ((+)-Ambrein) is a tricyclic triterpene compound. It is the main component of precious natural animal spices and traditional Chinese medicine ambergris. It is the source of the aroma of ambergris. Active ingredients for medicinal effects such as inflammation. Ambergol is generally used in the high-end perfume market. Because it is very rare in nature, the application of ambergol in the perfume industry and medicine is restricted. [0003] Ambergris alcohol was originally isolated from ambergris, the intestinal secretion of the sperm whale (Physeter macrocephalus), and it is considered to be a pathological calculus formed by incomplete digestion of food in the sperm whale. There are two ways to obtain ambergris, one is excreted from sperm w...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/113C12N15/90C12P7/02C12R1/19
CPCC12N9/1085C12N9/88C12N9/90C12N15/113C12N15/902C12P7/02C12Y205/01021C12Y504/99017C12Y402/0313
Inventor 卢文玉可迪
Owner TIANJIN UNIV
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