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Construction method of escherichia coli bacterial strain for generating lanosterol

A technology of Escherichia coli and lanosterol is applied in the field of genetic engineering to achieve the effect of increasing expression

Inactive Publication Date: 2020-03-27
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on lanosterol biosynthesis using Escherichia coli

Method used

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  • Construction method of escherichia coli bacterial strain for generating lanosterol
  • Construction method of escherichia coli bacterial strain for generating lanosterol
  • Construction method of escherichia coli bacterial strain for generating lanosterol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Synthesis and cloning of lanosterol synthesis pathway genes

[0023] 1. Codon optimization, synthesis and amplification of the gene sequence of squalene epoxidase.

[0024] According to the codons of Escherichia coli BL21 (DE3), the codon optimization of squalene epoxidase of Methylococcus capsulatus was performed using the online software DNAWorks. The optimized sequence (SEQ ID NO.1) was sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis. Using the optimized synthetic vector as a template, the squalene epoxidase gene fragment was amplified by PCR with Primstar DNA polymerase (TAKARA Co, Japan). The PCR products were separated using 1% agarose gel, and fragments of the target length were cut and recovered using the Axygen Gel Recovery Kit (Axygen Biotechnology (Hangzhou) Co., Ltd.).

[0025] Squalene epoxidase gene PCR amplification system:

[0026]

[0027] PCR program

[0028]

[0029] The name of the vector synthesized by the compa...

Embodiment 2

[0055] Embodiment 2: Construction of lanosterol expression vector

[0056] In this example, first, the fragments of squalene epoxidase, NADPH-cytochrome P450 reductase and lanosterol synthase were fused into a DNA fragment "SE-CPR-LS" by fusion PCR. And this fragment was cloned into the vector pet21c by the method of restriction restriction ligation, and the expression vector "pet21c-SE-CPR-LS" ( figure 2 A), its original complete sequence is shown as SEQ ID NO.13. Then, the squalene synthase gene fragment is amplified by PCR amplification method. And this fragment is cloned on the vector pACYCduet-1 by the method for homologous recombination, constructs expression vector " pACYCduet-SS " ( figure 2 B), its full sequence is shown in SEQ ID NO.14.

[0057] The specific implementation steps are:

[0058] 1. Construction of "pet21c-SE-CPR-LS" expression vector

[0059] (1) Fusion PCR to prepare DNA fragment "SE-CPR-LS"

[0060] Fusion PCR amplification system:

[0061] ...

Embodiment 3

[0101] Embodiment 3, construction and fermentation of lanosterol Escherichia coli synthetic strain

[0102] 1. Construction of a synthetic strain of Escherichia coli lanosterol

[0103] The vectors "pet21c-SE-CPR-LS" and "pACYCduet-SS" constructed in Example 2 were transformed into Escherichia coli BL21(DE3) competent cells by chemical transformation method. The specific operation is as follows:

[0104] 1) Thaw Escherichia coli BL21(DE3) competent cells on ice.

[0105] 2) Take 100 ng of pet21c-SE-CPR-LS and pACYCduet-SS plasmids and mix well, add to competent cells; let stand on ice for 30 minutes.

[0106] 3) Heat shock the competent and plasmid mixture at 42°C for 90 seconds. Immediately place on ice for 2 minutes.

[0107] 4) Add 800 μl LB liquid medium to the cells, and incubate at 37° C. and 200 rpm for 1 hour.

[0108] 5) Take 100 μl and spread on LB solid medium containing 100 μg / mL ampicillin and 170 μg / mL chloramphenicol, culture overnight at 37°C. The long cl...

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Abstract

The invention relates to a construction method of an escherichia coli bacterial strain for generating lanosterol. The construction method comprises the steps of optimizing a codon of squalene epoxidase of methylococcus capsulatus according to an escherichia coli codon, and performing PCR amplification to obtain a squalene epoxidase genetic fragment; performing PCR amplification to obtain NADPH-cytochrome P450 reductase, and lanosterol synthetase genetic fragments, performing merging to obtain a DNA fragment SE-CPR-LS, cloning the SE-CPR-LS fragment to a vector pet21c by a restriction enzyme cutting connection method, and constructing an expression vector pet21c-SE-CPR-LS; cloning the squalene synthase genetic fragment to the vector pACYCduet-1 by a homologous recombination method, and constructing an expression vector pACYCduet-SS; and transforming the vectors pet21c-SE-CPR-LS and pACYCduet-SS into escherichia coli competence cells, so as to obtain the escherichia coli bacterial strainfor generating lanosterol. Through an exogenous plasmid transforming method, an escherichia coli BL21(DE3) system is improved, so that synthesis of the lanosterol can be realized; and through a codonoptimization technique, the expression level of exogenous genes in an escherichia coli system can be increased.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing a lanosterol-producing Escherichia coli strain. Background technique [0002] Lanosterol, also known as isocholesterol, has a molecular formula of C 30 h 50 O. Soluble in ethanol, ether and chloroform; and crystallized in methanol and acetone. Lanosterol is a colorless powder that has many important biological activities and a wide range of pharmacological effects, such as lowering blood pressure, lowering blood sugar, anti-cancer, and treating cataracts. Moreover, lanosterol is also an intermediate in the biosynthesis of various important sterols, and is an important raw material in the chemical industry, medicine, and food industry. [0003] The development of synthetic biology provides a potential production route for the heterologous expression of biosynthetic genes using fast-growing microorganisms. The use of E. coli expression system has ma...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/54C12N15/61C12N15/70C12R1/19
CPCC12N9/0083C12N9/0081C12N9/1085C12N9/90C12N15/70C12Y114/99007C12Y114/15006C12Y205/01021C12Y504/99007C12N2800/22
Inventor 宛雯
Owner XUZHOU NORMAL UNIVERSITY
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