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Genetic engineering bacteria producing longifolene and construction method of genetic engineering bacteria and application

A technology of genetically engineered bacteria and longifolene, applied in the field of synthetic biology, can solve the problems of high cost pollution, low yield, and limited plant growth in chemical synthesis, and achieve good industrial application prospects

Inactive Publication Date: 2020-07-10
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The second object of the present invention is to provide a method for constructing the above-mentioned genetic engineering bacteria producing longifolene, which can overcome the disadvantages of natural plant extraction methods such as low yield, low efficiency, high cost, and limited plant growth. The chemical synthesis method has the disadvantages of high cost and large pollution

Method used

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  • Genetic engineering bacteria producing longifolene and construction method of genetic engineering bacteria and application
  • Genetic engineering bacteria producing longifolene and construction method of genetic engineering bacteria and application
  • Genetic engineering bacteria producing longifolene and construction method of genetic engineering bacteria and application

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Embodiment approach

[0072] As mentioned above, the existing methods for preparing longifolene and natural plant extraction methods have disadvantages such as low yield, low efficiency, high cost, and limited plant growth; chemical synthesis methods have high costs and large pollution; There are few reports on the synthesis of longifolene, and industrial production has not been realized. In view of this, the present inventors have conducted a lot of research on the method of biosynthesizing longifolene.

[0073] The inventors researched and designed and found that the longifolene synthase gene lgfS was synthesized by codon optimization using Escherichia coli as the expression host, so that the gene could be expressed more easily in heterologous hosts, Phosphate synthase gene ispA, thus can construct and obtain high-efficiency longifolene-producing genetically engineered bacteria. The inventors further studied the design and found that, through chassis modification, the precursor substance farnesy...

Embodiment 1

[0142] Embodiment 1: Construction of recombinant plasmid

[0143] The chemically synthesized and codon-optimized lgfS gene was cloned into the plasmid prsfDuet-1 through restriction sites BamHI and NcoI to obtain the plasmid prsf-lgfS. The genomes of Escherichia coli JM109 and Streptococcus pneumoniae were used as templates to amplify the genes ispA and idi, and the ispA-idi gene fragment was connected to prsf-lgfs in the form of Gibson self-assembly through the NaeI and PacI restriction sites to obtain the plasmid prsf- lgfs-ispA-idi; similarly, the genes erg20 and idi were amplified using the genomes of Saccharomyces cerevisiae and Streptococcus pneumoniae as templates, and the plasmid prsf-lgfs-erg20-idi was obtained in the same manner.

[0144] The mvaE and mvaS genes were amplified using the genome of Enterococcus faecalis as a template, and the gene mvaS was cloned into the first multiple cloning site of the plasmid prsf-lgfs-ispA-idi with BamHI and SalI, respectively, a...

Embodiment 2

[0147] Embodiment 2: Construction of recombinant bacterial strain

[0148] (1) Preparation of Competent Cells

[0149] Inoculate the glycerol bacteria of Escherichia coli stored at -80°C into 4mL LB test tubes, and after culturing overnight, insert a certain amount of inoculum into 20mL LB medium, so that the initial OD600 is about 0.2. Cultivate in a shaker at 37°C for about 2 hours, and take out when the OD600 reaches about 0.8 to prepare competent cells.

[0150] Put the cultured colony into a 1.5mL centrifuge tube, cool the cells to 0°C in an ice bath, then collect the cells by refrigerated centrifugation at 5000rpm at 4°C for 5 minutes, and resuspend the cells with 100 μL of pre-cooled 10% glycerol (four tubes The cells were mixed in one tube), and the cells were collected by refrigerated centrifugation at 5000 rpm for 5 min at 4°C. The washing was repeated three times, and after the washing was completed, the bacterial cells were resuspended with 100 μL of 10% sterile ...

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Abstract

The invention belongs to the field of synthetic biology, and relates to genetic engineering bacteria producing longifolene. The genetic engineering bacteria is a recombinant Escherichia coli containing related longifolene anabolic pathway genes (including farnesyl pyrophosphate synthetase gene ispA and longifolene synthase gene lgfS); and the recombinant Escherichia coli is subjected to base platemodification of optimized endogenous MEP synthesis pathway and the optimization of the metabolic pathway by introducing key enzyme genes in the heterologous MVA pathway, so that the obtained geneticengineering bacteria producing longifolene has a higher yield of longifolene. The genetic engineering bacteria is connected to a fermentation medium, an inducer IPTG is added, long-chain n-alkanes areadded during the fermentation and culture process for two-phase extraction fermentation, the yield of a longifolene shake flask reaches 1.77mg / L, and the industrial application prospects are good.

Description

technical field [0001] The invention belongs to the field of synthetic biology, and relates to a genetically engineered bacterium producing longifolene, a construction method and application thereof. Background technique [0002] Isoprene compounds, also known as terpenes or terpenoids, are the most diverse compounds that exist widely in nature. More than 40,000 different terpenoids have been isolated from plants, animals and microorganisms. The rich and diverse structures of these compounds provide a good compound screening library for solving human health and social problems, and some compounds have been widely used in medicine, health food, spices, agricultural chemicals, etc. However, the production of such compounds in natural hosts is extremely low, which largely limits the industrial production and application of such compounds. At present, one of the effective methods to solve this problem is to recombine and transform the metabolic pathways of heterologous microorg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/54C12N15/70C12P5/00C12R1/19
CPCC12N9/1085C12N15/70C12P5/002C12Y205/01068
Inventor 刘艳辉魏丽娟
Owner BEIJING UNIV OF CHEM TECH
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