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A kind of genetically engineered bacteria with high production of farnesene and its construction method and application

A technology of genetically engineered bacteria and construction method, which is applied in the field of genetically engineered bacteria with high production of farnesene and its construction, can solve the problems of host toxicity, low catalytic efficiency, etc. high purity effect

Active Publication Date: 2022-08-09
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of the low catalytic efficiency of the heterologous MVA downstream pathway in the process of E. coli synthesis of farnesene, the toxicity of intermediate metabolites such as IPP / DMAPP to the host, and how to further improve the production of farnesene, the technical scheme adopted by the present invention is as follows:

Method used

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  • A kind of genetically engineered bacteria with high production of farnesene and its construction method and application
  • A kind of genetically engineered bacteria with high production of farnesene and its construction method and application
  • A kind of genetically engineered bacteria with high production of farnesene and its construction method and application

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Embodiment 1

[0048] Example 1. Construction method of genetically engineered bacteria for synthesizing farnesene.

[0049]1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: After the β-farnesene synthase AaFS gene sequence from Artemisia annua was optimized by the codon bias of Escherichia coli, restriction enzymes BglII and XhoI were added at both ends respectively. The enzyme cleavage site was synthesized by the BGI gene, and the synthesized sequence was shown in SEQ ID No: 4, and cloned into the pUC57-simple vector to obtain the plasmid pUC57-AaFS. Plasmid pACYC-mvaE-mvaS-ispA-AaFS (attached figure 2 The construction shown in a) adopts the method of enzyme cleavage-ligation. Firstly, the plasmids pACYC-mvaE-mvaS-ispA-Sab1 and pUC57-AaFS were double digested with restriction enzymes BglII and XhoI respectively. The restriction enzyme digestion system is as follows:

[0050]

[0051] After digestion, the product was subjected to agarose gel electrophoresis and the target band was...

Embodiment 2

[0079] Example 2. Application of each genetically engineered bacteria constructed in Example 1 in the synthesis of farnesene.

[0080] The quantitative detection of farnesene by gas chromatography described in this example, the chromatographic column is an Agilent DB-5MS (30m × 0.25mm × 0.25μm) capillary column, and the column heating program is: initially at 60°C for 0.75min, and at 40°C for 0.75min. The temperature was raised to 300 °C for 2 min at a rate of °C / min, and then decreased to the initial temperature. Use β-farnesene standard as a standard curve (y=0.4582x+0.3383, x is the concentration of β-farnesene standard, the unit is g / L; y is the peak area of ​​β-farnesene) for quantification.

[0081] The primary seed medium described in this example is LB medium, and its components are: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract, and the rest is water.

[0082] Described shake flask fermentation medium composition is: 20g / L glucose, 9.8g / L K 2 HPO 4 , 5g / L beef e...

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Abstract

The invention relates to a genetically engineered bacterium with a high production of farnesene, a construction method and application thereof, and belongs to the technical field of microorganisms. In order to solve the problems of low catalytic efficiency of heterologous MVA downstream pathway and low farnesene production in the process of synthesizing farnesene in Escherichia coli, the present invention constructs plasmids pACYC-mvaE-mvaS-ispA-AaFS, pTrcLower-ΔIDI, pTrcLower- AaIDI and pET28a-AaFS-ispA-SlIDI / AaIDI (IDI genes are from tomato and Artemisia annua respectively), the above plasmids were combined and transformed into Escherichia coli, and the culture conditions were improved, and the prepared genetically engineered bacteria could significantly improve the synthetic yield of farnesene , which is conducive to promoting the industrialization process of biosynthesis of farnesene.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a genetically engineered bacterium with high production of farnesene and a construction method and application thereof. Background technique [0002] Farnesene, molecular formula C 15 H 24 , also known as farnesene, is a chain sesquiterpene. Farnesene can be used as an additive in daily chemical, pharmaceutical, food and other industries due to its aromatic odor and antioxidant activity; it can also be used as a pheromone for biological control of agricultural pests; in addition, farnesene is also a synthetic vitamin E An important intermediate in the side chain. In recent years, terpene-based biofuels have received increasing attention. The hydrogenation product of farnesene, farnesane, is considered to be a promising new biofuel for aerospace due to its high cetane number and low carbon emissions. [0003] Natural farnesene exists in a variety of plant e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/53C12N15/54C12N15/60C12N15/61C12P5/02C12R1/19
CPCC12N15/52C12N9/1029C12N9/0006C12N9/1025C12N9/1205C12N9/1229C12N9/88C12N9/90C12N9/1085C12N15/70C12P5/026C12Y203/01016C12Y101/01088C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002C12Y205/01092C12Y402/03047C12N2800/22Y02E50/10
Inventor 张海波门潇咸漠刘晋锋
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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