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Genetically engineered bacterium for high yield of farnesene and construction method and application thereof

A technology of genetically engineered bacteria and construction methods, applied in the field of genetically engineered bacteria with high farnesene production and its construction, can solve the problems of low catalytic efficiency and host toxicity

Active Publication Date: 2020-09-01
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • 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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  • Genetically engineered bacterium for high yield of farnesene and construction method and application thereof
  • Genetically engineered bacterium for high yield of farnesene and construction method and application thereof
  • Genetically engineered bacterium for high yield of farnesene and construction method and application thereof

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Embodiment 1

[0048] Embodiment 1. The construction method of the genetically engineered bacteria of synthesizing farnesene.

[0049]1) Construction of plasmid pACYC-mvaE-mvaS-ispA-AaFS: The gene sequence of β-farnesene synthase AaFS from Artemisia annua was optimized by codon preference in Escherichia coli, and restriction endonucleases BglII and XhoI were added at both ends The restriction site was synthesized by BGI. The synthesized sequence is 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 cutting-ligation. Firstly, the plasmids pACYC-mvaE-mvaS-ispA-Sab1 and pUC57-AaFS were double-digested with restriction endonucleases BglII and XhoI respectively. The restriction enzyme digestion system was as follows:

[0050]

[0051] The digested product was recovered by agarose gel electrophoresis and gel tapping of the target band. Th...

Embodiment 2

[0079] Embodiment 2. Application of each genetically engineered bacterium constructed in embodiment 1 in the synthesis of farnesene.

[0080] For 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. The temperature was raised to 300°C at a rate of ℃ / min and kept for 2 minutes, and then lowered to the initial temperature. The standard curve of β-farnesene (y=0.4582x+0.3383, x is the concentration of β-farnesene standard in g / L; y is the peak area of ​​β-farnesene) was used for quantification.

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

[0082] The shake flask fermentation medium composition is: 20g / L glucose, 9.8g / L K 2 HPO 4 , 5g / L beef extract, 0.3g / L ferric ammonium citrate, 2.1g / L citric acid monohydrate, 0.06g / L MgSO 4 , 1mL / L ...

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Abstract

The invention discloses a genetically engineered bacterium for high yield of farnesene and a construction method and application thereof, and belongs to the technical field of microorganisms. In orderto solve the problem that in the process of synthesizing farnesene from escherichia coli, the catalysis efficiency of a heterologous MVA downstream path is low, and the farnesene yield is low, plasmids pACYC-mvaE-mvaS-ispA-AaFS, pTrcLow-delta IDI, pTrcLower-AaIDI and pET28a-AaFS-ispA-SlIDI / AaIDI (IDI genes are respectively from tomatoes and artemisia apiacea) are constructed; after the plasmids are combined, escherichia coli is transformed, culture conditions are improved, the prepared genetically engineered bacterium can remarkably improve the farnesene synthesis yield, and the industrialization process of synthesizing farnesene by a biological method is promoted.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a genetically engineered bacterium with high yield of farnesene, its construction method and application. 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 because of its aromatic odor and anti-oxidation activity; it can also be used as a pheromone in the biological control of agricultural pests; in addition, farnesene is also a synthetic vitamin E An important intermediate of the side chain. In recent years, terpene-based biofuels have received increasing attention. Farnesane, the hydrogenation product of farnesene, is considered to be a promising new biofuel in the aerospace field because of its high cetane number and low carbon emissions. [0003] Natural farnesene exists in a variety o...

Claims

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Application Information

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Patent Type & Authority Applications(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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