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Escherichia coli for producting farnesene

A technology for producing farnesene and bacteria, which is applied in the field of genetic engineering and can solve problems such as low fermentation levels

Active Publication Date: 2019-04-12
ZHEJIANG MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the defect of low fermentation level of existing β-farnesene production strains, the present invention uses genetic engineering technology to transform isoprene-producing Escherichia coli, by enhancing genes related to β-farnesene production, replacing isoprene Diene Branch Metabolic Pathway, Obtaining a Production Strain with High Yield of β-Farnesene

Method used

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  • Escherichia coli for producting farnesene
  • Escherichia coli for producting farnesene
  • Escherichia coli for producting farnesene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Construction of plasmid pAKF

[0051] The primer sequence information used in the construction of plasmid pAKF is shown in Table 1.

[0052] Table 1. Primer sequences

[0053]

[0054] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0055] 1.1 Using the Escherichia coli MG1655 genome as a template and ispA-FG / ispAL-R2 as primers, PCR amplifies the ispAL fragment, about 969 bp (amplification of homology arm + ispA + linker). PCR conditions: denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 68°C for 1 min, 32 cycles, 10 min at 68°C, and 10 min at 16°C.

[0056] The ispAL fragment was recovered from the gel, and the ispAL fragment was used as a template and the ispA-FG / ispA-RG was used as a primer to amplify the ispALG fragment by PCR, which was about 1004bp (amplified homology arm+ispA+linker+homology arm). PCR conditions: denaturation at 95°C for 5 min,...

Embodiment 2

[0059] Embodiment 2: Construction of β-farnesene producing bacteria

[0060] 2.1 The plasmids PHGFH, PAGEs, and pAKF in Example 1 were directly co-transformed into Escherichia coli CIBTS1758 by electroporation, and the strain CIBTS1758 / PHGFH / PAGEs / pAKF was obtained, which was named CIBTS2509C.

[0061] 2.2 The plasmids PHGFH, PAGEs, and pAK in Example 1 were co-transformed into Escherichia coli CIBTS1758 by electroporation, and the strain CIBTS1758 / PHGFH / PAGEs / pAK was obtained as a control strain.

Embodiment 3

[0062] Example 3: Fermentative production of β-farnesene by bacterial strains

[0063] 3.1 Culture and fermentation of strain CIBTS2509C

[0064] Pick a single colony of CIBTS2509 into the LB medium containing 100 μg / ml ampicillin, 34 μg / ml chloramphenicol and 100 μg / ml spectinomycin, culture overnight at 37°C and 220 rpm; then inoculate at an inoculum of 8% v / v In the V7S medium containing the above three antibiotics, continue to culture at 37°C and 220rpm for 8-12h; then inoculate the V7E medium containing the above three antibiotics and 0.1mM IPTG according to the inoculum size of 8% v / v, at 37°C , 220rpm to continue culturing for 8-12h; add 1 / 5 volume of decane to continue culturing for 4h. Centrifuge at 12,000 rpm for 10 min, collect the decane phase, and send it to GC-MS for analysis.

[0065] The GC analysis method of farnesene is as follows: Agilent 7890A gas chromatograph equipped with 5975MS; chromatographic column Agilent HP-1 (30mx0.25mm, 0.25μm); injection volum...

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Abstract

The invention constructs a beta-farnesene producing bacterium through genetic engineering, and the beta-farnesene producing bacterium is preserved in the China General Microbiological Culture Collection Center, CGMCC, wherein the preservation number is CGMCC No.16709. The beta-farnesene producing bacterium can realize effective accumulation of beta-farnesene, the yield of the beta-farnesene is ashigh as 250mg / L, and the beta-farnesene producing bacterium has industrial prospects.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetic engineering production bacterium with high yield of β-farnesene. Background technique [0002] When aphids are frightened, they can secrete a kind of "alarm pheromone" from the abdominal tube, so that other aphids around can perceive and escape quickly, thus stopping the damage to crops. Bowers et al. isolated and identified the aphid alarm pheromone trans-β-farnesene (E-β-farnesene, referred to as EβF or EBF) for the first time in 1972. EβF is a sesquiterpenoid. Francis et al. found that EBF is the main or only component of 16 aphid alarm pheromones (Bowers et al., 1972; Francis et al., 2005). The application of this type of pheromone in the field can control the population density of aphids within a certain range within the threshold value. At this time, the insects and diseases have little impact on the crops. EβF is not only present in aphid alarm ho...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P5/02C12R1/19
CPCC12N9/88C12N15/70C12P5/026C12Y402/03047
Inventor 陈正杰蒋宇童阳阳许崇茂吴宇辉杨俊杰杨晟张芸赵梦凡朱丽
Owner ZHEJIANG MEDICINE CO LTD
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