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Method for producing isoprene by adopting cell fusion technology and fusant constructed by same

A technology of isoprene and microorganisms, which is applied in the field of isoprene production by cell fusion technology, can solve the problems of poor oil metabolism and low yield of isoprene, and achieve strong oil metabolism and convenient gene operation , the effect of clear genetic background

Active Publication Date: 2014-02-12
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] The purpose of the present invention is to overcome the problems of poor lipid metabolism and low yield of isoprene in Escherichia coli in the existing isoprene production process, and provide a fusion son with strong lipid metabolism

Method used

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  • Method for producing isoprene by adopting cell fusion technology and fusant constructed by same
  • Method for producing isoprene by adopting cell fusion technology and fusant constructed by same
  • Method for producing isoprene by adopting cell fusion technology and fusant constructed by same

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Alcaligenes eutropha with gentamicin resistance marker was inoculated in LB medium, cultured to the middle and late logarithmic growth period, the bacteria liquid was collected by centrifugation, and the bacteria were resuspended in 3 mL sucrose hypertonic solution Ⅰ (SMMP) , treated with lysozyme at a final concentration of 0.4g / L at 35°C for 8h. Protoplasts of Alcaligenes eutropha were prepared.

[0031]The medium SMMP used is 6g / L beef extract, 6g / L yeast extract, 20g / L peptone, 14g / L NaCl, 4g / L glucose, 14.7g / L K 2 HPO 4 , 5.4g / L KH 2 PO 4 , 170g / L sucrose, 2g / L maleic acid, 4g / L MgCl 2 6H 2 O, pH7.0.

[0032] The SMM used is 170g / L sucrose, 2g / L maleic acid, 4g / L MgCl 2 6H 2 O, pH7.0.

Embodiment 2

[0034] Inoculate engineered Escherichia coli (plasmid carrying isoprene synthesis pathway, see Yang, Plosone, 2012) in LB medium, culture until mid-to-late logarithmic growth, collect bacterial cells by centrifugation, and resuspend in 10 mL Sucrose hypertonic solution I (SMMP) was treated with lysozyme at a final concentration of 2g / L at 40°C for 16h. Preparation of E. coli protoplasts.

[0035] Then with the fusion and regeneration of the protoplasts of Alcaligenes eutropha obtained in Example 1. Add 14mL of 40% polyethylene glycol solution, mix well and incubate protoplasts at 30°C for 2 minutes, centrifuge at 3000r / min for 10 minutes, discard the supernatant, suspend fully with 2mL sucrose hypertonic solution II (SMM) solution, and then Spread in regeneration medium (CMR) containing 34mg / L chloramphenicol, 100mg / L penicillin and 10mg / L gentamicin, and culture at 30°C for 1-2 days. A total of 18 fusogens were obtained. Using lauric acid and peanut oil as carbon sources, t...

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Abstract

The invention discloses a method for producing isoprene by adopting a cell fusion technology and a fusant constructed by the same. The method comprises the following steps: screening by utilizing a protoplast fusion technology, taking escherichia coli and ralstonia eutropha as parents and taking lipid as a unique carbon source and a resistance maker plasmid to obtain the reconstituted cell of high-efficiency transformed lipid; and fermenting by utilizing the fusant to produce the isoprene. The method disclosed by the invention can increase the velocity of lipid metabolism, enhance the yield of the isoprene and reduce the fermentation cost.

Description

technical field [0001] The invention relates to a method for producing isoprene by fermentation, in particular to a method for producing isoprene by cell fusion technology. technical background [0002] The raw materials for producing isoprene by fermentation generally use glucose, glycerol, etc., and the highest level of isoprene yield in related reports is 11%, which cannot meet the requirements of industrial production. With glucose, glycerin and other raw materials, the theoretical yield of MVA pathway is 25%; with fatty acids and other oils as raw materials, the theoretical yield of MVA pathway is 68% (taking C12 as an example), much higher than other raw materials. Considering the characteristics of wide sources of cheap oil raw materials and high yield, compared with glucose and other raw materials, the conversion of oil to produce isoprene has more economic advantages, but there is no related report so far. [0003] Using oils as raw materials to synthesize other ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/03C12P5/00C12R1/19
Inventor 咸漠刘辉张海波邹慧斌
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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