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Escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and preparation method and application of escherichia coli genetically engineered bacteria

A technology of genetically engineered bacteria and isoprene, applied in the field of genetic engineering, can solve the problems of high glucose market price, large fluctuations, low yield and yield, etc.

Active Publication Date: 2016-01-20
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that using glucose as a carbon source to synthesize isoprene has low yield and yield, and the market price of glucose is higher than that of methanol and fluctuates greatly

Method used

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  • Escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and preparation method and application of escherichia coli genetically engineered bacteria
  • Escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and preparation method and application of escherichia coli genetically engineered bacteria
  • Escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and preparation method and application of escherichia coli genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The acquisition of embodiment 1 gene

[0041] In this embodiment, the gene sequence of methanol dehydrogenase gene MDH (gene accession number GI: 662720579) derived from methylotrophic Bacillus; ) gene sequence; the gene sequence of 6-phosphate-3-ketulose isomerase gene RmpB (gene accession number GI: 40074228); the gene sequence of methanol dehydrogenase activating protein gene ACT (gene accession number GI: 22654851) . The original sequences of the above genes were codon-optimized and synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The sequence of the optimized methanol dehydrogenase gene MDH is shown in SEQ ID NO.1; the sequence of the optimized 6-ketohexose phosphate synthase gene RmpA is shown in SEQ ID NO.2; The sequence of the constitutive enzyme gene RmpB is shown in SEQ ID NO.3; the sequence of the optimized methanol dehydrogenase activating protein gene ACT is shown in SEQ ID NO.4.

Embodiment 2

[0042] The preparation of embodiment 2 recombinant plasmids

[0043] Digest the methanol dehydrogenase gene MDH and plasmid pBAD18 obtained in Example 1 with EcoRI and SacI, and recover the target fragment with a gel recovery kit. After recovering the digested product, mix the carrier:target fragment at a molar ratio of 1 Mix at a ratio of 1, add T4 DNA ligase and ligate at 16°C for 6 hours to obtain recombinant plasmid pBAD18-1;

[0044] The 6-ketohexose phosphate synthase gene RmpA obtained in Example 1 and the plasmid pBAD18-1 were digested with SacI and KpnI, and the target fragment was recovered with a gel recovery kit. After the digested product was recovered, the vector: The fragments were mixed at a molar ratio of 1:1, and ligated at 16°C for 6 hours after adding T4 DNA ligase to obtain the recombinant plasmid pBAD18-2;

[0045] The 6-phosphate-3-ketulose isomerase gene RmpB obtained in Example 1 and the plasmid pBAD18-2 were digested with SalI and SphI, and the targe...

Embodiment 3

[0047] Example 3 Introducing the plasmid pBAD18-3 and the isoprene synthesis plasmid into Escherichia coli E.coliJM109(DE3) to synthesize isoprene

[0048] Add the recombinant plasmid pBAD18-3 obtained in Example 2 and the isoprene synthesis plasmid to E.coliJM109 (DE3) competent cells at the same time; ice-bath for 20 minutes, put the competent cells in a 42°C water bath to heat Shock for 45 s, and immediately ice-bath for 2 min after heat shock; add 600 μl sterilized LB liquid medium to the centrifuge tube where the competent cells are in the ultra-clean bench; place the centrifuge tube at 37°C for 1 h on a shaker; shaker culture Afterwards, centrifuge at 500rpm for 2min, suck out the supernatant with a pipette gun, leave a little supernatant to resuspend the cells, and spread it on the LB solid plate containing kanamycin and chloramphenicol; place the coated plate at 37 ℃ constant temperature incubator, and continue to cultivate until a single clone grows.

[0049] Add the...

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Abstract

The invention discloses escherichia coli genetically engineered bacteria for preparing isoprene through catalytic methanol and a preparation method and application of the escherichia coli genetically engineered bacteria, and belongs to the technical field of genetic engineering. The escherichia coli genetically engineered bacteria have the isoprene synthesizing approach, and meanwhile methanol dehydrogenase gene MDH, 3-hexulose-6-P synthase gene RmpA, 3-hexulose-6-P isomerase gene RmpB and methanol dehydrogenase activated protein gene Act are overexpressed. Meanwhile, the invention further provides a method and application method for establishing the escherichia coli genetically engineered bacteria. The escherichia coli genetically engineered bacteria can biologically synthesize isoprene with methanol as the substrate and has the advantages of being high in conversion efficiency, low in production cost and the like; the conversion rate of methanol-isoprene can reach 21.83%.

Description

technical field [0001] The invention relates to an Escherichia coli genetic engineering bacterium that catalyzes methanol to prepare isoprene, a preparation method and application thereof, and belongs to the technical field of genetic engineering. Background technique [0002] China is a big methanol country in the world, with production capacity exceeding 50% of the world's. In 2014, domestic production capacity exceeded 60 million tons, and in 2015 it is expected to exceed 65 million tons. According to international experience, maintaining the capacity utilization rate between 81% and 82% is the critical point to measure whether the industrial capacity is excess. Judging from the data on the average operating rate of the domestic methanol industry in recent years, the operating rate of methanol in my country has been below 60%. In 2013, the average operating rate of methanol was 57.13%, which is in a state of serious surplus. The overcapacity of domestic methanol and the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P5/02C12R1/19
CPCY02E50/30
Inventor 咸漠唐勇赵广廖本仁冯新军揭元萍刘会洲张春雷
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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