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A kind of isobutanol-producing Zymomonas mobilis genetically engineered bacteria and construction method thereof

A technology of Zymomonas and genetically engineered bacteria, applied in the field of genetically engineered bacteria producing isobutanol and its construction

Active Publication Date: 2016-05-11
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on isobutanol production by Zymomonas mobilis using genetic engineering

Method used

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  • A kind of isobutanol-producing Zymomonas mobilis genetically engineered bacteria and construction method thereof
  • A kind of isobutanol-producing Zymomonas mobilis genetically engineered bacteria and construction method thereof
  • A kind of isobutanol-producing Zymomonas mobilis genetically engineered bacteria and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 12

[0035] Example 12 - Amplification of Ketoisovalerate Decarboxylase and Alcohol Dehydrogenase Genes

[0036] (1) Culture of Lactococcus lactis and preparation of total DNA

[0037] Lactococcus lactis in MRS medium (peptone 10g / L, beef extract powder 10g / L, yeast powder 5g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, diamine hydrogen citrate 2g / L, sodium acetate 5g / L, magnesium sulfate 0.58g / L, manganese sulfate 0.25g / L, Tween 801mL / L, pH 7.0) at 30°C for static culture to the logarithmic phase, and then use the genome extraction kit to extract the total DNA.

[0038] (2) Amplification of 2-ketoisovalerate decarboxylase gene

[0039] The genome of Lactococcus lactis was extracted, and the primers were designed as follows according to the gene sequence of Lactococcus lactis 2-ketoisovalerate decarboxylase reported in GenBank:

[0040] Upstream primer k1: 5'-C GAGCTC AATAAAATATGGAGGAATGCGATG-3' restriction site is SacI;

[0041] Downstream primer k2: 5'-CGC GGATCC...

Embodiment 2

[0076] Example 2. Construction of recombinant plasmids

[0077] (1) Preparation of expression vector

[0078] In LB medium (10 g / L sodium chloride, 10 g / L peptone, 10 g / L yeast powder, pH 7.5) containing chloramphenicol (100 μg / mL), inoculate Escherichia coli DH5α carrying plasmid pZY507 at 37 Cultivate overnight at 200r / min. Extract the plasmid with a plasmid extraction universal kit.

[0079] (2) Construction of pZY507K recombinant plasmid

[0080] The vectors pZY507 and pMD19-kivd were double-digested with SacI and BamHI respectively, followed by agarose gel electrophoresis, and the required DNA fragments were recovered by gel cutting and ligated with T4 ligase. The double-digestion system was:

[0081] Plasmid 20μL

[0082] SacI1μL

[0083] BamHI 1 μL

[0084] 10×Buffer5μL

[0085] wxya 2 O23μL

[0086] Total volume50μL

[0087] Reaction at 37℃ for 1h

[0088] The above restriction fragments were purified using a DNA purification kit, and the resulting linear DN...

Embodiment 3

[0100] Embodiment 3. Construction of genetically engineered strains

[0101] Preparation of Competent Zymomonas mobilis:

[0102] (1) Pick a single colony from a freshly cultured agar plate and inoculate it in 10 mL of T medium (glucose 20 g / L, yeast powder 10 g / L, ammonium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 0.5g / L, pH6.5), cultured overnight at 30°C.

[0103] (2) Transfer the above culture into 500mL T liquid culture medium according to the inoculum amount of 1%, and culture it statically at 30°C.

[0104] (3) When the OD of the culture 600 When the temperature reaches 0.36, the culture is stopped, and the culture is quickly placed in an ice bath for 15-30 minutes to rapidly cool the culture.

[0105] (4) Transfer the bacterial solution to an ice-cold centrifuge tube, centrifuge at 5000 r / min at 4°C for 10 min, recover the cells, discard the supernatant, and wash the pellet three times with 10 mL of ice-cold sterile 10% glycerol.

[010...

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Abstract

The invention relates to a zymomonas mobilis gene engineering bacterium capable of producing isobutanol and a construction method of the zymomonas mobilis gene engineering bacterium. A 2-ketoisovalerate decarboxylase (kivd) gene and an alcohol dehydrogenase (adhA) gene in Lactococcus lactis 1.2829 can be obtained through cloning and connected to plasmid pZY507 with chloramphenicol resistance for construction to obtain recombinant plasmid pZY507KA, the recombinant plasmid is electro-transformed into zymomonas mobilis, and bacterial strains with chloramphenicol resistance are screened to be the target engineering bacterium. The obtained gene engineering bacterium can decompose glucose to synthesize the isobutanol after the engineering bacterium is induced, a zymomonas mobilis fermentation method is firstly used for producing the isobutanol, and simultaneously, huge potential and values of the zymomonas mobilis are shown.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and relates to a genetically engineered strain and a construction method thereof, in particular to a genetically engineered bacterium producing isobutanol and a construction method thereof. Background technique [0002] Isobutanol is a typical long-chain alcohol with high energy density, low corrosion, low hygroscopicity, easier blending with gasoline and diesel, and better compatibility with today's equipment compared to fuel ethanol . Therefore, isobutanol becomes a potential new renewable energy substitute and fuel additive. In addition to being a potential new gasoline alternative fuel, isobutanol also has many uses in the chemical market. Biomass, as a green renewable resource with abundant sources, can be widely used as a raw material for fermentation to prepare a variety of chemicals. Therefore, the production of isobutanol through the metabolism of renewable resou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
Inventor 高强朱燕孟庆艳潘超强
Owner TIANJIN UNIV OF SCI & TECH
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