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Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment

A technology of Zymomonas and movement, which is applied in fermentation, application, genetic engineering, etc., and can solve problems such as the speed of ethanol is not fast enough

Inactive Publication Date: 2005-03-30
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the rate of ethanol production by wild-type Zymomonas mobilis is still not fast enough, and the ethanol production reaches its maximum only after 72 hours of fermentation

Method used

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  • Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment
  • Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment
  • Clone and application of alcohol fermentation gene of unit cell fungus through motion ferment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of Zymomonas mobilis XW101 gene library

[0026] 1. Extraction of bacterial total DNA by CTAB method

[0027] 1) Take a single colony and inoculate it in 5ml of the corresponding culture medium, and cultivate it at a suitable temperature. Depending on the growth rate of the bacteria, the incubation time can range from a few hours to a few days.

[0028] 2) Take 1.0ml of bacterial liquid in a 1.5ml centrifuge tube, centrifuge at 13,000rpm for 2min, and discard the supernatant.

[0029] 3) The pellet was resuspended in 1.0 ml of 0.85% NaCl.

[0030] 4) Centrifuge at 13,000 rpm for 2 minutes at room temperature, and discard the supernatant.

[0031] 5) The pellet was resuspended in 550 μl 1×TE.

[0032] 6) Add 17 μl of lysozyme (35 mg / ml), and incubate at 37° C. for 30 minutes.

[0033]7) Add 3 μl of proteinase K (20 mg / ml), and incubate at 37° C. for 30 minutes.

[0034] 8) Add 30 μl of 10% SDS, and incubate at 37° C. for 30 minutes.

[0035]...

Embodiment 2

[0075] Example 2 Cloning of Alcohol Dehydrogenase II Gene

[0076] 1. PCR reaction system:

[0077] 10×PCR buffer 2μl

[0078] 2.5mMdNTP 2μl

[0079] 10×BSA 2μl

[0080] 50pM Primer 1 2μl

[0081] 50pM Primer 2 2μl

[0082] Template DNA 5ng

[0083] 5U / μl Taq enzyme 0.25μl

[0084] Add sterile water to 20 μl

[0085] 2. PCR reaction conditions:

[0086] 1. Pre-denaturation at 94°C for 20s

[0087] 2. 94°C 0s

[0088] 54°C 0s

[0089] 72℃ 40s

[0090] (25 cycles)

[0091] 3. 5min at 72°C

[0092] 3. DIG-DNA probe labeling

[0093] Main reagents:

[0094] 1) Sterilized dd-Water, 15-25°C, for diluting DNA

[0095] 2) EDTA: 0.2MpH 8.0 15-25°C, used to terminate the reaction

[0096] Template DNA: the purity requirement is purified with a kit (BioDev Company)

[0097] DNA fragment size: The template DNA is linear, and the size should be greater than 100bp. When the DNA template is greater than 5Kb, first nitrate it with a restriction endonuclease that recognizes 4...

Embodiment 3

[0143] Embodiment 3 Transformation of the plasmid with the gene of interest and its impact on ethanol yield

[0144] 1. Electroporation method: same as in Example 1.

[0145] 2. Determination of ethanol yield:

[0146] 1) Chromatographic conditions

[0147] The chromatographic column is 2m long and has an inner diameter of 4mm.

[0148] Stationary phase GDX-102, 60-80 mesh

[0149] The temperature of the gasification chamber is 190 degrees, the detector is 190 degrees, and the column temperature is 170 degrees.

[0150] Gas flow rate Carrier gas (N 2 ) 40ml / min.

[0151] 2) The injection volume is 5.0ul.

[0152] 3) Qualitative The ethanol standard peak time is used for qualitative determination. Inject 5.0ul of the ethanol standard application solution and the sample measurement solution, respectively, measure the retention time, and compare the sample with the standard peak time for qualitative determination.

[0153] 4) Quantitative

[0154] Determination of ethano...

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Abstract

This invention establishes a motion fermentation monocell XW 101 plasmid gene library, ethanol dehydrogenase II gene partial sequence is used as probe for screening or cloning ethanol dehydrogenase II gene from above said library, then connecting the same to shuttle plasmid carrier of broad host range, finally conducting to wild type motion fermentation monocell bacteria or colon bacillus. Said cloned ethanol dehydrogenase II coded gene sequence is as illustrated in SEQ ID No.1. The wild type motion fermentation monocell bacteria introduced with ethanol dehydrogenase II coded gene can greatly increase the prodn. rate of ethanol due to obviously speeding-up fermentation.

Description

Technical field: [0001] The invention relates to the construction of a genome library of Zymomonas mobilis, the cloning of functional genes involved in fermentation and the application of the corresponding genes in the construction of engineering strains. The invention also relates to the use of the Zymomonas mobilis engineering bacteria to ferment and produce ethanol. Background technique: [0002] Fuel ethanol is a kind of efficient, clean and environmentally friendly fuel that can supplement or replace gasoline. However, ethanol is expensive to produce and is not competitive with gasoline in price. In the process of producing ethanol by fermentation, the key problem that must be solved is to increase the conversion rate of sugar in order to reduce the production cost. [0003] The traditional yeast strain widely used in the research and application of ethanol fermentation is mainly Saccharomycescerevisiae. If alcohol is produced from sugary or starchy raw materials, th...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/09C12N15/31C12N15/53C12N15/70C12P7/06C12Q1/68
CPCY02E50/17Y02E50/10
Inventor 徐玉泉武志强张维于晓瑾李红权林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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