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Recombinant clostridium acetobutylicum for efficiently converting straw biomass carbon source as well as construction method and application of recombinant clostridium acetobutylicum

A biomass carbon source, the technology of Clostridium acetobutylicum, applied in the biological field, can solve the problems of low butanol yield and production intensity, low xylose conversion efficiency, long fermentation cycle, etc., to improve butanol yield and production Intensity, promotion of butanol synthesis and metabolism, and improvement of fermentation efficiency

Active Publication Date: 2020-11-24
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In fact, aerobic microorganisms such as Pseudomonas, Acetobacter, Aerobacter, Staphylococcus, and Erwinia can naturally synthesize xylose acid, and aerobic microorganisms such as Escherichia coli and yeast have been genetically engineered to be able to To promote the utilization of xylose by recombinant strains and the synthesis of xylose acid, the present invention expresses the xylose acid synthesis pathway heterologously for the first time in the strict anaerobic microorganism Clostridium acetobutylicum to solve the problem of the utilization of lignocellulosic biomass carbon by Clostridium acetobutylicum in the prior art. In the process of sourcing glucose and xylose, the conversion efficiency of xylose is low, the fermentation cycle is long, and the technical problems of butanol yield and production intensity are low

Method used

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  • Recombinant clostridium acetobutylicum for efficiently converting straw biomass carbon source as well as construction method and application of recombinant clostridium acetobutylicum
  • Recombinant clostridium acetobutylicum for efficiently converting straw biomass carbon source as well as construction method and application of recombinant clostridium acetobutylicum
  • Recombinant clostridium acetobutylicum for efficiently converting straw biomass carbon source as well as construction method and application of recombinant clostridium acetobutylicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the construction of recombinant plasmid and Clostridium acetobutylicum

[0050] 1. Gene fragment synthesis and recombinant plasmid construction

[0051] According to the information retrieved by NCBI (https: / / www.ncbi.nlm.nih.gov / gene), the involved promoter thl, Clostridium acetobutylicum xylose transporter gene xylT, Caulobacter crescentus xylose dehydrogenase and The sequence information of the xylolactonase-encoding genes xylB and xylC is shown below. The gene fragments xylT-thl and xylB-thl-xylC were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., where xylB-thl-xylC selects the enzyme cleavage site Point KpnI / EcoRI into the vector plasmid pIMP1 [Mermelstein L.D., Welker N.E., Bennett G.N., Papoutsakis E.T. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC824.Nature Biotechnology, 1992,10(2):190-5.], xylT-thl Select restriction site XbaI / BamHI to connect into vector plasmid pIMP1. It was verified by enz...

Embodiment 2

[0063]Example 2: Comparison of glucose and xylose fermentation between the control strain Clostridium acetobutylicum and the recombinant strain Clostridium acetobutylicum TXYL under uncontrolled pH conditions

[0064] Specifically, this embodiment includes the following steps:

[0065] 1. Activation culture: Inoculate the recombinant strain Clostridium acetobutylicum TXYL constructed in Example 1 and the control strain Clostridium acetobutylicum into the liquid activation medium (the experimental group contains erythromycin resistance) respectively, and place them in an anaerobic environment for static cultivation , the culture temperature is 37°C, and the static activation culture is used for seed culture for 24 hours;

[0066] 2. Seed culture: the activated bacterial classification in step 1 is inoculated in the liquid seed culture medium (the experimental group contains erythromycin resistance) by 10% (v / v) inoculum, and placed in an anaerobic environment for shaking flask ...

Embodiment 3

[0069] Example 3: Under the condition of adding 10g / L calcium carbonate, comparison of glucose and xylose fermentation by control strain Clostridium acetobutylicum and recombinant strain Clostridium acetobutylicum TXYL

[0070] Specifically, this embodiment includes the following steps:

[0071] 1. Activation culture: the same as the activation culture step in Example 2.

[0072] 2, seed cultivation: with the seed cultivation step of embodiment 2.

[0073] 3. Fermentation culture: Biotec-3BG-4 fermenter (Shanghai Baoxing Biological Equipment Engineering Co., Ltd.) was used for anaerobic fermentation. Controlled at 37°C, the stirring speed was 150rpm, and the fermenter was fed with N before inoculation. 2 to remove dissolved oxygen from the fermentation medium. After inoculation, 10g / L calcium carbonate was added to control the pH range of 5-6 throughout the fermentation process, and the fermentation was carried out for 72 hours.

[0074] Figure 4 The control strain Clost...

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Abstract

The invention provides recombinant clostridium acetobutylicum for efficiently converting a straw biomass carbon source as well as a construction method and application of the recombinant clostridium acetobutylicum. The recombinant clostridium acetobutylicum for efficiently converting the straw biomass carbon source is a clostridium acetobutylicum strain which is over-expressed by clostridium acetobutylicum xylose transporter protein, caulobacter crescentus xylose dehydrogenase and xylose lactonase in a synergistic manner. Specifically, the recombinant clostridium acetobutylicum is clostridiumacetobutylicum TXYL and was preserved in China Center for Type Culture Collection on May 8th 2020, and the preservation number of the strain is CCTCC NO: M 2020107. The recombinant clostridium acetobutylicum for efficiently converting the straw biomass carbon source is applied to fermentation of straw hydrolysate to produce butanol, the fermentation period is shortened to 24 hours, the consumptionrate of glucose and xylose is increased, the utilization rate of the xylose is increased, efficient conversion of straw glucose and xylose carbon sources is realized, and the yield of the butanol andthe production intensity are remarkably improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant Clostridium acetobutylicum capable of efficiently transforming straw biomass carbon sources and its construction method and application. Background technique [0002] Butanol is an important chemical raw material, which can be used as a precursor for the synthesis of industrial chemicals and an important solvent. It is widely used in the pharmaceutical industry, plastic industry, organic industry, printing and dyeing, etc. Butanol is also a recognized new type of renewable energy, which can effectively replace fossil fuels. It has the advantages of good compatibility with gasoline, high safety factor, high energy density and good anti-knock performance. Biobutanol produced by fermentation method As a green alternative to fossil fuels, alcohol has great development potential and can pave the way to get rid of fossil fuels. [0003] Lignocellulosic raw materi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/31C12N15/53C12N15/55C12P7/16C12R1/145
CPCC07K14/33C12N9/0006C12N9/18C12N15/74C12P7/16C12Y101/01C12Y301/01C12P2203/00Y02E50/10
Inventor 吴又多王振中薛闯
Owner DALIAN UNIV OF TECH
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