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Method for eliminating glucose inhibition effect of clostridium acetobutylicum

A technology for Clostridium acetobutylicum and glucose inhibition, which is applied in the field of genetic engineering to achieve the effect of increasing the concentration of ABE

Inactive Publication Date: 2011-05-04
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CCR effects have been more studied in Bacillus subtitles but less in Clostridium acetobutylicum

Method used

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  • Method for eliminating glucose inhibition effect of clostridium acetobutylicum
  • Method for eliminating glucose inhibition effect of clostridium acetobutylicum
  • Method for eliminating glucose inhibition effect of clostridium acetobutylicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 , Construction of pSY6-ccpA plasmid vector

[0052] Amplify the ccpA targetron fragment by PCR, then use XhoI and BsrG I to perform double digestion, and connect it to the pSY6 vector that has also been digested by XhoI and BsrG I to obtain the interrupted plasmid pSY6-ccpA, wherein the template for PCR amplification of ccpA targetron And the primer design method comes from the Targetron of Sigma-Aldrich company TM Gene Knockout System (TA0100) Kit, the specific steps are as follows:

[0053] 1.1. Primer design

[0054] ReferenceTargetron TM The method that Gene Knockout System (TA0100) Kit provides, respectively designs primer ccpA-IBS (as shown in sequence SEQ ID NO.: 1), ccpA-EBS1d (as shown in sequence SEQ ID NO.: 2) and ccpA-EBS2 ( The sequence shown as SEQ ID NO.: 3) was used to construct the pSY6-ccpA plasmid vector.

[0055] EBS universal required for PCR amplification by Targetron TM Gene Knockout System (TA0100) Kit comes with it.

[0056]...

Embodiment 2

[0065] Example 2 , Clostridium acetobutylicum ccpA-mutant strain construction and detection

[0066]After the pSY6-ccpA plasmid was methylated at the Cac8I site by E.coli ER2275 / pANS1, it was electroporated into Clostridium acetobutylicum ATCC 824. After recovery overnight, 200 μl of the cell solution was spread on a CGM plate supplemented with 40 μg / mL erythromycin , after culturing in an anaerobic box at 37°C for 48-96 hours, pick a single bacterium for colony PCR verification, the specific process is as follows:

[0067] 2.1. Methylation of pSY6-ccpA plasmid

[0068] To prevent exogenous DNA from being cut and degraded by its restriction system after entering c.acetobutylicum, the pSY6-ccpA plasmid needs to be methylated (Mermelstein, L.D and Papoutsakis, E.T.Appl Environ Microbiol.vol59.issue 4.page 1077-81).

[0069] The pANS1 plasmid was treated with CaCl 2 Transform into E.coli ER2275 by heat shock method to obtain strain E.coliER2275 / pANS1.

[0070] Transform the ...

Embodiment 3

[0083] Example 3 , Clostridium acetobutylicum ccpA-mutant strain fermentation

[0084] Get the Clostridium acetobutylicum strain Clostridium acetobutylicum ccpA that has interrupted the ccpA gene obtained in step 2.2 - Ferment in P2 medium, and detect fermented liquid, specific process is as follows:

[0085] Pick a single bacterium from the CGM plate and inoculate it into 5mL CGM liquid medium, cultivate it overnight, inoculate it into 50mL CGM medium with 1% inoculum, cultivate it for 8-10 hours to reach a bacterial concentration OD600 of 0.4, and inoculate it into P2 culture with 5% culture medium, and at 0, 12, 25, 34, 48, 60, and 72 o'clock, the fermentation broth was taken, and the OD600 was detected (the results were as follows: figure 2 Shown), residual sugar content (use the sugar-pak column of WATERS company to measure through Agela 1200HPLC, the result is as follows image 3 Shown) and acetone, butanol and ethanol content (using Agela 7890A gas chromatograph to...

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Abstract

The invention provides a method for eliminating glucose inhibition effect of clostridium acetobutylicum, and a recombinant strain of which the ccpA gene expression of clostridium acetobutylicum is inhibited in a genome. The method for eliminating the glucose inhibition effect of clostridium acetobutylicum is implemented by inhibiting the ccpA gene expression of clostridium acetobutylicum. By using the method provided by the invention, the ccpA gene expression of clostridium acetobutylicum is interrupted. In the strain provided by the invention, after the ccpA gene is subject to insertional inactivation through group II intron, the capability of the strain of synchronously utilizing xylose and glucose is obviously improved, and the concentration of converted ABE is correspondingly increased at the same time. The engineering strain constructed by the invention eliminates the glucose inhibition effect and can synchronously utilize glucose and xylose for fermenting, thus the strain has the potential in acetone-butanol fermentation with hydrolyzate of lignocelluloses.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for releasing the glucose inhibitory effect of Clostridium acetobutylicum. Background technique [0002] Due to the limitation of petroleum resources and the volatility of international crude oil prices, the use of renewable resources to produce bio-energy has attracted widespread attention from all over the world. Due to its excellent combustion characteristics and better storage and transportation characteristics than ethanol, butanol is expected to become a new generation of biofuel in the future. Since World War II, with the development of the petroleum industry, the large-scale fermentation of bio-butanol fermentation has been stagnant due to its high raw material cost, and with the continuous rise of food prices in recent years and the formulation of relevant national food security strategies, The use of grain fermentation to produce fuel b...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P7/28C12P7/16C12P7/06C12R1/145
CPCY02E50/10
Inventor 姜卫红任聪顾阳胡世元杨晟
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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