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Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar

A technology of Clostridium acetobutylicum and xylulokinase, which is applied in the fields of genetic engineering technology and fermentation and can solve problems such as reduction

Active Publication Date: 2012-11-28
南京食气生化科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the strategy of overexpressing all genes of xylose metabolism to improve xylose utilization has been reported in other microorganisms (Karhumaa, K., B. Hahn-Hagerdal, et al. (2005). Yeast 22(5):359-368; Zhang, M., C. Eddy et al. (1995). science 267(5195):240-243.), but it is currently technically impossible to express so many genes in Clostridium acetobutylicum, so it is necessary to confirm the real rate-limiting genes in the xylose pathway and reduce overexpression genes The number of this work is challenging (Nakotte, S., S. Schaffer et al. (1998). Appl Microbiol Biotechnol 50(5):564-567; Shao, L., S. Hu et al. (2007). Cell Research 17(11):963-965.)

Method used

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  • Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar
  • Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar
  • Method for improving sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] Example 1. Construction of pWJ1-glcG plasmid vector

[0166] Amplify the glcG targetron fragment by PCR, then use XhoI and BsrG I to perform double digestion, and connect it to the pWJ1 vector that has also been digested by XhoI and BsrG I, to obtain the interrupted plasmid pWJ1-glcG, in which, PCR amplifies the template of glcG 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:

[0167] 1.1PCR amplification primers

[0168] ReferenceTargetron TM According to the method provided by the Gene Knockout System (TA0100) kit, primers glcG-IBS (as shown in sequence SEQ ID NO.: 4), glcG-EBS1d (as shown in sequence SEQ ID NO.: 5) and glcG-EBS2 were designed respectively (as shown in the sequence SEQ ID NO.: 6), used to construct the pWJ1-glcG plasmid vector.

[0169] The EBS universal primers (EBS universal) required for PCR amplification were supplied by Target...

Embodiment 2

[0177] Example 2. Construction of Clostridium acetobutylicum glcG Mutant, Detection and Loss of Knockout Plasmid

[0178] After the pWJ1-glcG plasmid was methylated at the Cac8I site by Escherichia coli ER2275 / pANS1, it was electroporated into Clostridium acetobutylicum ATCC 824. After recovery overnight, 200 μl of the cell solution was applied to CGM with 20 μg / mL erythromycin On the plate, 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:

[0179] 2.1 Methylation of pWJ1-glcG plasmid

[0180] In order to prevent foreign DNA from entering Clostridium acetobutylicum and being cleaved and degraded by its restriction system, the pWJ1-glcG plasmid needs to be methylated (Mermelstein, L.D and Papoutsakis, E.T. Appl Environ Microbiol.vol 59.issue 4: p1077-81 ).

[0181] The pANS1 plasmid was treated with CaCl 2 Transform into Escherichia coli ER2275 by heat shock method to obtai...

Embodiment 3824

[0197] Fermentation of embodiment 3.824glcG mutant strain

[0198] Get the Clostridium acetobutylicum strain 824glcG that has interrupted the glcG gene in step 2.5 to ferment in P2 medium, and detect the fermentation broth, the specific process is as follows:

[0199] Pick a single bacterium from the CGM plate and insert it into 5mL CGM liquid medium, cultivate overnight, insert 1% inoculum into 50mL CGM medium, cultivate for 8~10hr, and make the bacterial concentration OD 600 Reach 0.4, insert in P2 substratum with 5% and cultivate and ferment, get fermented liquid and detect residual sugar content (use the sugar-park column of WATERS company to measure through Agela 1200HPLC, the result is as follows figure 1 shown), and Clostridium acetobutylicum ATCC 824 was used as a contrast, wherein the following pretreatment was required before the determination of the residual sugar content in the fermentation broth: after the fermentation broth was centrifuged, the supernatant was...

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Abstract

The invention discloses a method for improving the sugar utilization rate of clostridium acetobutylicum in fermentation of mixed sugar. The method comprises the following steps of: performing gene engineering modification on clostridium acetobutylicum, so that compared with wild type clostridium acetobutylicum, the clostridium acetobutylicum has the advantages that expression of g1cG gene can be inhibited, and the expression and activity of xylose transportprotein, xylose isomerase, and / or xylulokinase can be improved; and applying the obtained clostridium acetobutylicum which is subjected to gene engineering to fermentation of sugar. By the method, more xylose and arabinose can be used by clostridium acetobutylicum in the fermentation of the mixed sugar, a solvent product with high concentration can be produced, and the product yield can be improved; and the method has excellent industrial application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and fermentation technology. Specifically, the present invention relates to a method for improving the sugar utilization rate (especially the utilization rate of xylose and arabinose) of Clostridium acetobutylicum (Clostridium acetobutylicum) in mixed sugar fermentation, the bacterial strain used in the method, Its use and preparation method. Background technique [0002] Butanol is a bulk basic raw material with multiple uses. It can be used as a precursor for the synthesis of various organic compounds in the chemical and chemical fields such as dyes, paints, plastics, resins, and rubbers; it is essential in the production of antibiotics and synthetic drugs. Less solvent; it is also a food-grade extractant for food and spice industries. On the other hand, butanol is still a high-quality fuel and fuel additive with a higher octane number than gasoline, and its high boiling point (11...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/06C12P7/16C12P7/28C12R1/145
CPCC12P7/28C12Y207/01069C12N9/1205C12N9/92C12P7/065C12R1/145C12P7/16C12P7/06C07K14/33Y02E50/10Y02E50/17
Inventor 顾阳肖晗姜卫红宁媛媛李治林蒋宇孙喆杨晟
Owner 南京食气生化科技有限公司
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