Method for improving ratio of butanol produced from clostridium

A solvent-producing Clostridium and bacterial strain technology is applied in the field of bioengineering to achieve the effect of increasing the proportion of butanol

Inactive Publication Date: 2013-02-06
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

[0012] 2) Traditional mutagenesis method

Method used

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  • Method for improving ratio of butanol produced from clostridium
  • Method for improving ratio of butanol produced from clostridium
  • Method for improving ratio of butanol produced from clostridium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0052] The adc targetron fragment was amplified by PCR, digested with XhoI and BsrG I, and connected to the pSY6 vector cut with the same restriction enzymes to obtain the knockout plasmid pSY6-adc. Among them, the template and primer design of the amplified adc targetron are derived from the Targetron of Sigma-Aldrich Company TM Gene Knockout System (TA0100) Kit. Specific steps are as follows:

[0053] 1.1. Primer design

[0054] ReferenceTargetron TM The method provided by the Gene Knockout System (TA0100) Kit designed primers adc-IBS, adc-EBS1d and adc-EBS2 for the construction of the pSY6-adc plasmid vector respectively, and their sequences were as follows:

[0055] adc-IBS: 3'-aaaactcgagataattatccttattagtcaggtttgtgcgcccagatagggtg-5';

[0056] adc-EBS1d: 3'-cagattgtacaaatgtggtgataacagataagtcaggtttgataacttacctttctttgt-5';

[0057] adc-EBS2: 3'-tgaacgcaagtttctaatttcggttactaatcgatagaggaaagtgtct-5'.

[005...

Embodiment 2

[0067] Example 2 , Clostridium acetobutylicum ATCC824 adc gene knockout

[0068] After the pSY6-adc plasmid was methylated at the Cac8 I site by E.coli ER2275 / pANS, it was electroporated into Clostridiumacetobutylicum ATCC 824, and after recovery overnight, 100 μl of the cell liquid was applied to a 40 μg / ml Ery selective plate in an anaerobic box After culturing at 37°C for 48-96 hours, single bacteria were picked and verified by colony PCR. The specific steps are as follows:

[0069] 2.1. Methylation of pSY6-adc plasmid

[0070] Transform pANS into E.coli ER2275 to obtain E.coli ER2275 / pANS.

[0071] Prepare competent cells of E.coli ER2275 / pANS, and then transform the pSY6-adc plasmid into E.coliER2275 / pANS. Since the pANS plasmid has Spc resistance, put it on the LB medium plate containing 100μg / ml Amp and 50μg / mlSpc Cultivate overnight, and then pick a single colony into 4ml of LB medium supplemented with 100μg / ml Amp and 50μg / ml Spc, and culture overnight. Then, the...

Embodiment 3

[0090] Example 3 , Clostridium acetobutylicum CCTCC M 94061 adc gene knockout

[0091] Using the same method as in Example 2, the plasmid pSY6-adc with the methylated Cac824 I recognition site was electroporated into C. acetobutylicum CCTCC M 94061 to obtain the Ery-resistant C. acetobutylicum CCTCC M94061 transformant, and to The transformants were verified, and the verification results were as follows:

[0092] After colony PCR amplification of the transformants of C.acetobutylicum CCTCC M 94061, the PCR amplification products were verified by electrophoresis, and the verification results were as follows Figure 5 shown, according to Figure 5As a result, there were inserted recombinant bacteria in transformants 3, 4, 5, 6, 8, 9, 14, and 17, but because wild bacteria were mixed, the above 8 colonies needed to be further separated by streaking.

[0093] Perform colony PCR amplification of the single bacteria isolated by streaking, and verify the PCR product by electrophor...

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Abstract

The invention discloses a method for improving ratio of target product (butanol) in the fermentation products of clostridium acetobutylicum. The method inhibits the generation of acetone by effectively inhibiting the metabolic pathway from acetoacetyl-CoA to acetone in the clostridium acetobutylicum. The synthesis of acetone can be blocked by knocking out an acetoacetate decarboxylase gene so as to cause the acetoacetate decarboxylase to be inactive, thereby effectively improving the ratio of butanol in the fermentation products.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for increasing the ratio of butanol produced by Clostridium. 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 necessary in the production process of antibiotics and synthetic drugs. An indispensable solvent; it is also a food-grade extractant in the food and fragrance industries. More importantly, butanol is also a high-quality fuel and fuel additive with a higher octane number than ethanol. Studies have shown that the vapor pressure and heat of vaporization of butanol are 0.63psi and 141.3kcal kg respectively -1 , 2.25psi and 204.1kcal kg lower than ethanol -1 ; Its high boiling point (118°C) and low vapor pressure help the cold start of the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/16C12N1/21C12R1/01
Inventor 蒋宇赵蒙杨晟姜卫红何慧琪陈军杨蕴刘
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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