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Complete-biological synthesis method of adipic acid

A synthetic method, the technology of adipic acid, applied in the field of total biosynthesis of adipic acid, can solve the problems that the production capacity and efficiency of microbial transformation need to be further improved, and achieve the effect of high product quality and less pollution

Active Publication Date: 2015-12-02
黄山科宏生物香料股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the transformation production capacity and efficiency of microorganisms need to be further improved

Method used

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  • Complete-biological synthesis method of adipic acid
  • Complete-biological synthesis method of adipic acid
  • Complete-biological synthesis method of adipic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 50mL of wild-type thermophilic Bacillus tansica (WT) and metabolically engineered mutant (B6) were cultured in a 500mL Erlenmeyer flask with a stopper for pre-cultivation at 200rpm for 24 hours. In the fermentation test, 200mL of pre-cultured fermentation broth was inoculated into a 5L fermenter, the corncobs were crushed to a particle size of 200ˉ250μm, and treated with high-pressure steam for 1.5 hours, and then added to the medium. The cell culture conditions were: temperature 55°C , time 42-68 hours, micro-aerobic environment culture.

[0065] Take 6mL of cell culture solution in the double volume of bacterial RNA protection solution, briefly vortex to separate, let it stand at room temperature for at least 5 minutes (not more than 2 hours), centrifuge the sample, remove the supernatant, and place the lower layer in a 800ˉ1000uL Resuspend in RNA lysate, transfer the mixture to a blasting bead lysis tube, and centrifuge at a speed greater than 12,000xg / min for 1-2min...

Embodiment 2

[0070] 50mL of wild-type thermophilic Bacillus tansica (WT) and metabolically engineered mutant (B6) were cultured in a 500mL Erlenmeyer flask with a stopper for pre-cultivation at 200rpm for 24 hours. In the fermentation test, 200mL of pre-cultured fermentation broth was inoculated into a 5L fermenter, the corn cob was crushed to a particle size of 200-250μm, and treated with high-pressure steam for 1.5 hours, and added to the medium. The cell culture conditions were: temperature 55 ℃, time 42-68 hours, micro-aerobic environment culture.

[0071] Take 6mL of cell culture solution in the double volume of bacterial RNA protection solution, briefly vortex to separate, let it stand at room temperature for at least 5 minutes (not more than 2 hours), centrifuge the sample, remove the supernatant, and place the lower layer in a 800ˉ1000uL Resuspend in RNA lysate, transfer the mixture to a blasting bead lysis tube, and centrifuge at a speed greater than 12,000xg / min for 1-2min. Tran...

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Abstract

The present invention discloses a complete-biological synthesis method of adipic acid. The complete-biological synthesis method comprises: carrying out metabolic transformation on Thermobifida fusca to obtain a mutant strain B6 capable of producing adipic acid; determining the synthesis pathway of the adipic acid, transforming the gene of the pathway into escherichia coli, carrying out modular expression, and balancing the expression of a heterologous metabolic pathway; and regulating the expression level of the heterologous gene and the flow ratio of different modules to improve the accumulation of the adipic acid. According to the present invention, the bio-manufacturing of the adipic acid and the precursor thereof has characteristics of less pollution, high product quality, good development prospect, and the like.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for the total biosynthesis of adipic acid. Background technique [0002] Adipic acid (adipicacid or adipate), also known as fatty acid, is an important dicarboxylic acid, which can undergo salt-forming reactions, esterification reactions, amidation reactions, etc., and can polycondensate with diamines or diols into high molecular weight polymers. Currently the most important application field of adipic acid is the synthesis of nylon fibers (such as nylon 6,6). It is estimated that by 2018, the global market value of adipic acid will reach 6.3 billion US dollars, and the output will be about 2.56 billion kilograms, and it will continue to increase, and the new production capacity in the past two years will mainly be in China. [0003] The industrial production route of adipic acid is mainly produced by oxidizing the mixture of cyclohexanol and cyclohexanone (alcohol and ke...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12P7/44
Inventor 邓禹张晓娟毛银
Owner 黄山科宏生物香料股份有限公司
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