Method for biologically synthesizing glutaric acid

A biosynthesis, glutaric acid technology, applied in the biological field, can solve the problem that intermediate products cannot be used again

Active Publication Date: 2019-01-04
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Another object of the present invention is to use pentamethylenediamine transporter and 5-aminovaleric acid transporter to regulate the transport of intermediate products, which solves the problem that intermediate products accumulate in a large amount in the fermentation broth and cannot be used again, and obtain significant effect

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  • Method for biologically synthesizing glutaric acid
  • Method for biologically synthesizing glutaric acid
  • Method for biologically synthesizing glutaric acid

Examples

Experimental program
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Effect test

specific Embodiment 1

[0019] Modular optimization of glutaric acid production strains

[0020] Select the lysine decarboxylase cadA, pentane diamine aminotransferase patA, 5-aminovalerate semialdehyde dehydrogenase patD, 5-aminovalerate aminotransferase gabT, and glutaric acid semialdehyde dehydrogenase gabD from E. coli, obtained by PCR Gene fragment, then double-enzyme digestion of the fragment and vector with endonuclease, the digested fragment is recovered by gel or column, and then the target gene is inserted into the plasmid pZE12-luc (high copy), pCS27 (medium Copy), pSA74 (low copy). Regulate the expression of genes, detect the accumulation of target products and intermediate products, and optimize the host strain to obtain pSA-cadA, pCS-patAD-gabTD recombinant plasmids (Table 1).

[0021] Prepare competent cells by electroporation, and aliquot 100 μL into 1.5 mL EP tubes for transformation. Add 2 μL of the constructed recombinant plasmid pSA-cadA and 2 μL of pCS-patAD-gabTD into a 1.5 mL cent...

specific Embodiment 2

[0024] Co-expression of pentanediamine transporter potE and 5-aminovalerate transporter gabP accelerate glutaric acid synthesis

[0025] The potE, 5-aminovalerate transporter gabP from E. coli was selected, the fragments were obtained by PCR, and then the fragments and vectors were digested with endonuclease, and the digested fragments were recovered by gel or column, and then the target The genes potE and gabP were inserted into the plasmids pSA-cadA and pCS-patAD-gabTD to obtain pSA-cadA-potE and pCS-patAD-gabTDP recombinant plasmids (Table 1).

[0026] Prepare competent cells by electroporation, and aliquot 100 μL into 1.5 mL EP tubes for transformation. Add 2μL each of the constructed recombinant plasmids pSA-cadA-potE and pCS-patAD-gabTDP into a 1.5mL centrifuge tube containing 100μL of competent cells, and mix well. Then use an electroporator to electrotransform the plasmid into competent cells. After the electroporation is completed, add LB medium, and transfer the mixture...

specific Embodiment 3

[0029] Construction of metabolic pathway for de novo synthesis of glutaric acid by engineered strains

[0030] After the biotransformation of lysine into glutaric acid is achieved, the enzymes lysA, dapB, and lysC are used to enhance the upstream lysine flux fbr The expression of can realize the de novo synthesis of glutaric acid. The gene fragments were obtained by PCR, and then the fragments and the vector were digested with endonuclease. The digested fragments were recovered by gel or column, and then the target gene was inserted into the plasmid pZE12-luc to obtain pZE-lysA- dapB-lysC fbr (Table 1).

[0031] Prepare competent cells by electroporation, and aliquot 100μL into 1.5mL EP tubes for transformation. The constructed recombinant plasmid pZE-lysA-dapB-lysC fbr 2μL was added to a 1.5mL centrifuge tube containing 100μL of competent cells, and mixed well. Then use an electroporator to electrotransform the plasmid into competent cells. After the electroporation is c...

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Abstract

The invention discloses a method for biologically synthesizing glutaric acid. A synthetic route comprises five enzymes which include lysine decarboxylase (cadA), pentadiamine transaminase (patA), 5-aminovalerate hemialdehyde dehydrogenase (patD), 5-aminovalerate transaminase (gabT) and glutarate-semialdehyde dehydrogenase (gabD). Genes encoded by the enzymes are expressed in a host, and a production host for producing glutaric acid by lysine can be acquired. The genes are led into a high-yield strain of the lysine, so that denovo synthesis of the glutaric acid is achieved. The invention further discloses a method for reinforcing production of the glutaric acid. Pentane diamine transport protein and 5-aminovaleric acid transport protein co-expressed in engineering strains, intermediates outside cells are transported into the cells, so that the glutaric acid is efficiently produced. The method has application prospects in industrial production of the glutaric acid.

Description

Technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to a method for producing glutaric acid by biological method, and also relates to a method for co-expressing pentanediamine transporter and 5-aminovalerate transporter in engineered bacteria , To accelerate the production of glutaric acid. Background technique [0002] Glutarate (1,5-pentanedioic acid, Glutarate) is an aliphatic dicarboxylic acid whose molecular formula is C 5 H 8 O 4 , Molecular weight 132.11, structural formula: Easily soluble in water, ethanol, ether, etc., the solubility in water can reach 430g·L -1 . Among all the dicarboxylic acids, glutaric acid has the lowest melting point, 95-98℃. This good characteristic makes it more suitable for the C5 dibasic of important polymers such as nylon-4,5 and nylon-5,5. Carboxylic acid structural unit. At the same time, it is also the precursor of 1,5-pentanediol, which is a common plastici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/44
CPCC12N9/0008C12N9/1096C12N9/88C12P7/44C12Y102/0102C12Y206/01048C12Y206/01082C12Y401/01018
Inventor 袁其朋孙新晓李文娜李向来马琳
Owner BEIJING UNIV OF CHEM TECH
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