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Engineering bacterium for whole-cell catalytic production of 5-aminopentanoic acid and preparation method of 5-aminopentanoic acid

A technology of engineering bacteria and lysine, applied in the biological field, can solve the problems of the gap in yield level and the low catalytic performance of engineering bacteria cells

Pending Publication Date: 2020-10-30
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the existing technology of 5-aminovaleric acid biocatalysis has low catalytic performance of engineering bacteria cells, and there is a big gap between the production level and the realization of industrial application

Method used

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  • Engineering bacterium for whole-cell catalytic production of 5-aminopentanoic acid and preparation method of 5-aminopentanoic acid
  • Engineering bacterium for whole-cell catalytic production of 5-aminopentanoic acid and preparation method of 5-aminopentanoic acid
  • Engineering bacterium for whole-cell catalytic production of 5-aminopentanoic acid and preparation method of 5-aminopentanoic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The detection method of embodiment 1 5AVA

[0063] Take 10 μL of the diluted sample and add it to 990 μL of water, and take 1 μL for injection after filtering with an organic filter membrane.

[0064] Mobile phase A is 10mM Na 2 HPO 4 and 10mM Na 2 B 4 o 7 , pH8.2. To prepare 1L of mobile phase, weigh 1.4g of anhydrous Na 2 HPO 4 and 3.8gNa 2 B 4 o 7 10H 2 O, dissolve it in 1L of water. Adjust the pH to about 8.4 with 1.2 mL of concentrated hydrochloric acid, then add a few drops of acid and adjust the final pH to 8.2. Stir well before adjusting the pH to completely dissolve the borate crystals. Filtration was performed with a 0.45 m regenerated cellulose membrane (p / n 3150-0576). Mobile phase B is acetonitrile:methanol:water (45:45:10, v:v:v). All mobile phase solvents were HPLC grade. The injection diluent was 100mL mobile phase A and 0.4mL concentrated H 3 PO 4 . The solution was made up in 100 mL bottles and stored at 4°C. Derivatization reagents...

Embodiment 2

[0065] Example 2 Construction of γ-aminobutyraldehyde dehydrogenase abD, lysine dehydrogenase cadA, butanediamine oxidase puO expression vector and engineering bacteria

[0066] Using the vector carrying the abD gene sequence synthesized by codon optimization as a template, and using P1 and P2 as primers, PCR amplification was carried out as follows: denaturation at 98°C for 30 seconds, annealing at 65°C for 15 seconds, and extension at 72°C for 150 seconds, 26 cycle, amplify the abD-1 gene to obtain a 1431bp PCR product (SEQ ID NO.1); similarly use the E.coli MG1655 genome as a template, and use P3 and P4 as primers to amplify the cadA-1 gene to obtain a 2148bp The PCR product (SEQ ID NO.2); using the vector carrying the optimized and synthesized puO gene sequence as a template, and using P5 and P6 as primers to amplify the puO gene, a 1362bp PCR product (SEQ ID NO.3) was obtained.

[0067] The above three PCR products were purified.

[0068] Plasmid pETDuet-1 (purchased fro...

Embodiment 3

[0105] Example 3 Induced expression of AbD, CadA, and PuO proteins of engineering strains

[0106] The engineering bacteria EC7009(E.coli BL21(DE3) / pETDuet-PT7-abD-PT7-cadA-PT7-puO( Figure 5 abbreviated as pD T7 A T7 C T7 P)), EC7010 (E.coli BL21(DE3) / pETDuet-PT7-abD-PT7-cadA-PT7-His-puO)( Figure 5 Chinese abbreviation is pD T7 A T7 C T7 HIS P)), EC7011(E.coli BL21(DE3) / pETDuet-PT7-abD-PT7-cadA-PT7-MBP-puO( Figure 5 abbreviated as pD T7 A T7 C T7 MBP P)) and EC7012(E.coliBL21(DE3) / pETDuet-PT7-abD-PT7-cadA-PT7-BCD2-puO( Figure 5 abbreviated as pD T7 A T7 C T7 BCD2 P)), respectively streaked and inoculated on LB medium plates containing 100mg / L ampicillin, cultured in a 37°C incubator for 12 hours, scraped the cultured bacteria lawns on the plates and transferred them into 5mL liquid LB (containing 100mg / L L ampicillin) in a 50 mL shaker flask, placed on a shaker at 37°C at 200 rpm for 10 h. Take the cultured bacterial solution and transfer it into a 500mL...

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Abstract

The invention relates to an engineering bacterium for whole-cell catalytic production of 5-aminopentanoic acid and a preparation method of the 5-aminopentanoic acid. Compared with a starting strain, the engineering strain has the same expression as a gamma-aminobutyraldehyde dehydrogenase abD gene, a lysine dehydrogenase cadA gene and a butanediamine oxidase puO gene. The 5-aminopentanoic acid whole-cell catalytic engineering bacterium with high catalytic rate and high catalytic concentration is constructed through combination optimization of the expression of the gamma-aminobutyraldehyde dehydrogenase abD, the lysine dehydrogenase cadA and the butanediamine oxidase puO and selection of an optimal host bacterium.

Description

technical field [0001] The present invention generally relates to the field of biotechnology, in particular to an engineering bacterium that catalyzes the production of 5-aminovaleric acid by whole cells and a preparation method for 5-aminovaleric acid. Background technique [0002] 5-aminovalerate (5-aminovalerate, 5AVA) is a functional monomer raw material with odd number of carbons, which can produce δ-valerolactam (2-piperidone) through intramolecular dehydration and cyclization, which can be further processed into bio-based nylon-5 and nylon-65. [0003] Polyamide (Polyamide, PA), commonly known as nylon (Nylon), includes different forms such as monomers formed from the same molecule and copolymers formed from diamines or dibasic acids as monomers. It is widely used in textiles, automobiles, electronic appliances , bioplastics and pharmaceutical industries have a wide range of applications. The world mainly produces polyamides through petrochemical raw materials, and ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P13/00C12R1/19
CPCC12P13/001C12N9/0067C12N9/0016C12N9/0022C12Y102/01019C12Y104/01015
Inventor 温廷益米杰张芸刘树文
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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