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1,3-propylene glycol genetically engineered bacterium and method for producing 1,3-propylene glycol by converting same

A technology of genetically engineered bacteria and propylene glycol, applied in the field of bioengineering, can solve problems such as affecting the final product, impact, etc.

Active Publication Date: 2014-05-14
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that during the fermentation process of 1,3-PD engineering bacteria, 3-HPA tends to accumulate in large quantities, and at the same time, a variety of acidic substances are generated, which seriously affects the catalytic activity of PDOR, and the decline of PDOR activity will cause 3-HPA is further accumulated to form a vicious circle, and the growth of the fermentation strain is irreversibly stopped; in addition, the accumulation of 1,3-PD concentration in the fermentation broth will feedback the catalytic activity of PDOR, affecting the final product 1,3-PD The cumulative concentration of PD, which ultimately seriously affects the production of 1,3-PD

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of 1,3-propanediol oxidoreductase genetically engineered bacteria with high activity

[0045] According to the 1,3-propanediol oxidoreductase gene sequence of Lactobacillus brevis and the characteristics of the multiple cloning site on the expression vector pSE380, a synthetic primer was designed using bioinformatics software: primer1:5′-AT

[0046] T TCATGA AAATG CACCACCATCACCATCAT GCTGAACGTAGTTATGAC-3′ (with Pag Ⅰ restriction site), primer2: 5′-CCG GAATTC TTATTCAGCGTCGTAGG-3' (with EcoR Ⅰ restriction site).

[0047] Using Lactobacillus brevis genomic DNA as a template, the target gene was amplified by PCR.

[0048] PCR reaction parameters: pre-denaturation, 95°C 2min; denaturation, 94°C 30sec; annealing, 59°C 30sec; extension: 72°C 90sec; cycle: 30; stop extension: 72°C 10min; final 16°C incubation;

[0049] The obtained PCR product was detected by 1% agarose gel electrophoresis, and an electrophoretic band with a size of about 1.2K...

Embodiment 2

[0061] Example 2 Preparation of 1,3-propanediol by mixed transformation

[0062] (1) Lactobacillus brevis was first cultured in the seed liquid medium, and the slant colony of Lactobacillus breve with 2~3 rings was picked and placed in 10 ml seed liquid medium, and cultured with shaking at 37°C and 160 rpm for 16 hours. Seed liquid medium includes: 2g / L tryptone, 5g / L yeast extract, 12g / L beef extract, 16g / L glucose, 5g / L sodium acetate, 2.15g / L ammonium citrate, 1‰ Tween 80 , 0.58 g / L MgSO 4 ·7H 2 O, 0.05 g / L MnSO 4 · 4H 2 O, 2.0 g / L K 2 HPO 4 .

[0063] (2) The seed solution was inoculated into the expansion medium at an inoculation amount of 2%, and shaken at 37 °C and 160 rpm for 48 hours. The expansion medium includes: 2g / L tryptone, 5g / L yeast extract, 12g / L beef extract, 16g / L glucose, 5g / L sodium acetate, 2.15g / L ammonium citrate, 1‰ Tween 80, 0.58 g / L MgSO 4 ·7H 2 O, 0.05 g / L MnSO 4 · 4H 2 O, 2.0 g / L K 2 HPO 4 .

[0064] (3) Genetically engineered bac...

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PUM

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Abstract

The invention relates to a 1,3-propylene glycol genetically engineered bacterium and a method for producing 1,3-propylene glycol by mixing and converting the 1,3-propylene glycol genetically engineered bacterium, and belongs to the field of biotechnology. A PCR (Polymerase Chain Reaction) technology is used for cloning a 1,3-propylene glycol oxidation-reduction enzyme dhaT gene derived from lactobacillus brevis (Lactobacillibrevis CICC6239) so as to construct a genetically engineered strain E.coli-pSE-dhaT which is capable of efficiently and actively expressing the 1,3-propylene glycol oxidation-reduction enzyme (1,3-propanedioldehydrogenase, PDOR). The experiment proves that the enzymatic activity of the 1,3-propylene glycol oxidation-reduction enzyme expressed by the recombinant bacteria is increased by 16 times; the engineered bacterium and the lactobacillus brevis are mixed, stand still and are converted to glycerin, and the conversation rate from the glycerin to the 1,3-propylene glycol can reach 80.6%.

Description

[0001] invention technical field [0002] The invention relates to the technical field of bioengineering, and relates to the construction of a 1,3-propanediol genetically engineered bacterium and a method for producing 1,3-propanediol by mixing and transforming glycerol. Background technique [0003] 1,3-Propanediol (1,3-propanediol, 1,3-PD) is an important environmentally friendly chemical raw material, mainly used as a monomer of polyester, polyether and polyurethane, in chemical, textile, Food and other fields have broad applications, and are internationally recognized as one of the six new petrochemical products. In view of the many disadvantages of chemical synthesis of 1,3-PD, biological methods using cheap raw materials as substrates have received extensive attention. Glycerol is a natural substrate for the biotransformation of 1,3-PD producing bacteria. With the increase of biodiesel production in recent years, it is estimated that by 2020, the annual production of bi...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/74C12P7/18C12R1/19
Inventor 齐向辉王飞邓文颖林静王旭朱婧斐罗艳王亮孙文敬
Owner JIANGSU UNIV
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