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Genetically engineered strain and method for producing dihydroxyacetone by using the same

A technology of dihydroxyacetone and genetic engineering bacteria, applied in the field of genetic engineering, can solve the problems of dihydroxyacetone efficiency to be further improved, intolerance to high concentration of glycerin, etc., and achieve the effect of sufficient supply and low price

Inactive Publication Date: 2012-03-28
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the basis of Gluconobacter oxydans GDHE, the present inventor obtained a strain that can grow well on the medium with glucose as the only carbon source through further genetic engineering, but the GDHE strain is not tolerant to high concentrations of glycerol, and its transformation The efficiency of preparing dihydroxyacetone from glycerol needs to be further improved

Method used

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  • Genetically engineered strain and method for producing dihydroxyacetone by using the same
  • Genetically engineered strain and method for producing dihydroxyacetone by using the same
  • Genetically engineered strain and method for producing dihydroxyacetone by using the same

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preparation example Construction

[0060] In order to introduce the prepared recombinant vector into Gluconobacter oxidans, any known method reported so far can be used. For example, the sldAB gene is carried by the Escherichia coli-fungus shuttle vector to prepare the Escherichia coli transformant, and with the help of the helper bacteria, the zygote is obtained by three-parent conjugation with Gluconobacter oxydans. Also can use electrotransformation method, as with 1% inoculum size of Gluconobacter oxydans acceptor bacterium seed, in 100ml sorbitol culture medium (sorbitol 80g / L, yeast powder 20g / L, KH PO 1.5g / L, ( NH4)2SO4 1.5g / L and MgSO4·7H2O 0.5g / L), 30°C, 220rpm, cultivated for 18h, then centrifuged, washed with sterile 10% glycerol, centrifuged, repeated 3 times, and finally suspended in 1ml 10% glycerol , it is advisable to make the cell concentration reach 1010cells / ml, aliquot 50μl, and store at -80℃. Use the Gene-Pulser (Bio-Rad, MicroPulserTM, U.S.A.) electrotransformer to transform, mix 50 μl of...

Embodiment approach

[0065] A preferred embodiment of the DHA high-yield strain construction method of the present invention comprises the following steps:

[0066] 1. Construct madh gene knockout strain GDHEΔadh. Refer to the literature, use the plasmid pSUP202 as the carrier, connect the adh1 gene, the kana resistance gene, and the adh2 gene in sequence to obtain the plasmid pSUP202-3-adhA::Km (Wei, L.J., X.P.Yang, et al.Characterization of Enzymes in the Oxidation of 1,2-Propanediol to d-(-)-Lactic Acid by Gluconobacter oxydans DSM 2003. Molecular Biotechnology. 2010, 46(1):26-33.). Escherichia coli containing the single-gene knockout vector pSUP202-3-adhA::Km was used as the donor, Gluconobacter oxidans GDHE was used as the acceptor, and Escherichia coli containing the plasmid pRK2013 was used as the helper bacteria to carry out three-parent conjugation, and the obtained zygote was It is the madh gene knockout strain GDHEΔadh.

[0067] 2. Construct the Escherichia coli transformant containin...

Embodiment 1

[0074] Example 1 Construction of Knockout Strain GDHEΔadh

[0075] The membrane-bound alcohol dehydrogenase (madh) gene in the genome of Gluconobacter oxidans GDHE was knocked out by homology exchange. Membrane-bound alcohol dehydrogenase can oxidize ethanol to acetaldehyde, which is further oxidized to acetate by membrane-bound acetaldehyde dehydrogenase. details as follows:

[0076] 1.1 Construction of knockout vector pSUP202-3-adhA::Km

[0077] Refer to the literature, use the plasmid pSUP202 as the carrier, connect the adh1 gene, the kana resistance gene, and the adh2 gene in sequence to obtain the plasmid pSUP202-3-adhA::Km (Wei, L.J., X.P.Yang, et al.Characterization of Enzymes in the Oxidation of 1,2-Propanediol to d-(-)-Lactic Acid by Gluconobacter oxydans DSM 2003. Molecular Biotechnology. 2010, 46(1):26-33.).

[0078] 1.2 Introducing the knockout vector into the cells of Gluconobacter oxidans to complete gene knockout

[0079] ①Establishment of donor bacteria: Tr...

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Abstract

The invention discloses a strain in the field of genetic engineering and a method for producing dihydroxyacetone (DHA) by using the strain. The method is characterized in that: modified Gluconobacter oxydans is used to bioconvert glycerin into dihydroxyacetone; sldAB gene expression is added in the modified Gluconobacter oxydans through modification, and mgdh gene and madh gene are removed from the modified Gluconobacter oxydans; and the modified Gluconobacter oxydans is further subject to the adaptive evolution on a medium which uses glucose as the sole carbon source. The genetically engineered strain disclosed herein can grow well on the medium which uses glucose as the sole carbon source, overcomes the defects that wild type Gluconobacter oxydans can only use relatively expensive sorbitol and mannitol as the effective carbon source, and saves the production cost. In addition, compared with a genetically engineered strain GAN cultured on sorbitol, the genetically engineered strain GAN cultured on glucose has higher capability of producing DHA.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a bacterial strain in the technical field of genetic engineering and a method for producing dihydroxyacetone (DHA) by using the bacterial strain. Background technique [0002] Dihydroxyacetone (abbreviated as DHA) is an important chemical raw material, pharmaceutical and pesticide synthesis intermediate and functional additive, and has a wide range of uses. Dihydroxyacetone is an important raw material for the formulation of cosmetics, especially as a sunscreen that can prevent skin moisture loss and play the role of moisturizing, sunscreen and UV radiation protection. Dihydroxyacetone is an important intermediate product linking sugar metabolism and fat metabolism, and can effectively regulate the relationship between sugar metabolism and fat metabolism. In vitro supply of dihydroxyacetone can effectively slow down the rate of fat synthesis, thereby playing a role i...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N1/21C12N15/74C12R1/01
Inventor 花强韦柳静卢磊芳张敏华蒿珍珍张加奇马娜章辉潘鹏程
Owner EAST CHINA UNIV OF SCI & TECH
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