Construction and application of 1,3-dihydroxy acetone recombinant genetic engineering bacteria

A technology of dihydroxyacetone and genetically engineered bacteria, which is applied in the field of microbial fermentation, can solve the problems of difficulty in increasing the yield of DHA, cracking and inactivation of fermentation bacteria, and achieves the effects of low cost, broad application prospects, and simple construction methods

Inactive Publication Date: 2015-08-19
XUZHOU AOGEMAN NEW MATERIAL TECH CO LTD
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, the main problem of this method is that when the concentration of the substrate glycerol and the product DHA in the medium is too high, high osmotic pressure is generated, which makes the fermentation cells lyse and inactivate, thus making it difficult to increase the yield of DHA.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Plasma particles and strains:

[0029] Cloning vector pBluescript SK - , Escherichia coli E.Coli JM109 were purchased from commercial companies.

[0030] Restriction enzymes, Taq enzymes, and T4 DNA ligase were purchased from Dalian Bao Biological Company. Other chemical reagents are domestic analytical pure chemical reagents.

[0031]Aerobacter aerogenes isolation medium: distilled water 1000mL, glycerin 5g, NaCl 5g, yeast powder 5g, glucose 20g, agar powder 15g, adjust pH to 7.0, sterilize at 0.1MPa for 20min, cool to 35℃~45℃, add Ni Nile Red (Nile Red) 2mL / L (0.30mg Nile Red dissolved in 100mL dimethyl sulfoxide), poured into a petri dish under aseptic conditions, cooled and set aside.

[0032] Nutrient-enriched medium: distilled water 1000mL, yeast powder 10g, agar powder 10g, glycerin 3g, (NH 4 ) 2 SO 4 5g, adjust the pH to 7.0, and sterilize at 0.1MPa for 10min.

[0033] Product fermentation medium:

[0034] Preparation of phosphate buffer: dist...

Embodiment 2

[0048] The composition and culture conditions of each medium are the same as above.

[0049] Acquisition of dehydrogenase gene from Aerobacter aerogenes and construction of recombinant plasmid:

[0050] Through sequence analysis of 1,3-dihydroxyacetone synthase gldA, a conservative primer was designed: primer-I:5`-GGTGGGATCCTACATGCGCACTTATTTGAG-3`primer-II:5`-AATGCTCGAGCGAATTAACGCGCCAGCCAC-3`. A large fragment was amplified using the genomic DNA of Aerobacter aerogenes as a template, from which an open reading frame of 1133 bp in size was obtained from the DHA synthetase gene. The XbaI and EcoRI bits were introduced. The pBluescript SK- and PCR amplification products were treated with XbaI and EcoRI double enzymes, and after recovery, they were kept overnight at 16°C under the action of T4 DNA ligase to prepare for transformation.

[0051] PCR amplification: pre-denaturation at 97°C for 10 min, deformation at 94°C for 60 s, annealing at 58°C for 30 s, extension at 72°...

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Abstract

The invention discloses construction and an application of 1,3-dihydroxy acetone recombinant genetic engineering bacteria; with aerobacter aerogenes genome DNA as a template, PCR amplification is applied to obtain a gene (gldA) encoding glycerol dehydrogenase (GDH), the gene (gldA) is cloned onto an escherichia coli expression vector pBluescript SK<->, and a cloned vector pSK-gldA is constructed and is expressed successfully in E.Coli JM109. A method for expressing 1,3-dihydroxy acetone comprises that the engineering bacteria for expressing 1,3-dihydroxy acetone are fermented by using a glycerol-containing culture medium, and the product 1,3-dihydroxy acetone is obtained through filtering a fermented liquid, extracting with an organic solvent, recrystallizing a combined solvent and the like. The construction of the 1,3-dihydroxy acetone high-yielding recombinant genetic engineering bacteria has the advantages of being simple in method, high in yield and low in cost. Based on the above advantages, the engineering bacteria play an important role in biological preparation of the 1,3-dihydroxy acetone and have wide application prospects.

Description

[0001] Technical field: The present invention belongs to the technical field of microbial fermentation, and relates to the construction and application of recombinant genetically engineered bacteria of 1,3-dihydroxyacetone. Background technique [0002] 1,3-Dihydroxyacetone is an important chemical and biochemical raw material, a synthetic intermediate of medicine and pesticide, and a multifunctional food additive with a wide range of uses. The synthesis methods of dihydroxyacetone mainly include precious metal catalytic oxidation method and microbial method. The synthesis method of heavy metal catalyzed oxidation of glycerol, due to the inability to overcome the fatal shortcoming of poor catalytic selectivity, causes the conversion rate of glycerol to be lower than 40%, and the productive rate of DHA is lower than 25%. The industrial production method currently utilizes microbial batch fermentation. This method is to use the dehydrogenase produced by the bacteria to carry ou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12P7/26C12R1/19
Inventor 不公告发明人
Owner XUZHOU AOGEMAN NEW MATERIAL TECH CO LTD
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