Method for producing 3-acetoin from recombinant Escherichia coli by the aid of glycerin
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A technology of hydroxybutanone and Escherichia coli, which is applied in the field of recombinant Escherichia coli to produce 3-hydroxybutanone from glycerol, which can solve the problems such as the worrying prospect of fermentation production, and achieve the effect of stable output and reduced production cost
Inactive Publication Date: 2015-04-29
CHANGZHOU QIUHONG BIOTECH
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However, the production of 3-hydroxybutanone by these reported strains is based on sugar resources. Due to the shortage of food, the prospect of fermentation production using sugar as a substrate is worrying. Therefore, we used Escherichia coli that can utilize glycerol as a production strain
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Embodiment 1
[0049] 1) Cloning of gene fragments
[0050] Using primer sequence 1 and primer sequence 2, the gene fragment alsS was amplified, and the PCR amplification conditions were: 95° C. for 30 S, 62° C. for 20 S, 72° C. for 90 S, 30 cycles, and 72° C. for 10 min. Using primer sequence 3 and primer sequence 4, the gene fragment alsD was amplified, and the PCR amplification conditions were: 95°C 30S, 62C20S, 72°C 30S, 30 cycles, and 72°C extension for 10 min.
[0051] 2) Construction of plasmid
[0052] The amplified gene fragment alsS was digested with different restriction enzymes KpnI and SphI to obtain sticky ends, and the amplified gene fragment alsD was digested with different restriction enzymes SphI and XbaI, To obtain cohesive ends, the pUC19 vector was also digested with restriction enzymes KpnI and XbaI. The digestion conditions were all at 37°C and incubated for 2 hours. Recover all the obtained gene fragments and linearized vectors, mix the purified gene fragments and ...
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Abstract
The invention relates to a method for producing 3-acetoin from recombinant Escherichia coli by the aid of glycerin. The method comprises steps as follows: 1) cloning genes alsS and alsD involved in biosynthesis of the 3-acetoin; 2) connecting the genes with an Escherichia coli high-copy vector pUC19 to construct a recombinant vector of a coding gene containing protein required by biosynthesis of the 3-acetoin; 3) transferring a recombinant plasmid into the Escherichia coli BW25141, and obtaining a strain, which can realize the high yield of the 3-acetoin, of the Escherichia coli through selection marking by ampicillin; 4) producing the 3-acetoin through fermentation, and recovering the 3-acetoin from a fermentation liquid. The method has the advantages of environment-friendliness, high efficiency, stability, low cost and the like.
Description
Technical field: [0001] The invention belongs to the field of microbial genetic engineering. Specifically, two genes involved in the biosynthesis of 3-hydroxybutanone are cloned and connected to the high-copy vector pUC19 of Escherichia coli, and the recombinant plasmid is transferred into Escherichia coli, and ampicillin is used as a screening marker , to strengthen the expression of these two genes in Escherichia coli, so that 3-hydroxybutanone can be synthesized in a large amount in Escherichia coli. Background technique: [0002] 3-Hydroxybutanone (acetoin), also known as acetoin or methyl acetylmethanol, is widely found in many foods in nature, such as corn, grapes, cocoa, apples, bananas, cheese, meat, etc. Fragrance, so it is mostly used as a flavor enhancer for cream, cheese, coffee, and nuts. It can also be used to prepare cream, dairy products, yogurt, and strawberry flavors. In addition, beer flavor and cheese aroma are related to 3-hydroxybutanone. 3-Hydroxybut...
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