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Genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester, construction method and application thereof

A construction method, gene technology, applied in the fields of genetic engineering, fermentation engineering, and biotechnology, can solve problems such as reducing the growth rate of bacteria

Pending Publication Date: 2019-02-01
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Knockout of the glucose phosphotransferase system (PTS) in Escherichia coli can relieve the repression effect of carbon catabolites and realize the co-utilization of glucose and acetic acid, but the loss of PTS reduces the growth rate of bacteria

Method used

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  • Genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester, construction method and application thereof
  • Genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester, construction method and application thereof
  • Genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Construction of recombinant bacteria E.coli JM109-3 (pUC19-phaCAB-pct)

[0096] 1. Construction of recombinant expression vector pUC19-phaCAB-pct

[0097] 1. The DNA shown in sequence 1 in the sequence list is artificially synthesized, which contains the phaA expression cassette, the upstream is the KpnI site, and the downstream is the BamHI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-1251st nucleotide The nucleotides are the phaA gene sequence.

[0098] 2. The DNA shown in sequence 2 in the sequence list is artificially synthesized, which contains the phaB expression cassette, the upstream is the BamHI site, and the downstream is the SalI site, wherein the 9th-51st nucleotide is the promoter sequence, and the 70th-810th nucleotide Nucleotides are phaB gene sequences.

[0099] 3. The DNA shown in sequence 3 in the sequence list is artificially synthesized, which contains the phaC expression cassette, the upstream is the EcoR...

Embodiment 2

[0142] Example 2, Construction of recombinant bacteria E.coli MG1655-3 (pUC19-phaCAB-pct)

[0143] 1. Construction of recombinant expression vector pUC19-phaCAB-pct

[0144] As described in step one in Example 1.

[0145] 2. Construction of Escherichia coli E.coli MG1655-3

[0146] The method described in step 2 in Example 1 is basically the same, except that the recipient strain is replaced by E.coli MG1655 from E.coli JM109, and the gene knockout is also performed in the order of poxB, pflB, and aceEF genes to obtain E.coli. The poxB, pflB, and aceEF gene deletion mutant of coliMG1655 was named E.coli MG1655-3.

[0147] 3. Construction of recombinant bacteria E.coli MG1655-3 (pUC19-phaCAB-pct)

[0148] 1. Transform the recombinant plasmid pUC19-phaCAB-pct obtained in the above step 1 into the Escherichia coli E.coli MG1655-3 obtained in the above step 2 by electroporation, and spread it on the LB solid medium containing ampicillin, 37 Cultivate at ℃ for 24h.

[0149] 2....

Embodiment 3

[0152] Embodiment 3, the application of recombinant bacteria E.coli JM109-3 (pUC19-phaCAB-pct) in the production of lactic acid and 3-hydroxybutyric acid copolyester

[0153] 1. Shake flask experiment of E.coli JM109-3 mutant utilizing both glucose and acetic acid

[0154] 1. E. coli JM109-3 prepared in Step 2 of Example 1 was cultured in LB liquid medium for 16 hours at 37° C. at a rotational speed of 200 rpm, as a seed solution.

[0155] 2. Inoculate the seed liquid into the MM liquid culture medium according to the inoculation amount of 4% by volume. Each liter of culture medium contains 5 g of acetic acid and 5 g of glucose, and the liquid volume in a 250 ml shake flask is 50 ml. Under culture for 48h, during which the fermentation broth was collected.

[0156] 3. Quantitative detection of glucose and acetic acid consumption of E.coli JM109-3 mutant by high performance liquid chromatography. The specific conditions are as follows:

[0157] Instrument: Shimadzu Essentia ...

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Abstract

The invention discloses genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester, and a construction method and application thereof. The genetic engineering bacteria for producing lactic acid and 3-hydroxybutyrate copolyester are obtained by the following steps: knocking out the poxB gene, pflB gene and aceEF gene of a recipient bacterium; and increasing the contentof protein encoded by the phaA gene, phaB gene, phaC gene and pct gene. With the genetic engineering bacteria, lactic acid and 3-hydroxybutyrate copolyester can be synthesized by the use of glucose and acetic acid, and the polymer yield of the recombinant bacteria in the shake-flask culture can reach a high level. The ratio of lactic acid to 3-hydroxybutyrate monomer can be controlled by changingthe concentration of glucose and acetic acid. The invention has a good application prospect.

Description

technical field [0001] The invention belongs to the fields of biotechnology, genetic engineering and fermentation engineering, and relates to a genetically engineered bacterium for producing lactic acid and 3-hydroxybutyric acid copolyester and its construction method and application, more specifically, to a method of using glucose and acetic acid as A genetically engineered bacterium for producing lactic acid and 3-hydroxybutyric acid copolyester from a carbon source, its construction method and application. Background technique [0002] Acetic acid, also called acetic acid and glacial acetic acid, is the main component of vinegar and a common chemical product. In the industry, acetic acid is mainly produced by means of chemical synthesis such as acetaldehyde oxidation, liquid hydrocarbon liquid phase oxidation, and methanol carbonyl synthesis. In recent years, the acetic acid market has been in a downturn. If the downstream products of acetic acid can be developed and the...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/56C12P7/62C12R1/19
CPCC12N9/1029C12N9/13C12N15/70C12P7/56C12P7/625C12Y101/01036C12Y203/01085
Inventor 李正军史理陇笪央央普楠
Owner BEIJING UNIV OF CHEM TECH
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