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Recombinant strain for producing glycolate based polymers and application of recombinant strain

A recombinant bacteria and biosynthesis technology, applied in the biological field, can solve the problems of inability to study material properties, low biomass, difficult polymer extraction, etc., and achieve the effect of good industrial application prospects.

Active Publication Date: 2016-05-18
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low biomass and polymer content, the extraction of the polymer is very difficult and its material properties cannot be studied

Method used

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  • Recombinant strain for producing glycolate based polymers and application of recombinant strain
  • Recombinant strain for producing glycolate based polymers and application of recombinant strain
  • Recombinant strain for producing glycolate based polymers and application of recombinant strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, the construction of recombinant expression vector p19-PCAB

[0072] 1. Artificially synthesize the DNA shown in sequence 1 in the sequence list, wherein the 13th-55th nucleotides are the promoter sequence, the 72nd-650th nucleotides are the phaP gene sequence, and the 682-4395th nucleotides are phbCAB operator sequence.

[0073] 2. Digest the DNA sequence synthesized in step 1 with PstI and EcoRI, and recover a gene fragment with a size of about 4.4 kb.

[0074] 3. The vector pMD19-T (purchased from Takara, Japan) was double-digested with PstI and EcoRI, and a vector fragment with a size of about 2.6 kb was recovered.

[0075]4. The gene fragment obtained in step 2 is connected with the carrier fragment obtained in step 3 (solutionI connection kit of Takara), and the connection product is imported into E.coliJM109 (Promega Biotechnology Co., Ltd., catalogue) by chemical transformation No. P9751), spread on LB-Amp solid medium, and culture at 37°C for 16 ...

Embodiment 2

[0078] Embodiment 2, construction of recombinant expression vector pMCS-pctAAK

[0079] 1. The artificially synthesized DNA sequence shown in Sequence 2 in the sequence listing, wherein the 13th-55th nucleotides are the promoter sequence; the 72nd-1625th nucleotides are the pct gene sequence; the 1655th-2593rd nucleotides It is the ghrA gene sequence; the 2623-3927th nucleotide is the aceA gene sequence; the 4110-5826th nucleotide is the aceK gene sequence;

[0080] 2. Digest the DNA sequence synthesized in step 1 with EcoRI and XhoI, and recover a gene fragment with a size of about 5.8 kb;

[0081] 3. Digest the vector pBBR1MCS-2 with EcoRI and XhoI (the public can synthesize it by themselves according to the sequence of NCBI GenBank No. U23751), and recover the vector fragment with a size of about 5.1kb;

[0082] 4. Ligate the gene fragment obtained in step 2 with the carrier fragment obtained in step 3 (Takara's solutionI connection kit), and the obtained connection produc...

Embodiment 3

[0085] Embodiment 3, the construction of escherichia coli JM109ldhA

[0086] 1、合成ldhA基因敲除所用的引物ldhAF:5'-CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGCAATTCCGGGGATCCGTCGACC-3'和ldhAR:5'-TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGTGTAGGCTGGAGCTGCTTCG-3',以质粒pKD13为模板,采用ldhAF和ldhAR引物PCR扩增得到1.5kb左右的DNA Fragment, the sequence of the fragment is shown in SEQIDNo.3, and the obtained DNA fragment is purified by agarose gel electrophoresis;

[0087] 2. Cultivate Escherichia coli containing pKD46 in LB-Amp liquid medium at 30°C, extract plasmid pKD46, transform it into recipient strain E.coliJM109 by electroporation, and spread it on LB-Amp solid culture medium at 30°C for 24 hours to obtain a transformant, E.coliJM109(pKD46);

[0088] 3. Inoculate E.coliJM109(pKD46) into LB-Amp liquid medium, incubate at 30°C for 4h, add arabinose to a final concentration of 20g / L, continue to incubate for 1.5h, and then prepare the E.coliJM109(pKD46) sensation For competent cells, the DNA frag...

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Abstract

The invention provides a recombinant strain for producing glycolate based polymers and application of the recombinant strain. The recombinant strain is obtained through introducing PHB biosynthesis operons phbCAB, polyhydroxyalkanoate particle binding protein genes phaP, CoA-transferase genes pct, isocitrate lyase genes aceA, isocitrate dehydrogenase kinase genes aceK and glyoxylate reductase genes ghrA into host E.coli (Escherichia coli); and the E.coli is E.coli JM109 or E.coli JM109ldhA. The recombinant strain can be used for synthesizing two kinds of glycolate based polymers: glycolic acid, lactic acid and 3-hydroxybutyric acid copolyesters, and glycolic acid and 3-hydroxybutyric acid copolyesters; the biomass of the recombinant strain in the shaking culture and the yield of polymers can also achieve a higher level; and better industrial application prospects are realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacterium for producing glycolic acid-based polymers and its application. Background technique [0002] Glycolic acid (glycolic acid, hydroxyacetic acid), also known as glycolic acid or α-glycolic acid, is the simplest hydroxycarboxylic acid. As an important chemical product and organic synthesis intermediate, glycolic acid is widely used in many fields such as chemical industry, medicine, textile and metallurgy. For example, the mixed acid of 2% glycolic acid and 1% formic acid is an efficient and low-cost cleaning agent, which can be used for cleaning boilers, pipes, condensers and heat exchangers; glycolic acid has small molecules and can effectively penetrate pores to solve Skin aging, wrinkles, acne and other problems are often used as cosmetic additives; glycolic acid is often used as a cross-linking coupling agent for dyeing and finishing wool fibers and cellulo...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/62C12R1/19
Inventor 李正军郑伟涛张洁陈国强吴琼
Owner BEIJING UNIV OF CHEM TECH
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