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

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Problems solved by technology

Due to the low biomass and polymer content, the extraction of the po

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

Example Embodiment

[0071] Example 1. Construction of recombinant expression vector p19-PCAB

[0072] 1. Artificially synthesized the DNA shown in sequence 1 in the sequence table, in which nucleotides 13-55 are the promoter sequence, nucleotides 72-650 are the phaP gene sequence, and nucleotides 682-4395 are phbCAB operator sequence.

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

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

[0075] 4. Connect the gene fragment obtained in step 2 with the vector fragment obtained in step 3 (Takara's solutionI ligation kit), and the resulting ligation product is introduced into E.coli JM109 (Promega Biotechnology Co., Ltd., catalog by chemical transformation) No. P9751), and spread on LB-Amp solid medium, and cultivated at 37°C for 16 hours to obtain trans...

Example Embodiment

[0078] Example 2. Construction of recombinant expression vector pMCS-pctAAK

[0079] 1. Artificially synthesized DNA sequence shown in sequence 2 in the sequence table, in which nucleotides 13-55 are promoter sequences; nucleotides 72-1625 are pct gene sequences; nucleotides 1655-2593 Is the ghrA gene sequence; nucleotides 2623-3927 are the aceA gene sequence; nucleotides 4110-5826 are 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 5.8kb;

[0081] 3. Use EcoRI and XhoI to digest the vector pBBR1MCS-2 (the public can synthesize it according to the sequence of NCBIGenBank number U23751), and recover the vector fragment with a size of about 5.1kb;

[0082] 4. Connect the gene fragment obtained in step 2 with the vector fragment obtained in step 3 (Takara's solutionI ligation kit), and the resulting ligation product is introduced into E.coli JM109 by chemical transformation, and coated...

Example Embodiment

[0085] Example 3. Construction of E. coli JM109ldhA

[0086] 1. Synthesize the primers ldhAF used for the knockout of ldhA gene: 5'-CTCCCCTGGAATGCAGGGGAGCGGCAAGATTAAACCAGTTCGTTCGGGCAATTCCGGGGATCCGTCGACC-3' and ldhAR: 5'-TATTTTTAGTAGCTTAAATGTGATTCAACATCACTGGAGAAAGTCTTATGTGTAGGCTGCATGDNA is amplified by PCR with the left and right primers of ldh-TATTTTTAGTAGCTTAAATGTGATTTAGGCTGCATGCATGCATGCATGCATG-TATGAGGCTG-1.5kb. Fragment, the sequence of the fragment is shown in SEQ ID No. 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℃, extract plasmid pKD46, transform it into recipient strain E.coliJM109 by electrotransformation method, and spread it on LB-Amp solid Culture medium at 30°C for 24 hours to obtain transformants and obtain E.coli JM109 (pKD46);

[0088] 3. Inoculate E. coli JM109 (pKD46) into LB-Amp liquid medium, culture at 30°C for 4 hours, add arabinose to a final co...

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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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