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Method for polymerising glycolic acid with microorganisms

A technology of glycolic acid and microorganisms, applied in the direction of fermentation, etc., can solve problems such as the homopolymer method of glycolic acid that has not been reported

Inactive Publication Date: 2011-08-31
METABOLIC EXPLORER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] No prior art document available so far reports a process for the production of homopolymer glycolic acid (PGA) by microbial fermentation

Method used

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  • Method for polymerising glycolic acid with microorganisms
  • Method for polymerising glycolic acid with microorganisms
  • Method for polymerising glycolic acid with microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Construction of Recombinant Vector Containing Encoding Acyl-CoA Synthetase

[0095] Construction of pSCB-acs, pSCB-prpE and pSCB-prpEst

[0096] Glycolic acid is converted to glycolyl-CoA using two proteins, either propionyl-CoA synthase, prpE (from E. coli or S. typhimurium), or acetyl-CoA synthase, encoded by acs. Each gene was co-expressed intracellularly with the gene phaC1 encoding PHA synthase from R. eutrophica.

[0097] To amplify the acs and prpE genes, use the chromosomal DNA of E. coli as a template, and use the above primers (see Table 1), the primers for acs amplification are called acs F and acs R, and the primers for prpE amplification Called prpE F and prpE R, PCR was performed.

[0098] The PCR fragment of acs was cloned into the vector pSCB (Stratagene Blunt PCR Cloning KitCAT 240207-5) to obtain the plasmid pSCB-acs.

[0099] The PCR fragment of prpE was cloned into vector pSCB to obtain plasmid pSCB-prpE.

[0100] To amplify the gene prpE from Sa...

Embodiment 2

[0103] Construction of recombinant vectors containing genes encoding PHA synthase and acyl-CoA synthase

[0104] Construction of pMK-Ptrc01 / OP01 / RBS01-phaC1re-TT02

[0105] The plasmid carrying the gene phaC1 from R. eutrophica was provided by a company that synthesized the gene with an optimized sequence for optimal transcription rate in E. coli.

[0106] The relative frequency of codons varies widely depending on the organism and organelle. Many design programs for the synthesis of protein coding sequences allow selection of organisms. codon usage database Codon usage statistics are available for many commonly used and sequenced organisms such as E. coli.

[0107] The synthetic gene phaC1 encoding PHA synthase was provided ready-to-use by the company. The gene was cloned under the Ptrc01 promoter with an operator, the RBS sequence (SEQ NO: 1) located upstream of the gene, and the terminator sequence located downstream of phaC1re, resulting in plasmid pMK-Ptrc01 / OP01 / RBS0...

Embodiment 3

[0117] Construction of recombinant E. coli strains that produce PGA when cultured in the presence of glycolic acid and production of polyglycolic acid polymers

[0118] The vectors pMK-Ptrc01 / OP01 / RBS01-phaC1re-acs-TT02 and pMK-Ptrc01 / OP01 / RBS01-phaC1re-prpE-TT02 were introduced into Escherichia coli MG1655 wild-type strain by electroporation to obtain strain MG1655 (pMK-Ptrc01 / OP01 / RBS01-phaC1re-acs-TT02) and MG1655 (pMK-Ptrc01 / OP01 / RBS01-phaC1re-prpE-TT02).

[0119] The vectors pBBRMCS5-Ptrc01 / OP01 / RBS01-phaC1re-prpEst-TT02 and pUC19-Ptrc01 / OP01 / RBS01-phaC1re-prpEst-TT02 were introduced into the Escherichia coli MG1655 wild-type strain by electroporation, and the bacterial strain MG1655 (pBBRMCS5-Ptrc01 / OP01 / RBS01-phaC1re-prpEst-TT02) and MG1655 (pUC19-Ptrc01 / OP01 / RBS01-phaC1re-prpEst-TT02).

[0120] The resulting strain ( figure 2 ) were cultured in LB or MM medium containing about 5 g / L glycolic acid (the details of the conditions are in the following examples), and t...

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Abstract

The present invention relates to a method for producing and preparing polyglycolate (PGA) from genetically engineered organisms. More specifically, the invention relates to a method comprising two steps; 1) culturing, in a medium containing glycolic acid or not, the microorganism expressing at least one gene encoding an enzyme(s) that converts glycolate into glycolyl-CoA, and a gene encoding polyhydroxyalkanoate (PHA) synthase which uses glycolyl-CoA as a substrate, 2) recovering the polyglycolate polymer.

Description

field of invention [0001] The present invention relates to a process for the preparation of polyglycolic acid polymers known as PGA. More specifically, the invention relates to a method comprising the steps of: [0002] - Cultivation of a genetically engineered microorganism expressing a gene encoding an enzyme capable of converting glycolate into glycolyl-CoA and at least one gene encoding an enzyme involved in the synthesis of PHA, with a suitable carbon source including or excluding glycolic acid and [0003] - Recycling polyglycolic acid polymers. Background of the invention [0004] PLA and PGA polymers are biodegradable thermoplastic materials with a wide range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). [0005] These polyesters play an important role in applications, not only as industrial plastics, but also as pharmaceutical biopolymers, such as drug delivery carriers (Drug delivery and targeting. Nature 392, 5-10 (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62
CPCC12P7/625
Inventor 菲利普·索凯尔万达·迪斯彻特
Owner METABOLIC EXPLORER
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