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Poly(3-hydroxypropionate-b-lactate) block copolymer using microorganisms

A hydroxypropionate, block copolymer technology, applied in the directions of microorganisms, introduction of foreign genetic material using carriers, biochemical equipment and methods, etc., can solve problems such as poor brittleness and poor physical properties

Active Publication Date: 2020-05-22
LG CHEM LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the brittleness of this PLA is poor, so in order to improve the brittleness, it is reported that poly(3-hydroxypropionate-r- Lactate) (P(3HP-r-LA)) Random Copolymer
However, such poly(3-hydroxypropionate-r-lactate) has a problem of not crystallizing and thus having poor physical properties

Method used

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  • Poly(3-hydroxypropionate-b-lactate) block copolymer using microorganisms
  • Poly(3-hydroxypropionate-b-lactate) block copolymer using microorganisms
  • Poly(3-hydroxypropionate-b-lactate) block copolymer using microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1. Preparation of recombinant carrier for the preparation of 3-hydroxypropionate-lactate block copolymer

[0081] All DNA cloning experiments were performed according to standard methods (see J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular Cloning. A laboratory Manual, 2 nd ED. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0082] 1-1. Preparation of pCDFJ23-dhaB123-gdrAB-pduP-reC GK recombinant vector

[0083] pCDFduet TM -1 (Novagen, USA, 3.7 kb) contains two T7 promoters whose expression is induced by IPTG. In this experiment, it was deleted and two constantly expressed promoters were inserted. Digest pCDFduet with XbaI / XhoI TM -1, and a DNA fragment containing the sequences of the constantly expressed J23101 (SEQ ID NO: 19) and J23108 promoter (SEQ ID NO: 20) was inserted into the XbaI / XhoI recognition site. The size of the inserted DNA fragment (promoter) containing the sequences of the J23101 and J23108 promoters wa...

Embodiment 2

[0092] Example 2. Preparation of recombinant bacterial strains for the preparation of 3-hydroxypropionate-lactate block copolymer

[0093] 2.1. Preparation of ldhA gene knockout variants

[0094] In order to prepare lactate-free polymers based on E. coli XL1-Blue (Stratagene, USA), the D-lactate dehydrogenase gene from E. coli XL1-blue involved in the production of lactate during the metabolism of E. coli (ldhA; fermentative D-lactate dehydrogenase, NAD-dependent [Escherichia coli strain K-12 substrain] gene accession number: NC_000913.3, enzyme accession number: EC_1.1.1.28) was knocked out from genomic DNA to prepare E. coli variant, E. coli XL1-Blue ([Delta]ldhA) with deletion of ldhA was prepared. Gene deletions were performed using the red recombination method known in the art. The oligomer for deleting ldhA was synthesized from the base sequences of SEQ ID NO: 36 (5'-atcagcgtacccgtgatgctaacttctctctggaaggtctgaccggctttaattaaccctcactaaagggcg-3') and SEQ ID NO: 37 (5'-...

Embodiment 3

[0097] Example 3. Preparation of 3-hydroxypropionate-lactate block copolymer induced by IPTG

[0098] The recombinant strain prepared in Example 2.2 was cultured in two steps as follows to obtain a 3-hydroxypropionate-lactate block copolymer.

[0099] First, for the first step of cultivation, the transformed recombinant Escherichia coli prepared in Example 2.2 was inoculated into a mixture containing 100 mg / L of ampicillin, 25 mg / L of streptomycin, 20 g / L of glycerol, 0.5 mM of In 100ml MR medium of vitamin B12 and 10mg / L thiamine (every 1L medium contains KH 2 PO 4 6.67g, (NH 4 ) 2 HPO 4 4g, MgSO 4 ·7H 2 0.8g, citric acid 0.8g and trace metal solution 5mL; Wherein, every 1L described trace metal solution comprises 5M HCl 5mL, FeSO 4 ·7H 2 O 10g, CaCl 2 2g, ZnSO 4 ·7H 2 O 2.2g, MnSO 4 4H 2 O 0.5g, CuSO 4 ·5H 2 O 1g, (NH 4 ) 6 Mo 7 o 2 4H 2 O 0.1g, Na 2 B 4 o 2 10H 2 (0.02 g), and cultured while stirring at 30° C. and 250 rpm.

[0100] After 1 day ...

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Abstract

The present invention relates to a novel 3-hydroxypropionate-lactate block copolymer [P(3HP-b-LA)], and a method for preparing same, and more specifically, provides a method for preparing a 3-hydroxypropionate-lactate block copolymer, and a 3-hydroxypropionate-lactate block copolymer prepared thereby, the method comprising: a first culture step in which, by using recombinant E. coli enhanced so asto be incapable of biosynthesizing lactic acid, P(3HP) is biosynthesized at the early stage of culturing by having glycerol as a carbon source and through 3-hydroxypropionate-generating genes and anenhanced PHA synthase; and a second culture step in which P(3HP) production is inhibited by using a carbon catabolic repression system for selectively introducing only glucose into E. coli when glycerol and glucose are supplied together as carbon sources, and in which polylactate is biosynthesized to an interrupted P(3HP) terminus by the enabling of the expression of a lactate synthase and a lactyl-CoA converting enzyme through an IPTG induction system.

Description

technical field [0001] Cross References to Related Applications [0002] This application claims priority or rights to Korean Patent Application No. 10-2018-0030522 filed at the Korean Intellectual Property Office on March 15, 2018, the entire disclosure of which is incorporated herein by reference. [0003] The present invention relates to a kind of preparation method of poly(3-hydroxypropionate-b-lactate) block copolymer, more specifically, relate to a kind of preparation poly(3-hydroxypropionate-b-lactate) using recombinant microorganism Lactate) block copolymer method. Background technique [0004] Polylactate (PLA), a representative biodegradable polymer with lactate as a monomer, is a polymer with high applicability to general-purpose polymers or medical polymers. Currently, PLA is produced by polymerization of lactate esters produced by microbial fermentation, but direct polymerization of lactate esters only produces PLA with low molecular weight (1000 to 5000 Dalt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/62C12N9/04C08G63/06C08G63/08
CPCC08G63/08C12N15/70C12N9/0006C12Y101/01027C12Y102/01003C12N9/0008C12N9/1029C12Y203/01C12Y208/03001C12N9/13C12Y402/01036C12Y402/0103C12Y101/01028C12P7/625C08G63/06C12N1/20C12N2510/02C12N9/88
Inventor 姜惠玉姜东均金喆雄许仁荣崔祯允
Owner LG CHEM LTD
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