Recombinant bacteria for producing copolymer of 3-hydracrylic acid and 4-hydroxybutyric acid and application thereof

A technology of hydroxybutyric acid and hydroxypropionic acid, which is applied in the field of recombinant bacteria producing copolymers of 3-hydroxypropionic acid and 4-hydroxybutyric acid and its application field, to achieve the effect of excellent thermodynamic properties

Inactive Publication Date: 2012-09-19
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Recombinant bacteria for producing copolymer of 3-hydracrylic acid and 4-hydroxybutyric acid and application thereof
  • Recombinant bacteria for producing copolymer of 3-hydracrylic acid and 4-hydroxybutyric acid and application thereof
  • Recombinant bacteria for producing copolymer of 3-hydracrylic acid and 4-hydroxybutyric acid and application thereof

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Embodiment 1

[0050] Embodiment 1, the construction of expression engineering bacteria

[0051] 1. Construction of expression vector pZQ03-orfz

[0052] 1) Using plasmid pKSSE5.3 (Song, S.S., Hein, S., Steinbüchel, A., 1999. Production of poly(4-hydroxybutyric acid) by fed-batch cultures of recombinant strains of Escherichia coli. Biotechnol. Lett.21 , 193-197; the pKSSE5.3 plasmid is a well-known plasmid at present, and the public can obtain it from Tsinghua University) as a template, orfz Sense and orfz Antisense as primers, and use pfu enzyme to perform PCR reaction to amplify the target gene 4-hydroxybutyric acid Coenzyme A transferase gene (orfz), and designed to introduce enzyme cleavage sites at both ends of the fragment.

[0053] orfz Sense: 5'CGGTGT GGATCC CAAGGTGATG 3' (SEQ ID NO: 6)

[0054] Bam H I

[0055] orfz Ant i sense: 5' ATATA GGATCC CCGACTGGAAAGCG 3' (SEQ ID NO: 7)

[0056] Bam H I

[0057] PCR reaction conditions:

[0058] Pre-denaturation at 94°C for 10 minut...

Embodiment 2

[0070] Embodiment 2, the experiment of producing the copolymer P (3HP-co-4HB) of 3-hydroxypropionic acid and 4-hydroxybutyric acid with recombinant engineered bacteria

[0071] 1. Shake flask culture test

[0072] 1. Using TB as the medium to produce the copolymer P(3HP-co-4HB) of 3-hydroxypropionic acid and 4-hydroxybutyric acid

[0073] Recombinant engineering bacterium E.coliS17-1 (pZQ01, pZQ03-orfz) uses LB culture medium, at 37 ℃, 200rpm cultivates overnight; Then by 5% inoculum size (v / v, 10 11 cfu / ml), respectively inoculated into 50 mL of TB medium (BDO) containing 1,3-propanediol (PDO) and 1,4-butanediol (the amount of 1,3-propanediol, 1,4-butanediol (as described in Table 1), at 37° C., 200 rpm, shake the flask for 48 hours to obtain the bacterial liquid. Three sets of parallel experiments were set up for each experimental treatment. Use gas chromatography (GC, GC-2014, SHIMADZU) to verify the preliminary qualitative and quantitative analysis of polymers in cells ...

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Abstract

The invention discloses recombinant bacteria for producing a copolymer of 3-hydracrylic acid and 4-hydroxybutyric acid and application thereof. According to a method for constructing the recombinant bacteria, a 1,3-propanediol dehydrogenase encoding gene, an aldehyde dehydrogenase encoding gene, 3-hydracrylic acid coenzyme A ligase encoding gene, 4-hydroxybutyric acid coenzyme A transferase encoding gene and a polyhydroxyalkanoate polymerase encoding gene are imported into starting bacteria to obtain the recombinant bacteria. After engineering bacteria are produced in a 500-mL shake flask, the yield of P(3HP-co-4HB) can reach 6.2 g/L, and the maximum P(3HP-co-4HB) reaches 63 percent of the dry weight of cells. According to the recombinant bacteria, a production process is simple, and the thermodynamic property of the produced polymer can be changed by regulating the ratio of 3HP and 4HB, so the recombinant bacteria has bright application prospect.

Description

technical field [0001] The invention relates to a recombinant bacterium producing a copolymer of 3-hydroxypropionic acid and 4-hydroxybutyric acid (P(3HP-co-4HB)) and its application. Background technique [0002] Metabolic engineering of microorganisms has become a proven approach to produce chemical products as well as novel biomaterials. Polyhydroxyalkanoates (Polyhydroxyalkanoates or PHA) are a class of polymers that are synthesized and accumulated in microbial cells and are biodegradable and biocompatible (Steinbüchel, A., Fuchtenbusch, B., 1998.Bacterial and other biological systems for polyester production. Trends Biotechnol. 16, 419-427.). With its in-depth research, polyhydroxyalkanoate will be of great application value in environmental protection materials, biomedicine and bioenergy. [0003] According to the structural composition of monomers, PHA can be divided into short-chain and medium-long chains: the monomer composition of the former is C3 to C5, and the ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N1/21C12P7/62C12R1/19
Inventor 陈国强孟德川周琴石振宇吴琼
Owner TSINGHUA UNIV
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