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A method for efficiently synthesizing phb by knocking out the rfad gene

A high-efficiency, genetic technology, applied in the field of high-efficiency synthesis of PHB, can solve the problem that PHB cannot meet the needs of industrial production

Active Publication Date: 2021-02-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods make the improvement of PHB output still unable to meet the needs of industrial production

Method used

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  • A method for efficiently synthesizing phb by knocking out the rfad gene
  • A method for efficiently synthesizing phb by knocking out the rfad gene
  • A method for efficiently synthesizing phb by knocking out the rfad gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The construction of embodiment 1 engineering bacteria WJW00

[0046] The engineering bacteria WJW00 has been published in the SCI paper "Construction and Characterization of an Escherichia coli Mutant Producing Kdo 2 -Lipid A" (disclosure date: March 13, 2014). The specific construction process is as follows:

[0047] (1) Obtaining the rfaD gene knockout fragment

[0048] The knockout fragment of the rfaD gene was obtained by chemical total synthesis or step-by-step PCR amplification, and its two ends were the upstream and downstream homology arms of the rfaD gene, and the middle was a kan fragment with an FRT site. The nucleotide sequence of the rfaD gene knockout fragment is shown in SEQ ID NO.2. The rfaD gene knockout fragment was cloned into the plasmid pBlueScriptIISK(+) to obtain the recombinant plasmid pBlueScript IISK(+)-rfaDU-Fkan-rfaDD. Using the plasmid as a template, the knockout fragment rfaDU-Fkan-rfaDD can be amplified.

[0049] (2) Preparation and e...

Embodiment 2

[0055] Example 2 Construction of Engineering Strains W3110 / pDXW-8-phaCAB and WJW00 / pDXW-8-phaCAB

[0056] (1) Construction of plasmid

[0057] Plasmid pDXW-8-phaCAB has been co-produced with L-isoleucine in Corynebacterium glutamicum in the article "Ma, W., Wang, J., Li, Y., et.al. WM001[J].Microb Cell Fact,2018,17(1):93 10.1186 / s12934-018-0942-7" (publication date: 2018-12-31). The specific construction process is as follows:

[0058] The eutrophicum rosenbergii genome NC_008313.1 was used as a template, and the primers phaCAB-F / phaCAB-R were used to amplify the phaCAB gene cluster (see the accession number for the phaCAB gene cluster https: / / www.ncbi.nlm.nih.gov / nuccore / MH558939.1), the PCR product was digested with restriction endonucleases EcoRI and XhoI, and the vector pDXW-8 (patent: a kind of E. 04-14) It was also digested with EcoRI and XhoI. After purification, the digested product was ligated overnight at 22°C with T4 ligase, transformed into E. coli DH5α, and th...

Embodiment 3

[0065] Qualitative observation of embodiment 3 engineering bacterium fermentation production PHB

[0066] LB medium composition: yeast powder 5g / L, tryptone 10g / L and NaCl 10g / L.

[0067]The composition of M9G medium: 20g / L glucose (Glucose), 17.1g / L disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 12H 2 O), 3g / L potassium dihydrogen phosphate (KH 2 PO 4 ), 0.5g / L sodium chloride (NaCl), add 1mM magnesium sulfate (M gSO 4 ), 0.1mM calcium chloride (CaCl 2 ), 10mg / mL vitamin B1 (VB 1 ).

[0068] (1) Seed liquid culture

[0069] Recombinant bacteria W3110 / pDXW-8-phaCAB and WJW00 / pDXW-8-phaCAB1 ring lawns were picked respectively and added to 25mL LB medium, and 30μg / mL kanamycin was added, and cultured at 37°C and 200rpm for 5h to mid-logarithmic phase.

[0070] (2) Synthesis of PHB by fermentation

[0071] Culture method: the seed solution (OD 600 = about 1.8) According to the initial OD 600 =0.25 was transferred to conventional PHB fermentation medium M9G, and...

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Abstract

The invention discloses a method for efficiently synthesizing PHB by knocking out the rfaD gene, belonging to the fields of genetic engineering and fermentation engineering. In the invention, after knocking out the rfaD gene on the genome of Escherichia coli, the coding gene of the enzyme for synthesizing PHB is transformed into a bacterial strain to ferment and produce PHB. It was found that knockout of the rfaD gene could significantly improve the ability of Escherichia coli W3110, DH5α and JM109 to synthesize PHB, and the PHB volume production of WJW00 / pDXW‑8‑phaCAB was 3.95 and 3.66 times higher than that of the control bacteria W3110 / pDXW‑8‑phaCAB; WJD00 / The proportion of PHB synthesized by pDXW‑8‑phaCAB cells to cell dry weight increased by about 80% compared with the control bacteria, and the transformation rate increased by about 1.9 times; the ratio of PHB synthesized by WJJ00 / pDXW‑8‑phaCAB cells to cell dry weight increased by about 75% compared with the control bacteria , the conversion rate increased by about 1.8 times.

Description

technical field [0001] The invention relates to a method for efficiently synthesizing PHB by knocking out the rfaD gene, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Polyhydroxyalkanoates (PHA) are a class of renewable and degradable polymers with multiple material properties synthesized by microorganisms, and have broad application prospects in the fields of medicine, materials and environmental protection. Polyhydroxyalkanoate is widely present in microbial cells, mainly as a carbon source and energy storage carrier. The higher the ratio of C:N in the growth environment, the more favorable the synthesis of PHA. PHA exists in the form of hydrophobic particles in cells, and its content can exceed 90% of the dry weight of cells under certain conditions. According to different types of monomers and polymerization methods, PHA has a series of diverse material properties ranging from hard and brittle hard plastics t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/62C12N15/70C12R1/19
CPCC12N15/70C12P7/625
Inventor 王小元王建莉马文渐李烨
Owner JIANGNAN UNIV
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