Genetically engineered bacterium capable of generating polyhydroxybutyrate hydroxyvalerate and application method of genetically engineered bacterium

A technology of polyhydroxybutyrate hydroxyvalerate and genetically engineered bacteria, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of low mole fraction and increased fermentation production costs, and achieve the effect of reducing production costs

Active Publication Date: 2016-11-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, whether it is Gram-negative bacteria or Corynebacterium glutamicum, the vast majority of PHBV production is achieved by adding propionate; otherwise, without adding relevant carbon sources, the product is PHB instead of PHBV, which will increase the cost of fermentation production, and the mole fraction of 3HV in PHBV is still at a low level

Method used

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  • Genetically engineered bacterium capable of generating polyhydroxybutyrate hydroxyvalerate and application method of genetically engineered bacterium
  • Genetically engineered bacterium capable of generating polyhydroxybutyrate hydroxyvalerate and application method of genetically engineered bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The construction of embodiment 1 recombinant bacteria WM002

[0028] (1) Taking Eutropha rosenbergii genome NC_008313.1 as a template, primers phaCAB-F / phaCAB-R were used to amplify the phaCAB gene cluster, and the sequence of the primer phaCAB-F / phaCAB-R for amplifying the phaCAB gene cluster was

[0029] phaCAB-F: 5'-CTGGAATTCAGAAGGAGAATCAAATCATGGCGACCGG-3'

[0030] phaCAB-R: 5'-CCGCTCGAGAGGTCAGCCCATATGCAGG-3'

[0031] (2) Digest the PCR product with restriction endonucleases EcoRI and XhoI, the carrier pDXW-8 (the construction method of the carrier pDXW-8 refers to the application number 200910233618.1, and the name of the invention is a kind of Escherichia coli-coryneform bacteria shuttle type inducible expression carrier PDXW -8 and the patent application for its construction method) were digested with EcoRI and XhoI, and after purification of the digested products, they were treated overnight at 22°C with T4 ligase, transformed into Escherichia coli DH5α, and the...

Embodiment 2

[0033] Embodiment 2 uses Corynebacterium glutamicum genetically engineered bacteria WM002 shake flask fermentation to produce PHBV

[0034] (1) Shake flask fermentation of recombinant genetically engineered bacteria

[0035] Medium (g / 100mL): corn steep liquor 1.5, ammonium sulfate 3.5, magnesium sulfate 0.05, potassium dihydrogen phosphate 0.1, glucose 13, calcium carbonate 2, pH 7.2.;

[0036] Culture method: 31°C, 230rpm, culture for 96h.

[0037] Induction conditions: IPTG was added at a final concentration of 0.5 mM at 6 hours.

[0038] (2) Esterification of intracellular polyhydroxyalkanoate of genetically engineered bacteria WM002 and WM001 / pDXW-8 in Example 1: after the thalline in the obtained fermentation broth was centrifuged, wash two times with the phosphate buffer saline of pH 7.0 The second time, freeze-dry overnight in a vacuum freeze dryer, weigh about 20 mg of dried bacteria (mass is recorded as Wt), add it to the esterification tube, and then add 2 mL of c...

Embodiment 3

[0045] Example 3 Intracellular accumulation of Corynebacterium glutamicum genetically engineered bacteria ATCC13869 / pDXW-8-phaCAB shake flask fermentation is PHB instead of PHBV

[0046] (1) Shake flask fermentation of recombinant genetically engineered bacteria

[0047] The construction method of recombinant bacteria ATCC3869 / pDXW-8-phaCAB and ATCC3869 / pDXW-8 refers to (1) in Example 2;

[0048] (2) with (2) in embodiment 2;

[0049] (3) with (3) in embodiment 2;

[0050] (4) with (4) in embodiment 2; Draw the esterification product of genetic engineering bacterium ATCC3869 / pDXW-8-phaCAB to have only 1 characteristic peak (such as figure 1 Shown in C), can correspond to standard sample PHB characteristic peak (such as figure 1 Shown in A), there is no PHV characteristic peak in the PHBV standard sample (such as figure 1 Shown in B), the esterification product of ATCC3869 / pDXW-8 has no characteristic peak (as figure 1 Shown in D) shows that its intracellular synthetic pol...

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Abstract

The invention discloses a genetically engineered bacterium capable of generating polyhydroxybutyrate hydroxyvalerate and an application method of the genetically engineered bacterium, belonging to the fields of gene engineering and fermentation engineering. The constructed genetically engineered bacterium can be used for producing poly(3-hydrobutyrate-co-3-hydroxyvalerate) (PHBV) by utilizing a single carbon source, and the mole fraction of 3HV is 66%. With the adoption of the genetically engineered bacterium and the application method, the problem of high cost caused by adding of the auxiliary carbon source, namely, propionate during the production of PHBV is solved. Through the 130h fed batch fermentation by adopting a 3L fermentation tank, the corynebacterium glutamicum genetically engineered bacterium provided by the invention can generate PHBV with the weight accounting for 20% of the dry cell weight in the cells, the ratio of 3HV is 66%, and the corynebacterium glutamicum genetically engineered bacterium also can generate isoleucine.

Description

technical field [0001] The invention relates to a genetically engineered bacterium producing polyhydroxybutyrate hydroxyvalerate and an application method thereof, belonging to the fields of genetic engineering and fermentation engineering. Background technique [0002] Polyhydroxybutyrate hydroxyvalerate (PHBV) is an energy-storage biopolyester synthesized by microorganisms. It is a green and environmentally friendly medical and industrial biomaterial with good performance. Compared with brittle, the PHBV material added with hydroxyvaleric acid monomer is more flexible and plastic, and has greater advantages for its application in the fields of medicine and engineering, so most of the commercially available polyesters are PHBV. Microorganisms that can naturally synthesize polyhydroxyalkanoate (PHA) rarely accumulate PHBV due to the lack of intracellular propionyl-CoA, and most of them accumulate PHB. [0003] In order to realize the accumulation of PHBV in the cell, two mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P7/62C12R1/15
Inventor 王小元马文渐王建莉李烨林琳王甜忆黄舒亭张怡平
Owner JIANGNAN UNIV
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