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Recombined strain for producing polyhydroxy fatty ester and its construction and use

A technology of recombinant strains and fatty acids, applied in microorganism-based methods, biochemical equipment and methods, applications, etc., can solve the problems of increasing the difficulty of PHB, easy to break, low elongation, etc., and achieve the effect of broad practical application prospects.

Inactive Publication Date: 2006-03-22
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this product has the disadvantages of being brittle, easy to break, and low elongation, and will crack when the heating temperature is 10°C higher than the melting point (180°C). These shortcomings increase the difficulty of post-processing of PHB and greatly limit its scope of application

Method used

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  • Recombined strain for producing polyhydroxy fatty ester and its construction and use
  • Recombined strain for producing polyhydroxy fatty ester and its construction and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the acquisition of 3HB and 3HHx copolymer (PHBHHx) recombinant strain that produces high ratio 3HHx

[0027] 1. PCR amplification of DNA fragment containing yafH gene

[0028] Use the primer 5'-TATGGTACCGACCTGAAGTGCGGATAA and the primer 5'-AATCTCGAGGATGTGGTCCGAGAAATCTA to amplify the yafH gene from the Escherichia coli JM109 genome. The specific technical scheme is: add Ex Taq DNA polymerase to the PCR system, melt at 94 degrees Celsius for 30 seconds, and anneal at 60 degrees Celsius One minute, 72 degrees Celsius for 2 minutes, so 30 cycles.

[0029] 2. PCR product of restriction endonuclease cutting vector plasmid pBBR1MCS-2 (R.e) and yafH gene

[0030] 3. Use T4 ligase to connect the yafH gene into the plasmid vector at 16 degrees Celsius. The specific technical plan is to add 100ng of the vector plasmid pBBR1MCS-2 (R.e) fragment and 150ng of the PCR product fragment of the yafH gene to T4 ligase at 16 degrees Celsius. After connecting for 24 hours, ...

Embodiment 2

[0031] 4. The conjugative transformation method transforms the plasmid into the bacterial strain Aeromonas hydrophila (Aeromonas hydrophila) CGMCC1.1801. The specific technical scheme is to combine the donor bacterium Escherichia coli S17-1 and the recipient bacterium Aeromonas hydrophila ( Aeromonas hydrophila) CGMCC 1.1801 was cultured separately, and 0.2ml was added dropwise to the same point on the LB plate and cultured upright. After 24 hours of culture, the bacterial lawn was washed with a phosphate buffer solution of pH 7.0, and the washed bacterial liquid was coated with Spread on a double-resistant plate containing ampicillin and kanamycin, and pick a single clone for verification after 24 hours. Embodiment 2, using lauric acid and glucose as the carbon source fermentation of the recombinant Aeromonas hydrophila obtained in Example 1, producing 3-hydroxybutyric acid (3HB) and 3-hydroxybutyric acid (3HB) containing a high proportion of 3-hydroxyhexanoic acid (3HHx) Hyd...

Embodiment 3

[0036] Embodiment 3, the impact of the different concentrations of glucose carbon source on the ratio of 3HHx in the copolymer (PHBHHx)

[0037] According to the method of Example 2, other conditions remained unchanged, glucose was added as a co-substrate, and the concentration of glucose carbon source was adjusted. The culture results are shown in Table 1.

[0038] Glucose concentration (g / l)

[0039] As can be seen from the data in Table 1, when the glucose concentration increased from 3g / l to 8g / l, the content of 3HHx increased from 26.61mol% to 35.66mol%, and the content of 3HHx varied widely. When the glucose concentration was 8g / l l, the highest content of 3HHx.

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Abstract

The recombinant strain the present invention provides to produce 3-hydroxy butyric acid and 3-hydroxy hexylic acid copolymer with high hexylic acid ratio has introduced yafH gene of coding ester acyl CoA dehydrogenase and is microbe with capacity of synthesizing polyhydroxy aliphatic ester with aliphatic ester as substrate. The construction process of the recombinant strain includes inserting yafH gene into plasmid carrier to construct recombinant plasmid; and converting the strain with the recombinant plasmid to construct recombinant DNA strain. The present invention also provides the process of producing 3-hydroxy butyric acid and 3-hydroxy hexylic acid copolymer with high hexylic acid ratio with the recombinant strain. The present invention adopts gene recombination and microbial fermentation to produce 3-hydroxy butyric acid and 3-hydroxy hexylic acid copolymer with high hexylic acid ratio creatively and has wide practical application foreground.

Description

technical field [0001] The invention relates to a recombinant bacterial strain producing polyhydroxyalkanoate and its construction method and application in the field of genetic engineering and microbial fermentation, in particular to a 3-hydroxybutyric acid (3-hydroxybutyric acid) producing high proportion of 3-hydroxyhexanoic acid (3HHx) 3HB) and 3-hydroxyhexanoic acid (3HHx) copolymer (PHBHHx) recombinant strain and its construction method and application. Background technique [0002] Polyhydroxyalkanoates (polyhydroxyalkanoates, PHA) is a kind of polymer polyester synthesized by microorganisms, its molecular weight generally ranges from tens of thousands to several million, and widely exists in various microorganisms in nature. Some of its physical properties are similar to those of traditional petroleum-synthesized plastics such as polyethylene and polypropylene. It can be synthesized by renewable energy and can be completely degraded and enters the ecological cycle of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/11C12N15/31C12P7/62C12R1/01
Inventor 陈国强卢晓云
Owner TSINGHUA UNIV
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