Method for constructing genetic engineering bacteria and enhancing stress resistance of 1,3-propanediol producing strain

A technology of genetically engineered bacteria and production strains, applied in the field of constructing genetically engineered bacteria enhancement 1, to achieve the effects of reducing production costs, improving raw material utilization, and reducing fermentation abnormalities

Inactive Publication Date: 2009-03-11
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0025] There is no report on the introduction of PHB gene to i...

Method used

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  • Method for constructing genetic engineering bacteria and enhancing stress resistance of 1,3-propanediol producing strain
  • Method for constructing genetic engineering bacteria and enhancing stress resistance of 1,3-propanediol producing strain
  • Method for constructing genetic engineering bacteria and enhancing stress resistance of 1,3-propanediol producing strain

Examples

Experimental program
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Effect test

example 1

[0047] (1) Construction of genetically engineered bacteria

[0048] (i) Wild strain: Klebsiella AC01

[0049] (ii) Using the plasmid pBHR68 carrying the β-ketoacetyl-CoA thiolase (phbA), NADPH-dependent acetoacetyl-CoA reductase (phbB) and polyhydroxyfatty acid synthase (phbC) genes as a template, by PCR (polymerase chain reaction) method to completely clone its operon (phbCAB). PCR amplification conditions were as follows: pre-denaturation at 96°C for 8 min; denaturation at 96°C for 1 min, annealing at 62.5°C for 1 min, extension at 72°C for 4.5 min, 30 cycles; extension at 72°C for 10 min.

[0050] phbL: 5'-CCCGAATTC(EcoRI)CTGACGGCAGAGAGACAATC-3'

[0051] phbR: 5'-TATGGATCC(BamHI)TGCCGACTGGTTGAACCAG-3'

[0052] (iii) After purifying the PCR product β-ketoacetyl-CoA thiolase, NADPH-dependent acetoacetyl-CoA reductase and polyhydroxy fatty acid synthase gene fragments, they were connected to the cloning vector pMD18-T-vector and transformed into a competent state In E. col...

example 2

[0140] (1) Construction of genetically engineered bacteria

[0141] (i) Wild strain: Klebsiella HR521

[0142] (ii) with embodiment one

[0143] (iii) with embodiment one

[0144] (iv) with embodiment one

[0145] (v) with embodiment one

[0146] (vi) Extract the recombinant expression vector pDK-CAB from the positive clone, and transform it into the competent cells of wild-type Klebsiella HR521 by electroporation, identify and isolate the positive clone, which is the target strain HR521 -PHB.

[0147] (2) fermentation

[0148] (i) Strain: the constructed target strain HR521-PHB

[0149] (ii) culture medium: with embodiment one

[0150] (iii) Fermentation method: the constructed genetically engineered bacterium was cultivated on a solid medium for 24 hours, the bacterial classification was inserted into a seed medium containing 30g / L glycerol (250ml Erlenmeyer flask, 100ml liquid volume), and the culture temperature was 37 ℃, shaker speed 150rpm, aerobic culture for 18h....

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Abstract

The invention provides a method for constructing a gene engineering strain to enhance the resistance of a 1, 3-propanediol (PDO) production strain, which belongs to the technical field of biochemical engineering and comprises the step of: introducing Beta-ketothiolase PhbA, NADPH-dependent acetoacetyl CoA, PhbB and polyhydroxyalkanoates synthases, PhbC gene to a wild bacteria which can produce PDO to accumulate high concentration of PHB while the bacterial strain produces PDO so as obviously improve the tolerance of the bacterial strain to glycerol and 3-hydroxypropionaldehyde; the method can also be used to co-produce PHB and PDO. The method has the advantages that the constructed gene engineering strain improves the resistance of the bacterial strain to the high concentration of glycedrol and the intermediate product 3-hydroxypropionaldehyde and reduces the abnormal fermentation in industrial production; in addition, high concentration of PHB can be accumulated while PDO is produced, and PHB can be used as a separated product, thereby promoting the added valve of products and the utilization rate of materials and reducing the production cost.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and particularly relates to a method for constructing genetic engineering bacteria to enhance the stress resistance of 1,3-propanediol production strains. Background technique [0002] 1,3-Propanediol (PDO for short) is an important chemical raw material, which can be used as an organic solvent in ink, printing and dyeing, paint, lubricant, antifreeze and other industries. The main use of PDO is as a monomer for the synthesis of polyester and polyurethane, especially polytrimethylene terephthalate (PTT) produced by polymerization with terephthalic acid, which shows a higher ratio than 1,2-propanediol and butanediol Polymers synthesized from alcohols and ethylene glycol as monomers have better performance. At present, the world consumes tens of millions of tons of polyethylene terephthalate (PET) every year, and the chemical stability and biodegradability of PTT are comparable to PET...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/52C12N15/60C12N15/74C12P7/18C12P7/62C12R1/22C12R1/01C12R1/425
CPCY02E50/13Y02E50/10
Inventor 刘德华陈国强刘宏娟郭妮妮魏晓星
Owner TSINGHUA UNIV
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