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Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate as well as construction method and application thereof

A technology of polyhydroxybutyrate and genetically engineered bacteria, which is applied in the field of genetic engineering, can solve the problems of reducing, inhibiting the growth of bacteria, increasing the cost of raw materials, and separating the cost of downstream products, so as to achieve relatively comprehensive economic benefits, increase biomass, and alleviate Effects of overflooding of acetic acid

Inactive Publication Date: 2020-08-07
常州新东化工发展有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The accumulation of these by-products will not only reduce the yield of 1,3-propanediol to glycerol, but also lead to the inhibition of bacterial growth, increasing the cost of raw materials and downstream product separation costs in the fermentation process

Method used

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  • Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate as well as construction method and application thereof
  • Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate as well as construction method and application thereof
  • Genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: construct the recombinant Klebsiella pneumoniae that aldehyde / alcohol dehydrogenase gene (adhE), lactate dehydrogenase gene (ldhA) and fumarate reductase gene (frdA) delete

[0034] (1) Cloning of the upstream and downstream partial homologous sequences of the aldehyde / alcohol dehydrogenase gene (adhE)

[0035] Klebsiella pneumoniae ATCC 25955 genome was used as a template, primers were designed respectively, and the upstream homology arm and downstream homology arm of adhE gene were amplified by high-fidelity enzyme PCR.

[0036] Primers for PCR amplification of the upstream homology arm include adhE-F and adhE-F1:

[0037] adhE-F: ctatgacatgattacgaattcATGGCTGTTACTAATATCGCTGAACT;

[0038] adhE-F1: cattcgcgttatagcAGAGAAAATGATGGCGTTACGC.

[0039] The primers for PCR amplification of the homologous sequence of the downstream homology arm include adhE-R and adhE-R1:

[0040] adhE-R: tctctGCTATAACGCGAATGACAACCC;

[0041] adhE-R1: gccaagcttgcatgcctgcagTTTTT...

Embodiment 2

[0085] Example 2: Construction of recombinant plasmid pDK7-phbCBA

[0086] (1) Using the pBHR68 plasmid as a template to amplify the phbCBA gene (SEQ ID NO.4) derived from Cupriavidus necato, and design primers PHB-F and PHB-R required for PCR.

[0087] PHB-F: ggtacccggggatcctctagaGGGCAAGTACCTTGCCGAC,

[0088] PHB-R: tccgccaaaacagccaagcttCTTCTGAATCCATGACCAGCTGC.

[0089] The PCR system and amplification conditions are the same as step (1).

[0090] (2) Construction of expression plasmid pDK7-phbCBA

[0091] The pDK7 plasmid was digested with restriction endonucleases HindIII and XbaI, and the linearized pDK7 plasmid vector was recovered and purified by 1.0% agarose gel electrophoresis. The phbCBA gene fragment and the linearized pDK7 plasmid were ligated together using the ClonExpress MultiS OneStep Cloning Kit of Nanjing Nuoweizan Biotechnology Co., Ltd., and the ligated product was transformed into Escherichia coli DH5α (E.coli DH5α). 25 μg / mL) LB plates to screen positi...

Embodiment 3

[0092] Example 3: The recombinant plasmid pDK7-phbCBA was electrotransformed into recombinant Klebsiella pneumoniae Klebsiella pneumoniaeΔadhEΔldhAΔfrdA to construct Klebsiella pneumoniaeQ3-PHB

[0093] The recombinant plasmid pDK7-phbCBA was transformed into Klebsiella pneumoniaeΔadhEΔldhAΔfrdA using an electroporator, and the parameters of electroporation conditions were: voltage 2.5kv, resistance value 200Ω, pulse time 6.0msec. After electroporation, the electroporated Klebsiella pneumoniaeΔadhEΔldhAΔfrdA was washed out with pre-cooled liquid LB medium, cultured on a shaker at 37°C at 200rpm for 1-2 hours, and then spread on a medium containing 25μg / mL chloramphenicol. On the LB plate, colony PCR verification was performed with primers PHB-F and PHB-R, and the positive clone obtained was Klebsiella pneumoniaeQ3-PHB. The PCR verification of adhE gene, ldhA gene, frdA gene and phbCBA gene on Klebsiella pneumoniae Q3-PHB are all correct, the results are shown in figure 1 . ...

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Abstract

The invention provides a genetically engineered bacterium for co-production of 1, 3-propylene glycol and polyhydroxybutyrate as well as a construction method and application thereof, and belongs to the technical field of genetic engineering. The genetically engineered bacterium is classified and named as Klebsiella pneumoniae Q3-PHB, and the preservation registration number of the genetically engineered bacterium is CCTCC M 20191090. The construction method of the strain is simple; a batch feeding fermentation method is adopted; finally, the biomass of the thalli is greatly increased; the yield of the 1, 3-propylene glycol reaches 76.21 g / L; the conversion rate of 1, 3-propylene glycol is 0.56 mol / mol, the contents of ethanol and lactic acid are effectively controlled to below 1g / L; meanwhile, the thallus accumulation PHB accounts for 42.36% of the dry weight of thallus, so that the production cost is reduced, the downstream separation cost is greatly reduced, the comprehensive economic benefit is relatively high, and the industrial application value is achieved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a genetic engineering bacterium co-producing 1,3-propanediol and polyhydroxybutyrate (PHB), its construction method and application. Background technique [0002] 1,3-Propanediol (1,3-PD) is an important platform chemical, which has been widely used in medicine, cosmetics and polymer materials since it was developed in the 1990s. 1,3-propanediol is mainly used as a monomer to polymerize with terephthalic acid to form a new polyester material, polytrimethylene terephthalate (PTT). PTT has many superior properties, such as stretchability, flexibility, ease of processing, heat resistance and stability, etc. It is also based on these properties that PTT has extremely high application value in the field of synthetic fibers and engineering plastics, which further promotes the research and development of its synthetic monomer PDO. [0003] There are some microorganisms that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/18C12P7/62C12R1/22
CPCC07K14/195C12P7/18C12P7/625
Inventor 纪晓俊王维鉴陶春平徐俊蒋文彬
Owner 常州新东化工发展有限公司
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