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Genetic engineering strain for degrading PET (polyethylene terephthalate) plastics

A hydrolase and amino acid technology, applied in plastic recycling, bacteria, hydrolase and other directions, can solve the problems of slow growth of strains, low metabolic efficiency, difficult industrial application, etc., and achieve the effect of simple and efficient degradation

Active Publication Date: 2018-03-13
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although the discovery of Ideonella sakaiensis 201-F6 strain and the functional analysis of the key enzyme PET hydrolase (PETase) have brought hope for the biodegradation of PET plastics, the efficiency of Ideonella sakaiensis 201-F6 wild-type strains for the degradation of PET plastics is low, Moreover, the bacteria like amorphous PET polymers (while crystalline PET polymers are currently the main constituents of PET plastic waste), so it is difficult to directly realize the industrial application of PET plastic biodegradation
At the same time, the Ideonella sakaiensis 201-F6 strain grows slowly, has low metabolic efficiency (about 20 days for one generation), and has not been effectively confirmed for biological safety, and the functional analysis and activity of key enzymes (such as PETase) involved in PET degradation and metabolism in its cells Optimization, extraction and separation also need further research and optimization
These problems greatly limit the rapid and effective application of PET plastic biodegradation based on Ideonella sakaiensis 201-F6. It is urgent to transform it through genetic engineering technology to provide a feasible bioengineering strategy for the realization of efficient biodegradation of PET plastics.

Method used

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  • Genetic engineering strain for degrading PET (polyethylene terephthalate) plastics
  • Genetic engineering strain for degrading PET (polyethylene terephthalate) plastics
  • Genetic engineering strain for degrading PET (polyethylene terephthalate) plastics

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1. Design, construction and preparation of recombinant DNA vectors of Escherichia coli secreted type often expressing PET hydrolase (PETase)

[0041] Through artificial synthesis, Nanjing GenScript Biotechnology Co., Ltd. was entrusted to synthesize the PET hydrolase secreted normally expressed unit in pUC57-EcPETase:

[0042] EcCP_J23100+EcRBS_K1725305+EC_pelB+Ec_5D+Ec_PETase+EcT_B0015(Seq ID No. 1). This unit is composed of 6 sub-functional units. The specific design and specific structure of each sub-unit are described as follows:

[0043] 1), EcCP_J23100: Escherichia coli constitutive expression constant promoter, the nucleotide sequence is shown in the 1st to 35th position of Seq ID No.1;

[0044] 2), EcRBS_K1725305: Escherichia coli protein translation RBS sequence, the nucleotide sequence is shown in the 36th to 62nd position of Seq ID No.1;

[0045] 3), EC_pelB: Escherichia coli protein secretion and expression signal peptide, carry out codon optimizat...

Embodiment 2

[0050] Example 2. Evaluation of expression activity of pUC57-EcPETase recombinant DNA vector

[0051] Based on the constructed pUC57-EcPETase and the reported red fluorescent protein (RFP) reporter vector (pSB1C3-BBa_J04450), the RFP reporter gene was further introduced into the downstream of the PETase expression box of the pUC57-EcPETase recombinant DNA plasmid by a synthetic method, and the same Two EcPETase+RFP co-expression vectors initiated by the promoter (EcCP_J23100): 1) EcPETase and RFP have independent RBS sequences, and are transcribed at the same time, and the translation products are 2 independent proteins (EcPETase, RFP); 2) EcPETase and RFP expression The frame was fused using only 1 RBS sequence, and the translation product was 1 fusion protein (EcPETase-RFP fusion protein). The two EcPETase+RFP co-expression vectors were transformed into the expression strain E.coliBL21. After screening and identification, red recombinant colonies were seen on the LB plate at...

Embodiment 3

[0053] Example 3, pUC57-EcPETase genetic engineering Escherichia coli strain lipase activity and PET plastic degradation experiment

[0054] With reference to the pUC57-EcPETase genetically engineered Escherichia coli strain culture and sample preparation scheme in Example 2, a concentrated sample of the overnight culture product of the pUC57-EcPETase genetically engineered Escherichia coli strain was obtained, using pNPB as a substrate, reference literature: "oshida S , Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K. 2016. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351(6278):1196- 1199. "The experimental method disclosed in, testing the EcPETase in the culture solution of pUC57-EcPETase genetically engineered Escherichia coli strain. The experimental results showed that the pUC57-EcPETase genetically engineered Escherichia coli strain had significant lipase biological activity, and its pNPB h...

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Abstract

The invention belongs to the technical field of microbial gene engineering, aims to solve the problem of difficult degradation of the PET (polyethylene terephthalate) plastics, and provides a PET hydrolytic enzyme expression unit, a DNA (deoxyribonucleic acid) recombined vector built based on the unit and an application of the vector, wherein the vector can effectively secrete colibacillus expressing PET hydrolase (PETase). The DNA recombined vector can effectively achieve secreting type constant expression of the PET hydrolytic enzyme in colibacillus host bacteria, nutrient solution of the vector has remarkable lipase biological activity, PET plastic films can be degraded in continuous culture process, a genetic engineering approach is provided for biodegradation of the PET plastics, andthe vector has a good application prospect.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli genetic engineering, in particular to the construction, preparation, activity verification of a recombinant DNA expression vector based on PET hydrolase (PETase) and its application in PET plastic degradation. Background technique [0002] Plastic has many excellent properties such as strong corrosion resistance, waterproof, durability, and light weight, and has been widely used since its inception. With the development of society, the output of plastic is increasing year by year. According to the statistics of PlasticsEurope, from 1964 to 2014, the global plastic production increased from 15,000,000 tons in 1964 to 311,000,000 tons in 2014, a total of 20 times the global plastic production in 50 years. [0003] Polyethylene terephthalate (PET) is one of the most common plastic materials. In 2013, the annual output of PET reached 56 million tons, accounting for about 1 / 5 of the global pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C07K19/00C12N15/70C12N1/21B29B17/00C12R1/19
CPCB29B17/00C07K2319/02C12N9/18C12N15/70Y02W30/62
Inventor 张勇刘炳麟汤丽霞郑雪莲邓梅邹佳佳解富民
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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