Genetically engineered bacteria that degrade pet plastic

A hydrolase, Escherichia coli technology, applied in plastic recycling, bacteria, hydrolase and other directions, can solve the problems of slow growth of strains, fast restriction, effective application, difficult industrial application, etc., and achieve the effect of simple and efficient degradation

Active Publication Date: 2021-07-23
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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  • Genetically engineered bacteria that degrade pet plastic
  • Genetically engineered bacteria that degrade pet plastic
  • Genetically engineered bacteria that degrade pet plastic

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, the DNA recombinant vector design, construction, preparation 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 regular expression 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 the specific structure of each sub-unit are described as follows:

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

[0045] 3), EC_pelB: Escherichia coli protein secretion expression signal peptide, codon optimization fo...

Embodiment 2

[0050] Embodiment 2, expression activity evaluation 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 frame of the pUC57-EcPETase recombinant DNA plasmid by a synthetic method to obtain the same Two kinds of EcPETase+RFP co-expression vectors driven by the promoter (EcCP_J23100): 1) EcPETase and RFP have independent RBS sequences, which are transcribed simultaneously, and the translation products are two independent proteins (EcPETase, RFP); 2) EcPETase and RFP express The frame is fused, only one RBS sequence is used, and the translation product is one 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 could be se...

Embodiment 3

[0053] Example 3, pUC57-EcPETase Genetic Engineering Escherichia coli Strain Lipase Activity and PET Plastic Degradation Experiment

[0054] Referring to the pUC57-EcPETase genetically engineered Escherichia coli strain cultivation and sample preparation scheme in Example 2, the supernatant concentrated sample of the overnight culture product of the pUC57-EcPETase genetically engineered Escherichia coli strain was obtained, and pNPB was used as the substrate. Reference: "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 andassimilates poly(ethylene terephthalate).Science,351(6278):1196- 1199. ", test 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 culture fluid had significant lipase biological activity, and its pNPB hydrolysis efficiency was 10....

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Abstract

The invention belongs to the technical field of microbial genetic engineering. In order to solve the problem that PET plastic is difficult to degrade, it provides a PET hydrolase expression unit and an Escherichia coli DNA recombinant vector that can effectively secrete and express PETase (PETase) based on the unit. and its uses. The DNA recombination vector provided by the present invention can effectively realize the secreted regular expression of PET hydrolase in Escherichia coli host bacteria, and its culture medium has significant lipase biological activity, and can realize the degradation of PET plastic film in continuous culture, and become PET plastic Biodegradation provides a genetic engineering approach with good application prospects.

Description

technical field [0001] The invention belongs to the technical field of Escherichia coli genetic engineering, and in particular relates to the construction, preparation, activity verification and application of a PET hydrolase (PETase) recombinant DNA expression vector and its application in the degradation of PET plastics. 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, plastic production is increasing year by year. According to statistics from PlasticsEurope, from 1964 to 2014, global plastic production increased from 15 million tons in 1964 to 311 million 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 / ...

Claims

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Application Information

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Patent Type & Authority Patents(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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