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New method for in-situ evolution of target protein in escherichia coli cells

A technology of Escherichia coli and protein, which is applied in the biological field, can solve the problem that there is no in situ homologous recombination evolution of enzyme protein genes, etc., and achieve the effects of improving thermal stability and catalytic specificity, increasing specific enzyme activity, and improving catalytic efficiency

Inactive Publication Date: 2016-10-12
ZHEJIANG UNIV
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Problems solved by technology

It has been reported that many methods have been invented how to regulate the expression of target genes, but there is no method for in situ homologous recombination evolution of enzyme protein genes in E. coli cells

Method used

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  • New method for in-situ evolution of target protein in escherichia coli cells
  • New method for in-situ evolution of target protein in escherichia coli cells
  • New method for in-situ evolution of target protein in escherichia coli cells

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1, in situ evolution of pyrroloquinoline-dependent glucose dehydrogenase in Escherichia coli

[0031] Escherichia coli EcNR2 (Harris H. Wang et al. 2009 Nature. 460:894-890) can express the protein required for λ-Red recombination under the induction of temperature conditions, showing high recombination efficiency for 80-100nt oligonucleotides.

[0032] The pyrroloquinoline-dependent glucose dehydrogenase gene (gdh) was inserted into the genome of the above strain through λ-Red homologous recombination, and named as EcGDH. Two 90nt oligonucleotides containing merged bases were designed to correspond to the two active sites of the gdh gene: thermostability (S231, SEQ ID NO.1) and catalytic specificity (N452, SEQ ID NO.2), Construct an oligonucleotide mutation library, and use the mutation library to perform point saturation mutations on the genome. Simultaneously, a 90nt oligonucleotide (SEQ ID NO.3) used to restore the silenced chloramphenicol resistance gene o...

Embodiment 2

[0047] Example 2 In situ evolution of 1-deoxy-D-xylulose-5-phosphate synthase in Escherichia coli with double-stranded nucleotides

[0048] The efficiency of double-stranded nucleotides through λ-Red homologous recombination in E. coli is relatively low, only 0.01%. The study found that E. coli EcNR2 (Harris H. Wang et al. (2009) Nature.460:894-890) can express the protein required for λ-Red recombination under the induction of temperature conditions, which can be significantly improved by co-transformation with the resistance recovery fragment. Recombination efficiency.

[0049] 1-deoxy-D-xylulose-5-phosphate synthase (dxs) is a key enzyme in the isoprene synthesis pathway in Escherichia coli, which synthesizes 1-deoxy -D-xylulose-5-phosphate. The exogenous genes crtE, crtB, and crtI were introduced into Escherichia coli EcNR2, so that the Escherichia coli could produce lycopene, which was named EcLYC. Since lycopene has a visible red color, strains with increased lycopene...

Embodiment 3

[0066] Example 3 In situ evolution of 1-deoxy-D-xylulose-5-phosphate synthase in Escherichia coli with single-stranded nucleotides

[0067] Escherichia coli EcNR2 (Harris H.Wang et al. (2009) Nature.460:894-890) can express the protein required for λ-Red recombination under the induction of temperature conditions, and the long single Strands also have higher recombination efficiency, because there are fewer steps to change double strands into single strands during the recombination process.

[0068] 1-deoxy-D-xylulose-5-phosphate synthase (dxs) is a key enzyme in the isoprene synthesis pathway in Escherichia coli, which synthesizes 1-deoxy -D-xylulose-5-phosphate. The exogenous genes crtE, crtB, and crtI were introduced into Escherichia coli EcNR2, so that the Escherichia coli could produce lycopene, which was named as EcLYC as a screening marker.

[0069] In vitro preparation of dxs gene long single-chain with thio-modified 4 bases at the 5' end with mutation:

[0070] The...

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Abstract

The invention relates to a new method for in-situ evolution of protein in escherichia coli cells. The method includes: firstly preparing an oligonucleotide saturation mutagenesis single-chain library of specific amino acid site in a target protein molecule, or preparing a homoduplex or single-chain DNA mutant library of the target protein molecule; using the mutant library to convert escherichia coli containing the target protein or already integrating the target protein, and establishing an escherichia coli flora containing the target gene mutant library by lambda-Red homologous recombination technology; using the DNA mutant library to convert the obtained escherichia coli again so as to form an escherichia coli mutant library with greater library capacity; operating the process 3-30 times cyclically to construct an escherichia coli genome mutant library containing the target gene mutant library; and finally carrying out efficient screening or high-throughput screening to obtain a variety of target protein mutants. The technique can be applied to microbial single gene in-situ evolution or simultaneous in-situ evolution of multiple target genes to improve enzyme catalytic activity, and can be applied to improvement of the microbial metabolic product yield.

Description

field of invention [0001] The invention relates to a new method for in-situ evolution of proteins in Escherichia coli cells, belonging to the field of biotechnology. Background technique [0002] Directed evolution technology is a method of simulating Darwinian evolution in the laboratory to evolve proteins that do not exist in nature or have better properties, and can change the metabolic flow by changing the catalytic efficiency of enzymes, expand or build new metabolic pathways , weaken or eliminate unnecessary or harmful metabolic pathways, so as to achieve the purpose of increasing the yield of certain metabolites or degrading harmful substances. Directed evolution technology mainly has two steps: firstly, modern molecular biological methods are used to construct a gene mutation library, and then coupled screening method is used to screen the library. The construction of mutant library can provide enough diversity for screening, which is a key step in directed evolutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
CPCC12N9/0006C12N9/1022C12N15/70C12N2800/101C12N2800/30C12Y202/01007C12N1/205C12R2001/19
Inventor 徐志南濮悦黄磊杭宝建董昌蔡瑾
Owner ZHEJIANG UNIV
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