High-efficient expression D-amino acid oxidase methanol yeast, its construction and fermentation method

A methanol yeast, high-efficiency expression technology, applied in the field of bioengineering, can solve problems such as low yield and difficult post-processing, and achieve the effects of low cost and simple fermentation conditions

Inactive Publication Date: 2002-12-18
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The yield is improved, but the recombinant product of E. coli is higher, but the post-processing...

Method used

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  • High-efficient expression D-amino acid oxidase methanol yeast, its construction and fermentation method
  • High-efficient expression D-amino acid oxidase methanol yeast, its construction and fermentation method
  • High-efficient expression D-amino acid oxidase methanol yeast, its construction and fermentation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Acquisition of D-amino acid oxidase gene

[0018] According to the known 5' and 3' end sequences of Trichomonas oxidase gene, the primers were designed as follows:

[0019] 5'-primer (introduced BamH I restriction site):

[0020] 5'- GGATCC ATGGCTAAAATCGTTGT-3'

[0021] 3'-primer (introduced EcoR I restriction site):

[0022] 5'- GAATTC GTTGTTGATGGGAGGTAA-3'

[0023] Plasmid pAO6 containing the Trichomonas D-amino acid oxidase gene with the intron region removed was kindly provided by Prof. Dominguez. Using the plasmid pAO6 as a template, under the action of Taq DNA polymerase, the D-amino acid oxidase gene of Trichomonas spp. was transcribed and synthesized by PCR, and its 5' and 3' ends had BamHI and EcoR I restriction enzyme sites, respectively. The reaction conditions are:

[0024] 94°C, 30sec 5 cycles 94°C, 30sec 30 cycles 94°C 5min→→40°C, 30sec→→→→ 48°C, 30sec→→→→72°C, 10min

[0025] 72°C, 75sec 72°C, 75sec

[0026] After the P...

Embodiment 2

[0027] A total of 15ul of the above mixture was reacted overnight at 16°C. The resulting ligation product was transformed into E.coli TG1 competent cells, and spread on an ampicillin plate to screen positive clones. Plasmid DNA was extracted using a plasmid extraction kit (Promega product), and the obtained plasmid DNA was identified by PCR to prove that the recombinant plasmid was T-vector-DAAO.

Embodiment 3

[0028] Example 3 Construction of D-amino acid oxidase gene recombinant plasmid pTRCHISA-DAAO

[0029] Digest the T-vector-DAAO plasmid with BamH I, and cut out a DNA band with a size of about 1.3Kb, which proves that the D-amino acid oxidase gene is inserted in reverse. Digested with Kpn I, then incompletely digested with EcoR I, electrophoresed on 1% agarose, and a DNA gel recovery kit was used to recover a D-amino acid oxidase gene fragment of about 1.3Kb. The recovered fragment was ligated with the pTRCHISA plasmid cut with the same restriction enzymes, and the ligation reaction system was as follows:

[0030] pTRCHISA (Kpn I, EcoR I cut) 1ul

[0031]D-amino acid oxidase gene fragment 3ul

[0032] 5× ligation buffer 3ul

[0033] T4 DNA Ligase (1u / ul) 1.5ul

[0034] h 2 O 6.5ul

[0035] A total of 15ul of the above mixture was reacted overnight at 16°C. The resulting ligation product was transformed into E.coli TG1 competent cells, and spread on an ampicillin plate to...

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Abstract

The present invention discloses a methanol yeast gene engineering bacterium strain SIBAS 0111 originated from high-effective expression D-amino acid oxidase, its D-amino acids oxidase gene is originated from trigonopsis vriabllis. Said inventions is characterized by that it utilizes the construction, transformation and screening of recombinant expression plasmid pPIC 3.5K-DAAO to obtain recombinant gene engineering bacterium strain of high-yield D-amino acid oxidase. After fermentation expression the expression level of D-miano acid oxidase in fermentation liquor can be upto 23000 U/L.

Description

technical field [0001] The invention belongs to the technical field of bioengineering. Specifically, the invention relates to a methanol yeast genetic engineering strain SIBAS0111 highly expressing D-amino acid oxidase, its construction method and fermentation expression. Background technique [0002] Since the discovery of penicillin, β-lactam antibiotics have become one of the most abundant and effective drugs in clinical practice. However, with the increasing use of human beings, the resistance of bacteria has emerged and is becoming more and more serious. One of the most powerful contemporary means to combat bacterial resistance is the development of semi-synthetic β-lactam antibiotics (SSA) such as semi-synthetic penicillins (SSP) and semi-synthetic cephalosporins (SSC). The industrial production process of modern β-lactam antibiotics first relies on the fermentation method to produce antibiotics such as penicillin G, V and cephalosporin C (Cephalosporin C, CPC); Hydr...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/63C12N15/81
Inventor 袁中一于健李东阳杨晟
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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