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Gene for coding, 1,3-propylene glycol reductase in E.aero strain

A technology of propylene glycol and reductase, which is applied in the directions of oxidoreductase, genetic engineering, plant genetic improvement, etc., can solve problems such as low yield

Inactive Publication Date: 2006-03-15
迟乃玉
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the yield of 1,3-PD produced by C.but strains is low, and it is necessary to screen for safe and high-yield 1,3-PD strains

Method used

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  • Gene for coding, 1,3-propylene glycol reductase in E.aero strain
  • Gene for coding, 1,3-propylene glycol reductase in E.aero strain
  • Gene for coding, 1,3-propylene glycol reductase in E.aero strain

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Experimental program
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Embodiment 1

[0028] Embodiment 1: CGMCC 0532 bacterial strain genome DNA extraction

[0029]After CGMCC 0532 strain of the present invention is activated, inoculate to 8ml LB liquid culture medium (component concentration is 1% (W / V) peptone, 0.5% (W / V) yeast extract, 1% (W / V) ) NaCl) at 30°C overnight. The above-mentioned liquid seeds cultivated overnight are inoculated in 200 ml LB liquid medium according to the inoculum amount of 3-5%, and cultured at 30° C. to the mid-logarithmic growth phase. Centrifuge the above bacteria solution at 10000rpm at 4°C for 10-15min, and discard the supernatant. The collected bacteria were suspended in 2 ml of sterile pure water, centrifuged at 5000 rpm for 10 min at 4°C, discarded the supernatant, and repeated this operation twice. Add 400 μl sterile pure water to the collected bacteria, shake and suspend on a shaker, add 100 μl lysozyme (10 mg / ml) to make the final concentration reach 2 mg / ml, and keep the temperature in a super constant temperature w...

Embodiment 2

[0031] Embodiment 2: dhaT gene PCR cloning

[0032] 1. Cloning dhaT gene by PCR method

[0033] 1.1 Design and synthesis of primers

[0034] According to the dhaT gene of the K.pneu bacterial strain (as shown in SEQ ID NO: 3), design and synthesize the oligonucleotide primers used for dhaT gene amplification, and design a Hind III restriction site at the 5' end of the upstream primer , EcoR I restriction site was designed at the 5' end of the downstream primer.

[0035] PCR primers:

[0036] dhaT F 5′- AAG CTT ATG AGC TAT CGT ATG TTT GAT TAT CTG-3' (SEQ ID NO: 4)

[0037] dhaT R 5′- G AAT TC T CAG AAT GCC TGG CGG AAA AT-3' (SEQ ID NO: 5)

[0038] 1.2 PCR cloning dhaT gene

[0039] Use LA Taq TM , pyrobest Taq, EX Taq TM Kits (purchased from Treasure Bioengineering (Dalian) Co., Ltd., product numbers are: DRR002A or DRR002B; DR005A or DR005B; DRR001A or DRR001B or DRR001C), using CGMCC 0532 strain genomic DNA as a template, dhaTF / dhaTR as primers, as PCR.

[0040]...

Embodiment 3

[0071] Example 3: dhaT gene expression

[0072] Take the pMAL-c2X plasmid (purchased from New England Biolabs, product number: E8000S, see the structure Figure 4 ) constructed the fusion gene expression vector pMC58 (see the structure Figure 5 ). Sequencing results showed that the dhaT gene on the pMC58 expression vector was a full-length dhaT gene.

[0073] In 1 liter of LB medium, insert the culture of Escherichia coli JM109 containing pMC58 according to the inoculation amount of 1%, add IPTG to make the final concentration reach 0.4-0.8mM, the fusion protein expressed by the fusion gene composed of malE gene and dhaT gene Show that fusion protein molecular weight is 85KDa through SDS-PAGE electrophoresis, expression electrophoresis result (see Image 6 , band B). Image 6 Middle A: induced cells, B: purified fusion protein, C: purified dhaT protein, D: uninduced cells, Mh (kDa): 200, 160, 97, 4, 66, 45, ML (kDa): 97, 4, 66, 45, 31, 21.5, 14.5.

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Abstract

A coded 1,3-propylene-glycol redox enzyme gene of Eaero strain high-yielding mutant strain of aerogens 1.3-PD with number CGMCC 0532. The total length of the dhaT gene is 1164bp which codes 387 amino acid proteins, it begins with ATG initial codon and ends with TGA condon. The molecular weight of expressed protein is 43Kda and enzyme activity is 39muM / mg.

Description

technical field [0001] The present invention relates to the fields of genetic engineering, enzyme engineering, microbiology, biochemistry and the like, in particular to a high-yield 1,3-propanediol (abbreviated as 1,3-PD) Enterobacter aerogenes (abbreviated as E.aero) The bacterial strain (preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on January 16, 2001, with the preservation number CGMCC 0532, No. 13, North Zhongguancun, Haidian District, Beijing), is also involved in the oxidation of 1,3-propanediol encoded in the strain Reductase gene (dhaT). Background technique [0002] 1,3-PD microbial fermentation production can be divided into: one-step method and two-step method. One-step method refers to the direct conversion of corn sugar into 1,3-PD by engineering bacteria; two-step method refers to the conversion of corn sugar into glycerol by yeast, and then using glycerol as substrate Aerobic microbial ferment...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/53C12N9/02C12N15/63
Inventor 迟乃玉张庆芳
Owner 迟乃玉
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