E. coli K-12中 3- 羟基苯丙酸转运蛋白研究

报告人：陈 冰指导老师：周宁一研究员

2011.11.23

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中科院武汉病毒所 2009级硕士中期报告

Background

3-(3-hydroxyphenyl)propionate

(3-HPP)

From the degradation of lignin and other aromatic constituents of plants

COOH

OH

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Eduardo Díaz.,et al.2001. Biodegradation of Aromatic Compounds by Escherichia coli . Microbiology and Molecular Biology Reviews. p. 523-569, Vol. 65, No. 4

Pathway for the catabolism of 3-HPP in E.coli K-12

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Background

Transporter Classification system, TC

Transporters

Class

2

Subclass

2.A

Family

2.A.1

Subfamily 2.A.1.15

Electrochemical potential-driven transporters

Porters: uniporters, symporters and antiporters

The Major Facilitator Superfamily (MFS)

(Up to date :69)

The Aromatic Acid:H+ Symporter (AAHS) Family

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Background

  The Aromatic Acid:H+ Symporter (AAHS) Family:

MhpT : 3-Hydroxyphenyl propionate porter

PcaK : 4-Hydroxybenzoate/protocatachuate porter

TfdK : 2,4-Dichlorophenoxyacetate porter

BenK : Benzoate porter

GenK : gentisate porter

MhbT : 3-Hydroxybenzoate porter

VanK : Vanillate porter

MucK : cis,cis-muconate porter

NiaP or YceI : The putative niacin/nicotinamide uptake porter

Orf1 : Probable 1-hydroxy-2-naphthoate transporter

MmlH : Probable 4-methylmuconolactone transporterUp to date:11

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Background

E.coli K-12 W3110 can grow with 3-HPP as sole carbon source

The growth experiments of E.coli. K12 W3110

A: MM+MnSO4+E.coli K-12

B: MM+MnSO4+E.coli K-12+glycerol

C: MM+MnSO4+E.coli K-12+3-HPP (the final concertration:2mM)

D: MM+MnSO4+E.coli K-12+glucose

E: MM+MnSO4+E.coli K-12+glycerol+3-HPP

F: MM+MnSO4+E.coli K12+glycerol+glucose

Results

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MhpT-GFP is localized in the membrane

B

A

A: negative control, GFP -BL21 B: W3110-MhpT-GFP-BL21

A

Results

MhpT is a transmembrane protein by TMHMM topology prediction

A B

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Knockout of mhpT in Escherichia coli K-12

λ Red recombination system in Escherichia coli gene modification

Results

Two plasmids involved in the λ Red recombination system

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A simple gene disruption strategy

H1 and H2 refer to the homology extensions or regions.P1 and P2 refer to priming sites

Kirill A.,et al.2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.PNAS.6640-6645,vol.97

Results

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Verification of mhpT knock-out mutants

W3110 wild typeW3110△ mhpT::kan

Results

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Growth of Escherichia coli K-12 W3110 and mhpT mutant

Medium: Mineral medium (pH=7.0)

Results

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Growth of Escherichia coli K-12 W3110 and mhpT mutant

Medium: Na2HPO4/KH2PO4 buffer(pH=8.2)

pH=8.2

Results

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Construction of vectors to express MhpT

mhpT

Results

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Results

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wild typemutant

(mhpT-T31)

The growth curves of four different strains

mutant(T31)mutant

Site-directed mutagenesis of MhpT

Glu-27-Ala

Glu-27-Asp

Asp-75-Glu

Ala-272-His

Lys-276-Asp

Results

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Future work:

1.Uptake of the 14C-labelled 3-HPP in the wild type, mutant, overexpression and complementary strains 2. Activity analyses on MhpA to 3-HPP as initial substract in the wild type, mutant, overexpression and complementary strains

3. Detection of mhpT transcription by real-time PCR

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Thank you!

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