2. Background
COOH 3-(3-hydroxyphenyl)propionate
(3-HPP)
From the degradation of lignin and
other aromatic constituents of plants
OH
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3. Background
Pathway for the catabolism of 3-HPP in E.coli K-12
Eduardo Díaz.,et al.2001. Biodegradation of Aromatic Compounds by Escherichia coli .
Microbiology and Molecular Biology Reviews. p. 523-569, Vol. 65, No. 4
3/17
4. Background
Transporter Classification system, TC
Transporters
Class Electrochemical potential-driven transporters
2
Subclass
Porters: uniporters, symporters and antiporters
2.A
Family
The Major Facilitator Superfamily (MFS)
2.A.1
(Up to date :69)
Subfamily
2.A.1.15 The Aromatic Acid:H+ Symporter (AAHS) Family
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6. Results
E.coli K-12 W3110 can grow with 3-HPP as sole carbon source
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
The growth experiments of E.coli. K12 W3110
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7. Results
MhpT is a transmembrane protein by TMHMM topology prediction
A
A
MhpT-GFP is localized in the membrane
A B
B
A: negative control, GFP -BL21 B: W3110-MhpT-GFP-BL21
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8. Results
Two plasmids involved in the λ Red recombination system
Knockout of mhpT in Escherichia coli K-12
λ Red recombination system in
Escherichia coli gene modification
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9. Results 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 9/17
16. Future work:
•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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