The document describes metabolic engineering experiments to optimize Escherichia coli for efficient conversion of glucose to pyruvate. Key modifications included deleting the poxB gene encoding pyruvate oxidase to increase pyruvate production, and decreasing pyruvate dehydrogenase levels through deletion of pdhR and replacing its promoter. Initial experiments successfully deleted poxB but found contamination in strains. Batch cultures showed highest pyruvate concentrations of 6.14 g/L for strain R1 ΔpoxB. While results were mixed, the work provided experience with laboratory techniques and engineering a microbial strain through a full project.

1. METABOLIC ENGINEERING OF
ESCHERICHIA COLI FOR AN
EFFICIENT CONVERSION OF
GLUCOSE TO PYRUVATE
Practical work
5th year Biomolecular
engineering 2009
Bery Nicolas - Dernoncourt Emma - Ipas Hélène - Laborel Preneron Émeline

2. INTRODUCTION
Objective :
Engineer an E.coli strain for the
production of pyruvate
Conditions:
• Using glucose as substrate
• Reaching high yield
• Using a mineral medium
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3. INTRODUCTION
•Principles:
• Deletion of poxB gene (encoding the pyruvate oxidase)
• Decrease of the pyruvate dehydrogenase level
• By deletion of the pdhR gene
• And replacement of the natural promoter of the pdhR-aceEF-Ipd
operon by artificial promoters
• Strains
• R1 : MG1655 Δpts, ptrc-galP, ptrp-glk, ΔpflAB, IdhA::kana
• R2 : MG1655 Δpts, ptrc-galP, ptrp-glk, ΔpflAB, IdhA::kana Δack-pta
•Verifications:
• Pyruvate dehydrogenase enzymatic activity measurement
• Batch culture of recombinants strains 3

4. RESULTS AND DISCUSSIONS
DELETION OF POXB GENE BY TRANSDUCTION
(1) R1 after transduction
(2) R1 before transduction
(3) 1 Kb ladder
(4) R2 after transduction
1 2 3 (kb) 4 5 6 (5) R2 before transduction
10 (6) water
8
6
5
4 R1
3  1.879 kb
R2
2
1,5 The size difference between the wild
type sequence and the cassette : 0.5Kb
1
0.5 4

5. RESULTS AND DISCUSSIONS
ELIMINATION OF THE ANTIBIOTIC RESISTANCE CASSETTE
(1) 1 Kb ladder
(2) R1
(3) R2
(4) R1 after transformation
(5) R2 after transformation
(6) R1 ΔpoxB (with antibiotic cassette)
1 2 3 4 5 6 7
(7) R2 ΔpoxB (with antibiotic cassette).
10 kb
8 kb
6 kb R1 ΔpoxB without cassette
5 kb  0.42 kb
4 kb R2 ΔpoxB without cassette
3 kb
2 kb R1
 1.879 kb
1.5 kb R2
1kb
R1 ΔpoxB 5
 1.334 kb
0.5kb R2 ΔpoxB

6. RESULTS AND DISCUSSIONS
REPLACEMENT OF THE ACEE NATURAL PROMOTER
(1) Ladder
(2) R1
(3) R2
(4) R1 with antibiotic cassette
(5) R2 with antibiotic cassette
(6) R1 after transformation
(7) R2 after transformation
10 kb
8 kb
6 kb
5 kb
4 kb Same amplicon size than controls
3 kb selected clones are not transformants
2 kb
1,5 kb
1kb 6
1 2 3 4 5 6

7. RESULTS AND DISCUSSIONS
PYRUVATE DEHYDROGENASE ACTIVITY (PDA)
 Why ?
 Check the efficiency of the modifications
 Method to calculate PDA:
 Extraction
 Bradford dosage
 PDA dosage 7

8. RESULTS AND DISCUSSIONS
PYRUVATE DEHYDROGENASE ACTIVITY
 PDA decreased for reference strains only
 => Contaminants in our strains ? 8

9. RESULTS AND DISCUSSIONS
BATCH CULTURES ON MINERAL MEDIUM
 Why ?
 Compare pyruvate production of different strains
 Culture conditions :
 20 ml of mineral medium, 20 g/l glucose
 37°C, aerobic conditions, shaking
 Cell densities :
 R1 < R2
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10. RESULTS AND DISCUSSIONS
BATCH CULTURES ON MINERAL MEDIUM
 How ?
 HPLC analysis
 Standards: pyruvate, glucose, ethanol, succinic acid, acetic acid
 Pyruvate production
Highest pyruvate concentration
Strains Time (h)
measured (g/l)
R1 5,51 21,25
R2 5,18 15
R1 ΔpoxB 6,14 22,3
R2 ΔpoxB 5,92 22,3
R1 GI 1.5 (exp) 6,28 22,3
R1 GI 1.2 (exp) 6,19 22,3
10
R1 GI 1.2 (ref) 6,4 22,3
R2 GI 1.5 (ref) 6,08 22,3

11. CONCLUSION
 Mixed results:
 Successful deletion of poxB gene
 Contamination
 Surprising growth of R2 compared to R1
 Increase of the pyruvate production
 Repeat these experiments
 Accuracy
 Reproductivity
 Practical work:
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 Laboratory skills, techniques
 Follow a project from the beginning to the end

12. Thank you
for your attention
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