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Engineering E. coli for xylitol production during growth on xylose
Olubolaji Akinterinwa&
Patrick C. CirinoIBE 2008 Annual Meeting, March 7th
008-17 Advances in Engineering Microbial Metabolism
XylitolLow calorie sweetener
Promotes oral health
Non-insulin mediated metabolism
Obtained from reduction of xylose
Lactic acid
Propylene glycolMixture of Hydroxy furans
Glycerol
Ethylene glycol
Xylaric acid
H3C
OH
OH
H3COH
O
OH
HOOH
HO OH
OH
HO OH
OH OH
OHO O
O
HOOH
HO
HO OH
OH OH
OH
Xylitol
Some xylitol derivatives
Among Among ““Top Value Added Chemicals from BiomassTop Value Added Chemicals from Biomass””to drive to drive biorefinerybiorefinery economics (DOE, 2004)economics (DOE, 2004)
Source: www.sunopta.com/bioprocess/picture_gallery.aspx
Birch chipsand
Oat hullsPRETREATMENT EXTRACTION
ENZYMATIC HYDROLYSIS
XYLANS
XYLOSE
HYDROGENATION
WATER
LIGNIN/CELLULOSE
CATTLEFEED
CHROMATOGRAPHICSEPARATION
PURIFICATION
XYLITOL
Production of Xylitol
HYDROGENATIONHigh PHigh T
Raney Nickel Catalyst
BIOPRODUCTIONEnvironment - friendly
Potentially cheaper
in vitro in vivoYEASTSBACTERIAE. coli
E. coli
Well-studied organism
Metabolic network models
Easy to cultivate
Easy to manipulate
Gene knock-in and knock-out
Gene over-expression using plasmids
Able to utilize hexoses and pentoses
Example of a plasmid used
for gene expression in
E. coli
Diauxie in E. coli
Engineered strain for simultaneous uptake of sugars using cAMP-independent CRP mutant
=CRP*
Adenylyl cyclase
ATPcAMP + PPi
CRP-cAMP
CRP
(transcriptional activator:controls about 200 genes)
High glucoseLow/no glucose
Diauxie: preferential utilization of glucose in sugar mixtures
[Marschall & Hengge-Aronis, Molecular Microbiology (1995) 18, 175-184]
0
10
20
30
40
W3110 (wild type) PC05 (CRP*)
mM
XYLOSE CONSUMED
GLUCOSE CONSUMED
xylAxylBxylF xylG xylH xylR
xylAp
xylRpxylFp
E. coli’s Xylose Catabolic Operon
CRP-cAMP xylR
Note: XylB expression should not be affected in xylAdeletion strains and vice versa
Xylose transport into the cell
Transcriptional activator
Xylose Catabolism
Xylose Xylulose Xylulose-5-PxylA xylB Metabolism
Binding of xylR is enhanced in presence of xylose
Xylose
Xylitol
NADP+
CbXR
HeterologousReductaseCO2
Glucose
NADH
NAD+
NADPH
Platform for Xylitol Production in E. coliAim: Use glucose-xylose mixtures
Obtain reducing equivalents from glucoseUtilize xylose solely for xylitol production
Cofactors: serve as electron carriers
Xylulose
Xylulose-5-P
xylA
Metabolism
THD
xylB
∆XylA vs. ∆XylB vs. ∆XylA+B
XylB deletion is necessary for high xylitol titer in strains! Hypothesis:
Xylitol-5-phosphate (X-5-P) may be toxic to cells. Mechanism of toxicity?
crp*
Xylose Xylulose Xylulose-5-PxylA xylB Metabolism
0
100
200
300
0 48 96
Time (h)
Xylit
ol (m
M)
crp*
crp*, ∆xylA
crp*, ∆xylB
crp*, ∆xylAB
crp*, ∆xylA
crp*, ∆xylB
crp*, ∆xylA+B
Xylitol + ATP Xylitol-5-P + ADPXyl B
Shake-flask cultures in rich medium supplemented with glucose and xylose
Possible Mechanism for X-5-P Toxicity
0
2
4
6
8
0 8 16 24
Time (h)
OD
600
50mM G + 10mM xylitol
50mM G
50mM X + 10mM xylitol
50mM X
No xylB expression
No growth inhibition observed
xylB expressed
X-5-P produced
Growth inhibition observed
X-5-P may inhibit xylose transport
Growth of wildtype strain on minimal medium + sugar +/- xylitol
Concern: How can we produce xylitol solely from xylose?
Xylitol Production from Xylose
Tac promoter sequence
Multiple cloning site (MCS)
Desired gene sequence
Terminator sequence
Antibiotic resistance sequence
Origin of replication site
LacI (repressor) gene
Plasmid
Direction of transcription
Recruited xylulokinase (XK) from another organism
Xyl3 from Pichia stipitis yeast
Natural xylitol producer
23% Amino acid sequence similarity with XylB
Cloned P. stipitis xyl3 and E. colixylB to plasmids inducible by IPTG
Protein Expression AnalysisFunctional expression of XK confirmed in ∆xylB E. coli strain (PC07)
5.22PC07 + Xyl34.26PC07 + XylB
0.08PC07 + control plasmid
OD600t = 24Strain + cloned gene
< 0. 11.20 ± 0.26Xyl3
0.30 ± 0.111.63 ± 0.30XylB
< 0. 1< 0. 1No XK
Specific activity on xylitol
(units/mg protein)
Specific activity on xylulose
(units/mg protein)
Activity of XKs on xylulose (native substrate) and xylitol was investigated
ADP
XK
NADH NAD+
ATP
PK
LDH
Substrate Substrate Phosphate
PEP Pyruvate
Pyruvate Lactate
3 rxns coupled in assay
PK: Pyruvate kinase; LDH: Lactate dehydrogenase
X-5-P peak area ~4X higher with XylBthan Xyl3 * Peak area normalized w.r.t. cell OD
X-5-P Production Analysis Using 31P NMR
Xylitol-5-phosphate
Inorganic phosphate
HO OH
OH OH
OH
PO3
Chemical shift
Enzyme reactions performed in vitro
Xylitol + ATP X-5-P + ADPXK
Shake-Flask Cultures:Xylitol Titers & ODs
0
40
80
120
0 40 80 120
Time (h)
Xylit
ol (m
M)
W3110 + CbXR
PC07 + XylB + CbXR
PC07 + Xyl3 + CbXR
0
40
80
120
0 40 80 120
Time (h)
Xylit
ol (m
M)
0
4
8
12W3110 + CbXR
PC07 + Xyl B + CbXR
PC07 + Xyl 3 + CbXR
Open symbols indicate OD
Cells conditioned in glucose minimal medium. Cultures in xylose minimal medium
Spontaneous growth but not accompanied with
xylitol production!
Do cells relieve X-5-P toxicity by preventing xylitol production?
How?
OD
600
Conclusions
Deletion of xylB is necessary for obtaining high
xylitol titers in xylitol-producing E. coli strains
Expression of xylB results in the production of
toxic phosphorylated intermediate: X-5-P
X-5-P may inhibit xylose transport in E. coli
Xylitol production from xylose is enabled by
heterologous expression of P. stipitis xyl3
Dr. P Cirino
Cirino lab
Dr A. Benesi (PSU Chem.)
TW Jeffries, U Wisconsin
Acknowledgements
Metabolic Engineering Working Group, BES 0519516
Penn State Associate Vice President for Research
Questions
Thanks for Listening!
0
20
40
60
80
0 24 48 72 96
time (h)
mM
Ace
tate
crp*, ∆xylA
crp*, ∆xylB
crp*, ∆xylAB
crp*
Acetate overflow in strains expressing XylB
Acetate overflow: Shunting of carbons from glucose to acetate– Due to saturation of the respiratory pathways used in NADH reoxidation
Acetate overflow is relieved
X-5-P inhibits xylose transport?
HO OH
OH OH
OH
Lactic acid
Propylene glycolMixture of Hydroxy furans
Glycerol
Ethylene glycol
Xylaric acid
H3C
OH
OH
H3COH
O
OH
HOOH
HO OH
OH
HO OH
OH OH
OHO O
O
HOOH
HO
Xylitol
1319Soybean residue
1728Corn Stalks
1929Wheat Straw
622Softwoods
1726Hardwoods
XylanHemicellulosic sugar
% of total dry weight1
1 Jeffries TW: Advances in biochemical engineering/biotechnology 1983, 27:1-32
Batch cultures cont’dBatch culture inoculum conditioned on glucose minimal medium
Long growth lag phase
Switch to conditioning on xylose minimal medium
Results:
Growth lag reduced: growth in the first 24 hours
Xylitol production: xylose minimal medium
143.36PC07 + Xyl3 + CbXR0.00PC07 + XylB + CbXR0.53W3110 + CbXR
Xylitol t = 72 (mM)Strain Cells relieve growth inhibition/ production of
X-5-P by preventing xylitol production!
How?
Xylitol yield in other organisms
3.15 mol xylitol/mol glucose
LBC.tropicalis XRE.coliSuzuki et al.7
0.799 mol xylitol/mol xylose
ComplexD.hansenii XRDebaryomyces hansenii
Converti & Dominguez6
0.866 mol xylitol/mol xylose
DefinedC.tropicalis XRC.tropicalisKim & Oh5
1.32 mol xylitol/mol glucose
YPDP.stipitis XRS.cerevisiaeBae et al.4
0.921 mol xylitol/mol glucose
YPDP.stipitis XRS.cerevisiaeKim et al.3
0.474 mol xylitol/mol glucose
YPDS.cerevisiaeGRE3
S.cerevisiaeKim et al.3
0.436 mol xylitol/mol xylose
ComplexC.boidinii XRC.boidiniiWinkelhausen et al.2
0.199 mol xylitol/mol glucose
DefinedS.cerevisiaeGRE3
S.cerevisiaeLee et al.1
YieldMediaXylitol GeneHost OrganismAuthor
1) Lee et al. Process Biochemistry 35 (2000):1199-1203 2) Winkelhausen et al. Engineering in Life Sceinces (2004): 150-154.3) Kim et al. Enyme and Microbial Technology (2002): 862-866. 4) Bae et al. Enzyme and Microbial Technology (2004): 545-549.5) Kim & Oh. Biotechnology Letters (2003): 2085-2088. 6) Converti & Dominguez. Biotechnology and Bioengineering (2001):39-45.7) Suzuki et al. Journal of Biosceince and Bioengineering (1999): 280-284.
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