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.