James F. Preston, Guang Nong, Virginia Chow, Changhao Bi and

John D. Rice

University of Florida

Collaborators:

L. O. Ingram, MCS

K. T. Shanmugam, MCS

F. Altpeter, PCMB

G. Peter, PCMB

Bacterial Conversion of Hemicellulosic

Xylans to Biofuels and Chemicals

Objective

Genetically engineer bacteria for digestion and fermentation of

the hemicellulose fractions of agricultural residues (straw and

sugar cane bagasse) and energy crops (poplar, eucalyptus and

energy cane) to ethanol and lactic acid

Expected Outcome

Development of biocatalysts for the cost effective production

of fuel alcohols and bioplastics from underutilized renewable

resources

OBJECTIVE: Develop biocatalysts to provide maximal yields of alternative fuels and chemicals from lignocellulosics.

Propanediol

Cellulose

Hemicellulose

D-Glucose

D-Xylose

Butanediol

Ethanol

Lactic acid

Glycerol

Pyruvate

P-Glyceraldehyde

Hydrolysis/Saccharification Fermentation

SPECIFIC AIM: Develop bacterial biocatalysts that efficiently converthemicellulose-derived glucuronoxylan to ethanol and chemical feedstocks.

Cellulose fiber

32-48%

Methylglucuronoxylan

(MeGAXn) network

18-35%

Interaction of Major Polymeric Sugars in Lignocellulosic Biomass

Cellulose and glucuronoxylan are

tightly associated

Cellulose fibers form through hydrogen

bonding interactions between individual

cellulose stands

and cellulose fiber association by coating

oGlucuronoxylan acts to limit microfibril

r interacting with surface cellulose

strands

The noncarbohydrate polymer lignin

embeds the interacting cellulose and

glucuronoxylan through ester linkages to

glucuronoxylan

FeedstockGlucan

(cellulose)

Xylan

(hemicellulose)Lignin

Corn stover 37.5 22.4 17.6

Corn fiber 14.28 16.8 8.4

Pine wood 46.4 8.8 29.4

Popular 49.9 17.4 18.1

Wheat straw 38.2 21.2 23.4

Switch grass 31.0 20.4 17.6

Office paper 68.6 12.4 11.3

Composition of Selected Ligncellulosic Resouces, % dry weight

Adapted from N. Mosier et al., 2005. Biores Technol. 96:673-686

102030405060PPM708090100110

Xyl nrb C1,

int bC1

MeGA C4

MeGA C1

Acetone CH3

MeGA

OCH3

Xyl intC4

60.898.5

83.4

103.3 77.2

31.5

Quantification of MeGA substitution 4-O-methylglucuronoxylan by 13C-NMR

Sweetgum glucuronoxylan

Xylose/MeGA

Sweetgum 6-7

Yellow Poplar 6-7

Sugarcane bagasse >10

Dilute Acid Hydrolysis of Methylglucuronoxylan

Hydrolysis H2SO4 0.5%, 121 oC

(Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-Xyl-)n

MeGA MeGA

Xyl-Xyl

MeGA

Xyl+

}MeGA(X)n

20-31% Cfermxyl:MeGA=10-6

Xyl

MeGA

Fermentation products (mM)

Succinate Lactate Formate Acetate Butanediol Ethanol Ethanol yield

(% of theoretical)a

E. a. JDR-1 12.7 5.6 15.0 25.2 13.4 42.6 19.2

E. a. JDR-1

(pLOI555)2.2 1.2 3.6 4.2 ND 217.4 98.0

Xylose fermentation products from E.asburiae

JDR-1 and E.asburiae JDR-1 E1 (pfl- , pLOI155)

a: Yield of 100% theoretical defined as 5 mole ethanol formed/ 3 mole xylose

consumed.

• Gram-negative Enterobacter asburiae E1 (pLOI555) produced ethanol from glucuronoxylan hydrolysate in a theoretical yield of 98% compared to 63% for Gram-negative Escherichia coli KO11.

• The genetic basis for this metabolic potential may be transferred to E. coli and Klebsiella biocatalysts for more efficient bioconversion of hemicellulose hydrolysates to biofuels and chemicals.

Summary 1

Technologies for Saccharification of Hemicellulose

Treatment Xylose Aldouronate Inhibitors

Steam Explosion X,X2,X3 MeGAX1,2,3 < 0.1% Furfural

Dilute Acid, 140oC X MeGAX < 0.25% Furfural

AFEX, 160oC Xn MeGAXn

0.1 M Ca(OH)2GH10 xylanase

X2, X3 MeGAX3

Pretreatment of O-acetyl-arabinoglucuronoxylan and

enzymatic depolymerization of 4-O-methylglucuronoxylan

Properties of xylanolytic Paenibacillus sp.

JDR-2 selected for growth on MeGX

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25 30 35 40

Hours of Growth

OD

600 n

m

0.2% GAXn

0.2% GAX1

0.27% GlucoseMeGAXn

MeGAX

Glucose

Paenibacillus sp. strain JDR-2 (27227837)

Triplicate CBM 22 GH 10 CD CBM 9 SLH

Catalytic

Nucleophile

E262

Catalytic

Acid

E138

A

B DC

Modular architecture of XynA1 secreted by

Paenibacillus sp. JDR-2

yesN yesM ytcQ lplB ytcP xynA2aguA xynB

Predicted

polycistronic

mRNAs

= rho-independent terminator

= transcriptional promotor

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 kb

G GG

G = cre, catabolite repression motif

Aldouronate-

utilization operon

xynA1

G

Endoxylanase A1

G

Regulation of expression of aldouronate-utilization genes in Paenibacillus JDR-2

MeGAX

MeGA

Ethanol

MeGAX

MeGAX3

MeGAX3

ABC

ABC

X3

Xylose

AguA

XyloseXynA2

XynB

Fermentation

Lactate

MeGAXn

XynA1

H2SO4

Acetate

Succinate

Processing of Aldouronic Acids by Paenibacillus sp. JDR

Depolymerization of MeGXn with Xyl10B and AguA

from thermophilic Thermotoga maritima

Lane 1 and 7: standards

of MeGX1-4Lane 2 and 6: standards

of X1-4Lane 3: sweetgum with

the Xyl10B

Lane 4: sweetgum xylan

with Xyl10B and AguA

Lane 5. sweetgum xylan

1 2 3 4 5 6

7

Xyl10B + + -

AguA - + -

Summary 2

Cell-surface anchored GH10 xylanases catalyze depolymerization

of glucuronoxylan and vectoral processing of products.

Bacteria endowed with this capability may be developed for

direct conversion of hemicellulosic resources to biobased products

Thermophilic α-glucuronidases and endoxylanases produced in

planta may enhance xylan bioconversion by bacterial biocatalysts.

These systems may allow the application of alkaline pretreatment

protocols to reduce costs and increase yields of fuels and chemicals

from agicultural residues and energy crops.

Pretreatment

Agricultural

Residues

Energy

Crops

Lignin

Cellulose

Hemicellulose

Solid

Waste

Thermal

Conversion

Hydrogen

(Fuel)

Chemical

Feedstocks

Bioconversion

Fuels

(ethanol, butanol)

Chemical

Feedstocks

Options for Biomass and Solid Waste Conversion to Energy

Acknowledgements

Virginia Chow, Adnon Hasona, Guang Nong, John Rice, Franz St. John,

Lorraine Yomano, and Kheir Zuobi-Hasona

Dr. Sheilachu P. Gomez, Florida Center for Renewable Chemicals and Fuels,

University of Florida.

US Department of Energy Grants DE FC36-99GO10476 and DE FC36-

00GO10594.

The Consortium for Plant Biotechnology Research Project OR22072-94.

Current funding from the Florida Energy Systems Consortium

Institute of Food and Agricultural Sciences, University of Florida Experiment

Station, as CRIS Project MCS 3763.