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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.