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Genomic Science Program

2006 Awardee

Manipulation of Lignin Biosynthesis to MaximizeEthanol Production from Populus Feedstocks

INVESTIGATOR(S): Chapple, C.; Meilan, R.;Ladisch, M.

INSTITUTION: Purdue University

NON-TECHNICAL SUMMARY: High gasolineprices, global warming, national security, and thelimitations of global petroleum resources havereinvigorated worldwide interest in renewable resources asa feedstock for liquid transportation fuels, particularlythose derived from cellulose. As a perennial woody plant,hybrid poplar (genus Populus ) offers severaladvantages with regard to cellulosic biofuel productionincluding rapid growth rates, the ability to cyclenutrients, a wide geographic distribution, geneticdiversity, amenability to genetic engineering, and abundantgenomic resources. The phenolic cell wall polymer ligninconstitutes a significant barrier to biomass conversionbut, at the same time, it is essential to normal plantgrowth and development. Recent advances in ourunderstanding of how lignin monomers are synthesizedprovide us with an opportunity to modify the content andcomposition of the lignin polymer. The research to beconducted will enable us to rationally assess the costsavings that could result from using genetically engineeredpoplar, instead of corn, as a feedstock for producingbiofuels.

OBJECTIVES: 1) Generation of transgenicpoplar up- or down-regulated for four enzymes known toimpact lignin quantity and quality; 2) Development ofmetabolic profiling methods for poplar and theirapplication to greenhouse- and field-grown wild-type andtransgenic plants; 3) Morphometric analysis of transgeniclines grown in field plots; and 4) Cell wall deconstructionanalysis of wild-type and lignin-modified transgenic lines.

APPROACH: Obj. 1) The expression of fourenzymes in the lignin biosynthetic pathway will be up-and/or down-regulated. For each DNA construct, poplar cDNAwill be synthesized from young shoot RNA using reversetranscriptase and PCR-amplified with gene-specific primersdeveloped based on conserved regions within the genes'sequences identified from the poplar genome, theArabidopsis genome, and other plant sequences. Allconstructs will be transformed into clone NM-6 ( Populusnigra x P. maximowiczii ) using an Agrobacterium -mediated transformation protocol.Obj. 2) Transformants will be tested for changes in lignincomposition by a battery of lignin analyses (i.e., Klasonlignin, pyrolysis GC-MS, and DFRC analysis). At the sametime, HPLC and GC-MS will be used to assay total cellextracts and cell-wall hydrolysates from these plants todetermine whether perturbations in phenylpropanoid pathwaygene expression have led to alterations in free and/or cellwall-esterified phenolic compounds. Obj. 3) Morphometricanalyses will be conducted on all lines in the field trialto ensure the transgenes have no deleterious effects onphenotype. All plants will be visually examined at leasttwice during the first and second growing seasons,including at least once during leaf senescence in the falland bud flush in the spring. Pairs of ramets with anyunusual phenotypes will be photographed and measured forheight, diameter, branch length, crown width, and changesin phenology. Obj. 4) Pretreatment will be carried out inbatch-tube reactors. After pretreatment, an aliquot of theslurry in the tube will be collected and processed forimage analysis, and the rest of the slurry in the tube willbe processed by enzymatic hydrolysis using the NREL LAP009enzyme digestibility procedure with minor modifications.The liquid separated from the solids in each condition willbe filtered and analyzed via HPLC. These analyses willpermit us to determine the impact of lignin modification oncell wall degradability.


Name: Chapple, C.
Phone: 765-494-0494
Fax: 765-494-7897


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