title: Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane.
abstract: A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.
authors.affiliation: Syngenta Centre for Sugarcane Biofuels Development, Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Qld, Australia. md.harrison@qut.edu.au
authors.forename: Mark D
authors.initials: MD
authors.lastname: Harrison
authors.forename: Jason
authors.initials: J
authors.lastname: Geijskes
authors.forename: Heather D
authors.initials: HD
authors.lastname: Coleman
authors.forename: Kylie
authors.initials: K
authors.lastname: Shand
authors.forename: Mark
authors.initials: M
authors.lastname: Kinkema
authors.forename: Anthony
authors.initials: A
authors.lastname: Palupe
authors.forename: Rachael
authors.initials: R
authors.lastname: Hassall
authors.forename: Manuel
authors.initials: M
authors.lastname: Sainz
authors.forename: Robyn
authors.initials: R
authors.lastname: Lloyd
authors.forename: Stacy
authors.initials: S
authors.lastname: Miles
authors.forename: James L
authors.initials: JL
authors.lastname: Dale
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004789:Enzyme Activation; D057075:Enzyme Assays; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D031786:Saccharum / Q000201:enzymology* / Q000235:genetics; D019076:Transgenes; D003313:Zea mays / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase