title: Subcellular targeting is a key condition for high-level accumulation of cellulase protein in transgenic maize seed.
abstract: Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.
authors.affiliation: Arkansas State University, PO Box 2760, State University, AR 72467, USA. ehood@astate.edu
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Hood
authors.forename: Robert
authors.initials: R
authors.lastname: Love
authors.forename: Jeff
authors.initials: J
authors.lastname: Lane
authors.forename: Jeff
authors.initials: J
authors.lastname: Bray
authors.forename: Richard
authors.initials: R
authors.lastname: Clough
authors.forename: Kamesh
authors.initials: K
authors.lastname: Pappu
authors.forename: Carol
authors.initials: C
authors.lastname: Drees
authors.forename: Kendall R
authors.initials: KR
authors.lastname: Hood
authors.forename: Sangwoong
authors.initials: S
authors.lastname: Yoon
authors.forename: Atta
authors.initials: A
authors.lastname: Ahmad
authors.forename: John A
authors.initials: JA
authors.lastname: Howard
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000096:biosynthesis* / Q000191:economics / Q000235:genetics; D018390:Gene Targeting; D005822:Genetic Vectors; D030821:Plants, Genetically Modified / Q000201:enzymology* / Q000382:microbiology; D012639:Seeds / Q000201:enzymology*; D014170:Transformation, Genetic; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics / Q000382:microbiology
chemical_list: C101082:endocellulase E1; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase