title: In planta differential targeting analysis of Thermotoga maritima Cel5A and CBM6-engineered Cel5A for autohydrolysis.
abstract: The heterologous expression of glycosyl hydrolases in bioenergy crops can improve the lignocellulosic conversion process for ethanol production. We attempted to obtain high-level expression of an intact Thermotoga maritima endoglucanase, Cel5A, and CBM6-engineered Cel5A in transgenic tobacco plants for the mass production and autohydrolysis of endoglucanase. Cel5A expression was targeted to different subcellular compartments, namely, the cytosol, apoplast, and chloroplast, using the native form of the pathogenesis-related protein 1a (PR1a) and Rubisco activase (RA) transit peptides. Cel5A transgenic tobacco plants with the chloroplast transit peptide showed the highest average endoglucanase activity and protein accumulation up to 4.5% total soluble protein. Cel5A-CBM6 was targeted to the chloroplast and accumulated up to 5.2% total soluble protein. In terms of the direct conversion of plant tissue into free sugar, the Cel5A-CBM6 transgenic plant was 33% more efficient than the Cel5A transgenic plant. The protein stability of Cel5A and Cel5A-CBM6 in lyophilized leaf material is an additional advantage in the bioconversion process.
authors.affiliation: Department of Forest Products and Technology (BK21 Program), Chonnam National University, Gwangju, 500-757, Republic of Korea.
authors.forename: Shobana Arumugam
authors.initials: SA
authors.lastname: Mahadevan
authors.forename: Seung Gon
authors.initials: SG
authors.lastname: Wi
authors.forename: Yeon Ok
authors.initials: YO
authors.lastname: Kim
authors.forename: Kwang Ho
authors.initials: KH
authors.lastname: Lee
authors.forename: Hyeun-Jong
authors.initials: HJ
authors.lastname: Bae
mesh_terms: D056804:Biofuels; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering; D006868:Hydrolysis; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*
chemical_list: D056804:Biofuels; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C054765:rca protein, plant; D000431:Ethanol; C407024:endoglucanase Cel5A; D002480:Cellulase; D043364:Endo-1,4-beta Xylanases