title: Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification.
abstract: An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.
authors.affiliation: Chemical and Biological Processing Development Group, Process Science and Engineering Division, Pacific Northwest National Laboratory, Richland, WA 99352, USA. ziyu.dai@pnl.gov
authors.forename: Ziyu
authors.initials: Z
authors.lastname: Dai
authors.forename: Brian S
authors.initials: BS
authors.lastname: Hooker
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Quesenberry
authors.forename: Steven R
authors.initials: SR
authors.lastname: Thomas
mesh_terms: D020121:5' Untranslated Regions; D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D017794:Alfalfa mosaic virus / Q000235:genetics; D001483:Base Sequence; D044602:Cellulases / Q000096:biosynthesis* / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015388:Organelles / Q000201:enzymology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics*
chemical_list: D020121:5' Untranslated Regions; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D044602:Cellulases