Optimization of Acidothermus cellulolyticus endoglucanase (E1) production in transgenic tobacco plants by transcriptional, post-transcription and post-translational modification Ziyu Dai 1, *, Brian S. Hooker 1 , Ryan D. Quesenberry 1 & Steven R. Thomas 2 1 Chemical and Biological Processing Development Group, Process Science and Engineering Division, Pacific Northwest National Laboratory, Richland, WA, 99352, USA 2 National Renewable Energy Laboratory, 1617 Cole Boulevard, Golden, CO, 80401, USA Received 15 December 2004; accepted 12 April 2005 Key words: Acidothermus cellulolyticus, cellulases, E1 endoglucanase, post-transcriptional modification, sub-cellular compartmentation, transgenic tobacco 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 compartmen- tation for heterologous protein production in transgenic plants are discussed. Introduction The ability to use plant biomass as feedstock for conversion to other useful chemicals requires a complete hydrolysis of plant cell wall polysaccha- rides to fermentable sugars. Cellulose is an unbranched homopolymer of b-1, 4-linked glucose and it is the major polysaccharide component in plant biomass. Complete hydrolysis of cellulose requires at least three different hydrolytic enzymes: b-1, 4-endoglucanase (or endo-b-1, 4-glucanohy- drolase; E.C. 3.2.1.4), b-1, 4-exoglucanase (or *Author for correspondence E-mail: ziyu.dai@pnl.gov Transgenic Research (2005) 14:627–643 Ó Springer 2005 DOI 10.1007/s11248-005-5695-5