Expression and import of an active cellulase from a thermophilic bacterium into the chloroplast both in vitro and in vivo.
A bacterial thermostable cellulase, the endo-1,4-beta-D-glucanase E1 from Acidothermus cellulolyticus, was imported into chloroplasts, and an active enzyme was recovered both in vitro and in vivo. Precursor fusion proteins were synthesized with E1 or its catalytic domain, CD, fused to the transit peptide of ferredoxin or ribulose-bisphosphate carboxylase activase for stromal targeting. A spacer region of 1, 5 or 15 amino acids was included carboxy to the transit peptide. The efficiency of import and processing by the stromal processing peptidase depended on the nature of the transit peptide and the passenger protein, and increased with the length of the spacer between them. Besides finding E1 or CD in the stroma, protein was arrested in the envelope during import showing that structural features of E1 and CD, along with their proximity to the transit peptide, influence translocation. The cellulose binding domain and/or serine/proline/threoline-rich linker of E1 may impede efficient import. Significantly, most precursors for E1 and CD synthesized by in vitro translation possessed endoglucanse activity that was temperature-dependent, and required the residues AGGGY at the N-terminus of E1 and CD. Furthermore, activity was detected upon import into chloroplasts. Based on the in vitro analyses, five precursor fusion proteins were selected to determine if E1 and CD would be successfully targeted to chloroplasts in vivo. In transgenic tobacco plants, E1 and CD accumulated in both the stromal and membrane fractions and, importantly, chloroplast extracts showed endoglucanase activity.
Jin
Rongguan
R
Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
Richter
Stefan
S
Zhong
Rong
R
Lamppa
Gayle K
GK
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001692:Biological Transport; D020134:Catalytic Domain / Q000235:genetics; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000235:genetics
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D002480:Cellulase; D010447:Peptide Hydrolases