Dramatic effects of truncation and sub-cellular targeting on the accumulation of recombinant microbial cellulase in tobacco

The economical bioconversion of lignocellulosic biomass to ethanol is dependent on the availability of large quantities of inexpensive cellulase enzymes. One way to reduce the cost of such enzymes is to produce them in crop plants at high levels. In order to assess factors that limit recombinant cellulase expression in plants, we have introduced the gene encoding E1 endo-1,4-beta -glucanase (cellulase) of Acidothermus cellulolyticus into tobacco (Nicotiana tabacum) plants. Both the holoenzyme (E1) and catalytic domain (E1cd) were targeted to three sub-cellular compartments; the cytosol, chloroplast and apoplast. Accumulation of both E1 and E1cd was greatest in the apoplast, with levels more than 100-fold higher than observed for cytosolic accumulation. In all three compartments, E1cd accumulated to higher levels than the full-length enzyme. By combining truncation and apoplastic localization, an increase in expression of more than 500-fold was achieved, compared to cytosolic full-length E1. This effect is primarily post-transcriptional, since E1cd mRNA levels are very similar despite the range of E1cd accumulation observed. Recombinant E1cd, expressed at up to 1.6% total soluble protein, is extremely stable in both crude leaf extracts and dried leaf material.