Expression of thermostable microbial cellulases in the chloroplasts of nicotine-free tobacco

An inexpensive source of active cellulases is critical to efficient and cost-effective conversion of lignocellulosic biomass to ethanol. Transgenic plants expressing foreign cellulases are potential sources of cellulases for biomass conversion. A number of foreign proteins have been reported to accumulate to high levels when the transgene is incorporated into the chloroplast genome rather than into the nuclear genome. We developed plastid transformation vectors carrying two Thermobifida fusca thermostable cellulases, Cel6A and Cel6B, and expressed them in nicotine-free or nicotine-containing tobacco varieties following chloroplast transformation. We obtained homoplasmic tobacco plants expressing Cel6A or Cel6B. Maximum estimates of expression levels ranged from 2 to 4% of total soluble protein. Enzyme assays indicated that both Cel6A and Cel6B expressed in transplastomic tobacco were active in hydrolyzing crystalline cellulose. With further optimization, it may be feasible to produce bacterial cellulases in tobacco chloroplasts in large quantities.