High-level expression of a suite of thermostable cell wall-degrading enzymes from the chloroplast genome

The biological conversion of plant biomass into fermentable sugars is key to the efficient production of biofuels and other renewable chemicals from plants. As up to more than 90% of the dry weight of higher plants is fixed in the cell wall, this will require the low-cost production of large amounts of cell wall-degrading enzymes. Transgenic plants can potentially provide an unbeatably cheap production platform for industrial enzymes. Transgene expression from the plastid genome is particularly attractive, due to high-level foreign protein accumulation in chloroplasts, absence of epigenetic gene silencing and improved transgene containment. Here, we have explored the potential of transplastomic plants to produce large amounts of thermostable cell wall-degrading enzymes from the bacterium Thermobifida fusca. We show that a set of four enzymes that are required for efficient degradation of cellulose (and the hemicellulose xyloglucan) could be expressed successfully in transplastomic tobacco plants. However, overexpression of the enzymes (to between approximately 5 and 40% of the plant's total soluble protein) resulted in pigment-deficient mutant phenotypes. We demonstrate that the chloroplast-produced cellulolytic enzymes are highly active. Although further optimization is needed, our data indicate that transgenic plastids offer great potential for the production of enzyme cocktails for the bioconversion of cellulosic biomass.