High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass

Biofuels production from plant biomasses is a complex multi‐step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from hyperthermophilic Bacteria and Archaea and test their application in the bioconversion of an important industrially pretreated biomass feedstock for production of second‐generation biofuels. The selected enzymes, endoglucanase, endoxylanase and β‐glucosidase, were expressed in tobacco plastome with a protein yield upto 75 % of total soluble proteins. The accumulation of endoglucanase gave altered plant phenotypes whose severity was directly linked to the enzyme yield and that was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endoxylanase and β‐glucosidase, produced at very high level without detrimental effects on plant development, were enriched by heat treatment. Both biocatalysts retained the main features of the native or recombinantly expressed enzymes, but resulted more thermophilic than the E. coli recombinant counterparst. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid‐derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid‐derived xylanase, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β‐glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher. The very high production level of hyperthermophilic enzymes, their stability and bioconversion effciencies described in this study demonstrate that plastid transformation represents a real cost‐effective production platform for cellulolytic enzymes.