Subcellular targeting is a key condition for high‐level accumulation of cellulase protein in transgenic maize seed

Abstract

709 SummaryEthanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize ( Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-β -1,4-glucanase from Acidothermus cellulolyticus , was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei , also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall.With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T 2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…Comparison of a traditional pre-fermentation pipeline (purple boxes) and an approach based on in planta expression of hyperthermophilic lignocellulose digesting enzymes combining pretreatment and saccharification in a single reactor (green boxes). [15,38]. In planta expression of recombinant lignocellulosedigesting genes has involved a range of different promoters and other regulatory elements and targeting of the enzymes to the specific organelles via different transit peptides for accumulation and storage or targeting to the cell wall for immediate modification of the polysaccharides (Table S1 in the supplementary material online).…”

“…Although in planta expression of thermophilic endoglucanases has been achieved in the range of 10-26% TSP without any detrimental effects on transgenic plants [37][38][39][40][41][42][43][44][45][46][47][48], yields of 0.1-5% TSP are more typical, and several studies have described negative phenotypes associated with in planta expression of recombinant mesophilic cellulolytic enzymes [32,49].…”

“…N-terminal or C-terminal transit peptides generally possess the necessary information for the correct targeting of proteins to subcellular organelles. Subcellular targeting of heterologous proteins generally yields higher accumulation levels in transgenic plants than expression in the cytosol [32][33][34][35][36][37][38][39][40][41][42]47,51]. When expressed in subcellular compartments, heterologous proteins typically display correct folding and activity, appropriate glycosylation, reduced degradation, and increased stability, as compared to expression and accumulation in the cytosol [8,44].…”

“…Development of hyperthermophilic fermentation microbes would confer an additional advantage when using the in planta approach. Plants are increasingly being investigated as potential 'green bioreactors' for the cost-effective and large-scale production of biologically active recombinant enzymes [31][32][33][34][35][36], but the production of cellulolytic enzymes using transgenic plants has mostly been restricted to mesophilic and thermotolerant enzymes [37][38][39][40][41][42][43][44][45]. To the best of our knowledge, the effects of such expression on the efficiency of biomass conversion have been reported in few instances [15].…”

Expression of Glycosyl Hydrolases in Lignocellulosic Feedstock: An Alternative for Affordable Cellulosic Ethanol Production

“…For example, overexpression of Trichoderma reesei cellobiohydrolase Cel7A in Aspergillus niger resulted in an over-glycosylated version of the protein with a resultant low enzymatic activity toward known substrates [28]. However, Cel7A was successfully expressed in maize and shown to retain its activity [29].…”