Arabidopsis thaliana Rubisco small subunit transit peptide increases the accumulation of Thermotoga maritima endoglucanase Cel5A in chloroplasts of transgenic tobacco plants

Abstract

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.

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

“…Tissue-specific levels of Acidothermus cellulolyticus endoglucanase up to 16 % TSP have been achieved in maize using these promoters [29]. The green tissue specific promoter from the rubisco small subunit (rbcS) has also been successfully used for expression of cellulases [30,58] with TSP levels of 5.2 % in tobacco. Tissue-specific heterologous enzyme expression has certain conveniences for plant processing, including stability and convenience of harvest.…”

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

“…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].…”

“…In general, the most successful attempts to express hyperthermophilic enzymes in plant tissues (Table S1 in the supplementary material online) have come from targeted expression in the chloroplast [47,48]. For example, a hyperthermophilic endoglucanase (Cel5A) gene was targeted to Arabidopsis chloroplasts using the N-terminal transit peptides of nuclear-encoded plastid proteins, the lightharvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA).…”

“…For example, a hyperthermophilic endoglucanase (Cel5A) gene was targeted to Arabidopsis chloroplasts using the N-terminal transit peptides of nuclear-encoded plastid proteins, the lightharvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). An expression cassette containing the RS signal peptide driven by the RbcS promoter yielded the highest level of accumulation (5.2% of TSP) of Cel5A in tobacco chloroplasts, and the recombinant enzyme remained stable throughout the life cycle of the transgenic plants [48]. In a direct comparison of cytosolic and chloroplast expression of a hyperthermophilic cellulase, Bg1B, the highest enzyme activity and accumulation (5.8% of TSP) was achieved when Bg1B was directed to the chloroplast using the RA signal peptide and the RbcS promoter [35].…”

Overexpression of an endo-1,4-β-glucanase V gene (EGV) from Trichoderma reesei leads to the accumulation of cellulase activity in transgenic rice

“…The expression of cellulase in specific regions of plant cells can significantly improve the cellulase content, leading to the production of correctly folded, glycosylated cellulase with higher activity levels than cellulase expressed in the cytoplasm. This reduces hydrolysis and avoids the adverse effects of exogenous cellulase expression on the cellulose of plant cell walls (Ziegler et al, 2000;Ziegelhoffer et al, 2001;Hood et al, 2007;Kim et al, 2010).…”