Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

Abstract

SummaryA major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.

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

“…Promoters and enhancers (cis-acting regulatory elements) play a crucial role in regulating spatial and temporal patterns of transgene expression at particular stages or in response to particular conditions [50][51][52][53][54][55]. Several promoters and other cis-acting elements including signal peptides of plant, plant pathogen, or bacterial origin used in heterologous expression vectors have been described (Table S1 in the supplementary material online).…”

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

“…Expression levels of up to 26 % TSP in Arabidopsis have been achieved with this promoter. The synthetic Mac promoter is another commonly used constitutive promoter for hydrolytic protein production in various plant species including tobacco, potato, rice, sugarcane, and alfalfa [54][55][56]. Expression levels comparable to that of 35S have been achieved.…”

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

“…Other important conclusion is that the accumulation levels of vacuolar sorted foreign proteins were dependent of the developmental stage and physiological condition of leaves, therefore to achieve high yields samples should be collected prior senescence. 20,21 In conclusion, vacuolar sorting in vegetative plant tissues is a satisfactory strategy to enhance protein yields and the obtained results are superior than targeting to cytosol or to apoplast an could be also better than ER retention for cytotoxic proteins. For recombinant glycosylated proteins will be desirable to have a better understanding of the mechanism that control vacuolar delivery by the different targeting routes in order to predict glycosylation pattern.…”

“…Fungal cellobiohydrolase (CBH) I and II and bacterial endoglucanase (EG) accumulated to higher levels when fused to the barley polyamine oxidase Ct VSS compared to the fusion to an ERretention signal. 20 Yields of CBH I, CBH II and EG were reduced in senescent leaves probably due to endo-and exo-peptidases released during leaf senescence. 20,21 These results emphasize the importance of the development stage for a foreign protein deposition in leaves.…”

“…20 Yields of CBH I, CBH II and EG were reduced in senescent leaves probably due to endo-and exo-peptidases released during leaf senescence. 20,21 These results emphasize the importance of the development stage for a foreign protein deposition in leaves. Another toxic protein that was successfully expressed in Nicotiana benthamiana leaves is the bacteriophage CP933 endolysin (EL), an enzyme that hydrolyzes peptidoglycan.…”