Recombinant Cellulase Accumulation in the Leaves of Mature, Vegetatively Propagated Transgenic Sugarcane

Abstract

The cost of enzymes that hydrolyse lignocellulosic substrates to fermentable sugars needs to be reduced to make cellulosic ethanol a cost-competitive liquid transport fuel. Sugarcane is a perennial crop and the successful integration of cellulase transgenes into the sugarcane production system requires that transgene expression is stable in the ratoon. Herein, we compared the accumulation of recombinant fungal cellobiohydrolase I (CBH I), fungal cellobiohydrolase II (CBH II), and bacterial endoglucanase (EG) in the leaves of mature, initial transgenic sugarcane plants and their mature ratoon. Mature ratoon events containing equivalent or elevated levels of active CBH I, CBH II, and EG in the leaves were identified. Further, we have demonstrated that recombinant fungal CBH I and CBH II can resist proteolysis during sugarcane leaf senescence, while bacterial EG cannot. These results demonstrate the stability of cellulase enzyme transgene expression in transgenic sugarcane and the utility of sugarcane as a biofactory crop for production of cellulases.

Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

“…The high quantity of extractable juice with very low protein content and a wellestablished juice processing technology have encouraged research to express proteins in the sugarcane culm. Many attempts have been made to express and purify recombinant proteins from the sugarcane stem and leaves (Harrison et al, 2014;Jackson et al, 2007Jackson et al, , 2010Petrasovits et al, 2012). Initial studies that aimed at expressing the protein in cytoplasm and purifying it from leaves have resulted in poor yield as leaves contain many proteins other than the introduced ones, which may interfere in downstream processing (Fischer et al, 2004).…”

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

“…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.…”

“…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.…”

Green Production and Biotechnological Applications of Cell Wall Lytic Enzymes

“…Certain CWLEs such as cellobiohydrolase and endoglucanase accumulate at high yields in LVs [110]. However, vacuole sorting of CWLEs was strictly dependent on the development of LVs in different plant tissues, and protein accumulation was strongly reduced in both young and senescent leaves [111]. Interestingly, compartmentalized expression of CWLEs in crop plants concerns a very limited number of cellulases [102,104,105,[112][113][114].…”