Enhanced saccharification of rice straw by overexpression of rice exo-glucanase

Abstract

Background: Efficient production of carbon-neutral biofuels is key to resolving global warming and exhaustion of fossil fuels. Cellulose, which is the most abundant biomass, is physically strong and biochemically stable, and these characteristics lead to difficulty of efficient saccharification of cellulosic compounds for production of fermentable glucose and other sugars.

Results: We transformed rice with overexpressing constructs of rice genes encoding each of three classes of cellulases. The exo-glucanase overexpressing plants showed various abnormalities in leaf such as division of leaf blade, crack on leaf surface, excess lacunae in midrib structure and necrotic colour change. The overexpressing plants also showed sterility. Noticeably, these plants showed enhanced saccharification of stems after maturation. These results indicate that overexpression of the exo-glucanase gene brought about various developmental defects associated with modification of cell wall and enhanced saccharification in rice. On the other hand, endo-glucanase-overexpressing plants could not be obtained, and overexpression of β-glucosidase brought about no effect on plant growth and development.

Conclusions: Our results indicate that genetic engineering of cellulosic biomass plants by overexpressing cellulase genes will be one of the approaches to confer enhanced saccharification ability for efficient production of cellulosic biofuels such as ethanol.

Figure 1

Structure of the vectors used for rice transformation. Triangles: a promoter for a nopaline synthase gene (NOS), maize ubiqutin gene (Ubi), cauliflower mosaic virus 35S RNA (35S), rice acetolactate synthase gene (Als) or rice actin gene (Act); boxes: a coding sequence of exo-glucanase (EXG1), endo-glucanase (ENG1), β-glucosidase (BEG1), neomycin phosphotransferase II (NPTII), hygromycin phosphotransferase (HPT) or rice mutated bispyribac sodium-resistant acetolactate synthase (G95A); circles: a terminator for a nopaline synthase gene (T). RB and LB indicate right border and left border of T-DNA, respectively.

Figure 2

Generation of the EXG1 overexpressing plants. a and b Expression of EXG1 in the Pubi-EXG1 transgenic plants. RNAs isolated from leaves of the self-progenies of the Pubi-EXG1 primary transformants (Nipponbare in a and Taichung 65 in b) and the vector-transformed control plant were reverse-transcribed with the oligo(dT) primer and amplified by EXG1 or actin specific primers. RT– indicates that reverse-transcriptase was omitted from the reaction mixture. c Cellulase activities of the Pubi-EXG plants. Protein extracts prepared from transgenic leaf blade of young seedlings were incubated with a fluorescent substrate 4-methylumbelliferyl β-D-cellobioside. v: a vector-transformed control plant.

Figure 3

Phenotypes of the EXG1 -overexpressing plants. a Wild type leaf blade. b, c Division of leaf blade observed in the Pubi-EXG1 #4 (b) and #13 (c) plants (Nipponbare). Arrows indicate divided small leaf blade. d Necrotic colour change in leaf blade of the Pubi-EXG1 #10 plant (Nipponbare). e, f A transverse section of the leaf blade with two lacunae in the midvein of the control plant (e) or the leaf blade with four lacunae in the midvein of the Pubi-EXG1 #18 plant (Taichung 65) (f). Asterisks indicate lacunae. g, h Simplified SEM observation of the surface of the leaf blade of the control plant (g) or the Pubi-EXG1 #18 plant (h). i, j Panicle of the control plant (i) and the Pubi-EXG1 #18 plant (j). k, l TEM observation of chloroplast of the leaf blade of the control plant (k) or the Pubi-EXG1 #18 plant (l). Arrows indicate plastogranules. m, n Cell wall of the control plant (m) and the Pubi-EXG1 #18 plant (n). Bars = 1 cm in (a) to (d), (i) and (j), 100 μm in (e) to (h), and 1 μm in (k) to (n).

Figure 4

Saccharification of the EXG1 -overexpressing plants. a Glucose. b Reduced sugars. + and – indicate whether cellulases were added to or omitted from the reaction mixture. #18: the primary transformant of Pubi-EXG1 (Taichung 65), #21-3 and #27-4: the self-progenies of the primary transformants of Pubi-EXG1 (Taichung 65), v: the vector transformed control plant.