Genetic modification of plant cell walls has been implemented to reduce lignocellulosic recalcitrance for biofuel production. Plant glycoside hydrolase family 9 (GH9) comprises endo-β-1,4-glucanase in plants. Few studies have examined the roles of GH9 in cell wall modification. In this study, we independently overexpressed two genes from GH9B subclasses (OsGH9B1 and OsGH9B3) and examined cell wall features and biomass saccharification in transgenic rice plants. Compared with the wild type (WT, Nipponbare), the OsGH9B1 and OsGH9B3 transgenic rice plants, respectively, contained much higher OsGH9B1 and OsGH9B3 protein levels and both proteins were observed in situ with nonspecific distribution in the plant cells. The transgenic lines exhibited significantly increased cellulase activity in vitro than the WT. The OsGH9B1 and OsGH9B3 transgenic plants showed a slight alteration in three wall polymer compositions (cellulose, hemicelluloses, and lignin), in their stem mechanical strength and biomass yield, but were significantly decreased in the cellulose degree of polymerization (DP) and lignocellulose crystalline index (CrI) by 21–22%. Notably, the crude cellulose substrates of the transgenic lines were more efficiently digested by cellobiohydrolase (CBHI) than those of the WT, indicating the significantly increased amounts of reducing ends of β-1,4-glucans in cellulose microfibrils. Finally, the engineered lines generated high sugar yields after mild alkali pretreatments and subsequent enzymatic hydrolysis, resulting in the high bioethanol yields obtained at 22.5% of dry matter. Overproduction of OsGH9B1/B3 enzymes should have specific activity in the postmodification of cellulose microfibrils. The increased reducing ends of β-1,4-glucan chains for reduced cellulose DP and CrI positively affected biomass enzymatic saccharification. Our results demonstrate a potential strategy for genetic modification of cellulose microfibrils in bioenergy crops.

Rice is a major food crop containing large amounts of lignocellulose residues usable for biofuels. In this study, we collected transgenic rice plants that over-produced Trichoderma reesei β-1,4-D-glucosidase (BGL I) into the cell walls in the mature straws. Without any pretreatment, the transgenic rice straws showed a consistently higher biomass enzymatic saccharification than the wild-type (WT) cultivar, in particular when 1% Tween-80 or 0.5% PEG-4000 was co-supplied into the enzymatic hydrolysis. Notably, under mild alkali pretreatment (1% NaOH at 50 °C for 2 h), the desirable transgenic line exhibited complete biomass enzymatic hydrolysis, resulting in the highest bioethanol yield of 21% (% dry matter) when compared with the rice and other bioenergy crops subjected to stronger pretreatment conditions reported in previous studies. Meanwhile, despite relatively low hexose yields obtained under 1% H₂SO₄ pretreatment, the transgenic rice straw also showed high bioethanol production at 18% due to an almost complete sugar–ethanol conversion rate. Chemical analyses indicated that the transgenic rice straw had significantly increased biomass porosity and reduced cellulose features (CrI, DP), which contributed to the largely enhanced biomass enzymatic hydrolysis. In addition, the raised arabinose level in hemicellulose and the lignin H-monomer proportion may also positively affect the biomass enzymatic saccharification in the transgenic rice straw. Hence, this study demonstrated a cost-effective and green lignocellulose conversion technology for high bioethanol production in the transgenic rice straw. It also provided a strategy for the potential genetic modification of plant cell walls in bioenergy crops.

Although cellulose represents the most abundant biomass resource on Earth, its natural recalcitrance hinders its application for low-cost biofuels and high-value bioproducts on a large scale. In this study, we generated less recalcitrant cellulose nanofibril substrates by site-specific mutations and knockouts of three OsCESA4,7,9 isoforms that are essential as cellulose synthase complexes for the cellulose biosynthesis of secondary cell walls in rice. As a comparison with the wild type form, three genetically-engineered CESAs isoforms independently generated cellulose nanofibrils with the average lengths reduced by 63%, which accounts for the high density of the amorphous cellulose chains as the initial breakpoints for the consistently enhanced biomass enzymatic hydrolysis into fermentable sugars. The genetically modified cellulose nanofibrils efficiently produce ideal nanocrystals with the most reduced dimension to-date in three knockout lines, which are applicable as optimal intermediates for highly valuable bioproducts. The improved cellulose nanofibrils are also effective in inducing the secretion of lignocellulose-degradation enzymes from fungi (T. reesei) with the two cellulase (exoglucanases, β-glucosidases) activities elevated by 100% and 138%, and the total protein level increased by 44%. Our data thus reveal a novel green strategy for achieving high-quality diverse bioproduction by integrating the precise genetic modification of lignocellulose substrates with efficient biomass process technology.

Wheat is a major food crop providing substantial biomass straw applicable for heavy metal phytoremediation and cellulosic ethanol production. In this study, we observed that wheat mutant straw could accumulate a maximum cadmium (Cd) concentration of 8.29 µg g⁻¹ dry straw, whereas wheat wild type (WT) contained Cd at a concentration of 6.43 µg g⁻¹, while grown in soil pots supplied with 75 mg CdCl₂ per kg soil. Due to the Cd accumulation, the wheat mutant and WT straws distinctively showed a reduced cellulose level and degree of polymerization (DP) with increased soluble sugar stock compared to the control (without Cd), which led to much improved cellulose surface accessibility for relatively higher biomass enzymatic saccharification and bioethanol production under various chemical pretreatments. Notably, compared to the one-step pretreatment of a strong alkali or acid, the two-step pretreatment of a mild alkali and acid could cause an effective wall polymer extraction for a complete Cd release and collection from the mature straw typical in the wheat mutant. Therefore, this study has demonstrated a green-like technology for Cd collection and cellulosic ethanol co-production by employing a mild chemical pretreatment. It has also suggested a potential biotechnology for Cd phytoremediation by selecting cell wall-altered mutants and transgenic crops.