Upgraded cellulose and xylan digestions for synergistic enhancements of biomass enzymatic saccharification and bioethanol conversion using engineered Trichoderma reesei strains overproducing mushroom LeGH7 enzyme.

Crop straws provide enormous lignocellulose resources transformable for sustainable biofuels and valuable bioproducts. However, lignocellulose recalcitrance basically restricts essential biomass enzymatic saccharification at large scale. In this study, the mushroom-derived cellobiohydrolase (LeGH7) was introduced into Trichoderma reesei (Rut-C30) to generate two desirable strains, namely GH7-5 and GH7-6. Compared to the Rut-C30 strain, both engineered strains exhibited significantly enhanced enzymatic activities, with ß-glucosidases, endocellulases, cellobiohydrolases, and xylanase activities increasing by 113 %, 140 %, 241 %, and 196 %, respectively. By performing steam explosion and mild alkali pretreatments with mature straws of five bioenergy crops, diverse lignocellulose substrates were effectively digested by the crude enzymes secreted from the engineered strains, leading to the high-yield hexoses released for bioethanol production. Notably, the LeGH7 enzyme purified from engineered strain enabled to act as multiple cellulases and xylanase at higher activities, interpreting how synergistic enhancement of enzymatic saccharification was achieved for distinct lignocellulose substrates in major bioenergy crops. Therefore, this study has identified a novel enzyme that is active for simultaneous hydrolyses of cellulose and xylan, providing an applicable strategy for high biomass enzymatic saccharification and bioethanol conversion in bioenergy crops.

[Hemicellulose modification and cell wall genetic improvement in plants].

Hemicellulose, as a primary component of plant cell walls, constitutes approximately one third of cell wall dry matter and ranks as the second abundant renewable biomass resource in the nature after cellulose. Hemicellulose is tightly cross-linked with cellulose, lignin and other components in the plant cell wall, leading to lignocellulose recalcitrance. However, precise genetic modifications of plant cell walls can significantly improve the saccharification efficiency of lignocellulose while ensuring normal plant growth and development. We comprehensively review the research progress in the structural distribution of hemicellulose in plant cell walls, the cross-linking between hemicellulose and other components of the cell wall, and the impact of hemicellulose modification on the saccharification efficiency of the cell wall, proving a reference for the genetic improvement of energy crops.

Cellulose de-polymerization is selective for bioethanol refinery and multi-functional biochar assembly using brittle stalk of corn mutant.

As lignocellulose recalcitrance principally restricts for a cost-effective conversion into biofuels and bioproducts, this study re-selected the brittle stalk of corn mutant by MuDR-transposon insertion, and detected much reduced cellulose polymerization and crystallinity. Using recyclable CaO chemical for biomass pretreatment, we determined a consistently enhanced enzymatic saccharification of pretreated corn brittle stalk for higher-yield bioethanol conversion. Furthermore, the enzyme-undigestible lignocellulose was treated with two-step thermal-chemical processes via FeCl2 catalysis and KOH activation to generate the biochar with significantly raised adsorption capacities with two industry dyes (methylene blue and Congo red). However, the desirable biochar was attained from one-step KOH treatment with the entire brittle stalk, which was characterized as the highly-porous nanocarbon that is of the largest specific surface area at 1697.34 m2/g and 2-fold higher dyes adsorption. Notably, this nanocarbon enabled to eliminate the most toxic compounds released from CaO pretreatment and enzymatic hydrolysis, and also showed much improved electrochemical performance with specific capacitance at 205 F/g. Hence, this work has raised a mechanism model to interpret how the recalcitrance-reduced lignocellulose is convertible for high-yield bioethanol and multiple-function biochar with high performance.

Upgrading pectin methylation for consistently enhanced biomass enzymatic saccharification and cadmium phytoremediation in rice Ospmes site-mutants.

Crop straws provide enormous biomass residues applicable for biofuel production and trace metal phytoremediation. However, as lignocellulose recalcitrance determines a costly process with potential secondary waste liberation, genetic modification of plant cell walls is deemed as a promising solution. Although pectin methylation plays an important role for plant cell wall construction and integrity, little is known about its regulation roles on lignocellulose hydrolysis and trace metal elimination. In this study, we initially performed a typical CRISPR/Cas9 gene-editing for site mutations of OsPME31, OsPME34 and OsPME79 in rice, and then determined significantly upgraded pectin methylation degrees in the young seedlings of three distinct site-mutants compared to their wild type. We then examined distinctively improved lignocellulose recalcitrance in three mutants including reduced cellulose levels, crystallinity and polymerization or raised hemicellulose deposition and cellulose accessibility, which led to specifically enlarged biomass porosity either for consistently enhanced biomass enzymatic saccharification under mild alkali pretreatments or for cadmium (Cd) accumulation up to 2.4-fold. Therefore, this study proposed a novel model to elucidate how pectin methylation could play a unique enhancement role for both lignocellulose enzymatic hydrolysis and Cd phytoremediation, providing insights into precise pectin modification for effective biomass utilization and efficient trace metal exclusion.

Malic acid inhibits accumulation of cadmium, lead, nickel and chromium by down-regulation of OsCESA and up-regulation of OsGLR3 in rice plant.

Malic acid (MA) plays an important role in plant tolerance to toxic metals, but its effect in restricting the transport of harmful metals remains unclear. In this study, japonica rice NPB and its fragile-culm mutant fc8 with low cellulose and thin cell wall were used to investigate the influence of MA on the accumulation of 4 toxic elements (Cd, Pb, Ni, and Cr) and 8 essential elements (K, Mg, Ca, Fe, Mn, Zn, Cu and Mo) in rice. The results showed that fc8 accumulated less toxic elements but more Ca and glutamate in grains and vegetative organs than NPB. After foliar application with MA at rice anthesis stage, the content of Cd, Pb, Ni significantly decreased by 27.9-41.0%, while those of Ca and glutamate significantly increased in both NPB and fc8. Therefore, the ratios between Cd and Ca in grains of NPB (3.4‰) and fc8 (1.5‰) were greatly higher than that in grains of NPB + MA (1.1‰) and fc8+MA (0.8‰) treatments. Meanwhile, the expression of OsCEAS4,7,8,9 for the cellulose synthesis in secondary cell walls were down-regulated and cellulose content in vegetative organs of NPB and fc8 decreased by 16.7-21.1%. However, MA application significantly up-regulated the expression of GLR genes (OsGLR3.1-3.5) and raised the activity of glutamic-oxalacetic transaminease for glutamate synthesis in NPB and fc8. These results indicate that hazard risks of toxic elements in foods can be efficiently reduced through regulating cellulose biosynthesis and GLR channels in plant by combining genetic modification in vivo and malic acid application in vitro.

Cadmium pollution leads to selectivity loss of glutamate receptor channels for permeation of Ca2+/Mn2+/Fe2+/Zn2+ over Cd2+ in rice plant.

The selective permeation of glutamate receptor channels (GLRs) for essential and toxic elements in plant cells is poorly understood. The present study found that the ratios between cadmium (Cd) and 7 essential elements (i.e., K, Mg, Ca, Mn, Fe, Zn and Cu) in grains and vegetative organs increased significantly with the increase of soil Cd levels. Accumulation of Cd resulted in the significant increase of Ca, Mn, Fe and Zn content and the expression levels of Ca channel genes (OsCNGC1,2 and OsOSCA1.1,2.4), while remarkable reduction of glutamate content and expression levels of GLR3.1-3.4 in rice. When planted in the same Cd-polluted soil, mutant fc8 displayed significantly higher content of Ca, Fe, Zn and expression levels of GLR3.1-3.4 than its wild type NPB. On the contrary, the ratios between Cd and essential elements in fc8 were significantly lower than that in NPB. These results indicate that Cd pollution may damage the structural integrity of GLRs by inhibiting glutamate synthesis and expression levels of GLR3.1-3.4, which leads to the increase of ion influx but the decrease of preferential selectivity for Ca2+/ Mn2+/ Fe2+/ Zn2+ over Cd2+ through GLRs in rice cells.

Full-Chain FeCl3 Catalyzation Is Sufficient to Boost Cellulase Secretion and Cellulosic Ethanol along with Valorized Supercapacitor and Biosorbent Using Desirable Corn Stalk.

Cellulosic ethanol is regarded as a perfect additive for petrol fuels for global carbon neutralization. As bioethanol conversion requires strong biomass pretreatment and overpriced enzymatic hydrolysis, it is increasingly considered in the exploration of biomass processes with fewer chemicals for cost-effective biofuels and value-added bioproducts. In this study, we performed optimal liquid-hot-water pretreatment (190 °C for 10 min) co-supplied with 4% FeCl3 to achieve the near-complete biomass enzymatic saccharification of desirable corn stalk for high bioethanol production, and all the enzyme-undigestible lignocellulose residues were then examined as active biosorbents for high Cd adsorption. Furthermore, by incubating Trichoderma reesei with the desired corn stalk co-supplied with 0.05% FeCl3 for the secretion of lignocellulose-degradation enzymes in vivo, we examined five secreted enzyme activities elevated by 1.3-3.0-fold in vitro, compared to the control without FeCl3 supplementation. After further supplying 1:2 (w/w) FeCl3 into the T. reesei-undigested lignocellulose residue for the thermal-carbonization process, we generated highly porous carbon with specific electroconductivity raised by 3-12-fold for the supercapacitor. Therefore, this work demonstrates that FeCl3 can act as a universal catalyst for the full-chain enhancement of biological, biochemical, and chemical conversions of lignocellulose substrates, providing a green-like strategy for low-cost biofuels and high-value bioproducts.

BsEXLX of engineered Trichoderma reesei strain as dual-active expansin to boost cellulases secretion for synergistic enhancement of biomass enzymatic saccharification in corn and Miscanthus straws.

In this study, bacterial BsEXLE1 gene was overexpressed into T. reesei (Rut-C30) to generate a desirable engineered TrEXLX10 strain. While incubated with alkali-pretreated Miscanthus straw as carbon source, the TrEXLX10 secreted the ß-glucosidases, cellobiohydrolases and xylanses with activities raised by 34%, 82% and 159% compared to the Rut-C30. Supplying EXLX10-secreted crude enzymes and commercial mixed-cellulases for two-step lignocellulose hydrolyses of corn and Miscanthus straws after mild alkali pretreatments, this work measured consistently higher hexoses yields released by the EXLX10-secreted enzymes for synergistic enhancements of biomass saccharification in all parallel experiments examined. Meanwhile, this study detected that the expansin, purified from EXLX10-secreted solution, was of exceptionally high binding activities with wall polymers, and further determined its independent enhancement for cellulose hydrolysis. Therefore, this study raised a mechanism model to highlight EXLX/expansin dual-activation roles for both secretion of stable biomass-degradation enzymes at high activity and biomass enzymatic saccharification in bioenergy crops.

Single-molecular insights into the breakpoint of cellulose nanofibers assembly during saccharification.

Plant cellulose microfibrils are increasingly employed to produce functional nanofibers and nanocrystals for biomaterials, but their catalytic formation and conversion mechanisms remain elusive. Here, we characterize length-reduced cellulose nanofibers assembly in situ accounting for the high density of amorphous cellulose regions in the natural rice fragile culm 16 (Osfc16) mutant defective in cellulose biosynthesis using both classic and advanced atomic force microscopy (AFM) techniques equipped with a single-molecular recognition system. By employing individual types of cellulases, we observe efficient enzymatic catalysis modes in the mutant, due to amorphous and inner-broken cellulose chains elevated as breakpoints for initiating and completing cellulose hydrolyses into higher-yield fermentable sugars. Furthermore, effective chemical catalysis mode is examined in vitro for cellulose nanofibers conversion into nanocrystals with reduced dimensions. Our study addresses how plant cellulose substrates are digestible and convertible, revealing a strategy for precise engineering of cellulose substrates toward cost-effective biofuels and high-quality bioproducts.