Overproduction of native endo-β-1,4-glucanases leads to largely enhanced biomass saccharification and bioethanol production by specific modification of cellulose features in transgenic rice

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Bioethanol production; Biomass saccharification; Cellulose modification; Chemical pretreatment; Endo-β-1,4-glucanases; GH9B; Transgenic rice.

Fig. 1

Phylogenetic analysis of GH9B family and coexpression patterns of OsGH9B1 and OsGH9B3. a Phylogenetic tress of OsGH9Bs; b OsGH9B1 and OsGH9B3 coexpression profiling in all tissues covering almost entire life cycle of rice and a positive correlation between OsGH9B1 and OsGH9B3; ** as significant correlation at p < 0.01 level (n = 33)

Fig. 2

Transformation of OsGH9B1 and OsGH9B3 produced overexpressed proteins in the transgenic lines. a Constructs used for overexpression of the OsGH9B1 and OsGH9B3 genes. b Gene expression in WT (Nipponbare) plants and four transgenic lines. c Detection of OsGH9B1 and OsGH9B3 proteins in total proteins samples extracted from stem tissues. d The distribution of proteins in soluble protein samples (Sup), plasma membrane protein (PMy, and total protein samples (Res). e Observation of OsGH9B1/B3-eGFP in protoplast, scale bar as 10 μm. f Cellulase activity assay in vitro using total proteins extracted from stem tissues; Student’s t test performed for WT plants and transgenic lines as **p < 0.01 and *p < 0.05

Fig. 3

Biomass enzymatic saccharification in the OsGH9B1 and OsGH9B3 transgenic lines. a–c Hexoses yields released from enzymatic hydrolysis (% of total dry mass) without pretreatment, 0.5% H₂SO₄ pretreatment, and 0.5% NaOH pretreatment, respectively. d–f Total sugars yields released from pretreatment (if any) and subsequently enzyme hydrolysis (% of crude cell walls) without pretreatment, 0.5% H₂SO₄ pretreatment, and 0.5% NaOH pretreatment, respectively. Student’s t test performed for WT and transgenic plants as **p < 0.01

Fig. 4

Bioethanol productivity by yeast fermentation using total sugars released from enzymatic hydrolysis after 0.5% NaOH pretreatment as substrates in the transgenic lines. a Bioethanol yields (g/g % dry matter). b Sugar–ethanol conversion rates. Student’s t-test performed for WT and transgenic plants as **p < 0.01

Fig. 5

Phenotypes and mechanical strengths of the OsGH9B1 and OsGH9B3 transgenic plants. a Plant growths of representative transgenic plants and WT at filling stage, scale bar as 20 cm. b Plant height (WT: n = 46, OsGH9B1: n = 43, OsGH9B3: n = 28). c Breaking force (WT: n = 45, OsGH9B1: n = 43, OsGH9B3: n = 27) and extension force (WT: n = 38, OsGH9B1: n = 41, OsGH9B3: n = 26) of the stem tissues. d Dry biomass. Student’s t-test performed for WT and transgenic plants as **p < 0.01 and *p < 0.05

Fig. 6

Cell wall morphologies and compositions in the OsGH9B1 and OsGH9B3 transgenic lines. a Sclerenchyma cells with Calcofluor White staining observed under a fluorescence microscopy, scale bar as 50 μm. b Cell walls morphology of sclerenchyma cells under transmission electron microscopy, scale bar as 2 μm (up) and 0.5 μm (down). c The contents of three major cell wall polymers (% total). Student’s t test performed for WT and transgenic plants as *p < 0.05

Fig. 7

Cellulose DP and CrI in the OsGH9B1 and OsGH9B3 transgenic lines. a Schematic flow of crude cellulose extraction. b Crude cellulose DP. c Crude cellulose CrI. d Correlation of cellulase activity and cellulose CrI and DP (n = 15). e Glucose yield of cellobiose CBHI hydrolyzes using crude cellulose as samples in three time points. f Correlation of cellulose features (DP, CrI) and the glucose levels after CBHI hydrolysis (n = 15). g Correlation between cellulase activity and the glucose levels by CBHI hydrolysis (n = 15). Different grayscale circle in d, f and g indicate the biological data from WT (White), OsGH9B1 (Light gray), and OsGH9B3 (Dark gray), respectively

Fig. 8

Correlation analysis between enzymatic saccharification and cellulose features. * and ** Indicate the significant differences at p < 0.05 and p < 0.01, respectively (n = 15)

Fig. 9

Hypothetical model for the involvement of OsGH9B1 and OsGH9B3 in postmodification of cellulose microfibrils. Expression of OsGH9B1 and OsGH9B3 proteins remarkably increased the reducing ends of cellulose microfibrils, causing the reduced DP and CrI of cellulose, leading to the largely enhanced biomass saccharification and bioethanol production in the transgenic plants