Engineering the cell wall by reducing de-methyl-esterified homogalacturonan improves saccharification of plant tissues for bioconversion

Abstract

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.

Fig. 1.

Saccharification of leaf material from Arabidopsis PG or PMEI plants. Leaf material from untransformed Arabidopsis plants (WT) and from transgenic PG (A) and PMEI (B) plants were treated with Celluclast, and saccharification efficiency was measured after 24 hours. (C) Tissue maceration of representative samples of WT (Left) and transgenic (Right) PG57 and PMEI7 leaf material after 24 h of enzymatic saccharification. (D) Enzymatic saccharification efficiency of leaf tissue from WT, PG, and PMEI plants after dilute acid pretreatment. Numbers indicate different independent transgenic lines. Bars represent average saccharification efficiency ± SEM (N≥6). Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P < 0.05). These experiments were repeated at least twice with similar results.

Fig. 2.

Immunodot analysis of pectin in PG and PMEI plants. ChASS fractions were extracted from leaves of untransformed and transgenic Arabidopsis PG (A) and PMEI plants (B) and of untransformed and transgenic wheat AcPMEI plants (C). Numbers indicate different independent transgenic lines. The indicated amounts (in μg) of ChASS fraction were applied at a single point to a nitrocellulose membrane. Specific HGA epitopes were detected by using PAM1 and JIM5 monoclonal antibodies.

Fig. 3.

Saccharification of leaf material from tobacco PG and wheat PMEI plants. (A) Leaf material from tobacco untransformed (WT) and PG-expressing plants was treated with Celluclast, and saccharification efficiency was measured after 24 h. (B and C) Leaf material from untransformed (WT) and transgenic wheat plants overexpressing AcPMEI was treated with a mixture of Macerozyme R-10 and Celluclast 1.5 L without pretreatment (B) or after dilute acid pretreatment (C), and saccharification efficiency was measured after 24 h. Numbers indicate different independent transgenic lines. Bars represent average saccharification efficiency ± SEM (N≥6). These experiments were repeated at least twice with similar results. Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P < 0.01).

Fig. 4.

Saccharification of stem material from PG and PMEI plants. (A) Stem segments from Arabidopsis untransformed (WT), PG57, and PMEI7 plants were treated with Celluclast, and saccharification efficiency was measured after 24 h. (B and C) Stem segments from wheat untransformed (WT) and PMEI151 plants were treated with a mixture of Macerozyme and Celluclast without pretreatment (B) or after dilute acid pretreatment (C), and saccharification efficiency was measured after 24 h. Bars represent average saccharification efficiency ± SEM (N≥6). These experiments were repeated at least twice with similar results. Different letters indicate statistically significant differences, according to ANOVA followed by Tukey’s test (P < 0.01). Total sugars in stem material were the following (mg g^-1fresh weight ± SEM↦): Arabidopsis WT, 19.7 ± 0.5; PG57, 18.5 ± 0.04; PMEI7, 22.5 ± 1.6; wheat WT, 57.0 ± 4.8; PMEI151, 53.2 ± 4; pretreated wheat WT, 64.1 ± 3.3; and pretreated PMEI151, 64.8 ± 2.4. Differences in sugar content between transgenic and parental lines were not statistically different, according to Student’s t-test (P > 0.05).

Fig. 5.

Morphological analysis of Arabidopsis PMEI plants. (A) Photographs of representative Arabidopsis untransformed (Left) and PMEI7 (Right) plants grown for 30 days. (B) Transverse sections of hypocotyls of etiolated untransformed (Left) and PMEI7 (Right) seedlings were photographed in light microscopy. (Scale bar, 40 μm.)