Silencing of a BAHD acyltransferase in sugarcane increases biomass digestibility

Abstract

Background: Sugarcane (Saccharum spp.) covers vast areas of land (around 25 million ha worldwide), and its processing is already linked into infrastructure for producing bioethanol in many countries. This makes it an ideal candidate for improving composition of its residues (mostly cell walls), making them more suitable for cellulosic ethanol production. In this paper, we report an approach to improving saccharification of sugarcane straw by RNAi silencing of the recently discovered BAHD01 gene responsible for feruloylation of grass cell walls.

Results: We identified six BAHD genes in the sugarcane genome (SacBAHDs) and generated five lines with substantially decreased SacBAHD01 expression. To find optimal conditions for determining saccharification of sugarcane straw, we tried multiple combinations of solvent and temperature pretreatment conditions, devising a predictive model for finding their effects on glucose release. Under optimal conditions, demonstrated by Organosolv pretreatment using 30% ethanol for 240 min, transgenic lines showed increases in saccharification efficiency of up to 24%. The three lines with improved saccharification efficiency had lower cell-wall ferulate content but unchanged monosaccharide and lignin compositions.

Conclusions: The silencing of SacBAHD01 gene and subsequent decrease of cell-wall ferulate contents indicate a promising novel biotechnological approach for improving the suitability of sugarcane residues for cellulosic ethanol production. In addition, the Organosolv pretreatment of the genetically modified biomass and the optimal conditions for the enzymatic hydrolysis presented here might be incorporated in the sugarcane industry for bioethanol production.

Keywords: Biofuels; Biomass; Cell-wall acylation; Lignocellulosic feedstock; Sugarcane.

Fig. 1

Phylogenetic and expression analyses of candidate clade BAHD genes. a Phylogenetic tree of candidate BAHD genes identified in Arabidopsis (AT), Brachypodium (Bradi), maize (GRMZM), rice (LOC Os), sorghum (Sb), Setaria viridis (Sevir), Setaria italic (Si), and sugarcane (Sac, Riacho-Panõn and Matiello [32]; Sh, Garsmeur et al. [33]). Support for the topology is shown as fraction of bootstrap runs. BAHD names for each branch are based on Molinari et al. [46], and alternative AT names are based on Bartley et al. [20]. b Real-time qPCR analysis of the identified BAHD genes in sugarcane. Expression is relative to the high expressed reference genes GAPDH and EF1-α. Young and mature leaves correspond to tissues from three- and eight-month-old sugarcane plants, respectively (n = 5; error bars ± SEM)

Fig. 2

Cell-wall characterization and digestibility of sugarcane straw biomass. a Saccharification of sugarcane straw from SacBAHD01 RNAi lines and nontransformed (NT) plants after Organosolv pretreatment, using Cellic CTec 3 (Novozymes) at 15 FPU for 6 and 48 h. b Ester-linked contents of ferulate (FA) and p-coumarate acid (pCA) in the alcohol insoluble fraction (AIR) of sugarcane straw from three independent events of SacBAHD01 RNAi lines and NT plants. c HCA conjugates in supernatant following mild acidolysis of sugarcane straw AIR. The data are represented as relative peak areas of major peaks for p-coumarate (pCA)-Ara and ferulate (FA)-Ara, previously identified by LC–MS as described by de Souza et al. [1]. n = 4; error bars ± SEM; significance of difference of transgenic lines from NT plants indicated if difference in means > least significant difference from ANOVA at p < 0.01 (*) and p < 0.001 (**)

Fig. 3

2D-NMR heteronuclear single-quantum coherence (HSQC) partial spectra of sugarcane straw from nontransformed (NT) plants and the three transgenic lines (1, 2.2, and 2.4). Color-coding of the contours matches that of the assigned structures; where contour overlap occurs, the colorization is only approximate. The analytical data are from volume integrals of correlation peaks representing reasonably well-resolved (except for H) C/H pairs in similar environments; thus they are from S_2/6, G₂, H_2/6, FA₂, pCA_2/6, and T_2´6´, with correction for those units that have two C/H pairs per unit. All relative integrals are on a G + S = 100% basis; H-units are over-quantified due to an overlapping peak from protein phenylalanine (Phe) units [38]