Distinctively altered lignin biosynthesis by site‐modification of OsCAD2 for enhanced biomass saccharification in rice

Abstract Crop straws represent enormous biomass resource convertible for biofuels and bioproducts, but lignocellulose recalcitrance restricts its saccharification for commercial utility. Despite genetic modification of lignin biosynthesis being attempted to reduce recalcitrance in bioenergy crops, it remains challenging to optimize lignin deposition without an unacceptable yield penalty. Based on gene expression analysis and phylogenetic tree profiling, a cinnamyl alcohol dehydrogenase gene ( OsCAD2 ) as the target for genetic engineering of lignin biosynthesis in rice was selected in this study. Using CRISPR/Cas9 technology, independent homozygous transgenic lines with precise site mutation of OsCAD2 , which showed slightly reduced lignin levels but markedly decreased p ‐hydroxyphenyl (H) contents in lignin by 34% and increased guaiacyl (G) contents by 16%, compared to the wild type were generated in this study. Under mild alkali pretreatment (1% NaOH, 50°C), the OsCAD2 site‐modified lines showed effective lignin extraction up to 70% (of total lignin) from mature rice straws, which caused either significantly increased biomass porosity and cellulose accessibility or remarkably reduced cellulase adsorption to lignin in pretreated lignocellulose residues. These consequently led to almost complete biomass enzymatic saccharification with increased hexoses yields by 61%–72% in the modified lines, being much higher than those of the lignin‐altered lines reported in previous studies. Hence, this study has demonstrated a novel genetic engineering strategy to reduce lignocellulose recalcitrance with minimized biomass loss for cost‐effective biomass conversion to bioethanol in rice and bioenergy crops.