Improving lignocellulosic biofuel production by CRISPR/Cas9‐mediated lignin modification in barley

• Published in: Global change biology. Bioenergy 2021-04, Vol.13 (4), p.742-752
• Main Authors: Lee, Jae Hoon, Won, Hyo Jun, Hoang Nguyen Tran, Phuong, Lee, Sun‐mi, Kim, Ho‐Youn, Jung, Je Hyeong
• Format: Article
• Language: English
• Subjects: Barley; Biodiesel fuels; Biofuels; Biomass; Biorefineries; Biosynthesis; Caffeic acid; caffeic acid O‐methyltransferase; Cell walls; Cellulose; Cereal crops; Cloning; CRISPR; CRISPR/Cas9; Deoxyribonucleic acid; DNA; DNA methylation; Efficiency; Environmental conditions; Enzymes; Ethanol; Genetic engineering; Genomes; Hydrolysates; Lignin; lignin modification; Lignocellulose; lignocellulosic biofuel; lignocellulosic biomass; Methyltransferase; Mutagenesis; Mutants; Polymerase chain reaction; Raw materials; Regulatory approval; Saccharification; Sorghum; Sugarcane
• ISSN: 1757-1693; 1757-1707
• DOI: 10.1111/gcbb.12808

Barley is a major cereal crop with a wide ecological range, and its lignocellulosic residues have the potential to be used as a feedstock for various purposes, including biofuel production. Lignocellulosic biomass is an abundant renewable source of carbon energy. However, its heterogeneous properties and intrinsic recalcitrance caused by cell wall lignification have lowered the biorefinery efficiency. The reduced lignin content and/or altered lignin structure have been desirable traits for lignocellulosic feedstock. We report the reduction of lignin content in barley by CRISPR/Cas9‐mediated mutagenesis of caffeic acid O‐methyltransferase 1 (HvCOMT1), the lignin biosynthetic gene responsible for lignin syringyl unit formation. The transgene‐free, homozygous HvCOMT1 mutant was generated and analyzed for its cell wall composition, saccharification efficiency, and bioethanol production performance. The mutant had 14% lower total lignin content and 34% higher fermentable glucose recovery rate, compared to the wild‐type (WT). The bioethanol concentration and yield from the hydrolysates of the mutant biomass were 14.3 g/L and 0.46 g/g total sugar, respectively. This result was 34% and 12% higher than those obtained from WT (10.7 g/L and 0.41 g/g total sugar). Under controlled environmental conditions, the overall growth performance of the HvCOMT1 mutant was similar to WT, with no distinct morphological variations between them. The HvCOMT1 mutant barley could offer improved quality lignocellulosic feedstock for efficient lignocellulosic biofuel production. Barley is a major cereal crop worldwide, and its lignocellulosic residues are a promising feedstock for biofuel production. To offer improved quality of biomass with less lignin content, this study uses CRISPR/Cas9‐mediated mutagenesis to generate transgene‐free, homozygous barley mutant of caffeic acid O‐methyltransferase 1 (HvCOMT1), the lignin biosynthetic gene responsible for lignin syringyl unit formation. Compared to the wild‐type (WT), the mutant had reduced total lignin content, leading to increased fermentable sugar recovery rate and bioethanol production. Under controlled environmental conditions, the overall growth performance of the HvCOMT1 mutant was comparable to WT without distinct morphological variations.