Loss-of-function mutation of two phenylpropanoid pathway genes PAL1 and PAL2, coupled with MYB46 upregulation, increases fermentable sugar yield and ethanol production

Background: The recalcitrant nature of lignocellulosic biomass restricts the accessibility of hydrolytic enzymes to cellulose and hemicellulose. Although reducing lignin content improves the conversion of biomass to fermentable sugar, excessive reduction of lignin compromises cell wall integrity, lowering biomass yield. Results: This study aims to develop biotechnological means to genetically engineer the composition of lignocellulosic feedstock biomass for enhanced biomass conversion efficiency while minimizing adverse impacts of the genetic manipulation on plant growth. In order to test the concept, we produced transgenic Arabidopsis plants with reduced lignin content but enhanced cellulose and hemicellulose accumulation by combining CRISPR/Cas9-mediated knockout of two key phenylpropanoid biosynthetic genes PAL1 and PAL2 with the overexpression of MYB46, a central regulator of secondary wall biosynthesis. 35S::MYB46/pal1pal2 reduced lignin content by 36.2% while increasing cellulose content by 24.1% and hemicellulose content by 52.9%, compared to the wild type. In addition, 35S::MYB46/pal1pal2 increased cellulose content by 9.7% and hemicellulose content by 16.5%, compared to the pal1pal2 mutant. Enzymatic saccharification assays revealed that the biomass from the 35S::MYB46/pal1pal2 plants exhibited increased saccharification efficiency and glucose yield, compared to the wild type or the pal1pal2 mutant. Yeast-based ethanol fermentation assays further revealed that the elevated glucose yield resulted in a significant increase in fermentable sugar availability and ethanol production. Conclusion: This pathway-specific genetic engineering strategy provides a sustainable approach to bioenergy production by enhancing secondary cell wall integrity, compensating for reduced lignin through increased cellulose and hemicellulose accumulation, and improving bioethanol yields while minimizing adverse impacts on plant growth.