Abstract
Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.
Highlights
Plant lignocellulosic biomass is an important renewable and sustainable feedstock for the production of both biomaterials and biofuels [1,2]
Our results showed the significant reduction of total lignin contents in caffeoyl shikimate esterase (CSE)-clustered regularly interspaced short palindromic repeat (CRISPR) poplars up to 29.1% compared to BH poplars
Both CSE1-sg2 and CSE2-sg3 poplars exhibited collapses of xylem vessel cells (Figure 4), which is commonly found in plants that have defective accumulation of secondary wall components
Summary
Plant lignocellulosic biomass (i.e., wood) is an important renewable and sustainable feedstock for the production of both biomaterials and biofuels [1,2]. Biomass does not decompose due to the complex chemical and physical structure of the plant cell wall, which is referred to as biomass recalcitrance [6,7,8,9]. One of the major causes of biomass recalcitrance is the presence of lignin, a phenolic polymer that provides strength and hydrophobicity to the secondary cell wall. Lignin impedes the efficient enzymatic degradation of cellulose and hemicellulose into fermentable sugars by immobilizing hydrolytic enzymes and physically restricting access to the polysaccharide substrate [6,8,10,11]
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